WO2013151982A1 - Methods and compounds useful in treating pruritus, and methods for identifying such compounds - Google Patents

Methods and compounds useful in treating pruritus, and methods for identifying such compounds Download PDF

Info

Publication number
WO2013151982A1
WO2013151982A1 PCT/US2013/034925 US2013034925W WO2013151982A1 WO 2013151982 A1 WO2013151982 A1 WO 2013151982A1 US 2013034925 W US2013034925 W US 2013034925W WO 2013151982 A1 WO2013151982 A1 WO 2013151982A1
Authority
WO
WIPO (PCT)
Prior art keywords
antagonist
seq
test compound
receptor
protein
Prior art date
Application number
PCT/US2013/034925
Other languages
French (fr)
Inventor
Zheng Wei
Andew J. GROTTICK
David M. Mills
Brian M. Smith
Original Assignee
Arena Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Arena Pharmaceuticals, Inc. filed Critical Arena Pharmaceuticals, Inc.
Publication of WO2013151982A1 publication Critical patent/WO2013151982A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
    • C07D217/14—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders

Definitions

  • the present invention relates to methods of using histamine 3 receptor (H3R) antagonists to treat pruritus or itch in an individual and methods for using H3R to screen for compounds capable of treating pruritus in an individual and methods for using pruritus/itch model systems for screening H3R antagonists for their usefulness in treating pruritus.
  • H3R histamine 3 receptor
  • Pruritus or itch is the unpleasant sensation that leads to a desire to scratch (for reviews, see Journal of Investigative Dermatology (2006) 126: 1705-1718; and Lancet (2003) 361 : 690-94). It is a common and distressing symptom in a variety of conditions and diseases. Pruritus typically occurs in peripheral diseases, such as, allergic conjunctivitis, allergic rhinitis, hemorrhoids, atopic dermatitis, allergic dermatitis, acute and chronic urticaria (hives), psoriasis and dermatoses of fungal, allergic and non-allergic origin.
  • peripheral diseases such as, allergic conjunctivitis, allergic rhinitis, hemorrhoids, atopic dermatitis, allergic dermatitis, acute and chronic urticaria (hives), psoriasis and dermatoses of fungal, allergic and non-allergic origin.
  • Itching can also be a major symptom of many systemic diseases such as, Hodgkin's disease, chronic renal failure, polycythemia vera, hyperthyroidism, malignancy, infection, chronic cholestatic liver disease, and end-stage renal disease, and cholestasis.
  • systemic diseases such as, Hodgkin's disease, chronic renal failure, polycythemia vera, hyperthyroidism, malignancy, infection, chronic cholestatic liver disease, and end-stage renal disease, and cholestasis.
  • senile itch without an obvious cause, except perhaps xerosis, occurs in more than half of the population aged 70 years. In all cases chronic severe generalized itch can be disabling.
  • PBC primary biliary cirrhosis
  • PSC primary sclerosis cholangitis
  • Chronic renal disease epidural morphine
  • pregnancy diabetes mellitus, thyroid illness, hyperparathyroidism, iron deficiency anemia, viral infection, aquagenic pruritus, and psychogenic pruritus.
  • Pruritus causes sufferers to scratch, leading to skin damage, increased risk of skin infection, and worsening of inflammation.
  • the pathogenesis of pruritus is not well understood, and treatment options are limited (Paus, R., et al., J. Clin. Invest. 2006, 116: 1174-1185).
  • pruritus was once thought to be a subliminal form of pain (intensity theory), current evidence points to separate sensory neuronal systems mediating the two modalities.
  • pain and pruritus are dissociable. Pain and pruritus evoke different motor responses, scratching for pruritus and withdrawal for pain.
  • systemically-administered opioids have a dichotomous effect on these two sensory modalities.
  • ⁇ -Opioid receptor agonists reduce pain but can cause pruritus.
  • antagonizing the central mu-opioid receptors for example with naloxone or naltrexone, suppresses pruritus and at the same time may lower the pain threshold.
  • Pruritus due to skin inflammation is thought to be mediated at least partly by activation of skin mast cells, which release pruritogenic mediators to activate receptors on peripheral nerve endings to transmit itch signals.
  • histamine is a particularly potent pruritogen. Histamine injected into the skin causes strong itch sensations in humans and animals. Therefore, antagonists of the histamine receptors have been explored as pruritus treatments.
  • one aspect of the present invention relates to the inhibition of the H3R in an individual, such as by administration of a compound or agent of the present invention, can reduce itching or pruritus.
  • the G-protein coupled histamine 3 receptor H3R
  • the present invention provides a method of preventing and/or treating pruritus in an individual in need thereof by administering a therapeutically effective amount of a compound or agent that modulates the H3R.
  • peripherally restricted antagonists of H3R are capable of mediating the inhibition of itch. Accordingly, screening for peripherally restricted antagonists and the application of peripherally restricted antagonists in the various embodiments of the invention is contemplated. Peripherally restricted compounds may be advantageous to the extent that the peripheral restriction reduces the CNS effects of H3R inhibition. Such effects may include, for example, wakefulness.
  • H3R antagonists are peripherally restricted and may be assayed or screened based on their inability or reduced ability to inhibit H3R in the CNS (for example, in the brain).
  • the invention comprises a method for treating or preventing itching or the symptoms thereof in an individual wherein an antagonist of H3R is administered to the individual.
  • the invention disclosed herein is suitable for the prevention and/or treatment of pruritus that is associated with a disease or disorder, such as those described herein.
  • One aspect of the present invention encompasses every combination of one or more diseases or disorders for which itch is associated selected from the group: eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound
  • GPCR G protein-coupled receptor
  • GPCRs represent an important area for the development of pharmaceutical products. Drugs active at GPCRs have therapeutic benefit across a broad spectrum of human diseases as diverse as pain, cognitive dysfunction, hypertension, peptic ulcers, rhinitis, and asthma. Of the approximately 500 clinically marketed drugs, greater than 30% are modulators of GPCR function. These drugs exert their activity at approximately 30 well-characterized GPCRs. (See, e.g., Wise et al, Annu Rev Pharmacol Toxicol (2004) 44:43-66.)
  • GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane- 1 (TM-1), transmembrane-2 (TM-2), etc.).
  • the transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane- 3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or "extracellular" side, of the cell membrane (these are referred to as "extracellular" regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively).
  • transmembrane helices are also joined by strands of amino acids between transmembrane- 1 and transmembrane-2, transmembrane- 3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or "intracellular” side, of the cell membrane (these are referred to as "intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively).
  • the "carboxy" (“C”) terminus of the receptor lies in the intracellular space within the cell, and the "amino" (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
  • GPCR "promiscuous" with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, Life Sciences (1988) 43: 1095-1101. Although other G proteins exist, currently, Gq, Gs, Gi, Gz, and Go are G proteins that have been identified. Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as “signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Current understanding is that the IC-3 loop as well as the carboxy terminus of the receptor interacts with the G protein.
  • G proteins which appear to couple several classes of GPCRs to the phospholipase C pathway, such as G15 or G16 (Offermanns & Simon, J Biol Chem (1995) 270: 15175-80), or chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C (Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20: 118-24).
  • G15 or G16 Offermanns & Simon, J Biol Chem (1995) 270: 15175-80
  • chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C (Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20: 118-24).
  • GPCRs exist in the cell membrane in equilibrium between two different conformations: an "inactive" state and an “active” state.
  • a receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response.
  • Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
  • a receptor may be stabilized in an active state by a ligand or a compound such as a drug.
  • Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor.
  • GPCRs G-protein coupled receptors
  • At least four human and three rat splice variants have proven functional activity in pharmacological assays (Passani et al., Trends in Pharmacol. Sci. 2004, 25, 618-625).
  • Rat and human histamine H3 -receptors also show constitutive activity which means that they can transduce a signal even in the absence of a ligand. Histamine H3 -receptors also function as heteroreceptors, modulating the release of a number of other transmitter substances including serotonin, acetylcholine, dopamine and noradrenaline (see: Brown et al. Prog. Neurobiol. 2001, 63, 637-672).
  • the ligand functions as either an antagonist or inverse agonist (for reviews see: Leurs et al. Nat. Rev. Drug. Discov.
  • Antagonists of H1R also known as antihistamines, have long been used to treat allergies such as hay fever (rhinitis). Although they are also widely prescribed as anti -pruritus medications, their efficacy, particularly for chronic skin diseases such as atopic dermatitis, has been a subject of debate.
  • H4R has also recently been described to mediate itch. H4R deficient mice develop less severe pruritus compared to wild-type littermates, and the selective H4R receptor antagonist
  • JNJ7777120 inhibited pruritus in several mouse pruritus models (Dunford, P.J., et al., J. Allergy Clin. Immunol. 2007, 119: 176-83).
  • H3R inhibition does not reduce histamine-mediated pruritus. In fact, H3R inhibition has been linked to wakefulness and other related CNS mediated effects. Moreover, multiple groups (Br J Dermatol. 2003 Jul; 149 (1): 17-22 and Clin Exp Allergy. 2004 Mar;34 (3):456-9) have found that intradermal injection of H3R antagonists (i.e. thioperamide, iodophenpropit and clobenpropit) actually promote scratching behavior in mice. Therefore, prior to the present invention, existing reports suggested that H3R antagonism was not a viable treatment strategy for itch-associated disorders and, in fact, such treatment might actually be pruritogenic.
  • H3R antagonists i.e. thioperamide, iodophenpropit and clobenpropit
  • the present invention relates to the unexpected discovery that inhibition of the histamine 3 receptor (H3R) in an individual, such as by administration of a H3R antagonist, can reduce itching or pruritus.
  • the present invention describes methods relating to screening assays performed with H3R for identifying H3R antagonists, for example antagonists or inverse agonists, useful for treating a condition characterized by itching, methods for using H3R antagonists in model systems, for example pruritus model systems, for determining efficacy or usefulness, methods for using H3R antagonists (e.g. partial antagonists, full antagonists, and inverse agonists) in the treatment of a condition characterized by itching, and compounds useful for the treatment of a condition characterized by itching, for example, in an individual.
  • the individual is a human.
  • the present invention provides a method of treating pruritus in a subject in need thereof by administering a therapeutically effective amount of a compound or agent that modulates the H3R.
  • the compound or agent is an H3R antagonist.
  • the H3R antagonist is a H3R inverse agonist.
  • peripherally restricted antagonists of H3R are capable of mediating the inhibition of itch. Accordingly, screening for peripherally restricted antagonists (e.g.
  • H3R antagonists for example H3R inverse agonists, are peripherally restricted and may be assayed or screened based on their inability or reduced ability to inhibit H3R in the CNS (for example, in the brain).
  • H3R antagonists have been disclosed in the art. For example,
  • H3R antagonists are disclosed in the following PCT Publications. Accordingly, one aspect of the present invention encompasses every combination of one or more H3R antagonist selected from the H3R antagonists disclosed in each of the PCT Publications and pharmaceutically acceptable salts, solvates and hydrates thereof:
  • One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist.
  • One aspect of the present invention relates to use of a H3R antagonist in the manufacture of a medicament for treating pruritus.
  • One aspect of the present invention relates to H3R antagonists for use in a method for treating pruritus.
  • the invention disclosed herein is suitable for the treatment of pruritus that is associated with a disease or disorder selected from eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound
  • the invention disclosed herein utilizes a H3R antagonist, for example, a H3R antagonist from the genera described herein.
  • the invention disclosed herein utilizes a H3R antagonist, for example, a H3R antagonist found in Table A.
  • the H3R antagonist is peripherally restricted. In one embodiment, the H3R antagonist is applied topically to the site afflicted with itch. In another embodiment, the H3R antagonist is administered systemically. In one embodiment, the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of the H3R antagonist as the active ingredient, in admixture with a pharmaceutical carrier.
  • the H3R antagonist pharmaceutical composition can be formulated in a form suitable for topical application such as a skin patch.
  • the H3R antagonist is administered in conjunction with a pharmaceutically acceptable carrier.
  • the invention features a method for identifying a compound capable of inhibiting itch in an individual, the method comprising administering a H3R antagonist, such as an antagonist or an inverse agonist, to a mammal for which reduction of itch may be evaluated, and evaluating whether the administration of the compound results in reduced itch.
  • a H3R antagonist such as an antagonist or an inverse agonist
  • H3R antagonists may be screened in established models for evaluating itch (i.e. animal, cellular, in vitro or in vivo).
  • the invention features a method for identifying an antagonist (e.g. partial antagonists, full antagonists, and inverse agonists) of a H3R comprising: (a) contacting cells expressing the H3R in the presence of a known amount of histamine or surrogate thereof with a sample to be tested for the presence of a H3R antagonist; and (b) measuring at least one cellular function modulated by the binding of a ligand to the H3R, wherein the cellular function is selected from the group consisting of changes in intracellular second- messengerger levels, cell growth rates and hormone secretion; whereby the H3R antagonist in the sample is identified by measuring its effect on the cellular function compared to what would be measured in the absence of such antagonist.
  • an antagonist e.g. partial antagonists, full antagonists, and inverse agonists
  • the nucleotide sequence encoding human H3R polypeptide is given in SEQ ID NO: 1.
  • the amino acid sequence of the encoded human H3R polypeptide is given in SEQ ID NO:2.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • amino acids 2-445 of SEQ ID NO:2 with the proviso that the G protein-coupled receptor does not comprise the amino acid sequence of SEQ ID NO:2;
  • test compound wherein the ability of the test compound to decrease the response in a pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • the G protein-coupled receptor comprises the amino acid sequence of SEQ ID NO:2.
  • the variant of SEQ ID NO:2 is an allele of SEQ ID NO:2.
  • the variant of SEQ ID NO:2 is an ortholog of SEQ ID NO:2. In certain embodiments, the variant of SEQ ID NO:2 is a mammalian ortholog of SEQ ID NO:2.
  • the G protein-coupled receptor is recombinant.
  • the method is a method for identifying compounds useful for treating a condition characterized by itch.
  • the host cell is derived or obtained from a vertebrate.
  • the vertebrate is a mammal. In certain embodiments, the vertebrate is a non-human vertebrate. In certain embodiments, the mammal is a non-human mammal.
  • the invention additionally features a method for identifying compounds useful for treating a condition characterized by itch in an individual, optionally comprising steps (a) and (b) of the prior embodiments, and further comprising:
  • step (c) optionally synthesizing a compound which inhibits functionality of the receptor in step (b);
  • step (d) administering a compound which inhibits functionality of the receptor in step (b) to a vertebrate;
  • test compound wherein the ability of the test compound to treat or prevent pruritus in the vertebrate is indicative of the test compound being a compound useful for treating a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, further comprising the step of: determining the chemical structure of the anti-pruritic agent. It is understood that this step can occur at any time after the step of determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (H3R) or the step of determining whether less of the complex (i.e., between the known ligand and the G protein-coupled receptor (H3R)) is formed in the presence of the test compound than in the absence of the test compound.
  • this step can occur at any time after the step of determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (H3R) or the step of determining whether less of the complex (i.e., between the known ligand and the G protein-coupled receptor (H3R)) is formed in the presence of the test compound than in the absence of the test compound.
  • step (a) the steps of contacting a test compound with a host cell or with membrane of a host cell comprising a G protein-coupled receptor (i.e., step (a)) and determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (i.e., step (b)) are not required.
  • the determining comprises measuring a level of itch in the vertebrate.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • test compound wherein the ability of the test compound to reduce the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • test compound wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • test compound wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • optionally labeled known ligand refers to a known ligand that is optionally labeled, such as, a known ligand where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring).
  • Suitable radionuclides that may be incorporated in the known ligand include but are not limited to 3 H (also written as T for tritium), n C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 18 F, 35 S, 36 C1, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 1, 125 I, and 131 I.
  • 3 H also written as T for tritium
  • n C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 18 F 35 S, 36 C1, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 1, 125 I, and 131 I.
  • a know ligand that incorporates 3 H, 14 C, 35 S, 82 Br, 75 Br, 76 Br, 77 Br, 125 I, and 131 I will generally be most useful.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • test compound wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • test compound (d) observing a decreased response in a pruritus model test animal previously administered the test compound; wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
  • G protein-coupled receptor (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • test compound wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
  • One aspect of the present invention relates to methods for identifying an anti-pruritic agent for treating a condition characterized by itch in an individual, comprising the steps of:
  • H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent useful for the treatment of said condition characterized by itch in said individual.
  • two or more potential or actual H3R antagonists or anti-pruritic agents may be compared with each other in pruritus models. Such a comparison may be based, for example, on efficacy or brain to plasma distribution or any other aspect relevant to the H3R inhbitior or anti-pruritic agent(s) as described herein.
  • One aspect of the present invention relates to methods for evaluating two or more H3R antagonists comprising the steps of:
  • evaluating the two or more anti-pruritic agents by comparing the ability of each anti-pruritic agent to decrease a behavioral response to pruritus.
  • One aspect of the present invention relates to methods for evaluating a H3R antagonist at two or more different concentrations comprising the steps of:
  • H3R antagonist separately introducing or evaluating the H3R antagonist at two or more concentrations in a pruritus model
  • evaluating further comprises selecting the anti-pruritic agent or concentration of anti-pruritic agent with the best efficacy.
  • the G protein-coupled receptor is coupled to a G protein. In certain embodiments, activation of the G protein-coupled receptor decreases a level of intracellular cAMP. In certain embodiments, the G protein is Gi/o.
  • the G protein-coupled receptor variant is a variant that is encoded by a polynucleotide that is amplifiable by polymerase chain reaction of SEQ ID NO:2 or an allele thereof.
  • the G protein-coupled receptor encoded by a polynucleotide that is amplifiable by polymerase chain reaction is an allele of SEQ ID NO:2.
  • the G protein-coupled receptor encoded by a polynucleotide that is amplifiable by polymerase chain reaction exhibits a detectable level of constitutive activity.
  • the constitutive activity is for increasing a level of intracellular cAMP.
  • the constitutive activity is for causing melanophore cells to undergo pigment dispersion.
  • stringent hybridization conditions comprise hybridization at 42°C in a solution comprising 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/mL denatured, sheared salmon sperm DNA, followed by washing at 65°C in a solution comprising O.lxSSC.
  • Hybridization techniques are well known to the skilled artisan.
  • the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is SEQ ID NO:2 or an allele thereof.
  • the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an allele of SEQ ID NO:2. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an ortholog of SEQ ID NO:2. In some embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 exhibits a detectable level of constitutive activity. In some embodiments, the constitutive activity is for increasing a level of intracellular cAMP. In some embodiments, the constitutive activity is for causing melanophore cells to undergo pigment dispersion.
  • the G protein-coupled receptor is part of a fusion protein comprising a G protein.
  • Techniques for making a GPCR:G fusion construct are well known to the skilled artisan (see, e.g., International Application WO2002/42461).
  • the host cell comprises an expression vector, the expression vector comprising a polynucleotide encoding the G protein- coupled receptor.
  • the expression vector is pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 University Boulevard., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of
  • Microorganisms for the Purpose of Patent Procedure The DNA was tested by the ATCC and determined to be viable.
  • the ATCC has assigned the following deposit number to pCMV: ATCC #203351.
  • Other suitable expression vectors will be readily apparent to those of ordinary skill in the art, and a wide variety of expression vectors are commercially available (e.g., from Clontech, Palo Alto, CA; Stratagene, La Jolla, CA; and Invitrogen, Carlsbad, CA).
  • the activated G protein-coupled receptor is constitutively active.
  • the activated G protein-coupled receptor is activated by a ligand.
  • the receptor comprises the amino acid sequence of SEQ ID NO:2.
  • the step determining the ability of the test compound to inhibit the functionality of the receptor is through the measurement of a level of a second messenger.
  • the second messenger is selected from the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate ( ⁇ 3 ), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca 2+ .
  • cAMP cyclic AMP
  • cGMP cyclic GMP
  • DAG diacylglycerol
  • MEKK1 MAPK/ERK kinase kinase- 1
  • the second messenger is cAMP and the level of cAMP is decreased.
  • the level of cAMP is decreased.
  • the step determining is through the use of a Melanophore assay, or through the measurement of GTPyS binding to a membrane comprising said GPCR.
  • the anti-pruritic agent is a H3R antagonist.
  • the H3R antagonist is a H3R inverse agonist.
  • the H3R antagonist or anti-pruritic agent is peripherally restricted.
  • the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.5 or less after administration in a pruritus model.
  • the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less after administration in a pruritus model.
  • the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.1 or less after administration in a pruritus model.
  • the pruritus model is a vertebrate model.
  • the vertebrate model is a mammalian model.
  • the pruritus model is a primate model, a dog model, a guinea pig model, a mouse model, or a cat model.
  • the pruritus model is selected from the group: pruritogen injection model, passive cutaneous anaphylaxis model, allergic pruritus model, and spontaneous pruritus model.
  • the pruritus model is selected from the group: histamine induced pruritus model, DNP-Ovalbumin pruritus model, and DNFB pruritus model.
  • the response to pruritus is a behavioral response.
  • the individual is a non-human mammal.
  • the individual is a human.
  • One aspect of the present invention pertains to methods and processes, further comprising the step of subsequently admixing the anti-pruritic agent with a pharmaceutical carrier.
  • One aspect of the present invention pertains to methods and processes, further comprising the step of subsequently admixing the anti-pruritic agent with a pharmaceutical carrier to form an anti-pruritic pharmaceutical composition.
  • One aspect of the present invention pertains to methods and processes, further comprising the step of formulating the anti-pruritic pharmaceutical composition into a form suitable for oral application.
  • One aspect of the present invention pertains to methods and processes, further comprising the step of formulating said anti-pruritic pharmaceutical composition into a form suitable for topical application.
  • the anti-pruritic agent is subsequently admixed with a
  • the anti-pruritic agent is subsequently formulated with a pharmaceutical carrier.
  • the step determining the ability of the test compound to inhibit functionality of the receptor comprises comparing the functionality of the receptor in the presence and absence of the test compound and observing a decreased functionality in the presence of the test compound as compared to in the absence of the test compound.
  • the host cell is a vertebrate cell. In some embodiments, the host cell is mammalian. In some embodiments, the mammalian host cell is selected from the group consisting of 293, 293T, CHO, MCB3901, and COS-7. In some embodiments, the host cell is melanophore. Other suitable host cells will be readily apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.
  • the determining is consistent with the G protein-coupled receptor being a Gs-coupled receptor.
  • the determining is consistent with the G protein-coupled receptor being coupled through a promiscuous G protein, such as Gal5 or Gal6, to the phospholipase C pathway.
  • a promiscuous G protein such as Gal5 or Gal6
  • Promiscuous G proteins are well known to the skilled artisan (see, e.g., Offermanns et al., J Biol Chem (1995) 270: 15175-15180).
  • the determining is consistent with the G protein-coupled receptor being coupled through a chimeric G protein, e.g. to the
  • Chimeric G proteins are well known to the skilled artisan (see, e.g., Milligan et al., Trends in Pharmaceutical Sciences (1999) 20: 118-124; and WO2002/42461).
  • the determining is through the measurement of a level of a second messenger.
  • the determining is through the measurement of a level of a second messenger selected from the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate (IP 3 ), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca 2+ .
  • a second messenger is cAMP.
  • a level of intracellular cAMP is increased.
  • the determining is carried out using membrane comprising the G protein-coupled receptor.
  • the determining is through the use of a melanophore assay. In certain embodiments, a level of pigment dispersion is increased.
  • the determining is through the measurement of an activity mediated by increasing a level of intracellular cAMP.
  • the determining is through CRE-Luc reporter assay. In certain embodiments, a level of luciferase activity is increased.
  • the determining is through the measurement of GTPyS binding to membrane comprising the G protein-coupled receptor.
  • the GTPyS is labeled with [ 35 S].
  • the GTPyS binding to membrane comprising the GPCR is increased.
  • the test compound is a small molecule.
  • the test compound is a small molecule, with the proviso that the small molecule is not a polypeptide.
  • the test compound is a small molecule, with the proviso that the small molecule is not an antibody or an antigen-binding fragment thereof.
  • the test compound is a small molecule, with the proviso that the small molecule is not a lipid.
  • the test compound is a small molecule, with the proviso that the small molecule is not a polypeptide or a lipid. In some embodiments, the test compound is a polypeptide. In some embodiments, the test compound is a polypeptide, with the proviso that the polypeptide is not an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is a lipid. In some embodiments, the test compound is not an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is an anti-pruritic agent, a H3R antagonist, a H3R antagonist, or a H3R inverse agonist.
  • the test compound is an anti-pruritic agent. In some embodiments, the test compound is a H3R antagonist. In some embodiments, the test compound is a H3R antagonist. In some embodiments, the test compound is a H3R inverse agonist.
  • the known ligand is a ligand or agonist of an endogenous vertebrate, mammalian, or human H3R receptor. In certain embodiments, the known ligand is a known agonist of an endogenous vertebrate, mammalian, or human H3R receptor. In certain embodiments, the known ligand is a ligand or agonist of an endogenous human H3R receptor.
  • the known ligand is an endogenous ligand of an endogenous vertebrate, mammalian, or human H3R receptor.
  • the optionally labeled known ligand is a labeled known ligand.
  • the labeled known ligand is a radiolabeled known ligand. Techniques for radiolabeling a compound, such as for labeling a known ligand of a G protein-coupled receptor of the invention, are well known to the skilled artisan. See, e.g., International Application
  • the G protein-coupled receptor is part of a fusion protein comprising a G protein.
  • Techniques for making a GPCR:G fusion construct are well known to the skilled artisan (see, e.g., International Application WO2002/42461).
  • the determining is carried out using a host cell comprising the G protein-coupled receptor.
  • the host cell comprises an expression vector, the expression vector comprising a polynucleotide encoding the GPCR.
  • the expression vector is pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 University Boulevard., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of
  • Microorganisms for the Purpose of Patent Procedure The DNA was tested by the ATCC and determined to be viable.
  • the ATCC has assigned the following deposit number to pCMV: ATCC #203351.
  • Other suitable expression vectors will be readily apparent to those of ordinary skill in the art, and a wide variety of expression vectors are commercially available (e.g., from Clontech, Palo Alto, CA; Stratagene, La Jolla, CA; and Invitrogen, Carlsbad, CA).
  • the host cell is a vertebrate cell. In some embodiments, the host cell is mammalian. In some embodiments, the mammalian host cell is selected from the group consisting of 293, 293T, CHO, MCB3901, and COS-7. In some embodiments, the host cell is melanophore. Other suitable host cells will be readily apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.
  • the determining is carried out using membrane comprising the G protein-coupled receptor.
  • the method further comprises the step of optionally providing the name or structure of the compound useful for treating a condition characterized by itch in an individual.
  • the invention features a method comprising, having identified a compound for treating a condition characterized by itch in an individual according to the invention, formulating the compound for treating a condition characterized by itch in an individual into a pharmaceutical composition.
  • the invention features use of a G protein-coupled receptor to screen test compounds as anti-pruritic agents for treating a condition characterized by itch in an individual, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • polynucleotide that is amplifiable by polymerase chain reaction (PCR) on a human DNA sample
  • polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
  • the screen is for an antagonist of the receptor.
  • the screen is for a partial antagonist of the receptor.
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition
  • a pharmaceutical composition comprising formulating an anti-pruritic agent with a pharmaceutically acceptable carrier; wherein the anti-pruritic agent is a H3R antagonist.
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition
  • a pharmaceutical composition comprising formulating a H3R antagonist with a pharmaceutically acceptable carrier; wherein the H3R antagonist is an anti-pruritic agent.
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition
  • a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having contacted in vitro with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
  • test compound a biologically active fragment of any one of (i) to (v); wherein the ability of said test compound to inhibit the functionality of the activated G protein-coupled receptor in a host cell or with a membrane of a host cell is indicative of said test compound being an anti-pruritic agent;
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising: (a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having obtained a decreased response for said H3R antagonist in a pruritus model, wherein the ability of said H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent, said method comprising formulating said H3R antagonist having the effect of an anti-pruritic agent with a pharmaceutically acceptable carrier as a pharmaceutical composition.
  • One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
  • One aspect of the present invention relates to pharmaceutical compositions prepared by any of the methods or processes described herein.
  • One aspect of the present invention relates to pharmaceutical compositions obtained by any of the methods or processes described herein.
  • One aspect of the present invention relates to pharmaceutical compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is for the use in treating pruritus.
  • One aspect of the present invention relates to pharmaceutical compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is for the use in treating pruritus.
  • compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the H3R antagonist is identified using any of the methods or processes described herein.
  • One aspect of the present invention relates to pharmaceutical compositions according to claim 12, wherein the H3R antagonist is identified using any of the methods or processes described herein and the pharmaceutical composition is for the use in treating pruritus.
  • compositions comprising a
  • H3R antagonist of the present invention and a pharmaceutically acceptable carrier.
  • One aspect of the present invention pertains to methods for the treatment of pruritus in an individual, comprising administering to the individual in need thereof, a therapeutically effective amount of a H3R antagonist of the present invention or a pharmaceutical composition thereof.
  • One aspect of the present invention pertains to the use of a H3R antagonist of the present invention or a pharmaceutical composition thereof in the manufacture of a medicament for the treatment of pruritus.
  • One aspect of the present invention pertains to H3R antagonists of the present invention or a pharmaceutical composition thereof for use in a method of treatment of pruritus.
  • One aspect of the present invention pertains to H3R antagonists for preparing a composition comprising admixing a H3R antagonist of the present invention and a
  • Figure 1 shows the polynucleotide sequence of the human H3R.
  • Figure 2 shows the polypeptide sequence of the human H3R.
  • Figure 3 shows the polynucleotide sequence of the mouse H3R.
  • Figure 4 shows the polypeptide sequence of the mouse H3R.
  • Figure 5 shows that orally administered H3R antagonists are able to inhibit pruritus in a variety of pruritus animal models.
  • Figure 5A shows that oral administration of Compound B and Compound C inhibit histamine (HA) induced pruritus.
  • Figure 5B shows that oral administration of Compound A inhibits histamine-induced pruritus.
  • Figure 5C shows that oral administration of Compound A inhibits serotonin-induced pruritus.
  • Figure 5D shows that oral administration of Compound A inhibits Compound 48/80-induced pruritus.
  • Figure 6 shows that topical application of Compound A inhibits histamine-induced itch.
  • Figure 7 shows that administration of Compound A inhibits DNP-ovalbumin mediated allergic pruritus.
  • Figure 8 shows that central administration of a H3R antagonist, Compound A, does not inhibit histamine-induced pruritus.
  • Figure 8A shows ICV administration of Compound A increases cognitive function in mice.
  • Figure 8B shows that ICV administration of Compound A is unable to inhibit histamine-induced pruritus. Suggesting that brain exposure of a H3R antagonist is not sufficient and that histamine induced pruritus is peripherally mediated.
  • Figure 9 shows that administration of Compound 1 inhibits chronic DNFB -mediated allergic pruritus.
  • Figure 10 shows the data from the administration of Compound 1 in the histamine- induced pruritus mouse model. The data shows that Compound 1 effectively inhibits histamine- induced pruritus in mice.
  • Figure 11 shows the data from the administration of Compound 3 in the histamine- induced pruritus mouse model. The data shows that Compound 3 effectively inhibits histamine- induced pruritus in mice.
  • Figure 12 shows a general synthesis of biaryl derivatives useful as intermediates in the preparation of compounds of the present invention (Formula (Va)).
  • a mesylate derivative is coupled with an R 1 substituted pyrrolidine and subsequently converted to an aryl boronic acid by treatment of an aryl lithium intermediate with triisopropylborate followed by hydrolysis.
  • the aryl boronic acid is coupled with an aryl bromide in the presence of a palladium catalyst to prepare the biaryl derivatives.
  • Figure 13 shows general methods for preparing compounds of the present invention (Formula (Va)).
  • Method A shows the use of carboxylic acids in the presence of a coupling agent to prepare compounds of the invention
  • Method B shows the use of acid chlorides to prepare compounds of the invention, optionally in the presence of a base, such as an amine base.
  • Figure 14 shows a general method for preparing compounds of the present invention (Formula (Va)).
  • Method C shows the use of acid anhydrides in the presence of a base to prepare compounds of the invention.
  • the present invention features methods of using H3R receptor to identify compounds useful for treating pruritus.
  • Antagonists of H3R receptor are useful as therapeutic agents for treating a condition characterized by itch, such as pruritus.
  • the present invention is based, at least in part, on the surprising discovery by Applicant that administration of a H3R antagonist to an individual, such as by oral administration, can act at the H3R receptor to reduce, prevent or treat pruritus and its symptoms. Accordingly, the invention further includes methods of evaluating known H3R antagonists in a variety of pruritus models for their usefulness in the treatment or prevention of pruritus.
  • substituted indicates that at least one hydrogen atom of the chemical group is replaced by a non-hydrogen substituent or group, the non-hydrogen substituent or group can be monovalent or divalent. When the substituent or group is divalent, then it is understood that this group is further substituted with another substituent or group.
  • a chemical group herein when a chemical group herein is "substituted" it may have up to the full valance of substitution; for example, a methyl group can be substituted by 1, 2, or 3 substituents, a methylene group can be substituted by 1 or 2 substituents, a phenyl group can be substituted by 1, 2, 3, 4, or 5 substituents, a naphthyl group can be substituted by 1, 2, 3, 4, 5, 6, or 7 substituents, and the like.
  • substituted with one or more substituents refers to the substitution of a group with one substituent up to the total number of substituents physically allowed by the group. Further, when a group is substituted with more than one group they can be identical or they can be different.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. It is understood that the various tautomeric forms are within the scope of the compounds of the present invention.
  • compounds of Formulae (la), (Ila), (Ilia), (IVa), (IVb), and (Va) and formulae related thereto may have one or more chiral centers and therefore can exist as enantiomers and or diastereoisomers.
  • the invention is understood to extend to and embrace all such enantiomers, diastereoisomers and mixtures thereof, including but not limited to racemates.
  • compounds of Formulae (la), (Ha), (Ilia), (IVa), (IVb), and (Va) and other formulae used throughout this disclosure represent all individual enantiomers and mixtures thereof, unless stated or shown otherwise.
  • ligand shall mean a molecule (e.g., test compound) that specifically binds to a polypeptide, such as H3R.
  • a ligand may be, for example, a polypeptide, a lipid, a small molecule, an antibody.
  • An endogenous ligand is a ligand that is an endogenous, natural ligand for a native polypeptide, such as H3R.
  • a ligand may be an "antagonist”, “agonist”, “partial agonist”, or “inverse agonist”, or the like. Exemplary antagonists of the H3R receptor are described herein.
  • the H3R antagonist is a H3R inverse agonist.
  • One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2008/005338 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • H3R receptor ligands selected from compounds of Formula (la) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • R' andR 2 are each selected independently from the group consisting of H, C1-C6 acyl, Ci- C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C - alkylenyl, aryloxy-Ci-C 4 -alkylenyl, heteroaryl-Ci-C 4 -alkylenyl and heteroaryloxy-Ci-C 4 -alkylenyl, and each R 1 andR 2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C 2 -C 8 alkenyl, Ci-C6 alkoxy, Ci-C 8 alkyl, Ci-Cs alkylcarboxamide, C 2 -C 8 alkynyl, Ci
  • R 1 and R 2 together with the nitrogen atom to which they are both bonded form a C 3 -C 7 heterocyclyl or a C5-C10 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C 2 -C 8 alkenyl, C1-C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8
  • Ci-C 8 alkylsulfonamide Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; wherein each
  • R 2 is selected independently from the group consisting of H, C1-C6 acyl, Ci-C 8 alkyl, C 2 -C 8 alkenyl, C 2 -C 8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C -alkylenyl, aryloxy-Ci-C -alkylenyl, heteroaryl-Ci-C -alkylenyl and heteroaryloxy-Ci-C -alkylenyl, and each R 2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C 6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide
  • R 1 and R 12 together with the atoms to which they are both bonded form a C6-C 8 heterocyclyl group optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C 2 -C 8 alkenyl, Ci-C6 alkoxy, Ci-C 8 alkyl, Ci-Cs alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, aryl, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C 3 -C 7 cycloalkyl, C 2 -C 8 dial
  • J is -CH 2 CH 2 - or a l,2-C 3 -C 7 -cycloalkylenyl group, each optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of C 1 -C3 alkyl, C 1 -C4 alkoxy, carboxy, cyano, C 1 -C3 haloalkyl, halogen, hydroxyl and oxo;
  • R 3 , R 4 , R 5 , R 6 , R 7 , R 10 , R 11 and R 12 are each selected independently from the group consisting of H, C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C 6 -alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -
  • R 8 and R 9 are each selected independently from the group consisting of H, Ci-C 8 alkyl, C 2 - C 8 alkenyl, C 2 -C 8 alkynyl, C 3 -C 7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C 4 -alkylenyl, aryloxy-Ci-C 4 -alkylenyl, heteroaryl-Ci-C 4 -alkylenyl and heteroaryloxy-Ci-C 4 -alkylenyl, and each R 8 and R 9 is optionally substituted with 1 , 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C 6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, C C 8 alkyl, C C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsul
  • R 8 and R 9 together with the nitrogen atom to which they are both bonded form a C 3 -C 7 heterocyclyl or a C5-C 10 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8
  • alkylsulfonamide Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1 -C6 haloalkoxy, C 1 -C6 haloalkyl, C 1 -C6 haloalkylsulfinyl, C 1 -C6 haloalkylsulfonyl, C 1 -C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; provided that
  • One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2008/048609 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • H3R receptor ligands selected from compounds of Formula (Ha) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • R 1 is selected from the group consisting of H, C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 - C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C 3 -C 7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1 -C6 haloalkoxy, C
  • Ci-C 8 alkyl Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -
  • R 1 together with the W-S0 2 group and the ring atom to which the W-S0 2 group is bonded form a C5-C7 heterocyclic ring with Ring A whereby the C5-C7 heterocyclic ring and Ring A share two adjacent ring atoms, and the C5-C7 heterocyclic ring is optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8
  • alkylsulfonamide Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1 -C6 haloalkoxy, C 1 -C6 haloalkyl, C 1 -C6 haloalkylsulfinyl, C 1 -C6 haloalkylsulfonyl, C 1 -C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide;
  • W is C 1 -C4 alkylene, C 2 -C 4 alkenylene, C3-C7 cycloalkylene, C3-C7 heterocyclylene or phenylene, each optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 substituents selected
  • Ring A is 1,3-phenylene or 1,4-phenylene, each substituted with R 12 , R 13 , R 14 and R 15 , wherein R 12 , R 13 , R 14 and R 15 are each selected independently from the group consisting of H, C 1 -C6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, C C 8 alkylsulfonamide, C C 8 alkylsulfinyl, C C 8 alkylsulfonyl, C C 8 alkylthio, C C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C
  • Ring A is a 6-membered heteroarylene or a 5-membered heteroarylene, each optionally substituted with R 16 , R 17 and R 18 , wherein R 16 , R 17 and R 18 are each selected independently from the group consisting of Ci-C 6 acyl, Ci-C 6 acyloxy, C 2 -C 8 alkenyl, Ci-C 6 alkoxy, C C 8 alkyl, C C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C 6 - alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C
  • R 2 , R 3 , R 4 andR 5 are each selected independently from the group consisting of H, C 1 -C6 acyl, C 1 -C6 acyloxy, C 2 -C 8 alkenyl, C 1 -C6 alkoxy, Ci-C 8 alkyl, Ci-C 8 alkylcarboxamide, C 2 -C 8 alkynyl, Ci-C 8 alkylsulfonamide, Ci-C 8 alkylsulfinyl, Ci-C 8 alkylsulfonyl, Ci-C 8 alkylthio, Ci-C 8 alkylureyl, amino, Ci-C 8 alkylamino, C 2 -C 8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C 2 -C 8 dialkylcarboxamide, C 2 -C 8 dialkylsulfonamide, halogen, C 1
  • R 6 , R 7 , R 8 andR 9 are each selected independently from the group consisting of H, C 1 -C3 alkyl, C 1 -C4 alkoxy, carboxy, cyano, C 1 -C3 haloalkyl, halogen and hydroxyl; and
  • R 10 and R 11 together with the nitrogen atom to which they are both bonded form 2-methyl- pyrrolidin-l-yl;
  • Ring B and the sulfur of the R 1 -W-S(0)2- group are not bonded to adjacent ring atoms of Ring A;
  • Ring A is 1,3-phenylene or 1,4-phenylene
  • W is C3-C7 heterocyclylene
  • the ring atom of W that is directly bonded to the sulfur of the R 1 -W-S(0)2- group is other than nitrogen.
  • H3R receptor ligand selected from (R)- 1 - ⁇ 2- [4'-(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl ⁇ -2-methyl -pyrrolidine and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • H3R receptor ligands selected from the groups consisting of:
  • One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2009/058300 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • H3R receptor ligands selected from compounds of Formula (Ilia) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • ring A is heterocyclyl optionally substituted with one, two or three substituents selected from C 1 -C6 alkyl and oxo; wherein each Ci-C6 alkyl is optionally substituted with a Ci-C6 alkoxy substituent;
  • R 1 is H, C 1 -C6 alkoxy, Ci-C6 alkyl or halogen
  • R 2 is H, C 1 -C6 alkoxy, Ci-C6 alkyl or halogen
  • R 3 is H, C 1 -C6 alkoxy, Ci-C6 alkyl or halogen
  • R 4 is H or Ci-C 4 alkyl
  • n 0, 1 or 2.
  • One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2009/ 105206 pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • H3R receptor ligands selected from compounds of Formula (IVa) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • R 1 is H or C 1 -C4 alkyl
  • R 2 is H or halogen
  • R 3 is H, C 1 -C4 alkyl or C 3 -C6 cycloalkyl, and R 4 is H; or R 3 and R 4 together with the atom to which they are both bonded form a C 3 -C6 cycloalkyl;
  • R 5 is selected from: C 1 -C6 alkyl, aryl, C 3 -C6 cycloalkyl, heteroaryl and heterocyclyl; each of which is optionally substituted with one or more substituents selected from: C 1 -C6 alkoxy, halogen, heterocyclyl and hydroxyl;
  • R 6 , R 7 and R 8 are each independently selected from: H, Ci-Ce alkoxy, C 1 -C6 alkyl, amino, halogen, heterocyclyl and hydroxyl;
  • n 0 or 1
  • n 1 or 2;
  • V is CH 2 , O or absent.
  • H3R receptor ligands selected from compounds of Formula (IVb) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • R 5 is selected from: methyl, cyclopropyl, tetrahydropyran-4-yl, methoxymethyl, 2- methoxyethyl, 3-methoxypropyl, hydroxymethyl, 2-hydroxyethyl, tetrahydropyran-4-ylmethyl, 2,2- difluorocyclopropyl, 4-methoxyphenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, 6- hydroxypyridin-3-yl and 2-hydroxypyridin-4-yl.
  • Compound A (R)-2-hydroxy- l-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)ethanone (referred to as Compound A herein), see Compound 10 in PCT publication WO2009/105206.
  • H3R receptor ligands selected from (R)-2-hydroxy- 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)ethanone and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • One aspect of the present invention encompasses, inter alia, certain isoquinoline derivatives selected from compounds of Formula (Va) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • n, and p are each independently 0 or 1;
  • X is selected from the group: -S-, -0-, C3-C6 cycloalkylene, and CR C R C ;
  • R 1 is H or C 1 -C4 alkyl
  • R 2 is selected from the group: C 1 -C4 alkoxycarbonyl, carboxyl, and l/i-tetrazol-5-yl; and each R , R b , and R c is independently selected from the group: H, C 1 -C3 alkyl, amino, halogen, and hydroxyl.
  • One aspect of the present invention encompasses every combination of one or more H3R inhibitors selected from the following H3R inhibitors and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • One aspect of the present invention encompasses every combination of one or more H3R inhibitors selected from the H3R inhibitors in Table A and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • H3R receptor ligands selected from 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-lH-3-benzazepin-7-yl)oxy]-N-methyl-3- pyridinecarboxamide and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • H3R receptor ligands selected from 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-lH-3-benzazepin-7-yl)oxy]-N-methyl-3- pyridinecarboxamide hydrochloride and solvates and hydrates thereof.
  • the chemical structure for 4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine (referred to as Compound C herein) is as follows:
  • H3R receptor ligands selected from 4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine and pharmaceutically acceptable salts, solvates, and hydrates thereof.
  • antagonist includes partial antagonists, full antagonists, and inverse agonists, and shall mean any organic or inorganic molecule that does not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses of agonists and/or partial agonists. It is understood that an "antagonist” as defined herein can bind to the same active site as an agonist or the antagonist bind at a different site provided that the molecule is capable of inhibiting the response of a GPCR, such as, an allosteric antagonist. Another example includes nucleic acid molecules capable of interfering with the GPCR. Certainly, a H3R antagonist would include any anti-pruritic agent capable of inhibiting a response of the H3R. Inverse agonists are specific examples of H3R antagonists.
  • anti-pruritic agent shall mean an agent capable of treating one or more aspects of pruritus. Agents may include, for example, small molecules, large molecules, nucleic acids, synthetic nucleic acid analogs, polypeptide(s), antibodies, and other biologies. H3R antagonists and inverse agonists are examples of anti-pruritic agents.
  • agonist shall mean an agent (e.g., ligand, test compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response mediated by the GPCR.
  • partial agonist shall mean an agent (e.g., ligand, test compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response mediated by the GPCR, albeit to a lesser extent or degree than does a full agonist.
  • H3R antagonist refers to a compound that binds to H3R receptor and acts as an antagonist as defined herein.
  • the H3R antagonist is a selective antagonist.
  • the H3R antagonist has selectivity for H3R that is at least 10 fold greater than its selectivity for any one or more receptors selected from: HIR, H2R and H4R.
  • the H3R antagonist has a selectivity for H3R over HIR of at least about 10- fold.
  • the H3R antagonist has a selectivity for H3R over H2R of at least about 10-fold.
  • the H3R antagonist has a selectivity for H3R over H4R of at least about 10-fold.
  • the H3R antagonist has a selectivity for H3R over HIR, H2R, or H4R of at least about 10 fold. In various embodiments the H3R antagonist has in IC50 (H3R) of less than about 1 ⁇ , less than about 100 nM, less than about 10 nM or less than about 1 nM. In various embodiments the H3R antagonist has in IC50 (H3R) of about 1 ⁇ or less, about 100 nM or less, about 10 nM or less, or about 1 nM or less. In one embodiment, the H3R antagonist is an orally active H3R antagonist. In one embodiment, the H3R antagonist is peripherally restricted. In some embodiments, the H3R antagonist is an antagonist of the human
  • the H3R antagonist is administered orally and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R antagonist is applied topically and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R antagonist is in an amount sufficient to reduce itch in a human.
  • inverse agonist shall mean an agent (e.g., ligand, test compound) which binds to a GPCR and which inhibits the baseline intracellular response initiated by the active form of the receptor below the normal base level activity which is observed in the absence of an agonist or partial agonist.
  • H3R inverse agonist refers to a compound that binds to H3R receptor and acts as an inverse agonist.
  • the H3R inverse agonist is a selective inverse agonist.
  • the H3R inverse agonist has selectivity for H3R that is at least 10 fold greater than its selectivity for any one or more receptors selected from: HIR, H2R and H4R.
  • the H3R inverse agonist has a selectivity for H3R over HIR of at least about 10-fold.
  • the H3R inverse agonist has a selectivity for H3R over H2R of at least about 10-fold.
  • the H3R inverse agonist has a selectivity for H3R over H4R of at least about 10-fold. In one embodiments, the H3R inverse agonist has a selectivity for H3R over HIR, H2R, or H4R of at least about 10 fold. In various embodiments the H3R inverse agonist has in IC50 (H3R) of less than about ⁇ , less than about ⁇ , less than about ⁇ or less than about InM. In various embodiments the H3R inverse agonist has in IC50 (H3R) of about 1 ⁇ or less, about 100 nM or less, about 10 nM or less, or about 1 nM or less.
  • the H3R inverse agonist is an orally active H3R inverse agonist. In one embodiment, the H3R inverse agonist is peripherally restricted. In some embodiments, the H3R inverse agonist is an inverse agonist of the human H3R. In some embodiments, the H3R inverse agonist is orally active and has an IC50 of less than about 100 nM. In some embodiments, the H3R inverse agonist is administered orally and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R inverse agonist is applied topically and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R inverse agonist is in an amount sufficient to reduce itch in a human.
  • the individual is a fish.
  • the individual is an amphibian.
  • the individual is a reptile. In certain embodiments, the individual is a bird. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a mouse, a rat, a hamster, a rabbit, a pig, a dog, a cat, a horse, a cow, a sheep, a goat, a non-human primate, or a human (which may be included in embodiments of the invention individually or in any combination). In certain embodiments, the individual is a horse. In certain embodiments, the individual is a dog or a cat.
  • the individual is a human companion animal (such as a dog, a cat, etc.), a farm animal (such as a cow, a sheep, a goat, a pig, a chicken, etc.), a sports animal (such as a horse, a dog, etc.), a beast of burden (such as a mule, a camel, etc.) or an exotic animal (such as an animal found in a zoo, etc.), which may be included in embodiments of the invention individually or in any combination.
  • the individual is a non-human mammal.
  • the individual is a non-human primate (such as a rhesus monkey, a chimpanzee, etc.).
  • the individual is a human.
  • treating and “treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner in the case of humans; veterinarian in the case of non-human vertebrates, and in particular embodiment non-human mammals) that an individual requires or will benefit from treatment.
  • a caregiver e.g. physician, nurse, nurse practitioner in the case of humans; veterinarian in the case of non-human vertebrates, and in particular embodiment non-human mammals
  • terapéuticaally effective amount refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) Preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease,
  • Inhibiting the disease for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and
  • Ameliorating the disease for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
  • therapeutic efficacy refers to elicitation of the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:
  • Preventing the disease for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease,
  • Inhibiting the disease for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and
  • Ameliorating the disease for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
  • composition shall mean a material comprising at least one component.
  • active ingredient shall mean any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of a disease.
  • composition shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation and treatment in a mammal.
  • pharmaceutically acceptable it is meant that the carrier, vehicle, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • drug form shall mean the physical form in which a drug is produced and dispensed, such as a tablet, capsule, or an injectable.
  • endogenous shall mean a material that an individual naturally produces.
  • contact shall mean bringing at least two moieties together.
  • modulate shall be taken to refer to an increase or decrease in the amount, quality, or effect of a particular activity, function or molecule.
  • agonists, partial agonists, inverse agonists, and antagonists of a G protein-coupled receptor are modulators of the receptor.
  • small molecule shall be taken to mean a compound having a molecular weight of less than about 10,000 grams per mole, including a peptide, peptidomimetic, amino acid, amino acid analogue, polynucleotide, polynucleotide analogue, nucleotide, nucleotide analogue, organic compound or inorganic compound (i.e. including a heterorganic compound or organometallic compound), and salts, esters and other pharmaceutically acceptable forms thereof.
  • small molecules are organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole.
  • small molecules are organic or inorganic compounds having molecular weight of less than about 1,000 grams per mole.
  • small molecules are organic or inorganic compounds having a molecular weight of less than about 800 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 600 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 500 grams per mole.
  • itch refers to the sensation that elicits a reflex response to scratch. Itch can be a symptom of a disease, disorder or infection, or itch can arise spontaneously, without an underlying or identifiable physiological cause, known as idiopathic pruritus.
  • control of itch includes reducing, alleviating, relieving and numbing the sensation of itch. Control of itch also includes reducing the desire to scratch.
  • in need of treatment and the term “in need thereof” when referring to treatment are used interchangeably and refer to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, etc. in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver' s expertise, but that includes the knowledge that the individual or animal is ill, or will become ill, as the result of a disease, condition or disorder that is treatable by the compounds of the invention.
  • a caregiver e.g. physician, nurse, nurse practitioner, etc. in the case of humans; veterinarian in the case of animals, including non-human mammals
  • the compounds of the invention can be used in a protective or preventive manner; or compounds of the invention can be used to alleviate, inhibit or ameliorate the disease, condition or disorder and refers to all aspects of control of itching including prophylaxis and therapy.
  • Control of itch includes reducing, alleviating, relieving and numbing the sensation of itch.
  • Control of itch also includes reducing the desire to scratch.
  • antibody refers to immunoglobulin molecules and
  • immunologically active portions of immunoglobulin molecules i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen.
  • the terms also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms besides antibodies; including, for example, Fv, Fab, and F(ab)'2 as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.
  • polypeptide shall refer to a polymer of amino acids without regard to the length of the polymer.
  • peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides.
  • polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
  • antibody is intended herein to encompass monoclonal antibody and polyclonal antibody.
  • second messenger shall mean an intracellular response produced as a result of receptor activation.
  • a second messenger can include, for example, inositol 1,4,5-triphosphate (IP 3 ), diacylglycerol (DAG), cyclic AMP (cAMP), cyclic GMP (cGMP), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca 2+ .
  • Second messenger response can be measured for a determination of receptor activation.
  • receptor functionality shall refer to the normal operation of a receptor to receive a stimulus and moderate an effect in the cell, including, but not limited to regulating gene transcription, regulating the influx or efflux of ions, effecting a catalytic reaction, and/or modulating activity through G-proteins, such as eliciting a second messenger response.
  • “functionality of the receptor” shall mean that a response or a functionality of the receptor is increased in the presence of a compound as opposed to in the absence of the compound.
  • inhibitor or “inhibiting,” in relationship to the term “response” or “functionality of the receptor” shall mean that a response a functionality of the receptor is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
  • compound efficacy shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity.
  • the compound efficacy of different compounds may be evaluated with respect to each other or some other standard.
  • test compound used interchangeably herein with “candidate compound,” shall mean a molecule (for example, and not limitation, a chemical compound) which is amenable to a screening technique.
  • a test compound is an anti-pruritic agent.
  • a test compound is a H3R antagonist.
  • a test compound is a H3R inverse agonist.
  • directly identifying or “directly identified”, in relationship to the phrase “test compound,” shall mean the screening of a compound against a G protein-coupled receptor in the absence of a known ligand (e.g., a known agonist) to the G protein-coupled receptor.
  • a known ligand e.g., a known agonist
  • the term "pruritus model” shall mean an in vivo or in vitro model system which displays at least one aspect or element of pruritus and is amenable to or allows the evaluation of anti-pruritic agents.
  • the pruritus model is mammalian in origin.
  • peripherally restricted shall refer to a compound or agent that is restricted in some manner from crossing the blood brain barrier.
  • a peripherally restricted compound or agent may be less than 100% restricted to the periphery.
  • a peripherally restricted compound may be further referred to by a braimplasma ratio which will be less than 1 in at least one species.
  • H3R Histamine H3 Receptors
  • the human histamine H3 receptor (GenBank accession number: AF140538) is described in Figure 1 and Figure 2, and SEQ ID NO: 1 and SEQ ID NO:2 (Lovenberg, T.W., Roland, B.L., Wilson, S.J., Jiang, X., Pyati, J., Huvar, A., Jackson, M.R. and Erlander, M.G., Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 55 (6), 1101-1107 (1999)).
  • the human histamine H3 receptor gene contains three introns and 4 exons. Through alternative splicing, six receptor variants are known to be created.
  • the full-length receptor and two additional variants are known to bind histamine and other H3 agonists (Wellendorph P, Goodman MW, Burstein ES, Nash NR, Brann MR, Weiner DM. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor. Neuropharmacology.
  • mouse histamine H3 receptor (GeneBank accession number: AY142145) is described in Figure 3 and Figure 4 and SEQ ID NOS: 3 and 4 (Chen, J., Liu, C. and Lovenberg, T. W. Molecular and pharmacological characterization of the mouse histamine H(3) receptor. Eur J Pharmacol 467 (1-3), 57-65 (2003)).
  • H3R Use of species variants of H3R are envisioned to be within the scope of the invention. Allelic variants of H3R of SEQ ID NO:2 are envisioned to be within the scope of the invention. Human H3R is envisioned to be within the scope of the invention.
  • a variant which is a vertebrate ortholog of human H3R of SEQ ID NO:2 is envisioned to be within the scope of the invention.
  • a variant which is a mammalian ortholog of human H3R of SEQ ID NO:2 is envisioned to be within the scope of the invention.
  • mouse H3R, rat H3R, hamster H3R, dog H3R, and non-human primate H3R are envisioned to be within the scope of the invention.
  • the variant of SEQ ID NO:2 is a GPCR.
  • the GPCR comprises the amino acids 1-445 of SEQ ID NO:2.
  • the GPCR comprises the amino acids 2-445 of SEQ ID NO:2.
  • the GPCR comprises the amino acids 2-445 of SEQ ID NO:2, with the proviso that the GPCR does not comprise the amino acid sequence of SEQ ID NO: 2.
  • the GPCR comprises the amino acid sequence of a GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: l.
  • the GPCR comprises a variant of SEQ ID NO: 2.
  • a variant of SEQ ID NO:2 having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2 is envisioned to be within the scope of the invention.
  • the variant of SEQ ID NO:2 having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2 is a GPCR.
  • a variant GPCR that may be used in the subject methods has an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, of at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2.
  • a variant GPCR having, for example, 95% "identity" to SEQ ID NO:2 is meant that the amino acid sequence of the variant is identical to amino acids 1-335 of SEQ ID NO:2 except that it may include up to five amino acid alterations per each 100 amino acids of SEQ ID NO:2.
  • amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2 up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with amino acids 1-617 of SEQ ID NO: 2. These alternations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either subjectly among residues in the sequence or in one or more contiguous groups within the sequence.
  • a variant GPCR that may be used in the subject methods is a GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1.
  • the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an endogenous GPCR.
  • the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 and that is an endogenous GPCR is a mammalian endogenous GPCR.
  • the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an allele of SEQ ID NO:2. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an ortholog of SEQ ID NO:2.
  • the GPCR is screened in antagonist mode.
  • a GPCR of the invention forms part of a fusion protein with a G protein.
  • a preferred method for determining the best overall match between a query sequence e.g., the amino acid sequence of SEQ ID NO:2
  • a sequence to be interrogated also referred to as a global sequence alignment
  • FASTDB computer program based on the algorithm of Brutlag et al. (Comp App Biosci (1990) 6:237-245).
  • sequence alignment the query and interrogated sequences are both amino acid sequences.
  • the results of the global sequence alignment are in percent identity.
  • the results, in percent identity must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the interrogated sequence when calculating global percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the interrogated sequence, that are not matched/aligned with a corresponding interrogated sequence residue, as a percent of the total bases of the query sequence.
  • Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the interrogated sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query amino acid residues outside the farthest N- and C-terminal residues of the interrogated sequence.
  • a 90 amino acid residue interrogated sequence is aligned with a 100- residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the interrogated sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%.
  • a 90-residue interrogated sequence is compared with a 100-residue query sequence. This time the deletions are internal so there are no residues at the N- or C- termini of the interrogated sequence, which are not matched/aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected. No other corrections are made for the purposes of the present invention.
  • a polypeptide of interest is a fusion protein, and may contain, for example, an affinity tag domain or a reporter domain.
  • Suitable affinity tags include any amino acid sequence that may be specifically bound to another moiety, usually another polypeptide, most usually an antibody.
  • Suitable affinity tags include epitope tags, for example, the V5 tag, the FLAG tag, the HA tag (from hemagglutinin influenza virus), the myc tag, and the like, as is known in the art.
  • Suitable affinity tags also include domains for which, binding substrates are known, e.g., HIS, GST and MBP tags, as is known in the art, and domains from other proteins for which specific binding partners, e.g., antibodies, particularly monoclonal antibodies, are available.
  • Suitable affinity tags also include any protein-protein interaction domain, such as a IgG Fc region, which may be specifically bound and detected using a suitable binding partner, e.g. the IgG Fc receptor. It is expressly contemplated that such a fusion protein may contain a heterologous N-terminal domain (e.g., an epitope tag) fused in-frame with a GPCR that has had its N-terminal methionine residue either deleted or substituted with an alternative amino acid.
  • a heterologous N-terminal domain e.g., an epitope tag
  • Suitable reporter domains include any domain that can report the presence of a polypeptide. While it is recognized that an affinity tag may be used to report the presence of a polypeptide using, e.g., a labeled antibody that specifically binds to the tag, light emitting reporter domains are more usually used. Suitable light emitting reporter domains include luciferase (from, e.g., firefly, Vargula, Renilla reniformis or Renilla muelleri), or light emitting variants thereof.
  • reporter domains include fluorescent proteins, (from e.g., jellyfish, corals and other coelenterates as such those from Aequoria, Renilla, Ptilosarcus, Stylatula species), or light emitting variants thereof.
  • Light emitting variants of these reporter proteins are very well known in the art and may be brighter, dimmer, or have different excitation and/or emission spectra, as compared to a native reporter protein. For example, some variants are altered such that they no longer appear green, and may appear blue, cyan, yellow, enhanced yellow red (termed BFP, CFP, YFP eYFP and RFP, respectively) or have other emission spectra, as is known in the art.
  • reporter domains include domains that can report the presence of a polypeptide through a biochemical or color change, such as ⁇ -galactosidase, ⁇ - glucuronidase, chloramphenicol acetyl transferase, and secreted embryonic alkaline phosphatase.
  • an affinity tags or a reporter domain may be present at any position in a polypeptide of interest. However, in most embodiments, they are present at the C- or N-terminal end of a polypeptide of interest.
  • GPCR coding sequences may be isolated from a library of
  • GPCR coding sequence using any one or a combination of a variety of recombinant methods that do not need to be described herein. Subsequent substitution, deletion, and/or addition of nucleotides in the nucleic acid sequence encoding a protein may also be done using standard recombinant DNA techniques.
  • site directed mutagenesis and subcloning may be used to
  • nucleic acids encoding a polypeptide of interest may also be made by chemical synthesis entirely from oligonucleotides (e.g., Cello et al., Science (2002) 297: 1016-8).
  • the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly a mammalian, e.g., mouse, rat, hamster, non-human primate, or human, species.
  • the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly an amphibian species.
  • the invention further provides vectors (also referred to as "constructs") comprising a subject nucleic acid.
  • the subject nucleic acid sequences will be expressed in a host after the sequences have been operably linked to an expression control sequence, including, e.g. a promoter.
  • the subject nucleic acids are also typically placed in an expression vector that can replicate in a host cell either as an episome or as an integral part of the host chromosomal DNA.
  • expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein by reference).
  • Vectors, including single and dual expression cassette vectors are well known in the art
  • Suitable vectors include viral vectors, plasmids, cosmids, artificial chromosomes (human artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, etc.), mini-chromosomes, and the like. Retroviral, adenoviral and adeno-associated viral vectors may be used.
  • expression vectors are available to those in the art for purposes of producing a polypeptide of interest in a cell and include expression vectors which are commercially available (e.g., from Invitrogen, Carlsbad, CA; Clontech, Mountain View, CA; Stratagene, La Jolla, CA).
  • Commercially available expression vectors include, by way of non- limiting example, CMV promoter-based vectors.
  • One suitable expression vector is pCMV.
  • the expression vector may be adenoviral.
  • An exemplary adenoviral vector may be purchased as AdEasyTM from Qbiogene (Carlsbad, CA) (He TC et al, Proc Natl Acad Sci USA (1998) 95:2509-2514; and US Patent No. 5,922,576).
  • AdEasyTM from Qbiogene (Carlsbad, CA) (He TC et al, Proc Natl Acad Sci USA (1998) 95:2509-2514; and US Patent No. 5,922,
  • the subject nucleic acids usually comprise an single open reading frame encoding a subject polypeptide of interest, however, in certain embodiments, since the host cell for expression of the polypeptide of interest may be a eukaryotic cell, e.g., a mammalian cell, such as a human cell, the open reading frame may be interrupted by introns.
  • Subject nucleic acid are typically part of a transcriptional unit which may contain, in addition to the subject nucleic acid 3' and 5' untranslated regions (UTRs) which may direct RNA stability, translational efficiency, etc.
  • the subject nucleic acid may also be part of an expression cassette which contains, in addition to the subject nucleic acid a promoter, which directs the transcription and expression of a polypeptide of interest, and a transcriptional terminator.
  • Eukaryotic promoters can be any promoter that is functional in a eukaryotic host cell, including viral promoters and promoters derived from eukaryotic genes.
  • Exemplary eukaryotic promoters include, but are not limited to, the following: the promoter of the mouse
  • metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci.
  • a promoter is used that is a promoter of the target pathogen. Promoters for use in the present invention are selected such that they are functional in the cell type (and/or animal) into which they are being introduced. In certain embodiments, the promoter is a CMV promoter.
  • a subject vector may also provide for expression of a selectable marker.
  • Suitable vectors and selectable markers are well known in the art and discussed in Ausubel, et al, (Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995) and Sambrook, et al, (Molecular Cloning: A Laboratory Manual, Third Edition, (2001) Cold Spring Harbor, N.Y.).
  • a variety of different genes have been employed as selectable markers, and the particular gene employed in the subject vectors as a selectable marker is chosen primarily as a matter of convenience.
  • selectable marker genes include: the thymidine kinase gene, the dihydrofolate reductase gene, the xanthine-guanine phosphoribosyl transferase gene, CAD, the adenosine deaminase gene, the asparagine synthetase gene, the antibiotic resistance genes, e.g. tetr, ampr, Cmr or cat, kanr or neor (aminoglycoside phosphotransferase genes), the hygromycin B phosphotransferase gene, and the like.
  • polypeptides of interest may be fusion proteins that contain an affinity domain and/or a reporter domain.
  • Methods for making fusions between a reporter or tag and a GPCR, for example, at the C- or N-terminus of the GPCR, are well within the skill of one of skill in the art (e.g. McLean et al, Mol. Pharma. Mol Pharmacol. 1999 56: 1182-91 ; Ramsay et al., Br. J. Pharmacology, 2001, 315-323) and will not be described any further.
  • such a fusion protein may contain a heterologous N-terminal domain (e.g., an epitope tag) fused in-frame with a GPCR that has had its N-terminal methionine residue either deleted or substituted with an alternative amino acid.
  • a polypeptide of interest may first be made from a native polypeptide and then operably linked to a suitable reporter/tag as described above.
  • the subject nucleic acids may also contain restriction sites, multiple cloning sites, primer binding sites, ligatable ends, recombination sites etc., usually in order to facilitate the construction of a nucleic acid encoding a polypeptide of interest.
  • the invention further provides host cells comprising a vector comprising a subject nucleic acid.
  • Suitable host cells include prokaryotic, e.g., bacterial cells (for example E. coli), as well as eukaryotic cells e.g. an animal cell (for example an insect, mammal, fish, amphibian, bird or reptile cell), a plant cell (for example a maize or Arabidopsis cell), or a fungal cell (for example a S. cerevisiae cell).
  • prokaryotic e.g., bacterial cells (for example E. coli), as well as eukaryotic cells e.g. an animal cell (for example an insect, mammal, fish, amphibian, bird or reptile cell), a plant cell (for example a maize or Arabidopsis cell), or a fungal cell (for example a S. cerevisiae cell).
  • any cell suitable for expression of a polypeptide of interest-encoding nucleic acid may be used
  • an animal host cell line is used, examples of which are as follows: monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293 ["293”], Graham et al. J. Gen Virol. 36:59 (1977)); HEK-293T ["293T”] cells; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216, (1980); Syrian golden hamster cells
  • MCB3901 ATCC CRL-9595; mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); african green monkey kidney cells (VERO- 76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N. Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1).
  • melanophores are used.
  • Melanophores are skin cells found in lower vertebrates. Relevant materials and methods will be followed according to the disclosure of U.S. Patent Number 5,462,856 and U.S. Patent Number 6,051,386.
  • G protein receptor When a G protein receptor becomes active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, activated receptors continue to exchange GDP to GTP.
  • GTP GTPase
  • GTPase e.g., Gq, Gs, Gi, Gz, Go
  • candidate compounds are identified using the "generic" G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists or inverse agonists), in some embodiments further screening to confirm that the compounds have interacted at the receptor site is preferred.
  • a compound identified by the "generic” assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain.
  • Gs stimulates the enzyme adenylyl cyclase.
  • Gi and Gz and Go
  • Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP.
  • activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 ri Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992).
  • assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP).
  • a candidate compound e.g., an inverse agonist to the receptor
  • a variety of approaches known in the art for measuring cAMP can be utilized; in some embodiments a preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format.
  • Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes.
  • Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene.
  • Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., ⁇ -galactosidase or luciferase.
  • ⁇ -galactosidase or luciferase an activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein.
  • the reporter protein such as ⁇ -galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
  • Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP 2 , releasing two intracellular messengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ). Increased accumulation of IP 3 is associated with activation of Gq- and Go-associated receptors. See, generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron To Brain (3 Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992).
  • DAG diacylglycerol
  • IP 3 inositol 1,4,5-triphosphate
  • Assays that detect IP 3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP 3 ).
  • Gq-associated receptors can also been examined using an API reporter assay in that Gq-dependent phospholipase C causes activation of genes containing API elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression.
  • Commercially available assays for such detection are available.
  • an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR for use in screening of candidate compounds for the direct identification of inverse agonists or agonists provides an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto.
  • the receptor is active even in the absence of an endogenous ligand bound thereto.
  • an approach be utilized that can enhance such differentiation.
  • a preferred approach is the use of a GPCR Fusion Protein.
  • a non-endogenous GPCR has been constitutively activated using the assay techniques set forth above (as well as others known to the art-skilled)
  • screening take place using a mammalian or a melanophore expression system, as such a system will be expected to have endogenous G protein therein.
  • the non-endogenous, constitutively activated GPCR will continuously signal.
  • this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.
  • the GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the GPCR.
  • the GPCR Fusion Protein may be preferred for screening with either an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is generated in such screening techniques. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is preferred for the screening of candidate compounds as disclosed herein.
  • the construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator.
  • GPCR Fusion Protein construct Important criteria in the construction of such a GPCR Fusion Protein construct include but are not limited to, that the GPCR sequence and the G protein sequence both be in- frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence), and that the "stop" codon of the GPCR be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed.
  • the GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). Based upon convenience, it is preferred to use a spacer. In some embodiments, it is preferred that the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct.
  • activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging (i.e., the cAMP signal decreases upon activation, thus making the direct identification of, e.g., agonists (which would further decrease this signal) challenging).
  • the cAMP signal decreases upon activation, thus making the direct identification of, e.g., agonists (which would further decrease this signal) challenging.
  • it has been ascertained that for these types of receptors it is possible to create a GPCR Fusion Protein that is not based upon the GPCR's endogenous G protein, in an effort to establish a viable cyclase- based assay.
  • an endogenous Gi coupled receptor can be fused to a Gs protein -such a fusion construct, upon expression, "drives” or “forces” the endogenous GPCR to couple with, e.g., Gs rather than the "natural" Gi protein, such that a cyclase-based assay can be established.
  • a GPCR Fusion Protein is used and the assay is based upon detection of adenylyl cyclase activity, that the fusion construct be established with Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).
  • G Protein Fusion construct that utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein.
  • a preferred fusion construct can be accomplished with a Gq Protein wherein the first six (6) amino acids of the G-protein a-subunit ("Gaq") is deleted and the last five (5) amino acids at the C-terminal end of Gaq is replaced with the corresponding amino acids of the Ga of the G protein of interest.
  • a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct".
  • This fusion construct will force the endogenous Gi coupled receptor to couple to its non-endogenous G protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylglycerol, can be measured in lieu of cAMP production.
  • Gq non-endogenous G protein
  • a Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decreases the level of cAMP production, which can make the assessment of cAMP levels challenging.
  • an effective technique in measuring the decrease in production of cAMP as an indication of activation of a receptor that predominantly couples Gi upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non-endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I; see infra), with the Gi linked GPCR.
  • a signal enhancer e.g., a non-endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I; see infra)
  • activation of a Gs coupled receptor can be determined based upon an increase in production of cAMP.
  • Activation of a Gi coupled receptor leads to a decrease in production cAMP.
  • the co-transfection approach is intended to advantageously exploit these "opposite” affects.
  • co-transfection of a non- endogenous, constitutively activated Gs coupled receptor (the "signal enhancer") with expression vector alone provides a baseline cAMP signal (i.e., although the Gi coupled receptor will decrease cAMP levels, this "decrease” will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer).
  • the signal enhancer By then co- transfecting the signal enhancer with the "target receptor", an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal.
  • Candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or after screening against the co-transfected receptors).
  • IC50 value is determined using an assay selected from the group consisting of: IP3 assay carried out using transfected HEK293 cells expressing recombinant H3R polypeptide; and melanophore assay carried out using transfected melanophores expressing recombinant H3R polypeptide.
  • the test compound is an inverse agonist or antagonist with an IC50 of less than 10 ⁇ , of less than 1 ⁇ , of less than 100 nM, or of less than 10 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 10 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 9 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 8 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 7 ⁇ in the assay.
  • the test compound is an inverse agonist or antagonist with an IC50 of less than 6 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 5 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 4 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 3 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 2 ⁇ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 1 ⁇ in the assay.
  • the test compound is an inverse agonist or antagonist with an IC50 of less than 900 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 800 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 700 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 600 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 500 nM in the assay.
  • the test compound is an inverse agonist or antagonist with an IC50 of less than 400 nM n the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 300 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 200 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 100 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 90 nM in the assay.
  • the test compound is an inverse agonist or antagonist with an IC50 of less than 80 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 70 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 60 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 50 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 40 nM n the assay.
  • the test compound is an inverse agonist or antagonist with an IC50 of less than 30 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 20 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 10 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 10 ⁇ . In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 1 ⁇ .
  • the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 100 nM. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 10 nM. In some embodiments, the test compound is selective for the GPCR.
  • Melanophores are skin cells found in lower vertebrates. They contain pigmented organelles termed melanosomes. Melanophores are able to redistribute these melanosomes along a microtubule network upon G-protein coupled receptor (GPCR) activation. The result of this pigment movement is an apparent lightening or darkening of the cells.
  • GPCR G-protein coupled receptor
  • melanophores the decreased levels of intracellular cAMP that result from activation of a Gi-coupled receptor cause melanosomes to migrate to the center of the cell, resulting in a dramatic lightening in color. If cAMP levels are then raised, following activation of a Gs-coupled receptor, the melanosomes are re-dispersed and the cells appear dark again.
  • the increased levels of diacylglycerol that result from activation of Gq-coupled receptors can also induce this re-dispersion.
  • the technology is also suited to the study of certain receptor tyrosine kinases.
  • the response of the melanophores takes place within minutes of receptor activation and results in a simple, robust color change. The response can be easily detected using a conventional absorbance microplate reader or a modest video imaging system. Unlike other skin cells, the melanophores derive from the neural crest and appear to express a full complement of signaling proteins. In particular, the cells express an extremely wide range of G-proteins and so are able to functionally express almost all GPCRs.
  • Melanophores can be utilized to identify compounds, including natural ligands, which bind to and/or activate GPCRs.
  • This method can be conducted by introducing test cells of a pigment cell line capable of dispersing or aggregating their pigment in response to a specific stimulus and expressing an exogenous clone coding for the GPCR.
  • An initial state of pigment disposition can be set using, for example, using melatonin, MSH or light.
  • the test cells are then contacted with chemical compounds, and it is determined whether the pigment disposition in the cells changed from the initial state of pigment disposition. Dispersion of pigments cells due to the candidate compound, including but not limited to a ligand, coupling to the GPCR will appear dark on a petri dish, while aggregation of pigments cells will appear light.
  • Melanophores are transfected by electroporation with a plasmid which contains the coding sequence of mouse or human H3R.
  • the cells are plated in 96-well plates. 48 hours post- transfection, half of the cells on each plate are treated with lOnM melatonin.
  • Melatonin activates an endogenous Gi-coupled receptor in the melanophores and causes them to aggregate their pigment.
  • the remaining half of the cells are transferred to serum-free medium 0.7X L-15 (Gibco). After one hour, the cells in serum- free media remain in a pigment-dispersed state while the melatonin-treated cells are in a pigment-aggregated state.
  • the cells are treated with different compounds from a proprietary compound library containing 140,000-150,000 organic small molecule compounds. If H3R bound to the compound, the melanophores would be expected to undergo a color change, for example, due to pigment aggregation, in response to the compound.
  • a G protein-coupled receptor When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein.
  • the alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP.
  • the non-hydrolyzable GTP analog, [ 35 S]GTPyS can be utilized to demonstrate enhanced binding of [ 35 S]GTPyS to membranes expressing activated receptors.
  • the assay utilizes the ability of G protein coupled receptors to stimulate [ 35 S]GTPyS binding to membranes expressing the relevant receptors.
  • the assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous GPCRs and non- endogenous, constitutively activated GPCRs.
  • the assay is generic and has application to drug discovery at all G protein-coupled receptors.
  • the [ 35 S]GTPyS assay is incubated in 20 mM HEPES and between 1 and about 20mM MgCl 2 (this amount can be adjusted for optimization of results, although 20mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM [ 35 S]GTPyS (this amount can be adjusted for optimization of results, although 1.2 is preferred) and 12.5 to 75 ⁇ g membrane protein (e.g, 293 cells expressing the H3R; this amount can be adjusted for optimization) and 10 ⁇ GDP (this amount can be changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25 ⁇ ; Amersham) are then added and the mixture incubated for another 30 minutes at room temperature. The tubes are then centrifuged at 1500 x g for 5 minutes at room temperature and then counted in a scintillation counter.
  • a Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes.
  • the Flash Plate wells can contain a scintillant coating which also contains a specific antibody recognizing cAMP.
  • the cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express a receptor.
  • Transfected cells are harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10 mL of PBS is gently added to each dish of cells followed by careful aspiration. 1 mL of Sigma cell dissociation buffer and 3 mL of PBS are added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50 mL conical centrifuge tube. Cells are then centrifuged at room temperature at 1,100 rpm for 5 minutes. The cell pellet is carefully re-suspended into an appropriate volume of PBS (about 3 mL/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50 ⁇ ).
  • cAMP standards and Detection Buffer comprising ⁇ of tracer [ 125 ⁇ ] cAMP (50 ⁇ ) to 11 mL Detection Buffer
  • Assay Buffer is prepared fresh for screening and contains 50 ⁇ L ⁇ of Stimulation Buffer, 3 ⁇ L ⁇ of candidate compound (12 ⁇ final assay concentration) and 50 ⁇ L ⁇ cells.
  • Assay Buffer is stored on ice until utilized.
  • the assay preferably carried out, for example, in a 96-well plate, is initiated by addition of 50 ⁇ L ⁇ of cAMP standards to appropriate wells followed by addition of 50 ⁇ , of PBSA to wells HI 1 and H12.
  • Stimulation Buffer 50 ⁇ , of Stimulation Buffer is added to all wells.
  • DMSO or selected candidate compounds
  • DMSO or selected candidate compounds
  • the cells are then added to the wells and incubated for 60 minutes at room temperature.
  • 100 ⁇ L ⁇ of Detection Mix containing tracer cAMP is then added to the wells. Plates are then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are then extrapolated from a standard cAMP curve which is contained within each assay plate.
  • TSHR is a Gs coupled GPCR that causes the accumulation of cAMP upon activation.
  • TSHR can be constitutively activated by mutating amino acid residue 623 (i.e., changing an alanine residue to an isoleucine residue).
  • a Gi coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging.
  • An effective technique for measuring the decrease in production of cAMP as an indication of activation of a Gi coupled receptor can be accomplished by co-transfecting, non- endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baseline level of cAMP.
  • TSHR-A623I non- endogenous, constitutively activated TSHR
  • Gs coupled receptor an endogenous, constitutively active Gs coupled receptor
  • the target GPCR is then co-transfected with the signal enhancer, and it is this material that can be used for screening.
  • this approach is preferably used in the direct identification of candidate compounds against Gi coupled receptors. It is noted that for a Gi coupled GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP signal and an agonist will decrease the cAMP signal.
  • tube A is prepared by mixing 2 ⁇ g DNA of each receptor transfected into the mammalian cells, for a total of 4 ⁇ g DNA (e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPCR, etc.) in 1.2 mL serum free DMEM (Irvine Scientific, Irvine, CA); tube B is prepared by mixing 120 ⁇ L ⁇ lipofectamine (Gibco BRL) in 1.2 mL serum free DMEM.
  • Tubes A and B are then admixed by inversions (several times), followed by incubation at room temperature for 30-45minutes.
  • the admixture is referred to as the "transfection mixture”.
  • Plated 293 cells are washed with 1XPBS, followed by addition of 10 mL serum free DMEM.
  • 2.4 mL of the transfection mixture is then added to the cells, followed by incubation for 4 hours at 37°C/5% C0 2 .
  • the transfection mixture is then removed by aspiration, followed by the addition of 25 mL of DMEM/10% Fetal Bovine Serum. Cells are then incubated at 37°C/5% C0 2 . After 24 hours incubation, cells are harvested and utilized for analysis.
  • a Flash PlateTM Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is designed for cell-based assays, but can be modified for use with crude plasma membranes depending on the need of the skilled artisan.
  • the Flash Plate wells contain a scintillant coating which also contains a specific antibody recognizing cAMP.
  • the cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express a receptor of interest.
  • Transfected cells are harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10 mL of PBS is gently added to each dish of cells followed by careful aspiration. 1 mL of Sigma cell dissociation buffer and 3 mL of PBS is added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50 mL conical centrifuge tube. Cells are then centrifuged at room temperature at 1,100 rpm for 5 minutes. The cell pellet is carefully re-suspended into an appropriate volume of PBS (about 3 mL/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50 ⁇ /well).
  • cAMP standards and Detection Buffer comprising ⁇ of tracer [ 125 ⁇ ] cAMP (50 ⁇ ) to 11 mL Detection Buffer
  • Assay Buffer should be prepared fresh for screening and contain 50 ⁇ L ⁇ of Stimulation Buffer, 3 ⁇ L ⁇ of candidate compound (12 ⁇ final assay concentration) and 50 ⁇ L ⁇ cells.
  • Assay Buffer can be stored on ice until utilized. The assay can be initiated by addition of 50 ⁇ L ⁇ of cAMP standards to appropriate wells followed by addition of 50 ⁇ L ⁇ of PBS A to wells H-l 1 and H12. Fifty ⁇ L ⁇ o ⁇ Stimulation Buffer is added to all wells.
  • Selected compounds e.g., TSH
  • TSH Selected compounds
  • the cells are then added to the wells and incubated for 60 minutes at room temperature.
  • 100 ⁇ L ⁇ of Detection Mix containing tracer cAMP is then added to the wells. Plates are then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are extrapolated from a standard cAMP curve which is contained within each assay plate.
  • 293 or 293T cells are plated-out on 96 well plates at a density of 2 x 10 4 cells per well and are transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions.
  • BBL Lipofectamine Reagent
  • a DNA lipid mixture is prepared for each 6-well transfection as follows: 260ng of plasmid DNA in 100 ⁇ .
  • DMEM DMEM lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing lipid-containing 2 lipid.
  • the 260ng of plasmid DNA consists of 200ng of a 8xCRE-Luc reporter plasmid, 50ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and lOng of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)).
  • the 8XCRE-Luc reporter plasmid is prepared as follows: vector SRIF- -gal is obtained by cloning the rat somatostatin promoter (
  • Eight (8) copies of cAMP response element are obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, Suzuki et al., Hum Gene Ther 7: 1883-1893 (1996)) and cloned into the SRIF- -gal vector at the Kpn-BglV site, resulting in the 8xCRE- -gal reporter vector.
  • the 8xCRE-Luc reporter plasmid is generated by replacing the beta-galactosidase gene in the 8xCRE- -gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the Hindlll-BamHI site.
  • the DNA/lipid mixture is diluted with 400 ⁇ of DMEM and 100 ⁇ of the diluted mixture is added to each well.
  • 100 ⁇ of DMEM with 10% FCS are added to each well after a four hour incubation in a cell culture incubator.
  • the transfected cells are changed with 200 ⁇ /well of DMEM with 10% FCS.
  • the wells are changed to 100 ⁇ /well of DMEM without phenol red, after one wash with PBS.
  • Luciferase activity is measured the next day using the LucLite reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBetaTM scintillation and luminescence counter (Wallac).
  • a method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing API elements in their promoter.
  • a PathdetectTM AP-1 cis-Reporting System (Stratagene, Catalogue No. 219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate are 410 ng pAPl-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.
  • Gq-dependent phospholipase C One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter.
  • a PathdetectTM SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, for example, COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian TransfectionTM Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions.
  • 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
  • CMV-SEAP secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples
  • Cells are then lysed and assayed for luciferase activity using a LucliteTM Kit (Packard, Cat. No. 6016911) and "Trilux 1450 Microbeta" liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions.
  • the data can be analyzed using GraphPad PrismTM 2.0a (GraphPad Software Inc.).
  • cells comprising the receptor of interest can be plated onto 24 well plates, usually lxlO 5 cells/well (although this number can be optimized).
  • cells can be transfected by first mixing 0.25 ⁇ g DNA in 50 ⁇ serum free DMEM/well and 2 ⁇ lipofectamine in 50 ⁇ serum free DMEM/well. The solutions are gently mixed and incubated for 15-30 minutes at room temperature. Cells are washed with 0.5 mL PBS and 400 ⁇ of serum free media is mixed with the transfection media and added to the cells. The cells are then incubated for 3-4 hours at 37°C/5%C0 2 and then the transfection media is removed and replaced with 1 mL/well of regular growth media.
  • the cells are labeled with 3 H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 mL PBS. Then 0.5 mL inositol-free/serum free media (GIBCO BRL) is added/well with 0.25 ⁇ of 3 H-myo-inositol/ well and the cells are incubated for 16-18 hours overnight at 37°C/5%C0 2 .
  • GEBCO BRL inositol-free/serum free media
  • the cells are washed with 0.5 mL PBS and 0.45 mL of assay medium is added containing inositol-free/serum free media, 10 ⁇ pargyline, 10 mM lithium chloride or 0.4 mL of assay medium and 50 ⁇ L ⁇ of lOx ketanserin (ket) to final concentration of 10 ⁇ , if using a control construct containing a serotonin receptor.
  • the cells are then incubated for 30 minutes at 37°C.
  • the cells are then washed with 0.5 mL PBS and 200 ⁇ L ⁇ of fresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well.
  • the solution is kept on ice for 5-10 minutes or until cells were lysed and then neutralized by 200 ⁇ L ⁇ of fresh/ice cold neutralization sol. (7.5 % HCL).
  • the lysate is then transferred into 1.5 mL eppendorf tubes and 1 mL of chloroform/methanol (1:2) is added/tube.
  • the solution is vortexed for 15 seconds and the upper phase is applied to a Biorad AG1-X8TM anion exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1: 1.25 W/V and 0.9 mL of upper phase is loaded onto the column.
  • the column is washed with 10 mL of 5 mM myo-inositol and 10 mL of 5 mM Na- borate/60mM Na-formate.
  • the inositol tris phosphates are eluted into scintillation vials containing 10 mL of scintillation cocktail with 2 mL of 0.1 M formic acid/ 1 M ammonium formate.
  • the columns are regenerated by washing with 10 mL of 0.1 M formic acid/3 M ammonium formate and rinsed twice with dd H 2 0 and stored at 4°C in water.
  • membranes comprising the Target GPCR of interest for use in the identification of candidate compounds as, e.g.,. agonists, inverse agonists or antagonists are prepared as follows:
  • “Membrane Scrape Buffer” is comprised of 20mM HEPES and lOmM EDTA, pH 7.4;
  • “Membrane Wash Buffer” is comprised of 20mM HEPES and 0. lmM EDTA, pH 7.4;
  • “Binding Buffer” is comprised of 20mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.4.
  • the media is aspirated from a confluent monolayer of cells, followed by rinsing with lOmL cold PBS, followed by aspiration. Thereafter, 5 mL of Membrane Scrape Buffer is added to scrape cells; this is followed by transfer of cellular extract into 50 mL centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4°C). Thereafter, the supernatant is aspirated and the pellet is resuspended in 30 mL Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17 minutes at 4°C. The supernatant is then aspirated and the pellet resuspended in Binding Buffer. This is then homogenized using a Brinkman PolytronTM homogenizer (15-20 second bursts until the all material is in suspension). This is referred to herein as "Membrane Protein".
  • Protein concentration of the membranes is determined using the Bradford Protein Assay (protein can be diluted to about 1.5 mg/mL, aliquoted and frozen (-80°C) for later use; when frozen, protocol for use will be as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a Polytron at about 12 x 1,000 rpm for about 5-10 seconds; it is noted that for multiple preparations, the homogenizer should be thoroughly cleaned between homogenization of different preparations).
  • Binding Buffer (as per above); Bradford Dye Reagent; Bradford Protein Standard is utilized, following manufacturer instructions (Biorad, cat. no. 500-0006).
  • Duplicate tubes are prepared, one including the membrane, and one as a control "blank". Each tube contains 800 ⁇ L ⁇ Binding Buffer. Thereafter, 10 ⁇ L ⁇ of Bradford Protein Standard (Img/mL) is added to each tube, and 10 ⁇ L ⁇ of membrane Protein is then added to just one tube (not the blank). Thereafter, 200 ⁇ L ⁇ of Bradford Dye Reagent is added to each tube, followed by vortexing of each tube. After five (5) minutes, the tubes are re-vortexed and the material therein is transferred to cuvettes. The cuvettes are read using a CECIL 3041 spectrophotometer, at wavelength 595.
  • GDP Buffer consists of 37.5 mL Binding Buffer and 2 mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 ⁇ GDP (final concentration of GDP in each well is 0.1 ⁇ GDP); each well comprising a candidate compound has a final volume of 200 consisting of 100 ⁇ GDP Buffer (final concentration, ⁇ . ⁇ GDP), 50 ⁇ Membrane Protein in Binding Buffer, and 50 ⁇ [ 35 S]GTPyS (0.6 nM) in Binding Buffer (2.5 ⁇ [ 35 S]GTPyS per 10 mL Binding Buffer).
  • Candidate compounds can be screened using a 96-well plate format (these can be frozen at -80°C).
  • Membrane Protein or membranes with expression vector excluding the Target GPCR, as control), are homogenized briefly until in suspension. Protein concentration is be determined using the Bradford Protein Assay set forth above. Membrane Protein (and control) is diluted to
  • Binding Buffer 0.25mg/mL in Binding Buffer (final assay concentration, 12 ⁇ g/well). Thereafter, 100 ⁇ L ⁇ GDP Buffer is added to each well of a Wallac ScintistripTM (Wallac).
  • a 5 ⁇ ⁇ pin-tool is used to transfer 5 ⁇ L ⁇ of a candidate compound into such well (i.e., 5 ⁇ L ⁇ in total assay volume of 200 ⁇ L ⁇ is a 1:40 ratio such that the final screening concentration of the candidate compound is 10 ⁇ ).
  • the pin tool should be rinsed in three reservoirs comprising water (IX), ethanol (IX) and water (2X) - excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes.
  • Another assay approach for identifying candidate compounds as, e.g., agonists, inverse agonist, or antagonists can accomplished by utilizing a cyclase-based assay.
  • this assay approach can be utilized as an independent approach to provide confirmation of the results from the [ 35 S]GTPyS approach as set forth in the above example.
  • SMP004A can be utilized for direct identification of candidate compounds as inverse agonists and agonists to a receptor of interest in accordance with the following protocol.
  • Transfected cells are harvested approximately three days after transfection.
  • Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl 2 . Homogenization is performed on ice using a Brinkman PolytronTM for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 X g for 15 minutes at 4°C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 x g for 15 minutes at 4°C. The resulting pellet is then stored at -80°C until utilized.
  • the membrane pellet On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCl 2 , to yield a final protein concentration of 0.60 mg/mL (the resuspended membranes are placed on ice until use).
  • cAMP standards and Detection Buffer comprising 2 ⁇ of tracer [ 125 I]cAMP (100 ⁇ ) to 11 mL Detection Buffer] are prepared and maintained in accordance with the manufacturer' s instructions.
  • Assay Buffer is prepared fresh for screening and contains 20mM HEPES, pH 7.4, 10 mM MgCl 2 , 20mM phospocreatine (Sigma), 0.1 units/mL creatine phosphokinase (Sigma), 50 ⁇ GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer is then stored on ice until utilized.
  • Candidate compounds are added to, for example, 96-well plate wells (3 ⁇ /well; 12 ⁇ final assay concentration), together with 40 ⁇ Membrane Protein (30 ⁇ g/well) and 50 ⁇ of Assay Buffer. This admixture is then incubated for 30 minutes at room temperature, with gentle shaking.
  • Target Receptor (experimental) and pCMV (negative control) stably transfected cells from respective clonal lines are seeded into poly-D-lysine pretreated 96-well plates (Becton-Dickinson, #356640) at 5.5xl0 4 cells/well with complete culture medium (DMEM with 10% FBS, 2 mM L- glutamine, lmM sodium pyruvate) for assay the next day.
  • DMEM complete culture medium
  • a promiscuous G protein such as Gal 5, Gal6, or the chimeric Gq/Gi alpha subunit is available to cause a detectable calcium flux.
  • Fluo4-AM (Molecular Probe, #F14202) incubation buffer stock, 1 mg Fluo4-AM is dissolved in 467 ⁇ DMSO and 467 ⁇ Pluoronic acid (Molecular Probe, #P3000) to give a lmM stock solution that can be stored at -20°C for a month.
  • Fluo4-AM is a fluorescent calcium indicator dye.
  • Candidate compounds are prepared in wash buffer (IX HBSS/2.5mM Probenicid/20mM HEPES at pH 7.4).
  • culture medium is removed from the wells and the cells are loaded with ⁇ of 4 ⁇ Fluo4-AM/2.5 mM Probenicid (Sigma, #P8761)/20mM HEPES/complete medium at pH 7.4. Incubation at 37°C/5% C0 2 is allowed to proceed for 60 minutes.
  • the Fluo4-AM incubation buffer is removed and the cells are washed 2X with 100 ⁇ wash buffer. In each well is left 100 ⁇ wash buffer. The plate is returned to the incubator at 37°C/5% C0 2 for 60 minutes.
  • FLIPR Fluorometric Imaging Plate Reader
  • MAP kinase mitogen activated kinase
  • MAP kinase can be detected by several approaches.
  • One approach is based on an evaluation of the phosphorylation state, either unphosphorylated (inactive) or phosphorylated (active).
  • the phosphorylated protein has a slower mobility in SDS-PAGE and can therefore be compared with the unstimulated protein using Western blotting.
  • antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase.
  • cells are stimulated with the candidate compound and then extracted with Laemmli buffer. The soluble fraction is applied to an SDS- PAGE gel and proteins are transferred electrophoretically to nitrocellulose or Immobilin.
  • Immunoreactive bands are detected by standard Western blotting technique. Visible or chemiluminescent signals are recorded on film and can be quantified by densitometry.
  • Another approach is based on evalulation of the MAP kinase activity via a phosphorylation assay.
  • Cells are stimulated with the candidate compound and a soluble extract is prepared.
  • the extract is incubated at 30°C for 10 minutes with gamma- 32 P-ATP, an ATP regenerating system, and a specific substrate for MAP kinase such as phosphorylated heat and acid stable protein regulated by insulin, or PHAS-I.
  • the reaction is terminated by the addition of H 3 P0 4 and samples are transferred to ice.
  • An aliquot is spotted onto Whatman P81 chromatography paper, which retains the phosphorylated protein.
  • the chromatography paper is washed and counted for 32 P is a liquid scintillation counter.
  • the cell extract is incubated with gamma- 32 P-ATP, an ATP regenerating system, and biotinylated myelin basic proein bound by streptavidin to a filter support.
  • the myelin basic protein is a substrate for activated MAP kinase.
  • the phosphorylation reaction is carried out for 10 minutes at 30°C.
  • the extract can then be aspirated through the filter, which retains, the phosphorylated myelin basic protein.
  • the filter is washed and counted for 32 P by liquid scintillation counting.
  • pruritus model that displays at least one element or aspect of pruritus and are amenable to the evaluation of anti-pruritic agents.
  • the pruritus model is a behavioral model.
  • the pruritus model is a mechanistic or molecular model.
  • the pruritus model is a vertebrate model. In another embodiment, the pruritus model is a mammalian model. In another embodiment, the pruritus model is a primate model, a dog model, a guinea pig model, a mouse model, or a cat model.
  • animal models may be particularly useful in analyzing potential anti-pruritic agents.
  • animal pruritus models can be used to determine the in vivo efficacy of a H3R antagonist.
  • the pruritus model is behaviorally based and evaluation of such a model comprises evaluating the behavior of the organism that has been administered a test compound.
  • the pruritus model is selected from the group: pruritogen injection model, passive cutaneous anaphylaxis model, allergic pruritus model, and spontaneous pruritus model.
  • the pruritus model is selected from the group: histamine induced pruritus model, DNP-Ovalbumin pruritus model, and DNFB pruritus model.
  • the response to pruritus is a behavioral response.
  • the following systems are described for purposes of illustration and are not intended to be limiting.
  • itch is induced by an intradermal or subcutaneous injection of a pruritogen (i.e. histamine) into the rostral part of the back of mice. Mice are video recorded and the number of scratching of the injection site by a hind leg after pruritogen injection is counted.
  • a pruritogen i.e. histamine
  • Mice are video recorded and the number of scratching of the injection site by a hind leg after pruritogen injection is counted.
  • Several substances in addition to histamine can be injected to induce itch: serotonin, substance P, chloroquine, protease-activated receptor 2 (PAR-2) activating peptide, trypsin.
  • a test compound can be administered orally or by other routes shortly prior to itch induction, and the efficacy of the test compound compared to that of a sham treatment or that of a positive control drug.
  • itch is triggered not by direct injection of a pruritogen but rather by an anaphylactic reaction upon injection of an antigen to antibody-primed mice.
  • a small amount of IgE antibody directed against a known antigen i.e., dinitrophenyl-conjugated ovalbumin or DNP-OVA
  • the antigen is later given to induce an allergic reaction, which results in itch.
  • 20-100 ng of dinitrophenyl-IgE is injected into the rostral part of the back of the animals, and 24 hours laterlOO ug of antigen (DNP-OVA) is injected intraperitoneally.
  • a test compound can be dosed 30 minutes before antigen injection.
  • Allergic pruritus model differs from the passive cutaneous anaphylaxis model in that sensitization to an antigen develops through an active immunization process (i.e. hypersensitivity develops to a known antigen via an endogenously generated immune response).
  • an antigen DNP-OVA is administered by intraperitoneal injection in mice.
  • a booster immunization may be given a week later.
  • Pruritus is then induced two weeks after the initial immunization by injecting the antigen at the rostral part of the back. Again, a test compound is dosed shortly before itch induction.
  • antigen may be toluene-2,4-diisocyanate (TDI), 2,4-niditrochlorobenzene (DNCB), oxazolone, or picryl chloride.
  • Sensitization can be done by injection intradermally, intraperitoneally, subcutaneously or by other routes.
  • An antigen can be given by topical application, by intradermal or subcutaneous injection to induce an allergic reaction.
  • a test compound can be administered before or during the sensitization period or shortly before the allergen challenge.
  • mice develop pruritus due to ongoing inflammation without the need for pruritogen or antigen injection.
  • spontaneous models include the NC/Nga mice, interleukin-4 and interleukin-13 transgenic mice, and MRL/lpr mice.
  • NC/Nga mice develop skin lesions and pruritus after 8 weeks of age when kept in conventional vivarium conditions (Takano, N. et al., 2003, Eur. Pharmacol. 471: 223-228).
  • the interleukin-4 mice have been genetically manipulated to express high level of this Th2-bias cytokines in the skin. These mice develop skin lesions and pruritus with clinical features resembling those of human atopic dermatitis (Chan, L.S.
  • mice expressing high level of the Th2-bias cytokine IL-13 develop skin inflammation bearing the hallmarks of atopic dermatitis such as skin lesions, epidermal barrier breakdown and intense pruritus (Zheng, T. et al., J. Invest. Dermatol. 2008, Oct 16, online publication).
  • MRL lpr mice have systemic autoimmunity resulting from a mutation in the lymphocyte death promoting Fas gene. Spontaneous scratching and skin lesions develop in MRL/lpr mice at 18 weeks of age (Umeuchi, H et al 2005, Eur J Pharm.
  • mice that have already shown scratching behavior can be video recorded for one hour (or other length of time) to establish an itch intensity baseline.
  • the test compound can be administered orally or by other routes. Mice can be video recorded to determine the frequency of scratching.
  • the efficacy of a test compound can be compared to a sham treatment or a positive control.
  • a model for canine atopic dermatitis has been utilized through an environmental house dust mite challenge of high-IgE-producing beagles, mite hypersensitive dogs with atopic dermatitis and normal dogs (Vet Dermatol. 2006 Feb;17(l):24-35).
  • This model utilized high-IgE beagles epicutaneously sensitized to house dust mite (HDM) as a model for cAD.
  • HDM house dust mite
  • Six high-IgE beagles were environmentally challenged with HDM using various doses and protocols. Similar challenge protocols were used in positive and negative control dogs: three dogs with naturally occurring cAD and positive intradermal skin test (IDT) to HDM and three normal dogs without history of skin disease and negative IDT to HDM.
  • IDT intradermal skin test
  • a guinea pig pruritus model (Exp Dermatol. 2002 Aug;l 1(4):285-91) has been described using iontophoresis of histamine and capsaicin. It was also shown that contact sensitization with 2-4 dinitrochlorobenzene (DNCB) can be used as a simple assay for chronic itch allowing study of scratching over at least a 15-h period.
  • DNCB dinitrochlorobenzene
  • a primate pruritus model (J Neurosci. 2008 Jul 23;28(30):7659-69) has been described.
  • the existence of two peripheral pathways for itch has been suggested in some species: one pathway that is responsive to histamine and a second pathway that can be activated by nonhistaminergic pruritogens (e.g., cowhage spicules).
  • nonhistaminergic pruritogens e.g., cowhage spicules.
  • behavioral responses and neuronal activity in unmyelinated afferent fibers can be assessed in monkey after topical application of cowhage spicules or intradermal injection of histamine and capsaicin.
  • histamine induced itch can be analyzed in humans by injecting a pruritogen into the skin by iontophoresis.
  • a fresh solution of histamine in saline can be prepared shortly before the experiment.
  • a cotton disk can be soaked with this solution and mounted into the application chamber of an iontophoresis applicator.
  • An anodal current and a charge of 20 mC (1 mA, 20 s) can be used to deliver histamine iontophoretically into the skin.
  • the intensity of itch can be graded by subjects on a 100mm visual analogue scale (VAS) at 20s intervals for 5min after the injection of histamine.
  • VAS 100mm visual analogue scale
  • Compounds may be further selected based on their peripheral restriction (or lack thereof).
  • One aspect of the present invention relates to H3R antagonists that are peripherally restricted.
  • One aspect of the present invention relates to anti-pruritic agents that are peripherally restricted.
  • the Caco-2 cell line was purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and grown with Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, and 1% nonessential amino acids at 37°C in 5% C0 2 .
  • Multi-well insert systems with an array of 12 or 24 individual inserts were used in the permeability assay. Each multi-well insert unit has two compartments: the top compartment and the bottom compartment. The top compartment is the insert and is commonly referred to as the apical compartment or apical side (A).
  • the bottom compartment is the well of a multi-well plate where the insert is placed and is referred to as the basal compartment or basal side (B).
  • Basal compartment or basal side (B).
  • Caco-2 cells were seeded on a porous filter membrane. Cell culture medium was changed every two days. Cells were used for the transport experiments between 24 to 26 days after seeding.
  • AQ/At is the appearance rate (DPM/sec of radiolabeled test compound; umol/sec of non-labeled test compound) on the receiver side during the permeation process
  • A is the surface area of the cell monolayers
  • Co is the initial concentration (DPM/mL of radiolabeled test compound; umol/mL of non-labeled test compound) on the donor side.
  • Efflux ratio was defined as the ratio of Papp in the basolateral to apical direction over the Papp in the apical to basolateral direction.
  • Brain vs Plasma concentrations (or ratio) for administered compounds. For example this may be calculated based on standard curve concentration. After detection by LC/MS/MS, the final brain concentrations can be calculated. Plasma sample concentrations are calculated based on standard curve concentration. Brain/Plasma ratio can be calculated with the final brain and plasma data of each animal.
  • compounds are further selected based on desirable pharmacokinetic properties.
  • the compound would exhibit a Ki ⁇ 10 nM across species.
  • the compound exhibits selectivity across a broad receptor panel. In another embodiment, the compound does not exhibit CYP or hERG liability.
  • the compound is efficacious in a histamine-mediated itch model. In another embodiment, the compound is efficacious in a dermatitis-driven itch model. In another embodiment, the compound has at least 10X potency separation between wake promotion and itch models.
  • the compound or agent is restricted in some manner from crossing the blood brain barrier. In another embodiment, the compound or agent may be less than 100% restricted to the periphery. In one embodiment, the compound or agent has a braimplasma ratio (B/P) which will be less than 1 in at least one species.
  • B/P braimplasma ratio
  • the H3R antagonist or anti -pruritic agent has a brain to plasma ratio of about 0.5 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.4 or less. In another embodiment, the H3R antagonist or antipruritic agent has a brain to plasma ratio of about 0.3 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.15 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.1 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.01 or less.
  • the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.5 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.4 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti- pruritic agent has a brain to plasma ratio of about 0.3 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less after administration in a pruritus model animal.
  • the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.15 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti- pruritic agent has a brain to plasma ratio of about 0.1 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.01 or less after administration in a pruritus model animal.
  • the compound has good oral bioavailability.
  • the compound shows Tl/2>lh in microsomal stability across species.
  • the compound shows in vivo Tl/2 in animal species consistent with a QD or BID regimen.
  • the compound shows hERG > 10 uM.
  • the compound is Ames negative.
  • the compound has acceptable tolerability in rat repeat dosing.
  • a H3R antagonist or anti-pruritic agent such as an antagonist or an inverse agonist, can be formulated into pharmaceutical compositions and medicaments for use in accordance with the present invention using techniques well known in the art.
  • One aspect of the present invention relates to compositions comprising a H3R antagonist for use in treating pruritus/itch. Proper formulation is dependent on the route of administration chosen. In certain embodiments, the administration is to a non-human vertebrate or to a non-human mammal.
  • the anti-pruritic agent/H3R antagonist is subsequently admixed with a pharmaceutical carrier.
  • Pharmaceutical carriers are known in the art, such as pharmaceutical carriers described herein.
  • the H3R antagonist is formulated as a composition.
  • the H3R antagonist is a H3R inverse agonist.
  • the anti-pruritic agent is subsequently admixed with a
  • formulated compounds or agents can exhibit all the features previously described for compounds in relation to the screening assays or otherwise described in this application.
  • compounds for formulation, method of treatment or any of the other embodiments described herein may, for example, demonstrate the selectivity, B/P ratios, or potency as previously or subsequently described.
  • One aspect of the present invention relates to methods of preparing a pharmaceutical composition comprising a H3R antagonist for treating a condition characterized by itch, a method comprising:
  • the H3R antagonist is an antagonist of the human H3R.
  • the H3R antagonist is orally active.
  • the H3R antagonist is a selective H3R antagonist.
  • the H3R antagonist has a selectivity for H3R over H1R, H2R, or
  • H4R of at least about 10 to fold.
  • the H3R antagonist has an IC50 of less than about 1 ⁇ .
  • the H3R antagonist has an IC50 of less than about 100 nM.
  • the H3R antagonist has an IC50 of less than about 1 nM.
  • the H3R antagonist is a small molecule.
  • the mammal is a non-human mammal.
  • the mammal is selected from the group consisting of a mouse, a rat, a dog and a non-human primate.
  • the mammal is a human.
  • the pharmaceutical composition is in a dosage form.
  • the H3R antagonist has an IC50 of less than 1 ⁇ .
  • the H3R antagonist has an IC50 of less than about 10 nM.
  • the H3R antagonist has a selectivity for H3R over a H1R, H2R, or H4R of at least about 10 to fold.
  • the dosage form is in combination with a H1R antagonist.
  • the present invention provides a method of treating pruritus in a subject in need thereof by administering a therapeutically effective amount of a H3R antagonist.
  • the H3R antagonist is in an amount sufficient to reduce itch in an individual.
  • the H3R antagonist is selected from and the following compounds and pharmaceutically acceptable salts, solvates, and hydrates thereof:
  • the invention comprises conjugates of antibodies, e.g. Fv, Fab, and F(ab)'2, bifunctional hybrid antibodies and single chain antibodies that are known to one skilled it the art.
  • the method of treating pruritus in a subject in need thereof involves administering a therapeutically effective amount of a H3R antagonist in conjunction with a therapeutically effective amount of another compound known to facilitate the treatment of pruritus.
  • the methods disclosed herein can be used in conjunction with other known anti-pruritus therapies such as menthol and phenol, calamine, topical
  • antihistamines for uremia: dialysis, UVB phototherapy and for paraneoplasia: paroxetin.
  • the methods disclosed herein can be used in conjunction with therapies for eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound healing, itch associated with an insect bite, itch associated
  • compositions include solutions, suspensions, lotions, gels, creams, ointments, emulsions, skin patches, etc. All of these dosage forms, along with methods for their preparation, are well known in the pharmaceutical and cosmetic art: Harry's Cosmeticology (Chemical Publishing, 7th ed. 1982); Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th ed. 1990).
  • topical formulations contain the active ingredient in a concentration range of 0.001 to 10 mg/mL, in admixture with suitable vehicles.
  • Other desirable ingredients for use in such anti- pruritic preparations include preservatives, co-solvents, viscosity building agents, carriers, etc.
  • the carrier itself or a component dissolved in the carrier may have palliative or therapeutic properties of its own, including moisturizing, cleansing, or anti-inflammatory/anti-itching properties.
  • the H3R antagonists can be combined with a therapeutically effective amounts of anti-inflammation agents such as corticosteroids, fungicides, antibiotics, moisturizers or anti- itching compounds.
  • Penetration enhancers may, for example, be surface active agents; certain organic solvents, such as dimethyl sulfoxide and other sulfoxides, dimethyl-acetamide and pyrrolidone; certain amides of heterocyclic amines, glycols (e.g. propylene glycol); propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.
  • organic solvents such as dimethyl sulfoxide and other sulfoxides, dimethyl-acetamide and pyrrolidone
  • certain amides of heterocyclic amines such as glycols (e.g. propylene glycol); propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.
  • Topical administration of a pharmacologically effective amount may utilize transdermal delivery systems well known in the art.
  • the H3R antagonists may also be administered systemically, such as oral, parenteral, nasal inhalation, and intrarectal are also contemplated.
  • additional conventional pharmaceutical preparations such as tablets, granules, powders, capsules, and sprays may be preferentially required.
  • further conventional additives such as binding-agents, wetting agents, propellants, lubricants, and stabilizers may also be required.
  • Systemic administration preferably comprises ingestion of any solid or solution carriers containing a pharmacologically effective amount of one or more of the H3R antagonists.
  • Such solid or solution carriers may comprise pills, hard tablets, soft tablets, gums or ordinary liquids.
  • systemic administration of a pharmacologically effective amount may comprise invasive methodologies including intravenous, subcutaneous, intramuscular or intralesional injection of a suitable carrier, such as saline, containing a pharmacologically effective amount of one or more of the H3R antagonists.
  • the route of administration, dosage form, and the effective amount vary according to the potency of the selected H3R antagonist, its physicochemical characteristics, and according to the location of itch sensations.
  • the selection of proper dosage is well within the skill of an ordinary skilled physician.
  • Topical formulations are usually administered up to four-times a day.
  • H3R antagonists as anti -pruritics may be combined with, for example, Hl- antihistamines to provide superior therapy via additive or synergistic interaction.
  • the itch may be associated with a disease or disorder related from the group consisting of eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, lichen planus, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with haemodialysis, burns, scalds, sunburn, insect bites, urticaria, sweat gland abnormalities, bullous pemphigoid, photodermatoses, skin blisters, adult acne, chicken pox, and dermatitis herpetiformis.
  • H3R antagonists of the present invention can be administered in any suitable way.
  • Suitable routes of administration include oral, nasal, rectal, transmucosal, transdermal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art.
  • Other suitable routes of administration are aerosol and depot formulation. Sustained release formulations, particularly depot, of the invented medicaments are expressly contemplated.
  • the compounds according to the present invention are administered orally.
  • the compounds according to the present invention can be made up in solid or liquid form, such as tablets, capsules, powders, syrups, elixirs and the like, aerosols, sterile solutions, suspensions or emulsions, and the like.
  • the H3R antagonist is administered orally.
  • Formulations for oral administration may be in the form of aqueous solutions and suspensions, in addition to solid tablet and capsule formulations.
  • the aqueous solutions and suspensions may be prepared from sterile powders or granules.
  • the compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.
  • Other adjuvants are well and widely known in the art.
  • compositions of the H3R antagonist may be prepared by methods well known in the art, e.g., by means of conventional mixing, dissolving, granulation, dragee-making, levigating, emulsifying, encapsulating, entrapping, lyophilizing processes or spray drying.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers are available to those in the art (see, e.g., Remington: The Science and Practice of Pharmacy, (Gennaro et al., eds.), 20 th Edition, 2000, Lippincott Williams & Wilkins; and Handbook of Pharmaceutical Excipients (Rowe et al., eds), 4 th Edition, 2003, Pharmaceutical Press). Proper formulation is dependent upon the route of administration chosen.
  • carrier material or “excipient” material herein means any substance, not itself a therapeutic agent, used as a carrier and/or dilutent and/or adjuvant, or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition into a discrete article such as a capsule or tablet suitable for oral administration.
  • Excipients can include, by way of illustration and not limitation, diluents, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or counteract a disagreeable taste or odor, flavors, dyes, fragrances, and substances added to improved appearance of the composition.
  • Acceptable excipients include stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc, gelatin, acacia gum, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starches, gelatin, cellulosic materials, such as cellulose esters of alkanoic acids and cellulose alkyl esters, low melting wax cocoa butter or powder, polymers, such as polyvinylpyrrolidone, polyvinyl alcohol, and polytheylene glycols, and other pharmaceutically acceptable materials.
  • the components of the pharmaceutical composition can be encapsulated or tableted for convenient administration.
  • One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist.
  • H3R antagonist or a pharmaceutical composition as described herein.
  • One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist, wherein the H3R antagonist is identified by using any of the methods described herein.
  • the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
  • One aspect of the present invention relates to uses of a H3R antagonist in the manufacture of a medicament for treating pruritus.
  • One aspect of the present invention relates to uses of a H3R antagonist in the manufacture of a medicament for treating pruritus, wherein the H3R antagonist is identified by using any of the methods described herein.
  • One aspect of the present invention relates to H3R antagonists for use in a method for the treatment of pruritus.
  • One aspect of the present invention relates to H3R antagonists for use in a method for the treatment of pruritus, wherein the H3R antagonist is identified by using any of the methods described herein.
  • the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
  • One aspect of the present invention relates to H3R antagonists for use in a method for treating pruritus.
  • the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
  • the H3R antagonist is applied topically.
  • the H3R antagonist is applied topically to the site afflicted with itch.
  • the H3R antagonist is applied topically and has an IC50 (H3R) of about 100 nM or less.
  • the pharmaceutical composition is formulated in a form suitable for topical application.
  • the H3R antagonist is administered systemically.
  • the method further comprises administering a therapeutic amount of a H1R antagonist.
  • the antagonist is administered orally.
  • the H3R antagonist is peripherally restricted.
  • Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used which may optionally contain gum Arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycols, cremophor, capmul, medium or long chain mono-, di- or triglycerides.
  • Stabilizers may be added in these formulations, also.
  • a H3R antagonist may be delivered using a sustained-release system.
  • sustained-release materials have been established and are well known to those skilled in the art.
  • Sustained-release tablets or capsules are particularly preferred.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • the dosage form may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108, 4, 166,452, and 4,265,874 to form osmotic therapeutic tablets for controlled release.
  • therapies of the present invention may be administered or provided alone or in combination with one or more other pharmaceutically or physiologically acceptable compound.
  • the other pharmaceutically or physiologically acceptable compound i.e., second pharmaceutical agent
  • the present invention features a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention. In one aspect, the present invention features a pharmaceutical composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention and at least one pharmaceutically acceptable carrier.
  • the present invention relates to a composition
  • a composition comprising a compound identified according to one or more of the screening methods described herein.
  • the present invention features a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention.
  • the present invention features a pharmaceutical composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention and at least one pharmaceutically acceptable carrier.
  • the present invention also relates to a dosage form of the composition or of the pharmaceutical composition wherein the H3R antagonist is in an amount sufficient to give an effect in treating a condition characterized by itch.
  • the H3R antagonist is in an amount sufficient to reduce itch in an individual.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount to achieve their intended purpose.
  • a pharmaceutical composition of the present invention is understood to be useful for treating a condition characterized by itch, such as pruritus.
  • determination of the amount of a H3R antagonist sufficient to achieve an intended purpose according to the invention is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the data obtained from animal studies including but not limited to studies using mice, rats, rabbits, pigs, and non-human primates, can be used in formulating a range of dosage for use in humans.
  • one skilled in the art understands how to extrapolate in vivo data obtained in an animal model system to another, such as a human. In some circumstances, these extrapolations may merely be based on the weight of the animal model in comparison to another, such as a human; in other circumstances, these extrapolations are not simply based on weights but rather incorporate a variety of factors.
  • compositions of this invention are selected in accordance with a variety factors as cited above.
  • the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods of this invention.
  • An exemplary animal model system is mouse.
  • Dosage amount and interval may be adjusted in order to provide an intended therapeutic effect. It will be appreciated that the exact dosage of a H3R antagonist in accordance with the present invention will vary depending on the H3R antagonist, its potency, the mode of administration, the age and weight of the patient and the severity of the condition to be treated. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • an amount of a H3R antagonist in accordance with the present invention is less than about 0.001 mg/kg body weight, less than about 0.005 mg kg body weight, less than about 0.01 mg/kg body weight, less than about 0.05 mg/kg body weight, less than about 0.1 mg/kg body weight, less than about 0.5 mg/kg body weight, less than about 1 mg/kg body weight, less than about 5 mg/kg body weight, less than about 10 mg/kg body weight, less than about 50 mg/kg body weight, or less than about 100 mg/kg body weight.
  • an amount of a H3R antagonist in accordance with the present invention is less than about 0.001-100 mg/kg body weight, less than about 0.001-50 mg/kg body weight, less than about 0.001-10 mg/kg body weight, less than about 0.001-5 mg/kg body weight, less than about 0.001-1 mg/kg body weight, less than about 0.001 to 0.5 mg/kg body weight, less than about 0.001-0.1 mg/kg body weight, less than about 0.001-0.05 mg/kg body weight, less than about 0.001-0.01 mg/kg body weight, or less than about 0.001-0.005 mg/kg body weight.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of a H3R antagonist according to the present invention which achieve an intended therapeutic effect. Dosage intervals can also be determined using the value for a selected range of H3R antagonist concentration so as to achieve the intended therapeutic effect.
  • a H3R antagonist should be administered using a regimen that maintains plasma levels within the selected range of a H3R antagonist concentration for 10-90% of the time, preferably between 30-99% of the time, and most preferably between 50-90% of the time. In cases of local administration or selective uptake, the range of H3R antagonist concentration providing the intended therapeutic effect may not be related to plasma concentration.
  • composition administered will, of course, be dependent on the individual being treated, on the individual's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
  • the present invention accordingly features a method of treating a condition characterized by itch, such as pruritus, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention.
  • the composition is a pharmaceutical composition.
  • active ingredient is defined in the context of a
  • pharmaceutical composition and is intended to mean a component of a pharmaceutical composition that provides the primary pharmacological effect, as opposed to an "inactive ingredient” which would generally be recognized as providing no pharmaceutical benefit.
  • the amount of active ingredient, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of
  • compositions of this invention are selected in accordance with a variety factors as cited above.
  • the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods of this invention.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
  • the daily dose can be divided, especially when relatively large amounts are administered as deemed appropriate, into several, for example 2, 3 or 4 part administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the daily dose indicated.
  • the compounds of the present invention can be administrated in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt, solvate or hydrate of a compound of the invention.
  • a suitable pharmaceutically acceptable carrier can be either solid, liquid or a mixture of both.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules.
  • a solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier is a finely divided solid which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted to the desire shape and size.
  • the powders and tablets may contain varying percentage amounts of the active compound.
  • a representative amount in a powder or tablet may contain from 0.5 to about 90 percent of the active compound; however, an artisan would know when amounts outside of this range are necessary.
  • Suitable carriers for powders and tablets are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
  • preparation is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
  • carrier which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid forms suitable for oral administration.
  • a low melting wax such as an admixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogenous mixture is then poured into convenient sized molds, allowed to cool and thereby to solidify.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Liquid form preparations include solutions, suspensions and emulsions, for example, water or water-propylene glycol solutions.
  • parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injec tables.
  • the compounds according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • Aqueous formulations suitable for oral use can be prepared by dissolving or suspending the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired.
  • Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.
  • viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions and emulsions.
  • These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.
  • the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray.
  • the formulations may be provided in single or multi-dose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump.
  • Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant.
  • aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant.
  • the compounds of the present invention or pharmaceutical compositions comprising them are administered as aerosols, for example as nasal aerosols or by inhalation, this can be carried out, for example, using a spray, a nebulizer, a pump nebulizer, an inhalation apparatus, a metered inhaler or a dry powder inhaler.
  • Pharmaceutical forms for administration of the compounds of the present invention as an aerosol can be prepared by processes well known to the person skilled in the art.
  • solutions or dispersions of the compounds of the present invention in water, water/alcohol mixtures or suitable saline solutions can be employed using customary additives, for example benzyl alcohol or other suitable preservatives, absorption enhancers for increasing the bioavailability, solubilizers, dispersants and others and, if appropriate, customary propellants, for example include carbon dioxide, CFCs, such as,
  • the aerosol may conveniently also contain a surfactant such as lecithin.
  • the dose of drug may be controlled by provision of a metered valve.
  • the compound In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. When desired, formulations adapted to give sustained release of the active ingredient may be employed.
  • the active ingredients may be provided in the form of a dry powder, for example, a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • the powder carrier will form a gel in the nasal cavity.
  • the powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
  • the pharmaceutical preparations are preferably in unit dosage forms.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • Tablets or capsules for oral administration and liquids for intravenous administration are preferred compositions.
  • the compounds according to the invention may optionally exist as pharmaceutically acceptable salts including pharmaceutically acceptable acid addition salts prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Representative acids include, but are not limited to, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, dichloroacetic, formic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like.
  • Certain compounds of the present invention which contain a carboxylic acid functional group may optionally exist as pharmaceutically acceptable salts containing non-toxic, pharmaceutically acceptable metal cations and cations derived from organic bases.
  • Representative metals include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and the like. In some embodiments the pharmaceutically acceptable metal is sodium.
  • Organic bases include, but are not limited to, benzathine ( N2-dibenzylemane-l,2-diamine), chloroprocaine (2- (diethylamino)ethyl 4-(chloroamino)benzoate), choline, diethanolamine, ethylenediamine, meglumine ((2R,3R,4R,55)-6-(methylamino)hexane-l,2,3,4,5-pentaol), procaine (2- (diethylamino)ethyl 4-aminobenzoate), and the like.
  • Certain pharmaceutically acceptable salts are listed in Berge, et al., Journal of Pharmaceutical Sciences, 66: 1-19 (1977).
  • the acid addition salts may be obtained as the direct products of compound synthesis.
  • the free base may be dissolved in a suitable solvent containing the appropriate acid and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
  • the compounds of this invention may form solvates with standard low molecular weight solvents using methods known to the skilled artisan.
  • Pro-drugs refers to compounds that have been modified with specific chemical groups known in the art and when administered into an individual these groups undergo biotransformation to give the parent compound. Pro-drugs can thus be viewed as compounds of the invention containing one or more specialized non-toxic protective groups used in a transient manner to alter or to eliminate a property of the compound. In one general aspect, the "pro-drug” approach is utilized to facilitate oral absorption.
  • T. Higuchi and V. Stella Pro-drugs as Novel Delivery Systems Vol. 14 of the A.C.S. Symposium Series; and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
  • composition for "combination-therapy” comprising admixing at least one compound according to any of the compound embodiments disclosed herein, together with at least one known pharmaceutical agent as described herein and a pharmaceutically acceptable carrier.
  • H3R antagonists can be co-administered with other therapeutic agents to meet the treatment objectives.
  • a H3R antagonist may be combined with antagonists of other histamine receptor subtypes, particularly H1R and H4R antagonists.
  • H1R antagonists may include both sedation and non-sedation antihistamines such as diphenhydramine, cetirizine, hydroxyzine, chlorpheniramine, promethazine, fexofenadine, loratadine, and desloratadine.
  • the composition further comprises an H1R antagonist in an amount sufficient to reduce itch in an individual.
  • H4R antagonists to be combined may include compounds reported in the literature.
  • a H3R antagonist with therapeutic agents currently used to treat itch in patients with chronic metabolic diseases.
  • These drugs may include the bile resin cholestyramine and the antibiotic rifampicin, or opiate receptor antagonists naloxone, nalmefene and naltrexone.
  • opiate receptor antagonists naloxone, nalmefene and naltrexone.
  • H3R antagonists to be combined may include compounds reported in the literature.
  • H3R receptor modulators are utilized as active ingredients in a pharmaceutical composition, these are not intended for use only in humans, but in other non-human mammals as well. Indeed, recent advances in the area of animal health-care mandate that consideration be given for the use of active agents, such as H3R receptor modulators, for the treatment of a H3R-associated disease or disorder in companionship animals (e.g., cats, dogs, etc.) and in livestock animals (e.g., cows, chickens, fish, etc.) Those of ordinary skill in the art are readily credited with understanding the utility of such compounds in such settings.
  • companionship animals e.g., cats, dogs, etc.
  • livestock animals e.g., cows, chickens, fish, etc.
  • the dosage forms described herein may comprise, as the active component, either a compound described herein or a pharmaceutically acceptable salt or as a pharmaceutically acceptable solvate or hydrate thereof.
  • various hydrates and solvates of the compounds described herein and their salts will find use as intermediates in the manufacture of pharmaceutical compositions. Typical procedures for making and identifying suitable hydrates and solvates, outside those mentioned herein, are well known to those in the art; see for example, pages 202-209 of K.J. Guillory, "Generation of Polymorphs, Hydrates, Solvates, and Amorphous Solids," in: Polymorphism in Pharmaceutical Solids, ed. Harry G. England, Vol.
  • one aspect of the present invention pertains to methods of administering hydrates and solvates of compounds described herein and/or their pharmaceutical acceptable salts, that can be isolated and characterized by methods known in the art, such as, thermogravimetric analysis (TGA), TGA-mass spectroscopy, TGA-Infrared spectroscopy, powder X-ray diffraction (XRPD), Karl Fisher titration, high resolution X-ray diffraction, and the like.
  • TGA thermogravimetric analysis
  • TGA-mass spectroscopy TGA-Infrared spectroscopy
  • powder X-ray diffraction (XRPD) powder X-ray diffraction
  • Karl Fisher titration high resolution X-ray diffraction
  • the present disclosure includes all isotopes of atoms occurring in the present compounds, intermediates, salts and crystalline forms thereof.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • One aspect of the present invention includes every combination of one or more atoms in the present compounds, intermediates, salts, and crystalline forms thereof that is replaced with an atom having the same atomic number but a different mass number.
  • One such example is the replacement of an atom that is the most naturally abundant isotope, such as H or 12 C, found in one the present compounds, intermediates, salts, and crystalline forms thereof, with a different atom that is not the most naturally abundant isotope, such as 2 H or 3 H (replacing 3 ⁇ 4), or n C, 13 C, or 14 C (replacing 12 C).
  • a compound wherein such a replacement has taken place is commonly referred to as being an isotopically-labeled compound.
  • Isotopic-labeling of the present compounds, intermediates, salts, and crystalline forms thereof can be accomplished using any one of a variety of different synthetic methods know to those of ordinary skill in the art and they are readily credited with understanding the synthetic methods and available reagents needed to conduct such isotopic-labeling.
  • isotopes of hydrogen include 2 H (deuterium) and 3 H (tritium).
  • Isotopes of carbon include n C, 13 C, and 14 C.
  • Isotopes of nitrogen include 13 N and 15 N.
  • Isotopes of oxygen include 15 0, 17 0, and 18 C.
  • An isotope of fluorine includes 18 F.
  • An isotope of sulfur includes 35 S.
  • An isotope of chlorine includes 36 C1.
  • Isotopes of bromine include 75 Br, 76 Br, 77 Br, and 82 Br.
  • Isotopes of iodine include 123 1, 124 1, 125 I, and 131 I.
  • Another aspect of the present invention includes compositions, such as, those prepared during synthesis, preformulation, and the like, and pharmaceutical compositions, such as, those prepared with the intent of using in a mammal for the treatment of one or more of the disorders described herein, comprising one or more of the present compounds, intermediates, salts, and crystalline forms thereof, wherein the naturally occurring distribution of the isotopes in the composition is perturbed.
  • compositions and pharmaceutical compositions comprising compounds as described herein wherein the compound is enriched at one or more positions with an isotope other than the most naturally abundant isotope.
  • Methods are readily available to measure such isotope perturbations or enrichments, such as, mass spectrometry, and for isotopes that are radio-isotopes additional methods are available, such as, radio-detectors used in connection with HPLC or GC.
  • Example 1 Syntheses of Compounds of the Present Invention.
  • TLC Thin-layer chromatography
  • PK6F silica gel 60 A 1 mm plates (Whatman) and column chromatography was carried out on a silica gel column using Kieselgel 60, 0.063-0.200 mm (Merck). Evaporation was done under reduced pressure on a Biichi rotary evaporator.
  • LCMS spec HPLC-pumps: LC-10AD VP, Shimadzu Inc.; HPLC system controller: SCL- 10A VP, Shimadzu Inc; UV-Detector: SPD-IOA VP, Shimadzu Inc; Autosampler: CTC HTS, PAL, Leap Scientific; Mass spectrometer: API 150EX with Turbo Ion Spray source, AB MDS Sciex; Software: Analyst 1.2.
  • H3R antagonist of Formula (la) can be prepared according to procedures described in WO2008/005338, for example: 4'-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4-sulfonic acid 4-chloro-benzylamide can be prepared according to Example 1.9 (Cmpd 24), WO2008/005338; 4'- [2-((R)-2-Methyl-pyrrolidin- 1 -yl)-ethyl] -biphenyl-4-sulfonic acid amide can be prepared according to Example 1.23 (Cmpd 35), WO2008/005338; 4'-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]- biphenyl-4-sulfonic acid ethylamide can be prepared according to Example 1.21 (Cmpd 37), WO2008/005338; Propionic acid l-
  • Propionic acid 2-(l- ⁇ 4'-[2-((R)-2-methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4- sulfonyl ⁇ -piperidin-4-yl)-ethyl ester can be prepared according to Example 2.24 (Cmpd 63), WO2008/005338; and Propionic acid l- ⁇ 4'-[2-((R)-2-methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4- sulfonyl ⁇ -piperidin-4-ylmethyl ester can be prepared according to Example 2.23 (Cmpd 68), WO2008/005338.
  • H3R antagonist of Formula (Ila) can be prepared according to procedures described in WO2008/048609, for example: (R)-l-[2-(4'-Methanesulfonyl-biphenyl-4-yl)-ethyl]-2-methyl- pyrrolidine can be prepared according to Example 1.13 (Cmpd 1), WO2008/048609; (R)-l-[2-(4'- Ethanesulfonyl-biphenyl-4-yl)-ethyl]-2-methyl-pyrrolidine can be prepared according to Example 1.15 (Cmpd 2), WO2008/048609; (R)-l- ⁇ 2-[4'-(2-Methoxy-ethanesulfonyl)-biphenyl-4-yl]-ethyl ⁇ - 2-methyl-pyrrolidine can be prepared according to Example 1.5 (Cmpd 3), WO2008/048609; (R)- 2-Methyl-l- ⁇ 2-[4'-(propan
  • H3R antagonist of Formula (Ilia) can be prepared according to procedures described in WO2009/058300, for example: (,S , )-4-((4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4- yl)methyl)oxazolidin-2-one can be prepared according to Example 1.26 (Cmpd 1),
  • WO2009/058300; (,S , )-4-(4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)oxazolidin-2-one can be prepared according to Example 1.25 (Cmpd 2), WO2009/058300; (R)-4-((4'-(2-((R)-2- methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)methyl)oxazolidin-2-one can be prepared according to Example 1.4 (Cmpd 4), WO2009/058300; (R)-3-methyl-4-(4'-(2-((R)-2-methylpyrrolidin-l- yl)ethyl)biphenyl-4-yl)oxazolidin-2-one can be prepared according to Example 1.5 (Cmpd 5), WO2009/058300; (R)-3-isopropyl-4-(4'-
  • H3R antagonist of Formula (IVa) can be prepared according to procedures described in WO2009/105206, for example: (R)-3-methoxy-l-(7-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l//)-yl)propan-l-one can be prepared according to Example 1.19 (Cmpd 1), WO2009/105206; (R)-cyclopropyl(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)mefhanone can be prepared according to Example 1.10 (Cmpd 3), WO2009/105206; (R)-cyclopropyl(5 4 2 2-methylpyrrolidin-l-yl)ethyl)phenyl)isoindolin-2- yl)methanone can
  • Example 1.5-1 Preparation of Intermediate (/f)-6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l,2,3,4-tetrahydroisoquinoline.
  • Step A Preparation of (/f)-l-(4-Bromophenethyl)-2-methylpyrrolidine.
  • Step B Preparation of (/f)-4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenylboronic Acid.
  • Step C Preparation of Intermediate (fl)-6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l,2,3,4-tetrahydroisoquinoline.
  • 6-bromo-l,2,3,4-tetrahydroisoquinoline hydrochloride (2.00 g, 8.05 mmol)
  • (R)-4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenylboronic acid 2.063 g, 8.85 mmol
  • tetrakis(triphenylphosphine)palladium (0) 0.279 g, 0.241 mmol
  • benzene (30.00 mL), ethanol (10.00 mL), and 2.0 M aqueous solution of sodium bicarbonate (8.05 mL, 16.09 mmol).
  • the reaction mixture was refluxed for 6 h. Upon completion, water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na 2 S0 4 , and concentrated. The residue was taken up in 1 M HC1 solution and washed with ethyl acetate. The aqueous layer was basified with 10% aqueous NaOH to pH ⁇ l 1, extracted with ethyl acetate, and concentrated. The residue was purified by silica gel column, eluting with 5-10% 2.0 M ammonia in methanol/DCM to give a yellow solid (1.20 g).
  • Example 1.5-2 Preparation of (/f)-l-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)-2-(l/ -tetrazol-5-yl)ethanone (Compound 1).
  • Example 1.5-3 Preparation of (/f)-4-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-4-oxobutanoic Acid (Compound 2).
  • Example 1.5-4 Preparation of (/f)-2,2,3,3-tetrafluoro-4-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoic acid (Compound 8).
  • Example 1.5-5 Preparation of (/f)-3,3-Dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 3).
  • TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 4,4-dimethyldihydro-2/i-pyran-2,6(3//)-dione.
  • Example 1.5-6 Preparation of (/f)-2,2-Dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 4).
  • TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 3,3-dimethyldihydro-2/i-pyran-2,6(3H)-dione.
  • LCMS m/z 463.5 [M+H] + .
  • Example 1.5-7 Preparation of 3-Methyl-5-(6-(4-(2-((/f)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 5).
  • TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 4-methyldihydro-2/i-pyran-2,6(3//)-dione.
  • LCMS m/z 449.3 [M+H] + .
  • Example 1.5-8 Preparation of 3-hydroxy-5-(6-(4-(2-((/f)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic acid (Compound 6).
  • TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and l,4-oxathiane-2,6-dione.
  • LCMS m/z 453.5 [M+H] + .
  • Example 1.5-10 Preparation of (/f)-2 ⁇ -Dimethyl-4-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoic Acid (Compound 9).
  • TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 3,3-dimethyldihydrofuran-2,5-dione.
  • LCMS mJz 449.6 [M+H] + .
  • Example 1.5-12 Preparation of (/f)-5-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 11).
  • Example 1.5-13 Preparation of (fl)-Methyl 4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoate (Compound 12).
  • Example 1.5-15 Preparation of (/f)-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)(l/ -tetrazol-5-yl)methanone (Compound 14).

Abstract

The present invention relates to methods of using histamine 3 receptor (H3R) antagonists to treat pruritus or itch in an individual and methods for using H3R to screen for compounds capable of treating pruritus in an individual and methods for using pruritus/itch model systems for screening H3R antagonists for their usefulness in treating pruritus.

Description

METHODS AND COMPOUNDS USEFUL IN TREATING PRURITUS, AND METHODS FOR IDENTIFYING SUCH COMPOUNDS
FIELD OF THE INVENTION
The present invention relates to methods of using histamine 3 receptor (H3R) antagonists to treat pruritus or itch in an individual and methods for using H3R to screen for compounds capable of treating pruritus in an individual and methods for using pruritus/itch model systems for screening H3R antagonists for their usefulness in treating pruritus. BACKGROUND OF THE INVENTION
A. Pruritus or Itch
Pruritus or itch, is the unpleasant sensation that leads to a desire to scratch (for reviews, see Journal of Investigative Dermatology (2006) 126: 1705-1718; and Lancet (2003) 361 : 690-94). It is a common and distressing symptom in a variety of conditions and diseases. Pruritus typically occurs in peripheral diseases, such as, allergic conjunctivitis, allergic rhinitis, hemorrhoids, atopic dermatitis, allergic dermatitis, acute and chronic urticaria (hives), psoriasis and dermatoses of fungal, allergic and non-allergic origin. Itching can also be a major symptom of many systemic diseases such as, Hodgkin's disease, chronic renal failure, polycythemia vera, hyperthyroidism, malignancy, infection, chronic cholestatic liver disease, and end-stage renal disease, and cholestasis. In addition, senile itch without an obvious cause, except perhaps xerosis, occurs in more than half of the population aged 70 years. In all cases chronic severe generalized itch can be disabling.
Diseases or conditions associated with pruritus further include, for example, primary biliary cirrhosis (PBC), primary sclerosis cholangitis (PSC), chronic renal disease, epidural morphine, pregnancy, diabetes mellitus, thyroid illness, hyperparathyroidism, iron deficiency anemia, viral infection, aquagenic pruritus, and psychogenic pruritus. Pruritus causes sufferers to scratch, leading to skin damage, increased risk of skin infection, and worsening of inflammation. However, despite the prevalence of this clinically important symptom, the pathogenesis of pruritus is not well understood, and treatment options are limited (Paus, R., et al., J. Clin. Invest. 2006, 116: 1174-1185).
Itching can be elicited by chemical, electrical, mechanical and thermal stimulation. So far no morphological structure has been identified as a specific receptor for the itch sensation, but it is assumed that itch receptors are linked to the free nerve endings of C-fibers close to the dermo- epidermal junction. The impulses set up in the thin, non-myelinated, slowly conducting C-fibers enter the spinal cord via the dorsal horn, then ascend in the contralateral spinothalamic tract, pass via the thalamus and end in the somatosensory cortex of the post-central gyrus. Itching and pain are related phenomena, and it was previously believed that itching was equal to sub-threshold pain, i.e. with increased activity in the C-fibers the perceived sensation changed from itching to pain.
Although pruritus was once thought to be a subliminal form of pain (intensity theory), current evidence points to separate sensory neuronal systems mediating the two modalities. First, pain and pruritus are dissociable. Pain and pruritus evoke different motor responses, scratching for pruritus and withdrawal for pain. Second, based on clinical observations, systemically-administered opioids have a dichotomous effect on these two sensory modalities. μ-Opioid receptor agonists reduce pain but can cause pruritus. Furthermore, antagonizing the central mu-opioid receptors, for example with naloxone or naltrexone, suppresses pruritus and at the same time may lower the pain threshold.
Pruritus due to skin inflammation is thought to be mediated at least partly by activation of skin mast cells, which release pruritogenic mediators to activate receptors on peripheral nerve endings to transmit itch signals. Among the substances released from mast cells, histamine is a particularly potent pruritogen. Histamine injected into the skin causes strong itch sensations in humans and animals. Therefore, antagonists of the histamine receptors have been explored as pruritus treatments. There are several topical and systemic agents that suppress itching in selected clinical settings. Unfortunately, no universally effective anti -pruritic drug exists. Therefore, there is an urgent need for new approaches for managing pruritus.
Accordingly, one aspect of the present invention relates to the inhibition of the H3R in an individual, such as by administration of a compound or agent of the present invention, can reduce itching or pruritus. In Example 5 the G-protein coupled histamine 3 receptor (H3R) is shown as a key effector of the itch sensation. Accordingly, in one embodiment, the present invention provides a method of preventing and/or treating pruritus in an individual in need thereof by administering a therapeutically effective amount of a compound or agent that modulates the H3R.
In addition, peripherally restricted antagonists of H3R are capable of mediating the inhibition of itch. Accordingly, screening for peripherally restricted antagonists and the application of peripherally restricted antagonists in the various embodiments of the invention is contemplated. Peripherally restricted compounds may be advantageous to the extent that the peripheral restriction reduces the CNS effects of H3R inhibition. Such effects may include, for example, wakefulness. In one embodiment of this invention, H3R antagonists are peripherally restricted and may be assayed or screened based on their inability or reduced ability to inhibit H3R in the CNS (for example, in the brain).
In one embodiment, the invention comprises a method for treating or preventing itching or the symptoms thereof in an individual wherein an antagonist of H3R is administered to the individual.
In one embodiment, the invention disclosed herein is suitable for the prevention and/or treatment of pruritus that is associated with a disease or disorder, such as those described herein.
One aspect of the present invention encompasses every combination of one or more diseases or disorders for which itch is associated selected from the group: eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound healing, itch associated with an insect bite, itch associated with a flea bite, itch associated with an insect sting, itch associated with a mosquito sting, itch associated with a mite bite, urticaria, urticaria caused by a plant, urticaria caused by poison ivy, urticaria caused by stinging nettle, sweat gland abnormalities, bullous pemphigoid, photodermatoses, skin blisters, adult acne, chicken pox, and dermatitis herpetiformis.
B. G Protein-Coupled Receptors
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR) class. It is estimated that there are some 30,000-40,000 genes within the human genome, and of these, approximately 2% are estimated to code for GPCRs.
GPCRs represent an important area for the development of pharmaceutical products. Drugs active at GPCRs have therapeutic benefit across a broad spectrum of human diseases as diverse as pain, cognitive dysfunction, hypertension, peptic ulcers, rhinitis, and asthma. Of the approximately 500 clinically marketed drugs, greater than 30% are modulators of GPCR function. These drugs exert their activity at approximately 30 well-characterized GPCRs. (See, e.g., Wise et al, Annu Rev Pharmacol Toxicol (2004) 44:43-66.)
GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane- 1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane- 3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or "extracellular" side, of the cell membrane (these are referred to as "extracellular" regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane- 1 and transmembrane-2, transmembrane- 3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or "intracellular" side, of the cell membrane (these are referred to as "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The "carboxy" ("C") terminus of the receptor lies in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor lies in the extracellular space outside of the cell. Generally, when a ligand binds with the receptor (often referred to as "activation" of the receptor), there is a change in the conformation of the receptor that facilitates coupling between the intracellular region and an intracellular "G-protein." It has been reported that GPCRs are
"promiscuous" with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, Life Sciences (1988) 43: 1095-1101. Although other G proteins exist, currently, Gq, Gs, Gi, Gz, and Go are G proteins that have been identified. Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as "signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Current understanding is that the IC-3 loop as well as the carboxy terminus of the receptor interacts with the G protein.
There are also promiscuous G proteins, which appear to couple several classes of GPCRs to the phospholipase C pathway, such as G15 or G16 (Offermanns & Simon, J Biol Chem (1995) 270: 15175-80), or chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C (Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20: 118-24).
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an "inactive" state and an "active" state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by a ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor.
Stabilization by such ligand-independent means is termed "constitutive receptor activation."
C. Histamine Receptors
Histamine, 2-(imidazol-4-yl)ethylamine, exerts its physiological effects through four distinct G-protein coupled receptors (GPCRs), termed HI, H2, H3, and H4. The histamine H3- receptor was first identified in 1983, when it was determined that the H3-receptor acted as an autoreceptor controlling both the synthesis and release of histamine (see: Arrang et al. Nature 1983, 302, 832-7). At least four human and three rat splice variants have proven functional activity in pharmacological assays (Passani et al., Trends in Pharmacol. Sci. 2004, 25, 618-625). Rat and human histamine H3 -receptors also show constitutive activity which means that they can transduce a signal even in the absence of a ligand. Histamine H3 -receptors also function as heteroreceptors, modulating the release of a number of other transmitter substances including serotonin, acetylcholine, dopamine and noradrenaline (see: Brown et al. Prog. Neurobiol. 2001, 63, 637-672). Thus, there are a number of therapeutic applications for ligands that target the histamine H3- receptor, where the ligand functions as either an antagonist or inverse agonist (for reviews see: Leurs et al. Nat. Rev. Drug. Discov. 2005, 4, 107-120; Passani et al. Trends Pharmacol. Sci. 2004, 25, 618-625). Antagonists of H1R, also known as antihistamines, have long been used to treat allergies such as hay fever (rhinitis). Although they are also widely prescribed as anti -pruritus medications, their efficacy, particularly for chronic skin diseases such as atopic dermatitis, has been a subject of debate.
H4R has also recently been described to mediate itch. H4R deficient mice develop less severe pruritus compared to wild-type littermates, and the selective H4R receptor antagonist
JNJ7777120 inhibited pruritus in several mouse pruritus models (Dunford, P.J., et al., J. Allergy Clin. Immunol. 2007, 119: 176-83).
In contrast to the apparent dominant role for H4R, Dunford et al. reported that the H3R inhibition does not reduce histamine-mediated pruritus. In fact, H3R inhibition has been linked to wakefulness and other related CNS mediated effects. Moreover, multiple groups (Br J Dermatol. 2003 Jul; 149 (1): 17-22 and Clin Exp Allergy. 2004 Mar;34 (3):456-9) have found that intradermal injection of H3R antagonists (i.e. thioperamide, iodophenpropit and clobenpropit) actually promote scratching behavior in mice. Therefore, prior to the present invention, existing reports suggested that H3R antagonism was not a viable treatment strategy for itch-associated disorders and, in fact, such treatment might actually be pruritogenic.
SUMMARY OF THE INVENTION
The present invention relates to the unexpected discovery that inhibition of the histamine 3 receptor (H3R) in an individual, such as by administration of a H3R antagonist, can reduce itching or pruritus. The present invention describes methods relating to screening assays performed with H3R for identifying H3R antagonists, for example antagonists or inverse agonists, useful for treating a condition characterized by itching, methods for using H3R antagonists in model systems, for example pruritus model systems, for determining efficacy or usefulness, methods for using H3R antagonists (e.g. partial antagonists, full antagonists, and inverse agonists) in the treatment of a condition characterized by itching, and compounds useful for the treatment of a condition characterized by itching, for example, in an individual. In certain embodiments, the individual is a human.
The inventors have discovered that the G-protein coupled histamine 3 receptor is a key effector of the itch sensation. Accordingly, in one embodiment, the present invention provides a method of treating pruritus in a subject in need thereof by administering a therapeutically effective amount of a compound or agent that modulates the H3R. In one embodiment, the compound or agent is an H3R antagonist. In one embodiment, the H3R antagonist is a H3R inverse agonist. In addition, the inventors have discovered that peripherally restricted antagonists of H3R are capable of mediating the inhibition of itch. Accordingly, screening for peripherally restricted antagonists (e.g. partial antagonists, full antagonists, and inverse agonists), and the application of peripherally restricted antagonists in the various embodiments of the invention is contemplated. Peripherally restricted compounds may be advantageous to the extent that the peripheral restriction reduces the CNS effects of H3R inhibition. Such effects may include, for example, wakefulness. In one embodiment of this invention, H3R antagonists, for example H3R inverse agonists, are peripherally restricted and may be assayed or screened based on their inability or reduced ability to inhibit H3R in the CNS (for example, in the brain).
A number of H3R antagonists have been disclosed in the art. For example,
WO2008/005338, WO2008/048609, WO2008/153958, WO2009/128907, WO2009/058300, and WO2009/105206 disclose H3R antagonists. In addition, H3R antagonists are disclosed in the following PCT Publications. Accordingly, one aspect of the present invention encompasses every combination of one or more H3R antagonist selected from the H3R antagonists disclosed in each of the PCT Publications and pharmaceutically acceptable salts, solvates and hydrates thereof:
WO2010/052222 WO2010/151611 WO2010/086403 WO2010/129242 WO2010/01 1657 WO2010/011653 WO2010/007382 WO2010/000456 WO2009/150101 WO2009/147149 WO2009/142732 WO2009/135842 WO2009/121812 WO2009/105206 WO2009/097306 WO2009/095394 WO2009/097567 WO2009/092764 WO2009/071988 WO2009/063953 WO2009/058300 WO2009/055437 WO2009/036132 WO2009/030716 WO2009/024823 WO2009/012252 WO2009/009501 WO2009/005645 WO2009/005646 WO2009/005671 WO2008/154126 WO2008/115574 WO2008/059335 WO2008/045371 WO2008/032156 WO2008/005338 WO2008/048609 WO2007/137955 WO2007/138431 WO2007/137968 WO2007/127457 WO2007/122156 WO2007/105053 WO2007/099423 WO2007/095039 WO2007/088462 WO2007/088450 WO2007/082840 WO2007/075629 WO2007/075702 WO2007/076140 WO2007/071691 WO2007/069053 WO2007/063385 WO2007/049123 WO2007/048595 WO2007/025596 WO2007/009741 WO2007/009739 WO2007/005503 WO2007/002057 WO2007/001975 WO2006/136924 WO2006/125665 WO2006/124490 WO2006/103537 WO2006/103045 WO2006/103057 WO2006/103546 WO2006/101808 WO2006/097691 WO2006/078775 WO2006/071750 WO2006/061193 WO2006/058023 WO2006/052608 WO2006/046131 WO2006/044228 WO2006/040192 WO2006/029906 WO2006/023462 WO2006/019833 WO2006/018260 WO2005/123723 WO2005/121080 WO2005/097778 WO2005/097740 WO2005/097111 WO2005/089761 WO2005/087746 WO2005/082893 WO2005/058837 WO2005/056056 WO2005/053796 WO2005/040144 WO2005/014579 WO2005/014571 WO2005/009976 WO2004/108675 WO2004/101546 WO2004/089373 WO2004/069338 WO2004/066960 WO2004/056821 WO2004/056369 WO2004/055026 WO2004/054973 WO2004/037800 WO2004/037788 WO2004/035556; WO2004/035544; WO2004/026837; WO2004/018432; WO2004/009015; WO2004/000831; WO2003/104235; WO2003/103669; WO2003/099821; WO2003/088967; WO2003/066604; WO2003/064411; WO2003/042359; WO2003/040106; WO2003/033488; WO2003/031432; WO2003/024929; WO2003/024928; WO2002/076925; WO2002/072093; WO2002/064212; WO2002/056871; WO2002/044141; WO2002/032893; WO2002/024695; WO2002/024659; WO2002/024658; WO2002/024657; WO2002/012224; WO2002/012214; WO2002/012190; WO2001/074815; WO2001/074814; WO2001/074813; WO2001/074810; WO2001/074773; WO2001/068652; WO2001/066534; WO2000/053596; WO2000/042023; WO2000/023438; WO2000/006254; W099/42458; W099/24421; WO99/06377; WO98/06394; WO96/40126; W096/38141; W096/38142; W095/11894; WO95/06037; WO93/20062; WO93/20061;
WO93/12108; WO93/12107; WO93/12093; and W092/15567.
One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist.
One aspect of the present invention relates to use of a H3R antagonist in the manufacture of a medicament for treating pruritus.
One aspect of the present invention relates to H3R antagonists for use in a method for treating pruritus.
In one embodiment, the invention disclosed herein is suitable for the treatment of pruritus that is associated with a disease or disorder selected from eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound healing, itch associated with an insect bite, itch associated with a flea bite, itch associated with an insect sting, itch associated with a mosquito sting, itch associated with a mite bite, urticaria, urticaria caused by a plant, urticaria caused by poison ivy, urticaria caused by stinging nettle, sweat gland abnormalities, bullous pemphigoid, photodermatoses, skin blisters, adult acne, chicken pox, and dermatitis herpetiformis.
In one embodiment, the invention disclosed herein utilizes a H3R antagonist, for example, a H3R antagonist from the genera described herein.
In one embodiment, the invention disclosed herein utilizes a H3R antagonist, for example, a H3R antagonist found in Table A.
In one embodiment, the H3R antagonist is peripherally restricted. In one embodiment, the H3R antagonist is applied topically to the site afflicted with itch. In another embodiment, the H3R antagonist is administered systemically. In one embodiment, the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of the H3R antagonist as the active ingredient, in admixture with a pharmaceutical carrier. For topical application, the H3R antagonist pharmaceutical composition can be formulated in a form suitable for topical application such as a skin patch.
In one embodiment, the H3R antagonist is administered in conjunction with a pharmaceutically acceptable carrier.
In one embodiment, the invention features a method for identifying a compound capable of inhibiting itch in an individual, the method comprising administering a H3R antagonist, such as an antagonist or an inverse agonist, to a mammal for which reduction of itch may be evaluated, and evaluating whether the administration of the compound results in reduced itch.
In one embodiment, H3R antagonists may be screened in established models for evaluating itch (i.e. animal, cellular, in vitro or in vivo).
In one embodiment, the invention features a method for identifying an antagonist (e.g. partial antagonists, full antagonists, and inverse agonists) of a H3R comprising: (a) contacting cells expressing the H3R in the presence of a known amount of histamine or surrogate thereof with a sample to be tested for the presence of a H3R antagonist; and (b) measuring at least one cellular function modulated by the binding of a ligand to the H3R, wherein the cellular function is selected from the group consisting of changes in intracellular second-messenger levels, cell growth rates and hormone secretion; whereby the H3R antagonist in the sample is identified by measuring its effect on the cellular function compared to what would be measured in the absence of such antagonist.
The nucleotide sequence encoding human H3R polypeptide is given in SEQ ID NO: 1. The amino acid sequence of the encoded human H3R polypeptide is given in SEQ ID NO:2.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with membrane of a host cell comprising a G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the G protein-coupled receptor does not comprise the amino acid sequence of SEQ ID NO:2; (iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 ;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (vi); and (b) determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor;
wherein the ability of the test compound to decrease the response in a pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
In certain embodiments, the G protein-coupled receptor comprises the amino acid sequence of SEQ ID NO:2.
In certain embodiments, the variant of SEQ ID NO:2 is an allele of SEQ ID NO:2.
In certain embodiments, the variant of SEQ ID NO:2 is an ortholog of SEQ ID NO:2. In certain embodiments, the variant of SEQ ID NO:2 is a mammalian ortholog of SEQ ID NO:2.
In certain embodiments, the G protein-coupled receptor is recombinant.
In certain embodiments, the method is a method for identifying compounds useful for treating a condition characterized by itch.
In certain embodiments, the host cell is derived or obtained from a vertebrate.
In certain embodiments, the vertebrate is a mammal. In certain embodiments, the vertebrate is a non-human vertebrate. In certain embodiments, the mammal is a non-human mammal.
The invention additionally features a method for identifying compounds useful for treating a condition characterized by itch in an individual, optionally comprising steps (a) and (b) of the prior embodiments, and further comprising:
(c) optionally synthesizing a compound which inhibits functionality of the receptor in step (b);
(d) administering a compound which inhibits functionality of the receptor in step (b) to a vertebrate; and
(e) determining whether the compound treats or prevents pruritus in the vertebrate;
wherein the ability of the test compound to treat or prevent pruritus in the vertebrate is indicative of the test compound being a compound useful for treating a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, further comprising the step of: determining the chemical structure of the anti-pruritic agent. It is understood that this step can occur at any time after the step of determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (H3R) or the step of determining whether less of the complex (i.e., between the known ligand and the G protein-coupled receptor (H3R)) is formed in the presence of the test compound than in the absence of the test compound.
If a compound is known to be an antagonist of H3R, the steps of contacting a test compound with a host cell or with membrane of a host cell comprising a G protein-coupled receptor (i.e., step (a)) and determining the ability of the test compound to inhibit functionality of the G protein-coupled receptor (i.e., step (b)) are not required.
In certain embodiments, the determining comprises measuring a level of itch in the vertebrate.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of the test compound to inhibit the functionality of the receptor; and
(c) obtaining a decreased response for the test compound in a pruritus model;
wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2; (ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of the test compound to inhibit the functionality of the receptor; and
(c) detecting a decreased response for the test compound in a pruritus model;
wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of the test compound to inhibit the functionality of the receptor; and
(c) reducing a response in a pruritus model test animal previously administered the test compound;
wherein the ability of the test compound to reduce the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual. One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of the test compound to inhibit the functionality of the receptor; and
(c) determining a decreased response in a pruritus model test animal previously administered the test compound;
wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); (b) determining the ability of the test compound to inhibit the functionality of the receptor; and
(c) observing a decreased response in a pruritus model test animal previously administered the test compound;
wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: l;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of the test compound to inhibit the functionality of the receptor;
(c) introducing the test compound to a pruritus model; and
(d) observing a decreased response to pruritus;
wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2; (iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: l;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) detecting the complex between said known ligand and said receptor;
(c) determining whether less of said complex is formed in the presence of the test compound than in the absence of the test compound; and
(d) obtaining a decreased response for the test compound in a pruritus model;
wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
It is understood by one skilled in the art that the phrase "optionally labeled known ligand" refers to a known ligand that is optionally labeled, such as, a known ligand where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in the known ligand include but are not limited to 3H (also written as T for tritium), nC, 13C, 14C, 13N, 15N, 150, 170, 180, 18F, 35S, 36C1, 82Br, 75Br, 76Br, 77Br, 123I, 1241, 125I, and 131I. A know ligand that incorporates 3H, 14C, 35S, 82Br, 75Br, 76Br, 77Br, 125I, and 131I will generally be most useful.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: l;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); (b) detecting the complex between said known ligand and said receptor;
(c) determining whether less of said complex is formed in the presence of the test compound than in the absence of the test compound; and
(d) detecting a decreased response for the test compound in a pruritus model;
wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: l;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) detecting the complex between said known ligand and said receptor;
(c) determining whether less of said complex is formed in the presence of the test compound than in the absence of the test compound; and
(d) reducing a response in a pruritus model test animal previously administered the test compound;
wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2; (ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) detecting the complex between said known ligand and said receptor;
(c) determining whether less of said complex is formed in the presence of the test compound than in the absence of the test compound; and
(d) determining a decreased response in a pruritus model test animal previously administered the test compound;
wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) detecting the complex between said known ligand and said receptor;
(c) determining whether less of said complex is formed in the presence of the test compound than in the absence of the test compound; and
(d) observing a decreased response in a pruritus model test animal previously administered the test compound; wherein the ability of the test compound to decrease the response in the animal is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein the G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) detecting the complex between said known ligand and said receptor;
(c) determining whether less of said complex is formed in the presence of the test compound than in the absence of the test compound;
(d) introducing the test compound to a pruritus model; and
(e) observing a decreased response to pruritus;
wherein the ability of the test compound to decrease the response in the pruritus model is indicative of the test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
One aspect of the present invention relates to methods for identifying an anti-pruritic agent for treating a condition characterized by itch in an individual, comprising the steps of:
(a) administering a H3R antagonist to a pruritus model; and
(b) determining whether the H3R antagonist decreases a response in the pruritus model;
wherein the ability of said H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent useful for the treatment of said condition characterized by itch in said individual.
In certain embodiment, two or more potential or actual H3R antagonists or anti-pruritic agents may be compared with each other in pruritus models. Such a comparison may be based, for example, on efficacy or brain to plasma distribution or any other aspect relevant to the H3R inhbitior or anti-pruritic agent(s) as described herein.
One aspect of the present invention relates to methods for evaluating two or more H3R antagonists comprising the steps of:
separately introducing each H3R antagonist to or evaluating each H3R antagonist in a pruritus model;
determining the ability of each H3R antagonist to decrease a behavioral response to pruritus; and
evaluating the two or more anti-pruritic agents by comparing the ability of each anti-pruritic agent to decrease a behavioral response to pruritus.
One aspect of the present invention relates to methods for evaluating a H3R antagonist at two or more different concentrations comprising the steps of:
separately introducing or evaluating the H3R antagonist at two or more concentrations in a pruritus model;
determining the ability of each concentration of H3R antagonist to decrease a behavioral response to pruritus; and
evaluating the two or more concentrations of the H3R antagonist by comparing the ability of each concentration to decrease a behavioral response to pruritus.
In some embodiments, evaluating further comprises selecting the anti-pruritic agent or concentration of anti-pruritic agent with the best efficacy.
In certain embodiments, the G protein-coupled receptor is coupled to a G protein. In certain embodiments, activation of the G protein-coupled receptor decreases a level of intracellular cAMP. In certain embodiments, the G protein is Gi/o.
In certain embodiments, the G protein-coupled receptor variant is a variant that is encoded by a polynucleotide that is amplifiable by polymerase chain reaction of SEQ ID NO:2 or an allele thereof. In certain embodiments, the G protein-coupled receptor encoded by a polynucleotide that is amplifiable by polymerase chain reaction is an allele of SEQ ID NO:2. In some embodiments, the G protein-coupled receptor encoded by a polynucleotide that is amplifiable by polymerase chain reaction exhibits a detectable level of constitutive activity. In some embodiments, the constitutive activity is for increasing a level of intracellular cAMP. In some embodiments, the constitutive activity is for causing melanophore cells to undergo pigment dispersion.
In certain embodiments, stringent hybridization conditions comprise hybridization at 42°C in a solution comprising 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/mL denatured, sheared salmon sperm DNA, followed by washing at 65°C in a solution comprising O.lxSSC. Hybridization techniques are well known to the skilled artisan. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an allele of SEQ ID NO:2. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an ortholog of SEQ ID NO:2. In some embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 exhibits a detectable level of constitutive activity. In some embodiments, the constitutive activity is for increasing a level of intracellular cAMP. In some embodiments, the constitutive activity is for causing melanophore cells to undergo pigment dispersion.
In some embodiments, the G protein-coupled receptor is part of a fusion protein comprising a G protein. Techniques for making a GPCR:G fusion construct are well known to the skilled artisan (see, e.g., International Application WO2002/42461).
In certain embodiments, the host cell comprises an expression vector, the expression vector comprising a polynucleotide encoding the G protein- coupled receptor. In some embodiments, the expression vector is pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 University Blvd., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV: ATCC #203351. Other suitable expression vectors will be readily apparent to those of ordinary skill in the art, and a wide variety of expression vectors are commercially available (e.g., from Clontech, Palo Alto, CA; Stratagene, La Jolla, CA; and Invitrogen, Carlsbad, CA).
In some embodiments, the activated G protein-coupled receptor is constitutively active.
In some embodiments, the activated G protein-coupled receptor is activated by a ligand.
In some embodiments, the receptor comprises the amino acid sequence of SEQ ID NO:2.
In some embodiments, the step determining the ability of the test compound to inhibit the functionality of the receptor is through the measurement of a level of a second messenger.
In some embodiments, the second messenger is selected from the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate (ΓΡ3), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca2+.
In some embodiments, the second messenger is cAMP and the level of cAMP is decreased.
In some embodiments, the level of cAMP is decreased.
In some embodiments, the step determining is through the use of a Melanophore assay, or through the measurement of GTPyS binding to a membrane comprising said GPCR.
In some embodiments, the anti-pruritic agent is a H3R antagonist. In some embodiments, the H3R antagonist is a H3R inverse agonist.
In some embodiments, the H3R antagonist or anti-pruritic agent is peripherally restricted.
In some embodiments, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.5 or less after administration in a pruritus model.
In some embodiments, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less after administration in a pruritus model.
In some embodiments, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.1 or less after administration in a pruritus model.
In some embodiments, the pruritus model is a vertebrate model.
In some embodiments, the vertebrate model is a mammalian model.
In some embodiments, the pruritus model is a primate model, a dog model, a guinea pig model, a mouse model, or a cat model.
In some embodiments, the pruritus model is selected from the group: pruritogen injection model, passive cutaneous anaphylaxis model, allergic pruritus model, and spontaneous pruritus model.
In some embodiments, the pruritus model is selected from the group: histamine induced pruritus model, DNP-Ovalbumin pruritus model, and DNFB pruritus model.
In some embodiments, the response to pruritus is a behavioral response.
In some embodiments, the individual is a non-human mammal.
In some embodiments, the individual is a human.
One aspect of the present invention pertains to methods and processes, further comprising the step of subsequently admixing the anti-pruritic agent with a pharmaceutical carrier.
One aspect of the present invention pertains to methods and processes, further comprising the step of subsequently admixing the anti-pruritic agent with a pharmaceutical carrier to form an anti-pruritic pharmaceutical composition.
One aspect of the present invention pertains to methods and processes, further comprising the step of formulating the anti-pruritic pharmaceutical composition into a form suitable for oral application.
One aspect of the present invention pertains to methods and processes, further comprising the step of formulating said anti-pruritic pharmaceutical composition into a form suitable for topical application.
In some embodiments, the anti-pruritic agent is subsequently admixed with a
pharmaceutical carrier.
In some embodiments, the anti-pruritic agent is subsequently formulated with a pharmaceutical carrier.
In some embodiments, the step determining the ability of the test compound to inhibit functionality of the receptor comprises comparing the functionality of the receptor in the presence and absence of the test compound and observing a decreased functionality in the presence of the test compound as compared to in the absence of the test compound.
In some embodiments, the host cell is a vertebrate cell. In some embodiments, the host cell is mammalian. In some embodiments, the mammalian host cell is selected from the group consisting of 293, 293T, CHO, MCB3901, and COS-7. In some embodiments, the host cell is melanophore. Other suitable host cells will be readily apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.
In certain embodiments, the determining is consistent with the G protein-coupled receptor being a Gs-coupled receptor.
In some embodiments, the determining is consistent with the G protein-coupled receptor being coupled through a promiscuous G protein, such as Gal5 or Gal6, to the phospholipase C pathway. Promiscuous G proteins are well known to the skilled artisan (see, e.g., Offermanns et al., J Biol Chem (1995) 270: 15175-15180). In some embodiments, the determining is consistent with the G protein-coupled receptor being coupled through a chimeric G protein, e.g. to the
phospholipase C pathway. Chimeric G proteins are well known to the skilled artisan (see, e.g., Milligan et al., Trends in Pharmaceutical Sciences (1999) 20: 118-124; and WO2002/42461).
In some embodiments, the determining is through the measurement of a level of a second messenger.
In some embodiments, the determining is through the measurement of a level of a second messenger selected from the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate (IP3), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca2+. In some preferred embodiments, the second messenger is cAMP. In certain embodiments, a level of intracellular cAMP is increased.
In certain embodiments, the determining is carried out using membrane comprising the G protein-coupled receptor.
In certain embodiments, the determining is through the use of a melanophore assay. In certain embodiments, a level of pigment dispersion is increased.
In some embodiments, the determining is through the measurement of an activity mediated by increasing a level of intracellular cAMP.
In some embodiments, the determining is through CRE-Luc reporter assay. In certain embodiments, a level of luciferase activity is increased.
In some embodiments, the determining is through the measurement of GTPyS binding to membrane comprising the G protein-coupled receptor. In certain embodiments, the GTPyS is labeled with [35S]. In certain embodiments, the GTPyS binding to membrane comprising the GPCR is increased. In some embodiments, the test compound is a small molecule. In some embodiments, the test compound is a small molecule, with the proviso that the small molecule is not a polypeptide. In some embodiments, the test compound is a small molecule, with the proviso that the small molecule is not an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is a small molecule, with the proviso that the small molecule is not a lipid. In some embodiments, the test compound is a small molecule, with the proviso that the small molecule is not a polypeptide or a lipid. In some embodiments, the test compound is a polypeptide. In some embodiments, the test compound is a polypeptide, with the proviso that the polypeptide is not an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is a lipid. In some embodiments, the test compound is not an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is an antibody or an antigen-binding fragment thereof. In some embodiments, the test compound is an anti-pruritic agent, a H3R antagonist, a H3R antagonist, or a H3R inverse agonist. In some embodiments, the test compound is an anti-pruritic agent. In some embodiments, the test compound is a H3R antagonist. In some embodiments, the test compound is a H3R antagonist. In some embodiments, the test compound is a H3R inverse agonist.
In certain embodiments, the known ligand is a ligand or agonist of an endogenous vertebrate, mammalian, or human H3R receptor. In certain embodiments, the known ligand is a known agonist of an endogenous vertebrate, mammalian, or human H3R receptor. In certain embodiments, the known ligand is a ligand or agonist of an endogenous human H3R receptor.
In certain embodiments, the known ligand is an endogenous ligand of an endogenous vertebrate, mammalian, or human H3R receptor.
In certain embodiments, the optionally labeled known ligand is a labeled known ligand. In certain embodiments, the labeled known ligand is a radiolabeled known ligand. Techniques for radiolabeling a compound, such as for labeling a known ligand of a G protein-coupled receptor of the invention, are well known to the skilled artisan. See, e.g., International Application
WO2004/065380. Also see, e.g., Example 11, infra.
Techniques for detecting the complex between a G protein-coupled receptor and a compound known to be a ligand of the G protein-coupled receptor are well known to the skilled artisan. See, e.g., International Application WO2004/065380. Also see, e.g., Example 12, infra.
In some embodiments, the G protein-coupled receptor is part of a fusion protein comprising a G protein. Techniques for making a GPCR:G fusion construct are well known to the skilled artisan (see, e.g., International Application WO2002/42461).
In certain embodiments, the determining is carried out using a host cell comprising the G protein-coupled receptor. In certain embodiments, the host cell comprises an expression vector, the expression vector comprising a polynucleotide encoding the GPCR. In some embodiments, the expression vector is pCMV. This vector was deposited with the American Type Culture Collection (ATCC) on October 13, 1998 (10801 University Blvd., Manassas, VA 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV: ATCC #203351. Other suitable expression vectors will be readily apparent to those of ordinary skill in the art, and a wide variety of expression vectors are commercially available (e.g., from Clontech, Palo Alto, CA; Stratagene, La Jolla, CA; and Invitrogen, Carlsbad, CA).
In some embodiments, the host cell is a vertebrate cell. In some embodiments, the host cell is mammalian. In some embodiments, the mammalian host cell is selected from the group consisting of 293, 293T, CHO, MCB3901, and COS-7. In some embodiments, the host cell is melanophore. Other suitable host cells will be readily apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.
In certain embodiments, the determining is carried out using membrane comprising the G protein-coupled receptor.
In some embodiments, the method further comprises the step of optionally providing the name or structure of the compound useful for treating a condition characterized by itch in an individual.
In another embodiment, the invention features a method comprising, having identified a compound for treating a condition characterized by itch in an individual according to the invention, formulating the compound for treating a condition characterized by itch in an individual into a pharmaceutical composition.
In another embodiment, the invention features use of a G protein-coupled receptor to screen test compounds as anti-pruritic agents for treating a condition characterized by itch in an individual, wherein the G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(a) amino acids 1-445 of SEQ ID NO:2;
(b) amino acids 2-445 of SEQ ID NO:2;
(c) amino acids 2-445 of SEQ ID NO:2, wherein the GPCR does not comprise the amino acid sequence of SEQ ID NO:2;
(d) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide that is amplifiable by polymerase chain reaction (PCR) on a human DNA sample;
(e) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(f) a variant of SEQ ID NO:2; and (g) a biologically active fragment of any one of any one of (a) to (f).
In certain embodiments, the screen is for an antagonist of the receptor.
In certain embodiments, the screen is for a partial antagonist of the receptor.
One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising formulating an anti-pruritic agent with a pharmaceutically acceptable carrier; wherein the anti-pruritic agent is a H3R antagonist.
One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising formulating a H3R antagonist with a pharmaceutically acceptable carrier; wherein the H3R antagonist is an anti-pruritic agent.
One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having contacted in vitro with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); and determined to inhibit the functionality of the receptor; wherein the ability of said H3R antagonist to inhibit the functionality of the receptor is indicative of said H3R antagonist being an anti-pruritic agent, said method comprising formulating said H3R antagonist having the effect of an anti-pruritic agent with a pharmaceutically acceptable carrier as a
pharmaceutical composition.
One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
(a) determining the ability of a test compound to inhibit the functionality of an activated G protein-coupled receptor in a host cell or with a membrane of a host cell, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2; (iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); wherein the ability of said test compound to inhibit the functionality of the activated G protein-coupled receptor in a host cell or with a membrane of a host cell is indicative of said test compound being an anti-pruritic agent; and
(b) formulating said anti-pruritic agent with a pharmaceutically acceptable carrier as a pharmaceutical composition.
One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of said test compound to inhibit the functionality of the receptor; wherein the ability of said test compound to inhibit the functionality of the receptor is indicative of said test compound being an anti-pruritic agent; and
(c) formulating said anti-pruritic agent having the effect of a H3R antagonist with a pharmaceutically acceptable carrier as a pharmaceutical composition.
One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising: (a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of said test compound to inhibit the functionality of the receptor; wherein the ability of said test compound to inhibit the functionality of the receptor is indicative of said test compound being an anti-pruritic agent; and
(c) admixing said anti-pruritic agent with a pharmaceutically acceptable carrier. One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having obtained a decreased response for said H3R antagonist in a pruritus model, wherein the ability of said H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent, said method comprising formulating said H3R antagonist having the effect of an anti-pruritic agent with a pharmaceutically acceptable carrier as a pharmaceutical composition.
One aspect of the present invention pertains to methods of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1-445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 ;
(v) a variant of SEQ ID NO:2; and (vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of said test compound to inhibit the functionality of the receptor;
(c) obtaining a decreased response for said test compound in a pruritus model, wherein the ability of said test compound to decrease the response in said pruritus model is indicative of said test compound being an anti-pruritic agent; and
(d) formulating said anti-pruritic agent having the effect of a H3R antagonist with a pharmaceutically acceptable carrier as a pharmaceutical composition.
One aspect of the present invention relates to pharmaceutical compositions prepared by any of the methods or processes described herein.
One aspect of the present invention relates to pharmaceutical compositions obtained by any of the methods or processes described herein.
One aspect of the present invention relates to pharmaceutical compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is for the use in treating pruritus.
One aspect of the present invention relates to pharmaceutical compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is for the use in treating pruritus.
One aspect of the present invention relates to pharmaceutical compositions comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein the H3R antagonist is identified using any of the methods or processes described herein.
One aspect of the present invention relates to pharmaceutical compositions according to claim 12, wherein the H3R antagonist is identified using any of the methods or processes described herein and the pharmaceutical composition is for the use in treating pruritus.
One aspect of the present invention pertains to pharmaceutical compositions comprising a
H3R antagonist of the present invention and a pharmaceutically acceptable carrier.
One aspect of the present invention pertains to methods for the treatment of pruritus in an individual, comprising administering to the individual in need thereof, a therapeutically effective amount of a H3R antagonist of the present invention or a pharmaceutical composition thereof.
One aspect of the present invention pertains to the use of a H3R antagonist of the present invention or a pharmaceutical composition thereof in the manufacture of a medicament for the treatment of pruritus.
One aspect of the present invention pertains to H3R antagonists of the present invention or a pharmaceutical composition thereof for use in a method of treatment of pruritus.
One aspect of the present invention pertains to H3R antagonists for preparing a composition comprising admixing a H3R antagonist of the present invention and a
pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS
The invention is further illustrated in connection with the figures appended hereto in which:
Figure 1 shows the polynucleotide sequence of the human H3R.
Figure 2 shows the polypeptide sequence of the human H3R.
Figure 3 shows the polynucleotide sequence of the mouse H3R.
Figure 4 shows the polypeptide sequence of the mouse H3R.
Figure 5 shows that orally administered H3R antagonists are able to inhibit pruritus in a variety of pruritus animal models. Figure 5A shows that oral administration of Compound B and Compound C inhibit histamine (HA) induced pruritus. Figure 5B shows that oral administration of Compound A inhibits histamine-induced pruritus. Figure 5C shows that oral administration of Compound A inhibits serotonin-induced pruritus. Figure 5D shows that oral administration of Compound A inhibits Compound 48/80-induced pruritus.
Figure 6 shows that topical application of Compound A inhibits histamine-induced itch.
Figure 7 shows that administration of Compound A inhibits DNP-ovalbumin mediated allergic pruritus.
Figure 8 shows that central administration of a H3R antagonist, Compound A, does not inhibit histamine-induced pruritus. Figure 8A shows ICV administration of Compound A increases cognitive function in mice. Figure 8B shows that ICV administration of Compound A is unable to inhibit histamine-induced pruritus. Suggesting that brain exposure of a H3R antagonist is not sufficient and that histamine induced pruritus is peripherally mediated.
Figure 9 shows that administration of Compound 1 inhibits chronic DNFB -mediated allergic pruritus.
Figure 10 shows the data from the administration of Compound 1 in the histamine- induced pruritus mouse model. The data shows that Compound 1 effectively inhibits histamine- induced pruritus in mice.
Figure 11 shows the data from the administration of Compound 3 in the histamine- induced pruritus mouse model. The data shows that Compound 3 effectively inhibits histamine- induced pruritus in mice.
Figure 12 shows a general synthesis of biaryl derivatives useful as intermediates in the preparation of compounds of the present invention (Formula (Va)). First, a mesylate derivative is coupled with an R1 substituted pyrrolidine and subsequently converted to an aryl boronic acid by treatment of an aryl lithium intermediate with triisopropylborate followed by hydrolysis. Next the aryl boronic acid is coupled with an aryl bromide in the presence of a palladium catalyst to prepare the biaryl derivatives. Figure 13 shows general methods for preparing compounds of the present invention (Formula (Va)). A variety of methods can be used to acylate a secondary amine (Z = CH2). For example, Method A shows the use of carboxylic acids in the presence of a coupling agent to prepare compounds of the invention; and Method B shows the use of acid chlorides to prepare compounds of the invention, optionally in the presence of a base, such as an amine base.
Figure 14 shows a general method for preparing compounds of the present invention (Formula (Va)). A variety of methods can be used to acylate a secondary amine (Z = CH2) and the nitrogen of an amide group (Z = carbonyl, i.e., -(C=0)-). For example, Method C shows the use of acid anhydrides in the presence of a base to prepare compounds of the invention. When Z = CH2 the base is optional.
DETAILED DESCRIPTION OF THE INVENTION
The present invention features methods of using H3R receptor to identify compounds useful for treating pruritus. Antagonists of H3R receptor are useful as therapeutic agents for treating a condition characterized by itch, such as pruritus. The present invention is based, at least in part, on the surprising discovery by Applicant that administration of a H3R antagonist to an individual, such as by oral administration, can act at the H3R receptor to reduce, prevent or treat pruritus and its symptoms. Accordingly, the invention further includes methods of evaluating known H3R antagonists in a variety of pruritus models for their usefulness in the treatment or prevention of pruritus.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the chemical groups represented by the variables contained within the generic chemical formulae described herein, for example, (la), (Ila), (Ilia), (IVa), (IVb), and (Va), are specifically embraced by the present invention just as if each and every combination was individually and explicitly recited, to the extent that such combinations embrace compounds that result in stable compounds (i.e., compounds that can be isolated, characterized and tested for biological activity). In addition, all subcombinations of the chemical groups listed in the embodiments describing such variables, as well as all subcombinations of uses and medical indications described herein, such as those indications/disorders associated with pruritus/itch, are also specifically embraced by the present invention just as if each and every subcombination of chemical groups and subcombination of uses and medical indications was individually and explicitly recited herein. In addition, some embodiments include every combination of one or more second pharmaceutical agents either specifically disclosed herein or specifically disclosed in any reference recited herein just as if each and every combination was individually and explicitly recited.
As used herein, "substituted" indicates that at least one hydrogen atom of the chemical group is replaced by a non-hydrogen substituent or group, the non-hydrogen substituent or group can be monovalent or divalent. When the substituent or group is divalent, then it is understood that this group is further substituted with another substituent or group. When a chemical group herein is "substituted" it may have up to the full valance of substitution; for example, a methyl group can be substituted by 1, 2, or 3 substituents, a methylene group can be substituted by 1 or 2 substituents, a phenyl group can be substituted by 1, 2, 3, 4, or 5 substituents, a naphthyl group can be substituted by 1, 2, 3, 4, 5, 6, or 7 substituents, and the like. Likewise, "substituted with one or more substituents" refers to the substitution of a group with one substituent up to the total number of substituents physically allowed by the group. Further, when a group is substituted with more than one group they can be identical or they can be different.
Compounds of the invention can also include tautomeric forms, such as keto-enol tautomers and the like. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. It is understood that the various tautomeric forms are within the scope of the compounds of the present invention.
It is understood and appreciated that compounds of Formulae (la), (Ila), (Ilia), (IVa), (IVb), and (Va) and formulae related thereto may have one or more chiral centers and therefore can exist as enantiomers and or diastereoisomers. The invention is understood to extend to and embrace all such enantiomers, diastereoisomers and mixtures thereof, including but not limited to racemates. It is understood that compounds of Formulae (la), (Ha), (Ilia), (IVa), (IVb), and (Va) and other formulae used throughout this disclosure represent all individual enantiomers and mixtures thereof, unless stated or shown otherwise.
The term "ligand", as used herein, shall mean a molecule (e.g., test compound) that specifically binds to a polypeptide, such as H3R. A ligand may be, for example, a polypeptide, a lipid, a small molecule, an antibody. An endogenous ligand is a ligand that is an endogenous, natural ligand for a native polypeptide, such as H3R. A ligand may be an "antagonist", "agonist", "partial agonist", or "inverse agonist", or the like. Exemplary antagonists of the H3R receptor are described herein.
In some embodiments, the H3R antagonist is a H3R inverse agonist.
One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2008/005338 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
One aspect of the present invention encompasses certain H3R receptor ligands selected from compounds of Formula (la) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000033_0001
(la)
wherein:
R' andR2 are each selected independently from the group consisting of H, C1-C6 acyl, Ci- C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C - alkylenyl, aryloxy-Ci-C4-alkylenyl, heteroaryl-Ci-C4-alkylenyl and heteroaryloxy-Ci-C4-alkylenyl, and each R1 andR2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-Cs alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, aryl, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, heterocyclyl, hydroxyl, thiol, nitro and sulfonamide; wherein each Ci-C8 alkyl may be further substituted with hydroxy;
or
R1 and R2 together with the nitrogen atom to which they are both bonded form a C3-C7 heterocyclyl or a C5-C10 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8
alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; wherein each Ci-C8 alkyl and carboxy may be further substituted with C1-C6 acyloxy, C1-C6 alkoxy, aryl-Ci-C -alkylenyl, or hydroxy;
or
R2is selected independently from the group consisting of H, C1-C6 acyl, Ci-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C -alkylenyl, aryloxy-Ci-C -alkylenyl, heteroaryl-Ci-C -alkylenyl and heteroaryloxy-Ci-C -alkylenyl, and each R2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-Cs alkylthio, Ci-C8 alkylureyl, amino, aryl, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6- alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, heterocyclyl, hydroxyl, thiol, nitro and sulfonamide;
and
R1 and R12 together with the atoms to which they are both bonded form a C6-C8 heterocyclyl group optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-Cs alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, aryl, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, heterocyclyl, hydroxyl, thiol, nitro and sulfonamide;
J is -CH2CH2- or a l,2-C3-C7-cycloalkylenyl group, each optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of C1-C3 alkyl, C1-C4 alkoxy, carboxy, cyano, C1-C3 haloalkyl, halogen, hydroxyl and oxo;
R3, R4, R5, R6, R7, R10, R11 and R12 are each selected independently from the group consisting of H, C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
and
R8 and R9 are each selected independently from the group consisting of H, Ci-C8 alkyl, C2- C8 alkenyl, C2-C8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C4-alkylenyl, aryloxy-Ci-C4-alkylenyl, heteroaryl-Ci-C4-alkylenyl and heteroaryloxy-Ci-C4-alkylenyl, and each R8 and R9 is optionally substituted with 1 , 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, C C8 alkyl, C C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-Cs alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6- alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
or
R8 and R9 together with the nitrogen atom to which they are both bonded form a C3-C7 heterocyclyl or a C5-C10 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8
alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; provided that the compound is other than:
N-(3-cyanophenyl)-.V- [2- [4'- [ [( 1 , 1 -dimethylethyl)amino] sulfonyl] [1,1 '-biphenyl] -4- yl] ethyl] -glycine methyl ester;
N- [ [4 - [2-oxo-2-(phenylamino)ethyl] [1,1 '-biphenyl] -4-yl] sulfonyl] -D- valine 1,1- dimethylethylester;
N- [ [4 - [2-0X0-2- [(phenylmethyl)amino] ethyl] [1,1 -biphenyl] -4-yl] sulfonyl] -D-valine 1, 1- dimethylethyl ester;
N- [ [4 - [2-oxo-2-(phenylamino)ethyl] [1,1 '-biphenyl] -4-yl] sulfonyl] -D-valine; or
N- [ [4 - [2-0X0-2- [(phenylmethyl)amino] ethyl] [1,1 -biphenyl] -4-yl] sulfonyl] -D-valine.
One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2008/048609 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
One aspect of the present invention encompasses certain H3R receptor ligands selected from compounds of Formula (Ha) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000035_0001
(Ha)
wherein:
R1 is selected from the group consisting of H, C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2- C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, C3-C7 heterocyclyl, hydroxyl, thiol, nitro, phenyl and sulfonamide, and each is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, d-
C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-
C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide; or
R1 together with the W-S02 group and the ring atom to which the W-S02 group is bonded form a C5-C7 heterocyclic ring with Ring A whereby the C5-C7 heterocyclic ring and Ring A share two adjacent ring atoms, and the C5-C7 heterocyclic ring is optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8
alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide;
W is C1-C4 alkylene, C2-C4 alkenylene, C3-C7 cycloalkylene, C3-C7 heterocyclylene or phenylene, each optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 substituents selected
independently from the group consisting of C1-C3 alkyl, C1-C4 alkoxy, carboxy, cyano, C1-C3 haloalkyl, halogen, hydroxyl and oxo;
Ring A is 1,3-phenylene or 1,4-phenylene, each substituted with R12, R13, R14 and R15, wherein R12, R13, R14 and R15 are each selected independently from the group consisting of H, C1-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, C C8 alkylsulfonamide, C C8 alkylsulfinyl, C C8 alkylsulfonyl, C C8 alkylthio, C C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide; or
Ring A is a 6-membered heteroarylene or a 5-membered heteroarylene, each optionally substituted with R16, R17 and R18, wherein R16, R17 and R18 are each selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, C C8 alkyl, C C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6- alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
R2, R3, R4 andR5 are each selected independently from the group consisting of H, C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
R6, R7, R8 andR9 are each selected independently from the group consisting of H, C1-C3 alkyl, C1-C4 alkoxy, carboxy, cyano, C1-C3 haloalkyl, halogen and hydroxyl; and
R10 and R11 together with the nitrogen atom to which they are both bonded form 2-methyl- pyrrolidin-l-yl;
provided:
1) that Ring B and the sulfur of the R1-W-S(0)2- group are not bonded to adjacent ring atoms of Ring A; and
2) if Ring A is 1,3-phenylene or 1,4-phenylene, and W is C3-C7 heterocyclylene, then the ring atom of W that is directly bonded to the sulfur of the R1-W-S(0)2- group is other than nitrogen.
One aspect of the present invention encompasses a H3R receptor ligand selected from (R)- 1 - { 2- [4'-(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl -pyrrolidine and pharmaceutically acceptable salts, solvates, and hydrates thereof.
One aspect of the present invention encompasses certain H3R receptor ligands selected from the groups consisting of:
(R)- 1 - { 2-[4 '-(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl-pyrrolidine mono-citrate;
(R)- 1 - { 2-[4 '-(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl-pyrrolidine di-citrate;
(R)- 1 - { 2-[4' -(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl-pyrrolidine maleate; and
(R)- 1 - { 2-[4' -(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl-pyrrolidine hydrochloride.
One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2009/058300 and pharmaceutically acceptable salts, solvates, and hydrates thereof.
One aspect of the present invention encompasses certain H3R receptor ligands selected from compounds of Formula (Ilia) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000037_0001
(Ilia)
wherein: ring A is heterocyclyl optionally substituted with one, two or three substituents selected from C1-C6 alkyl and oxo; wherein each Ci-C6 alkyl is optionally substituted with a Ci-C6 alkoxy substituent;
R1 is H, C1-C6 alkoxy, Ci-C6 alkyl or halogen;
R2 is H, C1-C6 alkoxy, Ci-C6 alkyl or halogen;
R3 is H, C1-C6 alkoxy, Ci-C6 alkyl or halogen;
R4 is H or Ci-C4 alkyl; and
n is 0, 1 or 2.
One aspect of the present invention encompasses every combination of one or more H3R receptor ligands selected from the H3R receptor ligands found in PCT published patent application WO2009/ 105206 pharmaceutically acceptable salts, solvates, and hydrates thereof.
One aspect of the present invention encompasses certain H3R receptor ligands selected from compounds of Formula (IVa) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000038_0001
(IVa)
wherein:
R1 is H or C1-C4 alkyl;
R2 is H or halogen;
R3 is H, C1-C4 alkyl or C3-C6 cycloalkyl, and R4 is H; or R3 and R4 together with the atom to which they are both bonded form a C3-C6 cycloalkyl;
R5 is selected from: C1-C6 alkyl, aryl, C3-C6 cycloalkyl, heteroaryl and heterocyclyl; each of which is optionally substituted with one or more substituents selected from: C1-C6 alkoxy, halogen, heterocyclyl and hydroxyl;
R6, R7 and R8 are each independently selected from: H, Ci-Ce alkoxy, C1-C6 alkyl, amino, halogen, heterocyclyl and hydroxyl;
m is 0 or 1;
n is 1 or 2; and
V is CH2, O or absent.
One aspect of the present invention encompasses certain H3R receptor ligands selected from compounds of Formula (IVb) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000039_0001
wherein:
R5 is selected from: methyl, cyclopropyl, tetrahydropyran-4-yl, methoxymethyl, 2- methoxyethyl, 3-methoxypropyl, hydroxymethyl, 2-hydroxyethyl, tetrahydropyran-4-ylmethyl, 2,2- difluorocyclopropyl, 4-methoxyphenyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, 6- hydroxypyridin-3-yl and 2-hydroxypyridin-4-yl.
One such compound is (R)-2-hydroxy- l-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)ethanone (referred to as Compound A herein), see Compound 10 in PCT publication WO2009/105206.
One aspect of the present invention encompasses certain H3R receptor ligands selected from (R)-2-hydroxy- 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)ethanone and pharmaceutically acceptable salts, solvates, and hydrates thereof.
One aspect of the present invention encompasses, inter alia, certain isoquinoline derivatives selected from compounds of Formula (Va) and pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000039_0002
wherein:
m, n, and p are each independently 0 or 1;
X is selected from the group: -S-, -0-, C3-C6 cycloalkylene, and CRCRC;
Z is -CH2- or -(C=0)-;
R1 is H or C1-C4 alkyl;
R2 is selected from the group: C1-C4 alkoxycarbonyl, carboxyl, and l/i-tetrazol-5-yl; and each R , Rb, and Rc is independently selected from the group: H, C1-C3 alkyl, amino, halogen, and hydroxyl.
One aspect of the present invention encompasses every combination of one or more H3R inhibitors selected from the following H3R inhibitors and pharmaceutically acceptable salts, solvates, and hydrates thereof:
1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2( l//)-yl)-2- ( 1 /i-tetrazol-5 -yl)ethanone; 4- (6-(4-(2-(2-methylpyn-olidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)-4- oxobutanoic acid;
33-dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)-5-oxopentanoic acid;
2,2-dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)-5-oxopentanoic acid;
3-methyl-5-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)- yl)-5-oxopentanoic acid;
3-hydroxy-5-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)-5-oxopentanoic acid;
2 2 6 4 2 2-methylpyrrolidin -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)-2- oxoethylthio)acetic acid;
2,2,3,3-tetrafluoro-4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-4-oxobutanoic acid;
2,2-dimethyl-4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)-4-oxobutanoic acid;
2 2 6 4 2 2-methylpyrrolidin -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)-2- oxoethoxy)acetic acid;
5- (6-(4-(2-(2-methylpyn-olidin -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)-5- oxopentanoic acid;
methyl 4-(6-(4-(2-(2-methylpyrrolidin4-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)- yl)-4-oxobutanoate;
ethyl 2-amino-5-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2( l//)-yl)-5-oxopentanoate;
(6 4 2 2-methylpyrrolidin4-yl)e l)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)(l/i- tetrazol-5-yl)methanone;
ethyl 4-(6-(4-(2-(2-memylpyrrolidin4-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)- 4-oxobutanoate;
methyl 2-(6-(4-(2-(2-methylpyrrolidin4-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)- yl)-2-oxoacetate;
2-( 1 -(2-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2( l//)-yl)- 2-oxoethyl)cyclopentyl)acetic acid;
2-( 1 -(2-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2( l//)-yl)- 2-oxoethyl)cyclohexyl)acetic acid;
2-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)4 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclohexanecarboxylic acid; methyl 3-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)- y 1) - 3 -oxopropanoate;
methyl 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclopropanecarboxylate;
2-methyl- 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2( \H)- yl)-2-(l/i-tetrazol-5-yl)propan-l-one;
methyl 3,3-dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-5-oxopentanoate;
methyl 3-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclohexanecarboxylate;
methyl 4-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclohexanecarboxylate;
1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclopropanecarboxylic acid;
4-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclohexanecarboxylic acid;
3- (6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclohexanecarboxylic acid;
4- (6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l-oxo-3,4-dihydroisoquinolin-2(l//)-yl)- 4-oxobutanoic acid;
ethyl 2-( 1 -(2-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2( l//)-yl)-2-oxoethyl)cyclohexyl)acetate; and
methyl 2-(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2- carbonyl)cyclohexanecarboxylate.
One aspect of the present invention encompasses every combination of one or more H3R inhibitors selected from the H3R inhibitors in Table A and pharmaceutically acceptable salts, solvates, and hydrates thereof.
Table A
Figure imgf000041_0001
Figure imgf000042_0001
Cmpd
Chemical Structure/Chemical Name
No.
7
0 0
(R)-2-(2-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)-2-oxoethylthio)acetic acid
8 Ho rj
F F o
(R)-2,2,3,3-tetrafluoro-4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2( l//)-yl)-4-oxobutanoic acid
9
(R)-2,2-dimethyl-4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2( l//)-yl)-4-oxobutanoic acid
10
0 0
(R)-2-(2-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)-2-oxoethoxy)acetic acid
11
0 0
(R)-5-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)-5-oxopentanoic acid
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Cmpd
Chemical Structure/Chemical Name
No.
27
0
( 1 r,4r)-4-(6-(4-(2-((R)-2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4- tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic acid
28
O 0
(l.S,,3R)-3-(6-(4-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic acid
29
0 0
(R)-4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l-oxo-3,4- dihydroisoquinolin-2( l//)-yl)-4-oxobutanoic acid
30
(R)-efhyl 2-(l-(2-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-2-oxoethyl)cyclohexyl)acetate
31
MeO^O °
( lR,2R)-methyl 2-(6-(4-(2-((R)-2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4- tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylate
Giannoni and co-workers disclosed the compound 6-[(3-cyclobutyl-2,3,4,5-tetrahydro- l/i-3-benzazepin-7-yl)oxy]-Ai-methyl-3-pyridinecarboxamide (also referred to as GSK189254) as a non-imidazole histamine H3 receptor antagonist (J. Pharm. Exper. Therapeutics (2010), 332(1), 164-172). The chemical structure for 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-l#-3- benzazepin-7-yl)oxy]-Ar-methyl-3-pyridinecarboxamide (referred to as Compound B herein) is as follows:
Figure imgf000048_0001
One aspect of the present invention encompasses certain H3R receptor ligands selected from 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-lH-3-benzazepin-7-yl)oxy]-N-methyl-3- pyridinecarboxamide and pharmaceutically acceptable salts, solvates, and hydrates thereof.
One aspect of the present invention encompasses certain H3R receptor ligands selected from 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-lH-3-benzazepin-7-yl)oxy]-N-methyl-3- pyridinecarboxamide hydrochloride and solvates and hydrates thereof.
Galici and co-workers disclosed the compound 4-(3-(4-(piperidin-l-yl)but-l- ynyl)benzyl)morpholine (also referred to as JNJ-10181457) as a selective non-imidazole histamine H3 receptor antagonist (Neuropharmacology (2009), 56(8), 1131-1137). The chemical structure for 4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine (referred to as Compound C herein) is as follows:
Figure imgf000048_0002
One aspect of the present invention encompasses certain H3R receptor ligands selected from 4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine and pharmaceutically acceptable salts, solvates, and hydrates thereof.
The term "antagonist", as used herein, includes partial antagonists, full antagonists, and inverse agonists, and shall mean any organic or inorganic molecule that does not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular responses of agonists and/or partial agonists. It is understood that an "antagonist" as defined herein can bind to the same active site as an agonist or the antagonist bind at a different site provided that the molecule is capable of inhibiting the response of a GPCR, such as, an allosteric antagonist. Another example includes nucleic acid molecules capable of interfering with the GPCR. Certainly, a H3R antagonist would include any anti-pruritic agent capable of inhibiting a response of the H3R. Inverse agonists are specific examples of H3R antagonists.
The term "anti-pruritic agent" shall mean an agent capable of treating one or more aspects of pruritus. Agents may include, for example, small molecules, large molecules, nucleic acids, synthetic nucleic acid analogs, polypeptide(s), antibodies, and other biologies. H3R antagonists and inverse agonists are examples of anti-pruritic agents. The term "agonist", as used herein, shall mean an agent (e.g., ligand, test compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response mediated by the GPCR.
The term "partial agonist", as used herein, shall mean an agent (e.g., ligand, test compound) that by virtue of binding to a GPCR activates the GPCR so as to elicit an intracellular response mediated by the GPCR, albeit to a lesser extent or degree than does a full agonist.
The term "H3R antagonist" as used herein, refers to a compound that binds to H3R receptor and acts as an antagonist as defined herein. In one embodiment, the H3R antagonist is a selective antagonist. In one embodiment, the H3R antagonist has selectivity for H3R that is at least 10 fold greater than its selectivity for any one or more receptors selected from: HIR, H2R and H4R. In one embodiments, the H3R antagonist has a selectivity for H3R over HIR of at least about 10- fold. In one embodiments, the H3R antagonist has a selectivity for H3R over H2R of at least about 10-fold. In one embodiments, the H3R antagonist has a selectivity for H3R over H4R of at least about 10-fold. In one embodiments, the H3R antagonist has a selectivity for H3R over HIR, H2R, or H4R of at least about 10 fold. In various embodiments the H3R antagonist has in IC50 (H3R) of less than about 1 μΜ, less than about 100 nM, less than about 10 nM or less than about 1 nM. In various embodiments the H3R antagonist has in IC50 (H3R) of about 1 μΜ or less, about 100 nM or less, about 10 nM or less, or about 1 nM or less. In one embodiment, the H3R antagonist is an orally active H3R antagonist. In one embodiment, the H3R antagonist is peripherally restricted. In some embodiments, the H3R antagonist is an antagonist of the human
H3R. In some embodiments, the H3R antagonist is administered orally and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R antagonist is applied topically and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R antagonist is in an amount sufficient to reduce itch in a human.
The term "inverse agonist" shall mean an agent (e.g., ligand, test compound) which binds to a GPCR and which inhibits the baseline intracellular response initiated by the active form of the receptor below the normal base level activity which is observed in the absence of an agonist or partial agonist.
The term "H3R inverse agonist" as used herein, refers to a compound that binds to H3R receptor and acts as an inverse agonist. In one embodiment, the H3R inverse agonist is a selective inverse agonist. In one embodiment, the H3R inverse agonist has selectivity for H3R that is at least 10 fold greater than its selectivity for any one or more receptors selected from: HIR, H2R and H4R. In one embodiments, the H3R inverse agonist has a selectivity for H3R over HIR of at least about 10-fold. In one embodiments, the H3R inverse agonist has a selectivity for H3R over H2R of at least about 10-fold. In one embodiments, the H3R inverse agonist has a selectivity for H3R over H4R of at least about 10-fold. In one embodiments, the H3R inverse agonist has a selectivity for H3R over HIR, H2R, or H4R of at least about 10 fold. In various embodiments the H3R inverse agonist has in IC50 (H3R) of less than about ΙμΜ, less than about ΙΟΟηΜ, less than about ΙΟηΜ or less than about InM. In various embodiments the H3R inverse agonist has in IC50 (H3R) of about 1 μΜ or less, about 100 nM or less, about 10 nM or less, or about 1 nM or less. In one embodiment, the H3R inverse agonist is an orally active H3R inverse agonist. In one embodiment, the H3R inverse agonist is peripherally restricted. In some embodiments, the H3R inverse agonist is an inverse agonist of the human H3R. In some embodiments, the H3R inverse agonist is orally active and has an IC50 of less than about 100 nM. In some embodiments, the H3R inverse agonist is administered orally and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R inverse agonist is applied topically and has an IC50 (H3R) of about 100 nM or less. In some embodiments, the H3R inverse agonist is in an amount sufficient to reduce itch in a human.
The term "individual," as used herein, refers to a vertebrate, including but not limited to fish (such as commercially farmed fish, pet fish, etc.), amphibians (such as frogs, toads, pet amphibians, etc.), reptiles (such as snakes, lizards, turtles, pet reptiles, etc.), birds (such as chickens, turkeys, pet birds, etc.) and mammals (such as mice, rats, hamsters, rabbits, pigs, dogs, cats, horses, cows, sheep, goats, non-human primates, non-human mammals, pet non-human mammals, humans, etc.). In certain embodiments, the individual is a fish. In certain embodiments, the individual is an amphibian. In certain embodiments, the individual is a reptile. In certain embodiments, the individual is a bird. In certain embodiments, the individual is a mammal. In certain embodiments, the individual is a mouse, a rat, a hamster, a rabbit, a pig, a dog, a cat, a horse, a cow, a sheep, a goat, a non-human primate, or a human (which may be included in embodiments of the invention individually or in any combination). In certain embodiments, the individual is a horse. In certain embodiments, the individual is a dog or a cat. In certain embodiments, the individual is a human companion animal (such as a dog, a cat, etc.), a farm animal (such as a cow, a sheep, a goat, a pig, a chicken, etc.), a sports animal (such as a horse, a dog, etc.), a beast of burden (such as a mule, a camel, etc.) or an exotic animal (such as an animal found in a zoo, etc.), which may be included in embodiments of the invention individually or in any combination. In certain embodiments, the individual is a non-human mammal. In certain embodiments, the individual is a non-human primate (such as a rhesus monkey, a chimpanzee, etc.). In certain embodiments, the individual is a human.
The term "treating" and "treatment" as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner in the case of humans; veterinarian in the case of non-human vertebrates, and in particular embodiment non-human mammals) that an individual requires or will benefit from treatment.
The term "therapeutically effective amount" or "therapeutically effective dose" as used herein refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) Preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease,
(2) Inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and
(3) Ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
The term "therapeutic efficacy" as used herein refers to elicitation of the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:
(1) Preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease,
(2) Inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and
(3) Ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
The term "composition" shall mean a material comprising at least one component.
The term "active ingredient" shall mean any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of a disease.
The term "pharmaceutical composition" shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation and treatment in a mammal.
By "pharmaceutically acceptable" it is meant that the carrier, vehicle, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
The term "dosage form" shall mean the physical form in which a drug is produced and dispensed, such as a tablet, capsule, or an injectable.
The term "endogenous" shall mean a material that an individual naturally produces.
The term "contact" or "contacting" shall mean bringing at least two moieties together. The terms "modulate" or "modify" shall be taken to refer to an increase or decrease in the amount, quality, or effect of a particular activity, function or molecule. By way of illustration and not limitation, agonists, partial agonists, inverse agonists, and antagonists of a G protein-coupled receptor are modulators of the receptor.
The term "small molecule" shall be taken to mean a compound having a molecular weight of less than about 10,000 grams per mole, including a peptide, peptidomimetic, amino acid, amino acid analogue, polynucleotide, polynucleotide analogue, nucleotide, nucleotide analogue, organic compound or inorganic compound (i.e. including a heterorganic compound or organometallic compound), and salts, esters and other pharmaceutically acceptable forms thereof. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having molecular weight of less than about 1,000 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 800 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 600 grams per mole. In certain embodiments, small molecules are organic or inorganic compounds having a molecular weight of less than about 500 grams per mole.
As used herein, the term "itch", technically known as pruritus, refers to the sensation that elicits a reflex response to scratch. Itch can be a symptom of a disease, disorder or infection, or itch can arise spontaneously, without an underlying or identifiable physiological cause, known as idiopathic pruritus.
As used herein with respect to pruritus and itch, the term "treatment" refers to all aspects of control of itching including prophylaxis and therapy. Control of itch of includes reducing, alleviating, relieving and numbing the sensation of itch. Control of itch also includes reducing the desire to scratch.
The term "in need of treatment" and the term "in need thereof" when referring to treatment are used interchangeably and refer to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, etc. in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver' s expertise, but that includes the knowledge that the individual or animal is ill, or will become ill, as the result of a disease, condition or disorder that is treatable by the compounds of the invention. Accordingly, the compounds of the invention can be used in a protective or preventive manner; or compounds of the invention can be used to alleviate, inhibit or ameliorate the disease, condition or disorder and refers to all aspects of control of itching including prophylaxis and therapy. Control of itch includes reducing, alleviating, relieving and numbing the sensation of itch. Control of itch also includes reducing the desire to scratch. As used herein, the term "antibody" refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically bind an antigen. The terms also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms besides antibodies; including, for example, Fv, Fab, and F(ab)'2 as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., Immunology, Benjamin, N.Y., 2nd ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference).
The term "polypeptide" shall refer to a polymer of amino acids without regard to the length of the polymer. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides. For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
The term "antibody" is intended herein to encompass monoclonal antibody and polyclonal antibody.
The term "second messenger" shall mean an intracellular response produced as a result of receptor activation. A second messenger can include, for example, inositol 1,4,5-triphosphate (IP3), diacylglycerol (DAG), cyclic AMP (cAMP), cyclic GMP (cGMP), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca2+. Second messenger response can be measured for a determination of receptor activation.
The term "receptor functionality" shall refer to the normal operation of a receptor to receive a stimulus and moderate an effect in the cell, including, but not limited to regulating gene transcription, regulating the influx or efflux of ions, effecting a catalytic reaction, and/or modulating activity through G-proteins, such as eliciting a second messenger response.
The term "stimulate" or "stimulating," in relationship to the term "response" or
"functionality of the receptor" shall mean that a response or a functionality of the receptor is increased in the presence of a compound as opposed to in the absence of the compound.
The term "inhibit" or "inhibiting," in relationship to the term "response" or "functionality of the receptor" shall mean that a response a functionality of the receptor is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
The term "compound efficacy" shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity. The compound efficacy of different compounds may be evaluated with respect to each other or some other standard.
The term "test compound," used interchangeably herein with "candidate compound," shall mean a molecule (for example, and not limitation, a chemical compound) which is amenable to a screening technique. In one embodiment, a test compound is an anti-pruritic agent. In one embodiment, a test compound is a H3R antagonist. In one embodiment, a test compound is a H3R inverse agonist.
The term "directly identifying" or "directly identified", in relationship to the phrase "test compound," shall mean the screening of a compound against a G protein-coupled receptor in the absence of a known ligand (e.g., a known agonist) to the G protein-coupled receptor.
The term "pruritus model" shall mean an in vivo or in vitro model system which displays at least one aspect or element of pruritus and is amenable to or allows the evaluation of anti-pruritic agents. In one embodiment, the pruritus model is mammalian in origin.
The term "peripherally restricted" shall refer to a compound or agent that is restricted in some manner from crossing the blood brain barrier. A peripherally restricted compound or agent may be less than 100% restricted to the periphery. A peripherally restricted compound may be further referred to by a braimplasma ratio which will be less than 1 in at least one species.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Histamine H3 Receptors (H3R)
The human histamine H3 receptor (GenBank accession number: AF140538) is described in Figure 1 and Figure 2, and SEQ ID NO: 1 and SEQ ID NO:2 (Lovenberg, T.W., Roland, B.L., Wilson, S.J., Jiang, X., Pyati, J., Huvar, A., Jackson, M.R. and Erlander, M.G., Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 55 (6), 1101-1107 (1999)). The human histamine H3 receptor gene contains three introns and 4 exons. Through alternative splicing, six receptor variants are known to be created. The full-length receptor and two additional variants are known to bind histamine and other H3 agonists (Wellendorph P, Goodman MW, Burstein ES, Nash NR, Brann MR, Weiner DM. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor. Neuropharmacology.
42(7):929-40 (2002); Bongers G, Krueger KM, Miller TR, Baranowski JL, Estvander BR, Witte DG, Strakhova MI, van Meer P, Bakker RA, Cowart MD, Hancock AA, Esbenshade TA, Leurs R. An 80-Amino Acid Deletion in the Third Intracellular Loop of a Naturally Occurring Human Histamine H3 Isoform Confers Pharmacological Differences and Constitutive Activity. J Pharmacol Exp Ther 323(3):888-98 (2007)).
The mouse histamine H3 receptor (GeneBank accession number: AY142145) is described in Figure 3 and Figure 4 and SEQ ID NOS: 3 and 4 (Chen, J., Liu, C. and Lovenberg, T. W. Molecular and pharmacological characterization of the mouse histamine H(3) receptor. Eur J Pharmacol 467 (1-3), 57-65 (2003)).
Use of species variants of H3R are envisioned to be within the scope of the invention. Allelic variants of H3R of SEQ ID NO:2 are envisioned to be within the scope of the invention. Human H3R is envisioned to be within the scope of the invention.
A variant which is a vertebrate ortholog of human H3R of SEQ ID NO:2 is envisioned to be within the scope of the invention. A variant which is a mammalian ortholog of human H3R of SEQ ID NO:2 is envisioned to be within the scope of the invention. By way of illustration and not limitation, mouse H3R, rat H3R, hamster H3R, dog H3R, and non-human primate H3R are envisioned to be within the scope of the invention.
In certain embodiments, the variant of SEQ ID NO:2 is a GPCR. In some embodiments, the GPCR comprises the amino acids 1-445 of SEQ ID NO:2. In some embodiments, the GPCR comprises the amino acids 2-445 of SEQ ID NO:2. In some embodiments, the GPCR comprises the amino acids 2-445 of SEQ ID NO:2, with the proviso that the GPCR does not comprise the amino acid sequence of SEQ ID NO: 2. In some embodiments, the GPCR comprises the amino acid sequence of a GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: l. In some embodiments, the GPCR comprises a variant of SEQ ID NO: 2.
A variant of SEQ ID NO:2 having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2 is envisioned to be within the scope of the invention. In certain embodiments, the variant of SEQ ID NO:2 having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2 is a GPCR.
In certain embodiments, a variant GPCR that may be used in the subject methods has an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, of at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2. By a variant GPCR having, for example, 95% "identity" to SEQ ID NO:2 is meant that the amino acid sequence of the variant is identical to amino acids 1-335 of SEQ ID NO:2 except that it may include up to five amino acid alterations per each 100 amino acids of SEQ ID NO:2. Thus, to obtain for example an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2, up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with amino acids 1-617 of SEQ ID NO: 2. These alternations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either subjectly among residues in the sequence or in one or more contiguous groups within the sequence.
In some embodiments, a variant GPCR that may be used in the subject methods is a GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1. In some embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an endogenous GPCR. In some embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 and that is an endogenous GPCR is a mammalian endogenous GPCR. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an allele of SEQ ID NO:2. In certain embodiments, the GPCR encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 is an ortholog of SEQ ID NO:2.
In one embodiment, the GPCR is screened in antagonist mode.
In certain embodiments, a GPCR of the invention forms part of a fusion protein with a G protein.
Sequence Identity
A preferred method for determining the best overall match between a query sequence (e.g., the amino acid sequence of SEQ ID NO:2) and a sequence to be interrogated, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp App Biosci (1990) 6:237-245). In a sequence alignment the query and interrogated sequences are both amino acid sequences. The results of the global sequence alignment are in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group=25, Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or the lenth of the interrogated amino acid sequence, whichever is shorter.
If the interrogated sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, the results, in percent identity, must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the interrogated sequence when calculating global percent identity. For interrogated sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the interrogated sequence, that are not matched/aligned with a corresponding interrogated sequence residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the interrogated sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query amino acid residues outside the farthest N- and C-terminal residues of the interrogated sequence.
For example, a 90 amino acid residue interrogated sequence is aligned with a 100- residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the interrogated sequence and therefore, the FASTDB alignment does not match/align with the first residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%.
In another example, a 90-residue interrogated sequence is compared with a 100-residue query sequence. This time the deletions are internal so there are no residues at the N- or C- termini of the interrogated sequence, which are not matched/aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected. No other corrections are made for the purposes of the present invention.
Fusion Proteins
In certain embodiments, a polypeptide of interest is a fusion protein, and may contain, for example, an affinity tag domain or a reporter domain. Suitable affinity tags include any amino acid sequence that may be specifically bound to another moiety, usually another polypeptide, most usually an antibody. Suitable affinity tags include epitope tags, for example, the V5 tag, the FLAG tag, the HA tag (from hemagglutinin influenza virus), the myc tag, and the like, as is known in the art. Suitable affinity tags also include domains for which, binding substrates are known, e.g., HIS, GST and MBP tags, as is known in the art, and domains from other proteins for which specific binding partners, e.g., antibodies, particularly monoclonal antibodies, are available. Suitable affinity tags also include any protein-protein interaction domain, such as a IgG Fc region, which may be specifically bound and detected using a suitable binding partner, e.g. the IgG Fc receptor. It is expressly contemplated that such a fusion protein may contain a heterologous N-terminal domain (e.g., an epitope tag) fused in-frame with a GPCR that has had its N-terminal methionine residue either deleted or substituted with an alternative amino acid.
Suitable reporter domains include any domain that can report the presence of a polypeptide. While it is recognized that an affinity tag may be used to report the presence of a polypeptide using, e.g., a labeled antibody that specifically binds to the tag, light emitting reporter domains are more usually used. Suitable light emitting reporter domains include luciferase (from, e.g., firefly, Vargula, Renilla reniformis or Renilla muelleri), or light emitting variants thereof. Other suitable reporter domains include fluorescent proteins, (from e.g., jellyfish, corals and other coelenterates as such those from Aequoria, Renilla, Ptilosarcus, Stylatula species), or light emitting variants thereof. Light emitting variants of these reporter proteins are very well known in the art and may be brighter, dimmer, or have different excitation and/or emission spectra, as compared to a native reporter protein. For example, some variants are altered such that they no longer appear green, and may appear blue, cyan, yellow, enhanced yellow red (termed BFP, CFP, YFP eYFP and RFP, respectively) or have other emission spectra, as is known in the art. Other suitable reporter domains include domains that can report the presence of a polypeptide through a biochemical or color change, such as β-galactosidase, β- glucuronidase, chloramphenicol acetyl transferase, and secreted embryonic alkaline phosphatase.
Also as is known in the art, an affinity tags or a reporter domain may be present at any position in a polypeptide of interest. However, in most embodiments, they are present at the C- or N-terminal end of a polypeptide of interest.
Since the genetic code and recombinant techniques for manipulating nucleic acid are known, and the amino acid sequences of GPCR polypeptides of interest described as above, the design and production of nucleic acids encoding a GPCR polypeptide of interest is well within the skill of an artisan. In certain embodiments, standard recombinant DNA technology (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.) methods are used. For example, GPCR coding sequences may be isolated from a library of
GPCR coding sequence using any one or a combination of a variety of recombinant methods that do not need to be described herein. Subsequent substitution, deletion, and/or addition of nucleotides in the nucleic acid sequence encoding a protein may also be done using standard recombinant DNA techniques.
For example, site directed mutagenesis and subcloning may be used to
introduce/delete/substitute nucleic acid residues in a polynucleotide encoding a polypeptide of interest. In other embodiments, PCR may be used. Nucleic acids encoding a polypeptide of interest may also be made by chemical synthesis entirely from oligonucleotides (e.g., Cello et al., Science (2002) 297: 1016-8). In some embodiments, the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly a mammalian, e.g., mouse, rat, hamster, non-human primate, or human, species. In some embodiments, the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly an amphibian species.
Vectors
The invention further provides vectors (also referred to as "constructs") comprising a subject nucleic acid. In many embodiments of the invention, the subject nucleic acid sequences will be expressed in a host after the sequences have been operably linked to an expression control sequence, including, e.g. a promoter. The subject nucleic acids are also typically placed in an expression vector that can replicate in a host cell either as an episome or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein by reference). Vectors, including single and dual expression cassette vectors are well known in the art
(Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Suitable vectors include viral vectors, plasmids, cosmids, artificial chromosomes (human artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, etc.), mini-chromosomes, and the like. Retroviral, adenoviral and adeno-associated viral vectors may be used.
A variety of expression vectors are available to those in the art for purposes of producing a polypeptide of interest in a cell and include expression vectors which are commercially available (e.g., from Invitrogen, Carlsbad, CA; Clontech, Mountain View, CA; Stratagene, La Jolla, CA). Commercially available expression vectors include, by way of non- limiting example, CMV promoter-based vectors. One suitable expression vector is pCMV. The expression vector may be adenoviral. An exemplary adenoviral vector may be purchased as AdEasyTM from Qbiogene (Carlsbad, CA) (He TC et al, Proc Natl Acad Sci USA (1998) 95:2509-2514; and US Patent No. 5,922,576). Other suitable expression vectors will be readily apparent to those of ordinary skill in the art.
The subject nucleic acids usually comprise an single open reading frame encoding a subject polypeptide of interest, however, in certain embodiments, since the host cell for expression of the polypeptide of interest may be a eukaryotic cell, e.g., a mammalian cell, such as a human cell, the open reading frame may be interrupted by introns. Subject nucleic acid are typically part of a transcriptional unit which may contain, in addition to the subject nucleic acid 3' and 5' untranslated regions (UTRs) which may direct RNA stability, translational efficiency, etc. The subject nucleic acid may also be part of an expression cassette which contains, in addition to the subject nucleic acid a promoter, which directs the transcription and expression of a polypeptide of interest, and a transcriptional terminator.
Eukaryotic promoters can be any promoter that is functional in a eukaryotic host cell, including viral promoters and promoters derived from eukaryotic genes. Exemplary eukaryotic promoters include, but are not limited to, the following: the promoter of the mouse
metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81 :5951-59SS, 1984), the CMV promoter, the EF-1 promoter, Ecdysone-responsive promoter(s), tetracycline-responsive promoter, and the like. Viral promoters may be of particular interest as they are generally particularly strong promoters. In certain embodiments, a promoter is used that is a promoter of the target pathogen. Promoters for use in the present invention are selected such that they are functional in the cell type (and/or animal) into which they are being introduced. In certain embodiments, the promoter is a CMV promoter.
In certain embodiments, a subject vector may also provide for expression of a selectable marker. Suitable vectors and selectable markers are well known in the art and discussed in Ausubel, et al, (Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995) and Sambrook, et al, (Molecular Cloning: A Laboratory Manual, Third Edition, (2001) Cold Spring Harbor, N.Y.). A variety of different genes have been employed as selectable markers, and the particular gene employed in the subject vectors as a selectable marker is chosen primarily as a matter of convenience. Known selectable marker genes include: the thymidine kinase gene, the dihydrofolate reductase gene, the xanthine-guanine phosphoribosyl transferase gene, CAD, the adenosine deaminase gene, the asparagine synthetase gene, the antibiotic resistance genes, e.g. tetr, ampr, Cmr or cat, kanr or neor (aminoglycoside phosphotransferase genes), the hygromycin B phosphotransferase gene, and the like.
As mentioned above, polypeptides of interest may be fusion proteins that contain an affinity domain and/or a reporter domain. Methods for making fusions between a reporter or tag and a GPCR, for example, at the C- or N-terminus of the GPCR, are well within the skill of one of skill in the art (e.g. McLean et al, Mol. Pharma. Mol Pharmacol. 1999 56: 1182-91 ; Ramsay et al., Br. J. Pharmacology, 2001, 315-323) and will not be described any further. It is expressly contemplated that such a fusion protein may contain a heterologous N-terminal domain (e.g., an epitope tag) fused in-frame with a GPCR that has had its N-terminal methionine residue either deleted or substituted with an alternative amino acid. It is appreciated that a polypeptide of interest may first be made from a native polypeptide and then operably linked to a suitable reporter/tag as described above. The subject nucleic acids may also contain restriction sites, multiple cloning sites, primer binding sites, ligatable ends, recombination sites etc., usually in order to facilitate the construction of a nucleic acid encoding a polypeptide of interest.
Host cells
The invention further provides host cells comprising a vector comprising a subject nucleic acid. Suitable host cells include prokaryotic, e.g., bacterial cells (for example E. coli), as well as eukaryotic cells e.g. an animal cell (for example an insect, mammal, fish, amphibian, bird or reptile cell), a plant cell (for example a maize or Arabidopsis cell), or a fungal cell (for example a S. cerevisiae cell). In certain embodiments, any cell suitable for expression of a polypeptide of interest-encoding nucleic acid may be used as a host cell. Usually, an animal host cell line is used, examples of which are as follows: monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293 ["293"], Graham et al. J. Gen Virol. 36:59 (1977)); HEK-293T ["293T"] cells; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA) 77:4216, (1980); Syrian golden hamster cells
MCB3901 (ATCC CRL-9595); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); african green monkey kidney cells (VERO- 76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N. Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1).
In certain embodiments, melanophores are used. Melanophores are skin cells found in lower vertebrates. Relevant materials and methods will be followed according to the disclosure of U.S. Patent Number 5,462,856 and U.S. Patent Number 6,051,386.
Additional cell lines will become apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209.
Screening of Compounds
a. Generic GPCR screening assay techniques
When a G protein receptor becomes active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G protein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, activated receptors continue to exchange GDP to GTP. A non- hydrolyzable analog of GTP, [35S]GTPTS, can be used to monitor enhanced binding to membranes which express activated receptors. It is reported that [35S]GTPTS can be used to monitor G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, among other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995. A preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to all G protein- coupled receptors regardless of the particular G protein that interacts with the intracellular domain of the receptor.
b. Specific GPCR screening assay techniques
Once candidate compounds are identified using the "generic" G protein-coupled receptor assay (i.e., an assay to select compounds that are agonists or inverse agonists), in some embodiments further screening to confirm that the compounds have interacted at the receptor site is preferred. For example, a compound identified by the "generic" assay may not bind to the receptor, but may instead merely "uncouple" the G protein from the intracellular domain.
i. Gs, Gz and Gi.
Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on the other hand, inhibit adenylyl cyclase. Adenylyl cyclase catalyzes the conversion of ATP to cAMP; thus, activated GPCRs that couple the Gs protein are associated with increased cellular levels of cAMP. On the other hand, activated GPCRs that couple Gi (or Gz, Go) protein are associated with decreased cellular levels of cAMP. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3ri Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can be utilized to determine if a candidate compound is, e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP). A variety of approaches known in the art for measuring cAMP can be utilized; in some embodiments a preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format. Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene. Reporter systems can be constructed which have a promoter containing multiple cAMP response elements before the reporter gene, e.g., β-galactosidase or luciferase. Thus, an activated Gs-linked receptor causes the accumulation of cAMP that then activates the gene and expression of the reporter protein. The reporter protein such as β-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al. 1995).
ii. Go and Gq.
Gq and Go are associated with activation of the enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP2, releasing two intracellular messengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Increased accumulation of IP3 is associated with activation of Gq- and Go-associated receptors. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3 Ed.) Nichols, J.G. et al eds. Sinauer Associates, Inc. (1992). Assays that detect IP3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP3). Gq-associated receptors can also been examined using an API reporter assay in that Gq-dependent phospholipase C causes activation of genes containing API elements; thus, activated Gq-associated receptors will evidence an increase in the expression of such genes, whereby inverse agonists thereto will evidence a decrease in such expression, and agonists will evidence an increase in such expression. Commercially available assays for such detection are available.
c. GPCR Fusion Protein
The use of an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR, for use in screening of candidate compounds for the direct identification of inverse agonists or agonists provides an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, in order to differentiate between, e.g., the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound, with an aim of such a differentiation to allow for an understanding as to whether such compound may be an inverse agonist or agonist or have no affect on such a receptor, in some embodiments it is preferred that an approach be utilized that can enhance such differentiation. In some embodiments, a preferred approach is the use of a GPCR Fusion Protein.
Generally, once it is determined that a non-endogenous GPCR has been constitutively activated using the assay techniques set forth above (as well as others known to the art-skilled), it is possible to determine the predominant G protein that couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed. In some embodiments it is preferred that screening take place using a mammalian or a melanophore expression system, as such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the non-endogenous, constitutively activated GPCR will continuously signal. In some embodiments it is preferred that this signal be enhanced such that in the presence of, e.g., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularly in the context of screening, between the receptor when it is contacted with the inverse agonist.
The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the GPCR. The GPCR Fusion Protein may be preferred for screening with either an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR because such an approach increases the signal that is generated in such screening techniques. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is preferred for the screening of candidate compounds as disclosed herein. The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary skill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. Important criteria in the construction of such a GPCR Fusion Protein construct include but are not limited to, that the GPCR sequence and the G protein sequence both be in- frame (preferably, the sequence for the endogenous GPCR is upstream of the G protein sequence), and that the "stop" codon of the GPCR be deleted or replaced such that upon expression of the GPCR, the G protein can also be expressed. The GPCR can be linked directly to the G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). Based upon convenience, it is preferred to use a spacer. In some embodiments, it is preferred that the G protein that couples to the non-endogenous GPCR will have been identified prior to the creation of the GPCR Fusion Protein construct.
As noted above, activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging (i.e., the cAMP signal decreases upon activation, thus making the direct identification of, e.g., agonists (which would further decrease this signal) challenging). As will be disclosed herein, it has been ascertained that for these types of receptors, it is possible to create a GPCR Fusion Protein that is not based upon the GPCR's endogenous G protein, in an effort to establish a viable cyclase- based assay. Thus, for example, an endogenous Gi coupled receptor can be fused to a Gs protein -such a fusion construct, upon expression, "drives" or "forces" the endogenous GPCR to couple with, e.g., Gs rather than the "natural" Gi protein, such that a cyclase-based assay can be established. Thus, for Gi, Gz and Go coupled receptors, in some embodiments it is preferred that when a GPCR Fusion Protein is used and the assay is based upon detection of adenylyl cyclase activity, that the fusion construct be established with Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).
Table B
G protein Effect of cAMP Effect of IP3 Effect of cAMP Effect on IP3
Production upon Accumulation upon Production Accumulation Activation of Activation of GPCR upon contact upon contact GPCR (i.e., (i.e., constitutive with an Inverse with an Inverse constitutive activation or agonist Agonist Agonist activation or binding)
agonist binding)
Gs Increase N/A Decrease N/A Gi Decrease N/A Increase N/A
Gz Decrease N/A Increase N/A
Go Decrease Increase Increase Decrease
Gq N/A Increase N/A Decrease
Equally effective is a G Protein Fusion construct that utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein. In some embodiments a preferred fusion construct can be accomplished with a Gq Protein wherein the first six (6) amino acids of the G-protein a-subunit ("Gaq") is deleted and the last five (5) amino acids at the C-terminal end of Gaq is replaced with the corresponding amino acids of the Ga of the G protein of interest. For example, a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct". This fusion construct will force the endogenous Gi coupled receptor to couple to its non-endogenous G protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylglycerol, can be measured in lieu of cAMP production.
d. Co-transfection of a Target Gi Coupled GPCR with a Signal-Enhancer Gs Coupled GPCR (cAMP Based Assays)
A Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decreases the level of cAMP production, which can make the assessment of cAMP levels challenging. In certain embodiments, an effective technique in measuring the decrease in production of cAMP as an indication of activation of a receptor that predominantly couples Gi upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non-endogenous, constitutively activated receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I; see infra), with the Gi linked GPCR. As is apparent, activation of a Gs coupled receptor can be determined based upon an increase in production of cAMP. Activation of a Gi coupled receptor leads to a decrease in production cAMP. Thus, the co-transfection approach is intended to advantageously exploit these "opposite" affects. For example, co-transfection of a non- endogenous, constitutively activated Gs coupled receptor (the "signal enhancer") with expression vector alone provides a baseline cAMP signal (i.e., although the Gi coupled receptor will decrease cAMP levels, this "decrease" will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer). By then co- transfecting the signal enhancer with the "target receptor", an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal.
Candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or after screening against the co-transfected receptors).
In certain embodiments, IC50 value is determined using an assay selected from the group consisting of: IP3 assay carried out using transfected HEK293 cells expressing recombinant H3R polypeptide; and melanophore assay carried out using transfected melanophores expressing recombinant H3R polypeptide.
In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 10 μΜ, of less than 1 μΜ, of less than 100 nM, or of less than 10 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 10 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 9 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 8 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 7 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 6 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 5 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 4 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 3 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 2 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 1 μΜ in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 900 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 800 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 700 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 600 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 500 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 400 nM n the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 300 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 200 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 100 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 90 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 80 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 70 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 60 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 50 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 40 nM n the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 30 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 20 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 of less than 10 nM in the assay. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 10 μΜ. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 1 μΜ. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 100 nM. In some embodiments, the test compound is an inverse agonist or antagonist with an IC50 in the assay of a value selected from the interval of 1 nM to 10 nM. In some embodiments, the test compound is selective for the GPCR.
e. Functional Screening Assays
Melanophore Technology
Melanophores are skin cells found in lower vertebrates. They contain pigmented organelles termed melanosomes. Melanophores are able to redistribute these melanosomes along a microtubule network upon G-protein coupled receptor (GPCR) activation. The result of this pigment movement is an apparent lightening or darkening of the cells. In melanophores, the decreased levels of intracellular cAMP that result from activation of a Gi-coupled receptor cause melanosomes to migrate to the center of the cell, resulting in a dramatic lightening in color. If cAMP levels are then raised, following activation of a Gs-coupled receptor, the melanosomes are re-dispersed and the cells appear dark again. The increased levels of diacylglycerol that result from activation of Gq-coupled receptors can also induce this re-dispersion. In addition, the technology is also suited to the study of certain receptor tyrosine kinases. The response of the melanophores takes place within minutes of receptor activation and results in a simple, robust color change. The response can be easily detected using a conventional absorbance microplate reader or a modest video imaging system. Unlike other skin cells, the melanophores derive from the neural crest and appear to express a full complement of signaling proteins. In particular, the cells express an extremely wide range of G-proteins and so are able to functionally express almost all GPCRs.
Melanophores can be utilized to identify compounds, including natural ligands, which bind to and/or activate GPCRs. This method can be conducted by introducing test cells of a pigment cell line capable of dispersing or aggregating their pigment in response to a specific stimulus and expressing an exogenous clone coding for the GPCR. An initial state of pigment disposition can be set using, for example, using melatonin, MSH or light. The test cells are then contacted with chemical compounds, and it is determined whether the pigment disposition in the cells changed from the initial state of pigment disposition. Dispersion of pigments cells due to the candidate compound, including but not limited to a ligand, coupling to the GPCR will appear dark on a petri dish, while aggregation of pigments cells will appear light.
Materials and methods may be followed according to the disclosure of U.S. Patent Number 5,462,856 and U.S. Patent Number 6,051,386.
Melanophores are transfected by electroporation with a plasmid which contains the coding sequence of mouse or human H3R. The cells are plated in 96-well plates. 48 hours post- transfection, half of the cells on each plate are treated with lOnM melatonin. Melatonin activates an endogenous Gi-coupled receptor in the melanophores and causes them to aggregate their pigment. The remaining half of the cells are transferred to serum-free medium 0.7X L-15 (Gibco). After one hour, the cells in serum- free media remain in a pigment-dispersed state while the melatonin-treated cells are in a pigment-aggregated state. At this point, the cells are treated with different compounds from a proprietary compound library containing 140,000-150,000 organic small molecule compounds. If H3R bound to the compound, the melanophores would be expected to undergo a color change, for example, due to pigment aggregation, in response to the compound.
Assays for Determination of GPCR Activation
A variety of approaches are available for assessment of activation of human GPCRs. The following are illustrative; those of ordinary skill in the art are credited with the ability to determine those techniques that are preferentially beneficial for the needs of the artisan.
1. Membrane Binding Assays: [35S]GTPyS Assay
When a G protein-coupled receptor is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP. The non-hydrolyzable GTP analog, [35S]GTPyS, can be utilized to demonstrate enhanced binding of [35S]GTPyS to membranes expressing activated receptors. The advantage of using [35S]GTPyS binding to measure activation is that: (a) it is generically applicable to all G protein-coupled receptors; (b) it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade.
The assay utilizes the ability of G protein coupled receptors to stimulate [35S]GTPyS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous GPCRs and non- endogenous, constitutively activated GPCRs. The assay is generic and has application to drug discovery at all G protein-coupled receptors.
The [35S]GTPyS assay is incubated in 20 mM HEPES and between 1 and about 20mM MgCl2 (this amount can be adjusted for optimization of results, although 20mM is preferred) pH 7.4, binding buffer with between about 0.3 and about 1.2 nM [35S]GTPyS (this amount can be adjusted for optimization of results, although 1.2 is preferred) and 12.5 to 75 μg membrane protein (e.g, 293 cells expressing the H3R; this amount can be adjusted for optimization) and 10 μΜ GDP (this amount can be changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25 μί; Amersham) are then added and the mixture incubated for another 30 minutes at room temperature. The tubes are then centrifuged at 1500 x g for 5 minutes at room temperature and then counted in a scintillation counter.
2. Adenylyl Cyclase
A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes. The Flash Plate wells can contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express a receptor.
Transfected cells are harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10 mL of PBS is gently added to each dish of cells followed by careful aspiration. 1 mL of Sigma cell dissociation buffer and 3 mL of PBS are added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50 mL conical centrifuge tube. Cells are then centrifuged at room temperature at 1,100 rpm for 5 minutes. The cell pellet is carefully re-suspended into an appropriate volume of PBS (about 3 mL/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50 μίΛνεΙΙ).
cAMP standards and Detection Buffer (comprising ΙμΟ of tracer [125Γ] cAMP (50 μί) to 11 mL Detection Buffer) is prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer is prepared fresh for screening and contains 50 μL· of Stimulation Buffer, 3 μL· of candidate compound (12μΜ final assay concentration) and 50 μL· cells. Assay Buffer is stored on ice until utilized. The assay, preferably carried out, for example, in a 96-well plate, is initiated by addition of 50 μL· of cAMP standards to appropriate wells followed by addition of 50 μΐ, of PBSA to wells HI 1 and H12. 50 μΐ, of Stimulation Buffer is added to all wells. DMSO (or selected candidate compounds) is added to appropriate wells using a pin tool capable of dispensing 3 μL· of compound solution, with a final assay concentration of 12μΜ candidate compound and 100 μL· total assay volume. The cells are then added to the wells and incubated for 60 minutes at room temperature. 100 μL· of Detection Mix containing tracer cAMP is then added to the wells. Plates are then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are then extrapolated from a standard cAMP curve which is contained within each assay plate.
3. Cell-Based cAMP for Gi Coupled Target GPCRs
TSHR is a Gs coupled GPCR that causes the accumulation of cAMP upon activation. TSHR can be constitutively activated by mutating amino acid residue 623 (i.e., changing an alanine residue to an isoleucine residue). A Gi coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging. An effective technique for measuring the decrease in production of cAMP as an indication of activation of a Gi coupled receptor can be accomplished by co-transfecting, non- endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutively active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baseline level of cAMP. Upon creating an endogenous or non-endogenous version of the Gi coupled receptor, the target GPCR is then co-transfected with the signal enhancer, and it is this material that can be used for screening. In some embodiments, this approach is preferably used in the direct identification of candidate compounds against Gi coupled receptors. It is noted that for a Gi coupled GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP signal and an agonist will decrease the cAMP signal.
On day one, 2xl04 293 cells/well are plated out. On day two, two reaction tubes are prepared (the proportions to follow for each tube are per plate): tube A is prepared by mixing 2μg DNA of each receptor transfected into the mammalian cells, for a total of 4μg DNA (e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPCR, etc.) in 1.2 mL serum free DMEM (Irvine Scientific, Irvine, CA); tube B is prepared by mixing 120 μL· lipofectamine (Gibco BRL) in 1.2 mL serum free DMEM. Tubes A and B are then admixed by inversions (several times), followed by incubation at room temperature for 30-45minutes. The admixture is referred to as the "transfection mixture". Plated 293 cells are washed with 1XPBS, followed by addition of 10 mL serum free DMEM. 2.4 mL of the transfection mixture is then added to the cells, followed by incubation for 4 hours at 37°C/5% C02. The transfection mixture is then removed by aspiration, followed by the addition of 25 mL of DMEM/10% Fetal Bovine Serum. Cells are then incubated at 37°C/5% C02. After 24 hours incubation, cells are harvested and utilized for analysis.
A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No. SMP004A) is designed for cell-based assays, but can be modified for use with crude plasma membranes depending on the need of the skilled artisan. The Flash Plate wells contain a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express a receptor of interest.
Transfected cells are harvested approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10 mL of PBS is gently added to each dish of cells followed by careful aspiration. 1 mL of Sigma cell dissociation buffer and 3 mL of PBS is added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50 mL conical centrifuge tube. Cells are then centrifuged at room temperature at 1,100 rpm for 5 minutes. The cell pellet is carefully re-suspended into an appropriate volume of PBS (about 3 mL/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50 μΕ/well).
cAMP standards and Detection Buffer (comprising ΙμΟ of tracer [125Γ] cAMP (50 μί) to 11 mL Detection Buffer) is prepared and maintained in accordance with the manufacturer' s instructions. Assay Buffer should be prepared fresh for screening and contain 50 μL· of Stimulation Buffer, 3 μL· of candidate compound (12μΜ final assay concentration) and 50 μL· cells. Assay Buffer can be stored on ice until utilized. The assay can be initiated by addition of 50 μL· of cAMP standards to appropriate wells followed by addition of 50 μL· of PBS A to wells H-l 1 and H12. Fifty μL· oί Stimulation Buffer is added to all wells. Selected compounds (e.g., TSH) are added to appropriate wells using a pin tool capable of dispensing 3 μL· of compound solution, with a final assay concentration of 12μΜ candidate compound and 100 μL· total assay volume. The cells are then added to the wells and incubated for 60 minutes at room temperature. 100 μL· of Detection Mix containing tracer cAMP is then added to the wells. Plates are then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are extrapolated from a standard cAMP curve which is contained within each assay plate.
4. Reporter-Based Assays
a. CRE-LUC Reporter Assay (Gs-associated receptors)
293 or 293T cells are plated-out on 96 well plates at a density of 2 x 104 cells per well and are transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions. A DNA lipid mixture is prepared for each 6-well transfection as follows: 260ng of plasmid DNA in 100 μΐ. of DMEM is gently mixed with 2 μΕ of lipid in 100 μΕ of DMEM (the 260ng of plasmid DNA consists of 200ng of a 8xCRE-Luc reporter plasmid, 50ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and lOng of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8XCRE-Luc reporter plasmid is prepared as follows: vector SRIF- -gal is obtained by cloning the rat somatostatin promoter (-71/+51) at BglV-Hindni site in the p gal-Basic Vector (Clontech). Eight (8) copies of cAMP response element are obtained by PCR from an adenovirus template AdpCF126CCRE8 (see, Suzuki et al., Hum Gene Ther 7: 1883-1893 (1996)) and cloned into the SRIF- -gal vector at the Kpn-BglV site, resulting in the 8xCRE- -gal reporter vector. The 8xCRE-Luc reporter plasmid is generated by replacing the beta-galactosidase gene in the 8xCRE- -gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at the Hindlll-BamHI site. Following 30 minutes incubation at room temperature, the DNA/lipid mixture is diluted with 400 μΕ of DMEM and 100 μΕ of the diluted mixture is added to each well. 100 μΕ of DMEM with 10% FCS are added to each well after a four hour incubation in a cell culture incubator. The following day the transfected cells are changed with 200 μΕ/well of DMEM with 10% FCS. Eight (8) hours later, the wells are changed to 100 μΕ /well of DMEM without phenol red, after one wash with PBS. Luciferase activity is measured the next day using the LucLite reporter gene assay kit (Packard) following manufacturer instructions and read on a 1450 MicroBeta™ scintillation and luminescence counter (Wallac).
b. API reporter assay (Gq-associated receptors)
A method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing API elements in their promoter. A Pathdetect™ AP-1 cis-Reporting System (Stratagene, Catalogue No. 219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate are 410 ng pAPl-Luc, 80 ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.
c. SRF-LUC Reporter Assay (Gq-associated receptors)
One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes containing serum response factors in their promoter. A Pathdetect™ SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gq coupled activity in, for example, COS7 cells. Cells are transfected with the plasmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian Transfection™ Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is measured in the media of transfected cells to control for variations in transfection efficiency between samples) are combined in a calcium phosphate precipitate as per the manufacturer's instructions. Half of the precipitate is equally distributed over 3 wells in a 96-well plate and kept on the cells in a serum free media for 24 hours. The last 5 hours the cells are incubated with, for example, ΙμΜ, candidate compound. Cells are then lysed and assayed for luciferase activity using a Luclite™ Kit (Packard, Cat. No. 6016911) and "Trilux 1450 Microbeta" liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions. The data can be analyzed using GraphPad Prism™ 2.0a (GraphPad Software Inc.).
d. Intracellular IP3 Accumulation Assay (Gq-associated receptors)
On day 1, cells comprising the receptor of interest (endogenous or non-endogenous) can be plated onto 24 well plates, usually lxlO5 cells/well (although this number can be optimized). On day 2 cells can be transfected by first mixing 0.25μg DNA in 50 μΕ serum free DMEM/well and 2 μΕ lipofectamine in 50 μΕ serum free DMEM/well. The solutions are gently mixed and incubated for 15-30 minutes at room temperature. Cells are washed with 0.5 mL PBS and 400 μΕ of serum free media is mixed with the transfection media and added to the cells. The cells are then incubated for 3-4 hours at 37°C/5%C02 and then the transfection media is removed and replaced with 1 mL/well of regular growth media. On day 3 the cells are labeled with 3H-myo-inositol. Briefly, the media is removed and the cells are washed with 0.5 mL PBS. Then 0.5 mL inositol-free/serum free media (GIBCO BRL) is added/well with 0.25 μθ of 3H-myo-inositol/ well and the cells are incubated for 16-18 hours overnight at 37°C/5%C02 . On Day 4 the cells are washed with 0.5 mL PBS and 0.45 mL of assay medium is added containing inositol-free/serum free media, 10 μΜ pargyline, 10 mM lithium chloride or 0.4 mL of assay medium and 50 μL· of lOx ketanserin (ket) to final concentration of 10μΜ, if using a control construct containing a serotonin receptor. The cells are then incubated for 30 minutes at 37°C. The cells are then washed with 0.5 mL PBS and 200 μL· of fresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) is added/well. The solution is kept on ice for 5-10 minutes or until cells were lysed and then neutralized by 200 μL· of fresh/ice cold neutralization sol. (7.5 % HCL). The lysate is then transferred into 1.5 mL eppendorf tubes and 1 mL of chloroform/methanol (1:2) is added/tube. The solution is vortexed for 15 seconds and the upper phase is applied to a Biorad AG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resin is washed with water at 1: 1.25 W/V and 0.9 mL of upper phase is loaded onto the column. The column is washed with 10 mL of 5 mM myo-inositol and 10 mL of 5 mM Na- borate/60mM Na-formate. The inositol tris phosphates are eluted into scintillation vials containing 10 mL of scintillation cocktail with 2 mL of 0.1 M formic acid/ 1 M ammonium formate. The columns are regenerated by washing with 10 mL of 0.1 M formic acid/3 M ammonium formate and rinsed twice with dd H20 and stored at 4°C in water.
[35S]GTPyS Assay
A. Membrane Preparation
In some embodiments membranes comprising the Target GPCR of interest for use in the identification of candidate compounds as, e.g.,. agonists, inverse agonists or antagonists, are prepared as follows:
a. Materials
"Membrane Scrape Buffer" is comprised of 20mM HEPES and lOmM EDTA, pH 7.4; "Membrane Wash Buffer" is comprised of 20mM HEPES and 0. lmM EDTA, pH 7.4; "Binding Buffer" is comprised of 20mM HEPES, 100 mM NaCl, and 10 mM MgCl2, pH 7.4.
b. Procedure
All materials are kept on ice throughout the procedure. Firstly, the media is aspirated from a confluent monolayer of cells, followed by rinsing with lOmL cold PBS, followed by aspiration. Thereafter, 5 mL of Membrane Scrape Buffer is added to scrape cells; this is followed by transfer of cellular extract into 50 mL centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4°C). Thereafter, the supernatant is aspirated and the pellet is resuspended in 30 mL Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17 minutes at 4°C. The supernatant is then aspirated and the pellet resuspended in Binding Buffer. This is then homogenized using a Brinkman Polytron™ homogenizer (15-20 second bursts until the all material is in suspension). This is referred to herein as "Membrane Protein".
Bradford Protein Assay Following the homogenization, protein concentration of the membranes is determined using the Bradford Protein Assay (protein can be diluted to about 1.5 mg/mL, aliquoted and frozen (-80°C) for later use; when frozen, protocol for use will be as follows: on the day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a Polytron at about 12 x 1,000 rpm for about 5-10 seconds; it is noted that for multiple preparations, the homogenizer should be thoroughly cleaned between homogenization of different preparations).
a. Materials
Binding Buffer (as per above); Bradford Dye Reagent; Bradford Protein Standard is utilized, following manufacturer instructions (Biorad, cat. no. 500-0006).
b. Procedure
Duplicate tubes are prepared, one including the membrane, and one as a control "blank". Each tube contains 800 μL· Binding Buffer. Thereafter, 10 μL· of Bradford Protein Standard (Img/mL) is added to each tube, and 10 μL· of membrane Protein is then added to just one tube (not the blank). Thereafter, 200 μL· of Bradford Dye Reagent is added to each tube, followed by vortexing of each tube. After five (5) minutes, the tubes are re-vortexed and the material therein is transferred to cuvettes. The cuvettes are read using a CECIL 3041 spectrophotometer, at wavelength 595.
Identification Assay
a. Materials
GDP Buffer consists of 37.5 mL Binding Buffer and 2 mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 μΜ GDP (final concentration of GDP in each well is 0.1 μΜ GDP); each well comprising a candidate compound has a final volume of 200 consisting of 100 μΕ GDP Buffer (final concentration, Ο.ΙμΜ GDP), 50 μΕ Membrane Protein in Binding Buffer, and 50 μΕ [35S]GTPyS (0.6 nM) in Binding Buffer (2.5 μΕ [35S]GTPyS per 10 mL Binding Buffer).
b. Procedure
Candidate compounds can be screened using a 96-well plate format (these can be frozen at -80°C). Membrane Protein (or membranes with expression vector excluding the Target GPCR, as control), are homogenized briefly until in suspension. Protein concentration is be determined using the Bradford Protein Assay set forth above. Membrane Protein (and control) is diluted to
0.25mg/mL in Binding Buffer (final assay concentration, 12^g/well). Thereafter, 100 μL· GDP Buffer is added to each well of a Wallac Scintistrip™ (Wallac). A 5 μΐ^ pin-tool is used to transfer 5 μL· of a candidate compound into such well (i.e., 5 μL· in total assay volume of 200 μL· is a 1:40 ratio such that the final screening concentration of the candidate compound is 10μΜ). Again, to avoid contamination, after each transfer step the pin tool should be rinsed in three reservoirs comprising water (IX), ethanol (IX) and water (2X) - excess liquid should be shaken from the tool after each rinse and dried with paper and kimwipes. Thereafter, 50 μL· of Membrane Protein is added to each well (a control well comprising membranes without the Target GPCR is also utilized), and pre-incubated for 5-10 minutes at room temperature. Thereafter, 50 μΐ^ of [35S]GTPyS (0.6 nM) in Binding Buffer is added to each well, followed by incubation on a shaker for 60 minutes at room temperature (plates are covered with foil). The assay is then stopped by spinning of the plates at 4000 RPM for 15 minutes at 22°C. The plates are aspirated with an 8 channel manifold and sealed with plate covers. The plates are read on a Wallac 1450 using setting "Prot. #37" (as per manufacturer's instructions).
Cyclic AMP Assay
Another assay approach for identifying candidate compounds as, e.g., agonists, inverse agonist, or antagonists, can accomplished by utilizing a cyclase-based assay. In addition to direct identification, this assay approach can be utilized as an independent approach to provide confirmation of the results from the [35S]GTPyS approach as set forth in the above example.
A modified Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.
SMP004A) can be utilized for direct identification of candidate compounds as inverse agonists and agonists to a receptor of interest in accordance with the following protocol.
Transfected cells are harvested approximately three days after transfection. Membranes are prepared by homogenization of suspended cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl2. Homogenization is performed on ice using a Brinkman Polytron™ for approximately 10 seconds. The resulting homogenate is centrifuged at 49,000 X g for 15 minutes at 4°C. The resulting pellet is then resuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by centrifugation at 49,000 x g for 15 minutes at 4°C. The resulting pellet is then stored at -80°C until utilized. On the day of direct identification screening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCl2, to yield a final protein concentration of 0.60 mg/mL (the resuspended membranes are placed on ice until use).
cAMP standards and Detection Buffer (comprising 2 μθ of tracer [125I]cAMP (100 μΕ) to 11 mL Detection Buffer] are prepared and maintained in accordance with the manufacturer' s instructions. Assay Buffer is prepared fresh for screening and contains 20mM HEPES, pH 7.4, 10 mM MgCl2, 20mM phospocreatine (Sigma), 0.1 units/mL creatine phosphokinase (Sigma), 50 μΜ GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer is then stored on ice until utilized.
Candidate compounds are added to, for example, 96-well plate wells (3 μΕ/well; 12μΜ final assay concentration), together with 40 μΕ Membrane Protein (30μg/well) and 50 μΕ of Assay Buffer. This admixture is then incubated for 30 minutes at room temperature, with gentle shaking.
Following the incubation, 100 μΕ of Detection Buffer is added to each well, followed by incubation for 2-24 hours. Plates are then counted in a Wallac MicroBeta™ plate reader using "Prot. #31" (as per manufacturer's instructions). Fluorometric Imaging Plate Reader (FLIPR) Assay for the Measurement of Intracellular Calcium Concentration
Target Receptor (experimental) and pCMV (negative control) stably transfected cells from respective clonal lines are seeded into poly-D-lysine pretreated 96-well plates (Becton-Dickinson, #356640) at 5.5xl04 cells/well with complete culture medium (DMEM with 10% FBS, 2 mM L- glutamine, lmM sodium pyruvate) for assay the next day. A promiscuous G protein such as Gal 5, Gal6, or the chimeric Gq/Gi alpha subunit is available to cause a detectable calcium flux. To prepare Fluo4-AM (Molecular Probe, #F14202) incubation buffer stock, 1 mg Fluo4-AM is dissolved in 467 μΐ DMSO and 467 μΐ Pluoronic acid (Molecular Probe, #P3000) to give a lmM stock solution that can be stored at -20°C for a month. Fluo4-AM is a fluorescent calcium indicator dye.
Candidate compounds are prepared in wash buffer (IX HBSS/2.5mM Probenicid/20mM HEPES at pH 7.4).
At the time of assay, culture medium is removed from the wells and the cells are loaded with ΙΟΟμΙ of 4μΜ Fluo4-AM/2.5 mM Probenicid (Sigma, #P8761)/20mM HEPES/complete medium at pH 7.4. Incubation at 37°C/5% C02 is allowed to proceed for 60 minutes.
After the 1 hour incubation, the Fluo4-AM incubation buffer is removed and the cells are washed 2X with 100 μΐ wash buffer. In each well is left 100 μΐ wash buffer. The plate is returned to the incubator at 37°C/5% C02 for 60 minutes.
FLIPR (Fluorometric Imaging Plate Reader; Molecular Device) is programmed to add 50 μΐ candidate compound on the 30th second and to record transient changes in intracellular calcium concentration ([Ca2+]) evoked by the candidate compound for another 150 seconds. Total fluorescence change counts are used to determine agonist activity using the FLIPR software. The instrument software normalizes the fluorescent reading to give equivalent initial readings at zero.
Although the foregoing provides a FLIPR assay for agonist activity using stably transfected cells, a person of ordinary skill in the art would readily be able to modify the assay in order to characterize antagonist activity. The person of ordinary skill in the art would also readily appreciate that, alternatively, transiently transfected cells could be used.
MAP Kinase Assay
MAP kinase (mitogen activated kinase) can be monitored to evaluate receptor activation.
MAP kinase can be detected by several approaches. One approach is based on an evaluation of the phosphorylation state, either unphosphorylated (inactive) or phosphorylated (active). The phosphorylated protein has a slower mobility in SDS-PAGE and can therefore be compared with the unstimulated protein using Western blotting. Alternatively, antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase. In either method, cells are stimulated with the candidate compound and then extracted with Laemmli buffer. The soluble fraction is applied to an SDS- PAGE gel and proteins are transferred electrophoretically to nitrocellulose or Immobilin.
Immunoreactive bands are detected by standard Western blotting technique. Visible or chemiluminescent signals are recorded on film and can be quantified by densitometry.
Another approach is based on evalulation of the MAP kinase activity via a phosphorylation assay. Cells are stimulated with the candidate compound and a soluble extract is prepared. The extract is incubated at 30°C for 10 minutes with gamma-32P-ATP, an ATP regenerating system, and a specific substrate for MAP kinase such as phosphorylated heat and acid stable protein regulated by insulin, or PHAS-I. The reaction is terminated by the addition of H3P04 and samples are transferred to ice. An aliquot is spotted onto Whatman P81 chromatography paper, which retains the phosphorylated protein. The chromatography paper is washed and counted for 32P is a liquid scintillation counter. Alternatively, the cell extract is incubated with gamma-32P-ATP, an ATP regenerating system, and biotinylated myelin basic proein bound by streptavidin to a filter support. The myelin basic protein is a substrate for activated MAP kinase. The phosphorylation reaction is carried out for 10 minutes at 30°C. The extract can then be aspirated through the filter, which retains, the phosphorylated myelin basic protein. The filter is washed and counted for 32P by liquid scintillation counting.
f. Assays and Model Systems Relating to Itch
One of skill in the art will recognize the usefulness and applicability of a pruritus model that displays at least one element or aspect of pruritus and are amenable to the evaluation of anti-pruritic agents.
In one embodiment, the pruritus model is a behavioral model.
In one embodiment, the pruritus model is a mechanistic or molecular model.
In one embodiment, the pruritus model is a vertebrate model. In another embodiment, the pruritus model is a mammalian model. In another embodiment, the pruritus model is a primate model, a dog model, a guinea pig model, a mouse model, or a cat model.
In that respect, animal models may be particularly useful in analyzing potential anti-pruritic agents. For example, animal pruritus models can be used to determine the in vivo efficacy of a H3R antagonist. In one embodiment, the pruritus model is behaviorally based and evaluation of such a model comprises evaluating the behavior of the organism that has been administered a test compound.
In some embodiments, the pruritus model is selected from the group: pruritogen injection model, passive cutaneous anaphylaxis model, allergic pruritus model, and spontaneous pruritus model.
In some embodiments, the pruritus model is selected from the group: histamine induced pruritus model, DNP-Ovalbumin pruritus model, and DNFB pruritus model.
In some embodiments, the response to pruritus is a behavioral response. The following systems are described for purposes of illustration and are not intended to be limiting.
Pruritogen injection model
In this model, itch is induced by an intradermal or subcutaneous injection of a pruritogen (i.e. histamine) into the rostral part of the back of mice. Mice are video recorded and the number of scratching of the injection site by a hind leg after pruritogen injection is counted. Several substances in addition to histamine can be injected to induce itch: serotonin, substance P, chloroquine, protease-activated receptor 2 (PAR-2) activating peptide, trypsin. A test compound can be administered orally or by other routes shortly prior to itch induction, and the efficacy of the test compound compared to that of a sham treatment or that of a positive control drug.
Passive cutaneous anaphylaxis model
In this model, itch is triggered not by direct injection of a pruritogen but rather by an anaphylactic reaction upon injection of an antigen to antibody-primed mice. To perform this model, a small amount of IgE antibody directed against a known antigen (i.e., dinitrophenyl-conjugated ovalbumin or DNP-OVA) is injected intradermally into mice to prime the animals. The antigen is later given to induce an allergic reaction, which results in itch. In a typical experiment, 20-100 ng of dinitrophenyl-IgE is injected into the rostral part of the back of the animals, and 24 hours laterlOO ug of antigen (DNP-OVA) is injected intraperitoneally. A test compound can be dosed 30 minutes before antigen injection.
Allergic pruritus model
Allergic pruritus model differs from the passive cutaneous anaphylaxis model in that sensitization to an antigen develops through an active immunization process (i.e. hypersensitivity develops to a known antigen via an endogenously generated immune response). In this model, an antigen DNP-OVA is administered by intraperitoneal injection in mice. A booster immunization may be given a week later. Pruritus is then induced two weeks after the initial immunization by injecting the antigen at the rostral part of the back. Again, a test compound is dosed shortly before itch induction.
The parameters in the allergic model may vary considerably with respect to the choice of antigens, procedures for sensitization, and time of test compound administration. For example, antigen may be toluene-2,4-diisocyanate (TDI), 2,4-niditrochlorobenzene (DNCB), oxazolone, or picryl chloride. Sensitization can be done by injection intradermally, intraperitoneally, subcutaneously or by other routes. An antigen can be given by topical application, by intradermal or subcutaneous injection to induce an allergic reaction. A test compound can be administered before or during the sensitization period or shortly before the allergen challenge.
Spontaneous pruritus model
In this model mice develop pruritus due to ongoing inflammation without the need for pruritogen or antigen injection. Examples of spontaneous models include the NC/Nga mice, interleukin-4 and interleukin-13 transgenic mice, and MRL/lpr mice. NC/Nga mice develop skin lesions and pruritus after 8 weeks of age when kept in conventional vivarium conditions (Takano, N. et al., 2003, Eur. Pharmacol. 471: 223-228). The interleukin-4 mice have been genetically manipulated to express high level of this Th2-bias cytokines in the skin. These mice develop skin lesions and pruritus with clinical features resembling those of human atopic dermatitis (Chan, L.S. et al., 2001, J. Invest. Dermatol. 117:977-83). Similarly, transgenic mice expressing high level of the Th2-bias cytokine IL-13 develop skin inflammation bearing the hallmarks of atopic dermatitis such as skin lesions, epidermal barrier breakdown and intense pruritus (Zheng, T. et al., J. Invest. Dermatol. 2008, Oct 16, online publication). MRL lpr mice have systemic autoimmunity resulting from a mutation in the lymphocyte death promoting Fas gene. Spontaneous scratching and skin lesions develop in MRL/lpr mice at 18 weeks of age (Umeuchi, H et al 2005, Eur J Pharm.
518: 133-139), and are associated with elevated serum IgGl and IgG2a levels.
To evaluate compound efficacy in a spontaneous model, mice that have already shown scratching behavior can be video recorded for one hour (or other length of time) to establish an itch intensity baseline. The test compound can be administered orally or by other routes. Mice can be video recorded to determine the frequency of scratching. The efficacy of a test compound can be compared to a sham treatment or a positive control.
Additional animal models are available. For example, a model for canine atopic dermatitis has been utilized through an environmental house dust mite challenge of high-IgE-producing beagles, mite hypersensitive dogs with atopic dermatitis and normal dogs (Vet Dermatol. 2006 Feb;17(l):24-35). This model utilized high-IgE beagles epicutaneously sensitized to house dust mite (HDM) as a model for cAD. Six high-IgE beagles were environmentally challenged with HDM using various doses and protocols. Similar challenge protocols were used in positive and negative control dogs: three dogs with naturally occurring cAD and positive intradermal skin test (IDT) to HDM and three normal dogs without history of skin disease and negative IDT to HDM. All high-IgE beagles and all atopic dogs developed severe cutaneous lesions and pruritus after challenge. Lesions were erythematous papules and macules in contact areas such as face, ears, ventral abdomen, groin, axillae and feet. They were first visible after 6 h and increased in severity over time. No normal dog developed pruritus or lesions. Biopsies of representative lesions in the high-IgE beagles were taken for histopathology and immunohistochemistry. There was superficial perivascular dermatitis with mononuclear infiltrates and spongiosis. Lymphocytes and eosinophils accumulated in small epidermal micro-abscesses with hyperplasia of epidermal IgE-bearing dendritic cells. These findings suggest that this colony of high-IgE beagles develops a dermatitis that clinically, histopathologically and immunologically resembles the naturally occurring canine disease. It is also concluded that this modality of challenge is not irritating to normal dogs but induces flare-ups in hypersensitive atopic dogs. Further, a cat pruritus model (Acta Vet Scand. 2009 Oct 20;51:40) has been described in which the cats suffer from D. gatoi instigated pruritus.
Further, a guinea pig pruritus model (Exp Dermatol. 2002 Aug;l 1(4):285-91) has been described using iontophoresis of histamine and capsaicin. It was also shown that contact sensitization with 2-4 dinitrochlorobenzene (DNCB) can be used as a simple assay for chronic itch allowing study of scratching over at least a 15-h period.
Further, a primate pruritus model (J Neurosci. 2008 Jul 23;28(30):7659-69) has been described. The existence of two peripheral pathways for itch has been suggested in some species: one pathway that is responsive to histamine and a second pathway that can be activated by nonhistaminergic pruritogens (e.g., cowhage spicules). For example, behavioral responses and neuronal activity in unmyelinated afferent fibers can be assessed in monkey after topical application of cowhage spicules or intradermal injection of histamine and capsaicin.
Further, histamine induced itch can be analyzed in humans by injecting a pruritogen into the skin by iontophoresis. A fresh solution of histamine in saline can be prepared shortly before the experiment. A cotton disk can be soaked with this solution and mounted into the application chamber of an iontophoresis applicator. An anodal current and a charge of 20 mC (1 mA, 20 s) can be used to deliver histamine iontophoretically into the skin. The intensity of itch can be graded by subjects on a 100mm visual analogue scale (VAS) at 20s intervals for 5min after the injection of histamine.
g. Optional Further Selection of Compounds
Compounds may be further selected based on their peripheral restriction (or lack thereof). One aspect of the present invention relates to H3R antagonists that are peripherally restricted. One aspect of the present invention relates to anti-pruritic agents that are peripherally restricted.
Identification of peripherally restricted H3R antagonists can be performed, for example, using a caco-2 permeability assay: The Caco-2 cell line was purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and grown with Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 1% nonessential amino acids at 37°C in 5% C02. Multi-well insert systems with an array of 12 or 24 individual inserts were used in the permeability assay. Each multi-well insert unit has two compartments: the top compartment and the bottom compartment. The top compartment is the insert and is commonly referred to as the apical compartment or apical side (A). The bottom compartment is the well of a multi-well plate where the insert is placed and is referred to as the basal compartment or basal side (B). In each insert, Caco-2 cells were seeded on a porous filter membrane. Cell culture medium was changed every two days. Cells were used for the transport experiments between 24 to 26 days after seeding.
The bidirectional transport of H3R compounds (final concentration: 10 μΜ) and the known P-gp substrate 3H-digoxin (-15 nM) across Caco-2 cell monolayers was determined in the absence and presence of the known P-gp inhibitor cyclosporin A (5 μΜ). All samples from the permeability experiments of H3R compounds were mixed with acetonitrile containing internal standard. After protein precipitation by centnfugation, supernatant was analyzed using LC/MS MS. Samples from 3H-digoxin transport experiment were mixed with liquid scintillation counting cocktail and counted with a liquid scintillation counter.
The apparent permeability coefficients (Papp) were calculated using the following equation:
Figure imgf000081_0001
where AQ/At is the appearance rate (DPM/sec of radiolabeled test compound; umol/sec of non-labeled test compound) on the receiver side during the permeation process, A is the surface area of the cell monolayers, and Co is the initial concentration (DPM/mL of radiolabeled test compound; umol/mL of non-labeled test compound) on the donor side.
Efflux ratio was defined as the ratio of Papp in the basolateral to apical direction over the Papp in the apical to basolateral direction.
One of skill in the art would understand and be capable of performing techniques for determining the Brain vs Plasma concentrations (or ratio) for administered compounds. For example this may be calculated based on standard curve concentration. After detection by LC/MS/MS, the final brain concentrations can be calculated. Plasma sample concentrations are calculated based on standard curve concentration. Brain/Plasma ratio can be calculated with the final brain and plasma data of each animal.
In one embodiment, compounds are further selected based on desirable pharmacokinetic properties. For example, in one preferred embodiment, the compound would exhibit a Ki <10 nM across species.
In another embodiment, the compound exhibits selectivity across a broad receptor panel. In another embodiment, the compound does not exhibit CYP or hERG liability.
In another embodiment, the compound is efficacious in a histamine-mediated itch model. In another embodiment, the compound is efficacious in a dermatitis-driven itch model. In another embodiment, the compound has at least 10X potency separation between wake promotion and itch models.
In one embodiment, the compound or agent is restricted in some manner from crossing the blood brain barrier. In another embodiment, the compound or agent may be less than 100% restricted to the periphery. In one embodiment, the compound or agent has a braimplasma ratio (B/P) which will be less than 1 in at least one species.
In another embodiment, the H3R antagonist or anti -pruritic agent has a brain to plasma ratio of about 0.5 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.4 or less. In another embodiment, the H3R antagonist or antipruritic agent has a brain to plasma ratio of about 0.3 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.15 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.1 or less. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.01 or less.
In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.5 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.4 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti- pruritic agent has a brain to plasma ratio of about 0.3 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.2 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.15 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti- pruritic agent has a brain to plasma ratio of about 0.1 or less after administration in a pruritus model animal. In another embodiment, the H3R antagonist or anti-pruritic agent has a brain to plasma ratio of about 0.01 or less after administration in a pruritus model animal.
In another embodiment, the compound has good oral bioavailability.
In another embodiment, the compound shows Tl/2>lh in microsomal stability across species.
In another embodiment, the compound shows in vivo Tl/2 in animal species consistent with a QD or BID regimen.
In another embodiment, the compound shows hERG > 10 uM.
In another embodiment, the compound is Ames negative.
In another embodiment, the compound has acceptable tolerability in rat repeat dosing.
Composition/Formulation and Methods of Treatment
A H3R antagonist or anti-pruritic agent, such as an antagonist or an inverse agonist, can be formulated into pharmaceutical compositions and medicaments for use in accordance with the present invention using techniques well known in the art. One aspect of the present invention relates to compositions comprising a H3R antagonist for use in treating pruritus/itch. Proper formulation is dependent on the route of administration chosen. In certain embodiments, the administration is to a non-human vertebrate or to a non-human mammal.
Once the anti-pruritic agent H3R antagonist has been identified using any of the methods described herein, the anti-pruritic agent/H3R antagonist is subsequently admixed with a pharmaceutical carrier. Pharmaceutical carriers are known in the art, such as pharmaceutical carriers described herein.
In some embodiments, the H3R antagonist is formulated as a composition. Γη some embodiments, the H3R antagonist is a H3R inverse agonist.
In some embodiments, the anti-pruritic agent is subsequently admixed with a
pharmaceutical carrier.
The formulated compounds or agents can exhibit all the features previously described for compounds in relation to the screening assays or otherwise described in this application. For example, compounds for formulation, method of treatment or any of the other embodiments described herein may, for example, demonstrate the selectivity, B/P ratios, or potency as previously or subsequently described.
One aspect of the present invention relates to methods of preparing a pharmaceutical composition comprising a H3R antagonist for treating a condition characterized by itch, a method comprising:
(a) determining a reduction of itch in a mammal, the mammal having been administered with the a H3R antagonist, wherein the ability of the H3R antagonist to reduce itch in the mammal is indicative of the H3R antagonist being useful for treating a condition characterized by itch; and
(b) admixing the H3R antagonist with a pharmaceutically acceptable carrier. In some embodiments, the H3R antagonist is an antagonist of the human H3R.
In some embodiments, the H3R antagonist is orally active.
In some embodiments, the H3R antagonist is a selective H3R antagonist.
In some embodiments, the H3R antagonist has a selectivity for H3R over H1R, H2R, or
H4R of at least about 10 to fold.
In some embodiments, the H3R antagonist has an IC50 of less than about 1 μΜ.
In some embodiments, the H3R antagonist has an IC50 of less than about 100 nM.
In some embodiments, the H3R antagonist has an IC50 of less than about 1 nM.
In some embodiments, the H3R antagonist is a small molecule.
In some embodiments, the mammal is a non-human mammal.
In some embodiments, the mammal is selected from the group consisting of a mouse, a rat, a dog and a non-human primate.
In some embodiments, the mammal is a human.
In some embodiments, the pharmaceutical composition is in a dosage form.
In some embodiments, the H3R antagonist has an IC50 of less than 1 μΜ.
In some embodiments, the H3R antagonist has an IC50 of less than about 10 nM.
In some embodiments, the H3R antagonist has a selectivity for H3R over a H1R, H2R, or H4R of at least about 10 to fold.
In some embodiments, the dosage form is in combination with a H1R antagonist.
In one embodiment, the present invention provides a method of treating pruritus in a subject in need thereof by administering a therapeutically effective amount of a H3R antagonist. Γη one embodiment, the H3R antagonist is in an amount sufficient to reduce itch in an individual.
In some embodiments, the H3R antagonist is selected from and the following compounds and pharmaceutically acceptable salts, solvates, and hydrates thereof:
(R)- 1 - { 2-[4'-(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl-pyrrolidine;
(R)-2-hydroxy- 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin- 2(l//)-yl)ethanone;
6-[(3-cyclobutyl-2,3,4,5-tetrahydro-l/i-3-benzazepin-7-yl)oxy]-Ai-methyl-3- pyridinecarboxamide; and
4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine.
In one embodiment, the invention comprises conjugates of antibodies, e.g. Fv, Fab, and F(ab)'2, bifunctional hybrid antibodies and single chain antibodies that are known to one skilled it the art.
In one embodiment, the method of treating pruritus in a subject in need thereof involves administering a therapeutically effective amount of a H3R antagonist in conjunction with a therapeutically effective amount of another compound known to facilitate the treatment of pruritus.
In another embodiment, the methods disclosed herein can be used in conjunction with other known anti-pruritus therapies such as menthol and phenol, calamine, topical
antihistamines, local anesthetics, capsaicin, strontium nitrate, HI -receptor antagonists, H2- receptor antagonists, doxepin, ondansetron, paroxetine, mirtazapine, opioid antagonists, dry skin: emollient cream, cholestasis: cole sty ramine, rifampicin, opioid antagonists, androgens, for uremia: dialysis, UVB phototherapy and for paraneoplasia: paroxetin.
In another embodiment, the methods disclosed herein can be used in conjunction with therapies for eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound healing, itch associated with an insect bite, itch associated with a flea bite, itch associated with an insect sting, itch associated with a mosquito sting, itch associated with a mite bite, urticaria, urticaria caused by a plant, urticaria caused by poison ivy, urticaria caused by stinging nettle, sweat gland abnormalities, bullous pemphigoid, photodermatoses, skin blisters, adult acne, chicken pox, and dermatitis herpetiformis. In accordance with the present invention, the H3R antagonists may be applied topically to the site afflicted with itch in therapeutically effective amount in admixture with
pharmaceutical carriers, in the form of topical pharmaceutical compositions. Such compositions include solutions, suspensions, lotions, gels, creams, ointments, emulsions, skin patches, etc. All of these dosage forms, along with methods for their preparation, are well known in the pharmaceutical and cosmetic art: Harry's Cosmeticology (Chemical Publishing, 7th ed. 1982); Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th ed. 1990). Typically, such topical formulations contain the active ingredient in a concentration range of 0.001 to 10 mg/mL, in admixture with suitable vehicles. Other desirable ingredients for use in such anti- pruritic preparations include preservatives, co-solvents, viscosity building agents, carriers, etc. The carrier itself or a component dissolved in the carrier may have palliative or therapeutic properties of its own, including moisturizing, cleansing, or anti-inflammatory/anti-itching properties. The H3R antagonists can be combined with a therapeutically effective amounts of anti-inflammation agents such as corticosteroids, fungicides, antibiotics, moisturizers or anti- itching compounds.
Penetration enhancers may, for example, be surface active agents; certain organic solvents, such as dimethyl sulfoxide and other sulfoxides, dimethyl-acetamide and pyrrolidone; certain amides of heterocyclic amines, glycols (e.g. propylene glycol); propylene carbonate; oleic acid; alkyl amines and derivatives; various cationic, anionic, nonionic, and amphoteric surface active agents; and the like.
Topical administration of a pharmacologically effective amount may utilize transdermal delivery systems well known in the art.
In addition to topical therapy, the H3R antagonists may also be administered systemically, such as oral, parenteral, nasal inhalation, and intrarectal are also contemplated. For these uses, additional conventional pharmaceutical preparations such as tablets, granules, powders, capsules, and sprays may be preferentially required. In such formulations further conventional additives such as binding-agents, wetting agents, propellants, lubricants, and stabilizers may also be required.
Systemic administration preferably comprises ingestion of any solid or solution carriers containing a pharmacologically effective amount of one or more of the H3R antagonists. Such solid or solution carriers may comprise pills, hard tablets, soft tablets, gums or ordinary liquids. Additionally, systemic administration of a pharmacologically effective amount may comprise invasive methodologies including intravenous, subcutaneous, intramuscular or intralesional injection of a suitable carrier, such as saline, containing a pharmacologically effective amount of one or more of the H3R antagonists.
The route of administration, dosage form, and the effective amount vary according to the potency of the selected H3R antagonist, its physicochemical characteristics, and according to the location of itch sensations. The selection of proper dosage is well within the skill of an ordinary skilled physician. Topical formulations are usually administered up to four-times a day.
The use of H3R antagonists as anti -pruritics may be combined with, for example, Hl- antihistamines to provide superior therapy via additive or synergistic interaction.
The itch may be associated with a disease or disorder related from the group consisting of eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, lichen planus, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with haemodialysis, burns, scalds, sunburn, insect bites, urticaria, sweat gland abnormalities, bullous pemphigoid, photodermatoses, skin blisters, adult acne, chicken pox, and dermatitis herpetiformis.
H3R antagonists of the present invention can be administered in any suitable way. Suitable routes of administration include oral, nasal, rectal, transmucosal, transdermal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocular injections using methods known in the art. Other suitable routes of administration are aerosol and depot formulation. Sustained release formulations, particularly depot, of the invented medicaments are expressly contemplated. In certain preferred embodiments, the compounds according to the present invention are administered orally. The compounds according to the present invention can be made up in solid or liquid form, such as tablets, capsules, powders, syrups, elixirs and the like, aerosols, sterile solutions, suspensions or emulsions, and the like. In certain embodiments, the H3R antagonist is administered orally.
Formulations for oral administration may be in the form of aqueous solutions and suspensions, in addition to solid tablet and capsule formulations. The aqueous solutions and suspensions may be prepared from sterile powders or granules. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. Other adjuvants are well and widely known in the art.
Pharmaceutical compositions of the H3R antagonist may be prepared by methods well known in the art, e.g., by means of conventional mixing, dissolving, granulation, dragee-making, levigating, emulsifying, encapsulating, entrapping, lyophilizing processes or spray drying.
Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable pharmaceutically acceptable carriers are available to those in the art (see, e.g., Remington: The Science and Practice of Pharmacy, (Gennaro et al., eds.), 20th Edition, 2000, Lippincott Williams & Wilkins; and Handbook of Pharmaceutical Excipients (Rowe et al., eds), 4th Edition, 2003, Pharmaceutical Press). Proper formulation is dependent upon the route of administration chosen. The term "carrier" material or "excipient" material herein means any substance, not itself a therapeutic agent, used as a carrier and/or dilutent and/or adjuvant, or vehicle for delivery of a therapeutic agent to a subject or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition into a discrete article such as a capsule or tablet suitable for oral administration. Excipients can include, by way of illustration and not limitation, diluents, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or counteract a disagreeable taste or odor, flavors, dyes, fragrances, and substances added to improved appearance of the composition. Acceptable excipients include stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc, gelatin, acacia gum, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starches, gelatin, cellulosic materials, such as cellulose esters of alkanoic acids and cellulose alkyl esters, low melting wax cocoa butter or powder, polymers, such as polyvinylpyrrolidone, polyvinyl alcohol, and polytheylene glycols, and other pharmaceutically acceptable materials. The components of the pharmaceutical composition can be encapsulated or tableted for convenient administration.
One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist.
One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a
H3R antagonist; or a pharmaceutical composition as described herein.
One aspect of the present invention relates to methods for treating pruritus in an individual comprising administering to the individual in need thereof a therapeutically effective amount of a H3R antagonist, wherein the H3R antagonist is identified by using any of the methods described herein.
In some embodiments, the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
One aspect of the present invention relates to uses of a H3R antagonist in the manufacture of a medicament for treating pruritus.
One aspect of the present invention relates to uses of a H3R antagonist in the manufacture of a medicament for treating pruritus, wherein the H3R antagonist is identified by using any of the methods described herein.
One aspect of the present invention relates to H3R antagonists for use in a method for the treatment of pruritus. One aspect of the present invention relates to H3R antagonists for use in a method for the treatment of pruritus, wherein the H3R antagonist is identified by using any of the methods described herein.
In some embodiments, the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
One aspect of the present invention relates to H3R antagonists for use in a method for treating pruritus.
In some embodiments, the H3R antagonist is administered in the form of a pharmaceutical composition comprising a therapeutically effective amount of said H3R antagonist as active ingredient, in admixture with a pharmaceutical carrier.
In some embodiments, the H3R antagonist is applied topically.
In some embodiments, the H3R antagonist is applied topically to the site afflicted with itch.
In some embodiments, the H3R antagonist is applied topically and has an IC50 (H3R) of about 100 nM or less.
In some embodiments, the pharmaceutical composition is formulated in a form suitable for topical application.
In some embodiments, the H3R antagonist is administered systemically.
In some embodiments, the method further comprises administering a therapeutic amount of a H1R antagonist.
In some embodiments, the antagonist is administered orally.
In some embodiments, the H3R antagonist is peripherally restricted.
Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum Arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycols, cremophor, capmul, medium or long chain mono-, di- or triglycerides. Stabilizers may be added in these formulations, also.
Additionally, a H3R antagonist may be delivered using a sustained-release system. Various sustained-release materials have been established and are well known to those skilled in the art. Sustained-release tablets or capsules are particularly preferred. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. The dosage form may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108, 4, 166,452, and 4,265,874 to form osmotic therapeutic tablets for controlled release.
It is expressly contemplated that therapies of the present invention, namely therapies relating to a H3R antagonist, may be administered or provided alone or in combination with one or more other pharmaceutically or physiologically acceptable compound. In one aspect of the present invention, the other pharmaceutically or physiologically acceptable compound (i.e., second pharmaceutical agent) is not a H3R antagonist.
In one aspect, the present invention features a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention. In one aspect, the present invention features a pharmaceutical composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention and at least one pharmaceutically acceptable carrier.
In one embodiment, the present invention relates to a composition comprising a compound identified according to one or more of the screening methods described herein.
In one aspect, the present invention features a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention. In one aspect, the present invention features a pharmaceutical composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention and at least one pharmaceutically acceptable carrier. The present invention also relates to a dosage form of the composition or of the pharmaceutical composition wherein the H3R antagonist is in an amount sufficient to give an effect in treating a condition characterized by itch.
In one embodiment, the H3R antagonist is in an amount sufficient to reduce itch in an individual.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an amount to achieve their intended purpose. In some embodiments, a pharmaceutical composition of the present invention is understood to be useful for treating a condition characterized by itch, such as pruritus. As relates to the present invention, determination of the amount of a H3R antagonist sufficient to achieve an intended purpose according to the invention is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. The data obtained from animal studies, including but not limited to studies using mice, rats, rabbits, pigs, and non-human primates, can be used in formulating a range of dosage for use in humans. In general, one skilled in the art understands how to extrapolate in vivo data obtained in an animal model system to another, such as a human. In some circumstances, these extrapolations may merely be based on the weight of the animal model in comparison to another, such as a human; in other circumstances, these extrapolations are not simply based on weights but rather incorporate a variety of factors. Representative factors include the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized, on whether an acute or chronic disease state is being treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds of the present invention and as part of a drug combination. The dosage regimen for treating a disease condition with the compounds and/or compositions of this invention is selected in accordance with a variety factors as cited above. Thus, the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods of this invention.
An exemplary animal model system is mouse.
Dosage amount and interval may be adjusted in order to provide an intended therapeutic effect. It will be appreciated that the exact dosage of a H3R antagonist in accordance with the present invention will vary depending on the H3R antagonist, its potency, the mode of administration, the age and weight of the patient and the severity of the condition to be treated. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. By way of illustration and not limitation, an amount of a H3R antagonist in accordance with the present invention is less than about 0.001 mg/kg body weight, less than about 0.005 mg kg body weight, less than about 0.01 mg/kg body weight, less than about 0.05 mg/kg body weight, less than about 0.1 mg/kg body weight, less than about 0.5 mg/kg body weight, less than about 1 mg/kg body weight, less than about 5 mg/kg body weight, less than about 10 mg/kg body weight, less than about 50 mg/kg body weight, or less than about 100 mg/kg body weight. In certain embodiments, an amount of a H3R antagonist in accordance with the present invention is less than about 0.001-100 mg/kg body weight, less than about 0.001-50 mg/kg body weight, less than about 0.001-10 mg/kg body weight, less than about 0.001-5 mg/kg body weight, less than about 0.001-1 mg/kg body weight, less than about 0.001 to 0.5 mg/kg body weight, less than about 0.001-0.1 mg/kg body weight, less than about 0.001-0.05 mg/kg body weight, less than about 0.001-0.01 mg/kg body weight, or less than about 0.001-0.005 mg/kg body weight. Dosage amount and interval may be adjusted individually to provide plasma levels of a H3R antagonist according to the present invention which achieve an intended therapeutic effect. Dosage intervals can also be determined using the value for a selected range of H3R antagonist concentration so as to achieve the intended therapeutic effect. A H3R antagonist should be administered using a regimen that maintains plasma levels within the selected range of a H3R antagonist concentration for 10-90% of the time, preferably between 30-99% of the time, and most preferably between 50-90% of the time. In cases of local administration or selective uptake, the range of H3R antagonist concentration providing the intended therapeutic effect may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the individual being treated, on the individual's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
In one aspect, the present invention accordingly features a method of treating a condition characterized by itch, such as pruritus, comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising or consisting essentially of an amount of a H3R antagonist according to the present invention. In certain embodiments, the composition is a pharmaceutical composition.
Compounds of the present invention or a solvate, hydrate or physiologically functional derivative thereof can be used as active ingredients in pharmaceutical compositions, specifically as H3R receptor antagonists. By the term "active ingredient" is defined in the context of a
"pharmaceutical composition" and is intended to mean a component of a pharmaceutical composition that provides the primary pharmacological effect, as opposed to an "inactive ingredient" which would generally be recognized as providing no pharmaceutical benefit.
The amount of active ingredient, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of
administration, the nature of the condition being treated and the age and condition of the patient and will ultimately be at the discretion of the attendant physician or clinician. In general, one skilled in the art understands how to extrapolate in vivo data obtained in a model system, typically an animal model, to another, such as a human. In some circumstances, these extrapolations may merely be based on the weight of the animal model in comparison to another, such as a mammal, preferably a human, however, more often, these extrapolations are not simply based on weights, but rather incorporate a variety of factors. Representative factors include the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized, on whether an acute or chronic disease state is being treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds of the present invention and as part of a drug combination. The dosage regimen for treating a disease condition with the compounds and/or compositions of this invention is selected in accordance with a variety factors as cited above. Thus, the actual dosage regimen employed may vary widely and therefore may deviate from a preferred dosage regimen and one skilled in the art will recognize that dosage and dosage regimen outside these typical ranges can be tested and, where appropriate, may be used in the methods of this invention.
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. The daily dose can be divided, especially when relatively large amounts are administered as deemed appropriate, into several, for example 2, 3 or 4 part administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the daily dose indicated.
The compounds of the present invention can be administrated in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt, solvate or hydrate of a compound of the invention.
For preparing pharmaceutical compositions from the compounds of the present invention, the selection of a suitable pharmaceutically acceptable carrier can be either solid, liquid or a mixture of both. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.
In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted to the desire shape and size.
The powders and tablets may contain varying percentage amounts of the active compound. A representative amount in a powder or tablet may contain from 0.5 to about 90 percent of the active compound; however, an artisan would know when amounts outside of this range are necessary. Suitable carriers for powders and tablets are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose, a low melting wax, cocoa butter and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as an admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool and thereby to solidify.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Liquid form preparations include solutions, suspensions and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injec tables.
The compounds according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
Aqueous formulations suitable for oral use can be prepared by dissolving or suspending the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired.
Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.
For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch.
Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multi-dose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump.
Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant. If the compounds of the present invention or pharmaceutical compositions comprising them are administered as aerosols, for example as nasal aerosols or by inhalation, this can be carried out, for example, using a spray, a nebulizer, a pump nebulizer, an inhalation apparatus, a metered inhaler or a dry powder inhaler. Pharmaceutical forms for administration of the compounds of the present invention as an aerosol can be prepared by processes well known to the person skilled in the art. For their preparation, for example, solutions or dispersions of the compounds of the present invention in water, water/alcohol mixtures or suitable saline solutions can be employed using customary additives, for example benzyl alcohol or other suitable preservatives, absorption enhancers for increasing the bioavailability, solubilizers, dispersants and others and, if appropriate, customary propellants, for example include carbon dioxide, CFCs, such as,
dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane; and the like. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.
In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. When desired, formulations adapted to give sustained release of the active ingredient may be employed.
Alternatively the active ingredients may be provided in the form of a dry powder, for example, a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
Tablets or capsules for oral administration and liquids for intravenous administration are preferred compositions.
The compounds according to the invention may optionally exist as pharmaceutically acceptable salts including pharmaceutically acceptable acid addition salts prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Representative acids include, but are not limited to, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, dichloroacetic, formic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like. Certain compounds of the present invention which contain a carboxylic acid functional group may optionally exist as pharmaceutically acceptable salts containing non-toxic, pharmaceutically acceptable metal cations and cations derived from organic bases. Representative metals include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and the like. In some embodiments the pharmaceutically acceptable metal is sodium. Representative organic bases include, but are not limited to, benzathine ( N2-dibenzylemane-l,2-diamine), chloroprocaine (2- (diethylamino)ethyl 4-(chloroamino)benzoate), choline, diethanolamine, ethylenediamine, meglumine ((2R,3R,4R,55)-6-(methylamino)hexane-l,2,3,4,5-pentaol), procaine (2- (diethylamino)ethyl 4-aminobenzoate), and the like. Certain pharmaceutically acceptable salts are listed in Berge, et al., Journal of Pharmaceutical Sciences, 66: 1-19 (1977).
The acid addition salts may be obtained as the direct products of compound synthesis. In the alternative, the free base may be dissolved in a suitable solvent containing the appropriate acid and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent. The compounds of this invention may form solvates with standard low molecular weight solvents using methods known to the skilled artisan.
Compounds of the present invention can be converted to "pro-drugs." The term "prodrugs" refers to compounds that have been modified with specific chemical groups known in the art and when administered into an individual these groups undergo biotransformation to give the parent compound. Pro-drugs can thus be viewed as compounds of the invention containing one or more specialized non-toxic protective groups used in a transient manner to alter or to eliminate a property of the compound. In one general aspect, the "pro-drug" approach is utilized to facilitate oral absorption. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems Vol. 14 of the A.C.S. Symposium Series; and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
Some embodiments of the present invention include a method of producing a
pharmaceutical composition for "combination-therapy" comprising admixing at least one compound according to any of the compound embodiments disclosed herein, together with at least one known pharmaceutical agent as described herein and a pharmaceutically acceptable carrier.
H3R antagonists can be co-administered with other therapeutic agents to meet the treatment objectives. For example, a H3R antagonist may be combined with antagonists of other histamine receptor subtypes, particularly H1R and H4R antagonists. H1R antagonists may include both sedation and non-sedation antihistamines such as diphenhydramine, cetirizine, hydroxyzine, chlorpheniramine, promethazine, fexofenadine, loratadine, and desloratadine.
In one embodiment, the composition further comprises an H1R antagonist in an amount sufficient to reduce itch in an individual.
H4R antagonists to be combined may include compounds reported in the literature. In addition, it may also be desirable to co-administer a H3R antagonist with therapeutic agents currently used to treat itch in patients with chronic metabolic diseases. These drugs may include the bile resin cholestyramine and the antibiotic rifampicin, or opiate receptor antagonists naloxone, nalmefene and naltrexone. It may also be desirable to combine a H3R antagonist with existing treatments of atopic dermatitis particularly glucocorticoids and immunomodulators pimecrulimus and tacrulimus.
It is noted that when the H3R receptor modulators are utilized as active ingredients in a pharmaceutical composition, these are not intended for use only in humans, but in other non-human mammals as well. Indeed, recent advances in the area of animal health-care mandate that consideration be given for the use of active agents, such as H3R receptor modulators, for the treatment of a H3R-associated disease or disorder in companionship animals (e.g., cats, dogs, etc.) and in livestock animals (e.g., cows, chickens, fish, etc.) Those of ordinary skill in the art are readily credited with understanding the utility of such compounds in such settings.
Hydrates and Solvates It is understood that when the phrase "pharmaceutically acceptable salts, solvates, and hydrates" or the phrase "pharmaceutically acceptable salt, solvate, or hydrate" is used when referring to compounds described herein, it embraces pharmaceutically acceptable solvates and/or hydrates of the compounds, pharmaceutically acceptable salts of the compounds, as well as pharmaceutically acceptable solvates and/or hydrates of pharmaceutically acceptable salts of the compounds. It is also understood that when the phrase "pharmaceutically acceptable solvates and hydrates" or the phrase "pharmaceutically acceptable solvate or hydrate" is used when referring to salts described herein, it embraces pharmaceutically acceptable solvates and/or hydrates of such salts.
It will be apparent to those skilled in the art that the dosage forms described herein may comprise, as the active component, either a compound described herein or a pharmaceutically acceptable salt or as a pharmaceutically acceptable solvate or hydrate thereof. Moreover, various hydrates and solvates of the compounds described herein and their salts will find use as intermediates in the manufacture of pharmaceutical compositions. Typical procedures for making and identifying suitable hydrates and solvates, outside those mentioned herein, are well known to those in the art; see for example, pages 202-209 of K.J. Guillory, "Generation of Polymorphs, Hydrates, Solvates, and Amorphous Solids," in: Polymorphism in Pharmaceutical Solids, ed. Harry G. Britain, Vol. 95, Marcel Dekker, Inc., New York, 1999. Accordingly, one aspect of the present invention pertains to methods of administering hydrates and solvates of compounds described herein and/or their pharmaceutical acceptable salts, that can be isolated and characterized by methods known in the art, such as, thermogravimetric analysis (TGA), TGA-mass spectroscopy, TGA-Infrared spectroscopy, powder X-ray diffraction (XRPD), Karl Fisher titration, high resolution X-ray diffraction, and the like. There are several commercial entities that provide quick and efficient services for identifying solvates and hydrates on a routine basis. Example companies offering these services include Wilmington PharmaTech (Wilmington, DE), Avantium
Technologies (Amsterdam) and Aptuit (Greenwich, CT).
Isotopes
The present disclosure includes all isotopes of atoms occurring in the present compounds, intermediates, salts and crystalline forms thereof. Isotopes include those atoms having the same atomic number but different mass numbers. One aspect of the present invention includes every combination of one or more atoms in the present compounds, intermediates, salts, and crystalline forms thereof that is replaced with an atom having the same atomic number but a different mass number. One such example is the replacement of an atom that is the most naturally abundant isotope, such as H or 12C, found in one the present compounds, intermediates, salts, and crystalline forms thereof, with a different atom that is not the most naturally abundant isotope, such as 2H or 3H (replacing ¾), or nC, 13C, or 14C (replacing 12C). A compound wherein such a replacement has taken place is commonly referred to as being an isotopically-labeled compound. Isotopic-labeling of the present compounds, intermediates, salts, and crystalline forms thereof can be accomplished using any one of a variety of different synthetic methods know to those of ordinary skill in the art and they are readily credited with understanding the synthetic methods and available reagents needed to conduct such isotopic-labeling. By way of general example, and without limitation, isotopes of hydrogen include 2H (deuterium) and 3H (tritium). Isotopes of carbon include nC, 13C, and 14C. Isotopes of nitrogen include 13N and 15N. Isotopes of oxygen include 150, 170, and 18C. An isotope of fluorine includes 18F. An isotope of sulfur includes 35S. An isotope of chlorine includes 36C1. Isotopes of bromine include 75Br, 76Br, 77Br, and 82Br. Isotopes of iodine include 1231, 1241, 125I, and 131I. Another aspect of the present invention includes compositions, such as, those prepared during synthesis, preformulation, and the like, and pharmaceutical compositions, such as, those prepared with the intent of using in a mammal for the treatment of one or more of the disorders described herein, comprising one or more of the present compounds, intermediates, salts, and crystalline forms thereof, wherein the naturally occurring distribution of the isotopes in the composition is perturbed. Another aspect of the present invention includes compositions and pharmaceutical compositions comprising compounds as described herein wherein the compound is enriched at one or more positions with an isotope other than the most naturally abundant isotope. Methods are readily available to measure such isotope perturbations or enrichments, such as, mass spectrometry, and for isotopes that are radio-isotopes additional methods are available, such as, radio-detectors used in connection with HPLC or GC.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, practice the present invention to its fullest extent. The following detailed examples are to be construed as merely illustrative, and not limitations of the preceding disclosure in any way whatsoever. Those skilled in the art will promptly recognize appropriate variations from the procedures.
EXAMPLES
Example 1: Syntheses of Compounds of the Present Invention.
Compounds of the present invention can be prepared by methods readily know by those skilled in the art, for example, those methods described in WO2008/005338, WO2008/048609, WO2009/058300, WO2009/105206, and like references.
The following examples are provided to further define the invention without, however, limiting the invention to the particulars of these examples. The compounds described herein, supra and infra, are named according to the CS ChemDraw Ultra Version 7.0.1, AutoNom version 2.2, or CS ChemDraw Ultra Version 9.0.7. In certain instances common names are used and it is understood that these common names would be recognized by those skilled in the art.
Chemistry: Proton nuclear magnetic resonance (¾ NMR) spectra were recorded on a Bruker Avance-400 equipped with a QNP (Quad Nucleus Probe) or a BBI (Broad Band Inverse) and z-gradient. Chemical shifts are given in parts per million (ppm) with the residual solvent signal used as reference. NMR abbreviations are used as follows: s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dt = doublet of triplets, t = triplet, td = triplet of doublets, tt = triplet of triplets, q = quartet, m = multiplet, bs = broad singlet, bt = broad triplet. Microwave irradiations were carried out using a Smith Synthesizer™ or an Emrys Optimizer™ (Biotage). Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Merck), preparatory thin-layer chromatography (prep TLC) was preformed on PK6F silica gel 60 A 1 mm plates (Whatman) and column chromatography was carried out on a silica gel column using Kieselgel 60, 0.063-0.200 mm (Merck). Evaporation was done under reduced pressure on a Biichi rotary evaporator.
LCMS spec: HPLC-pumps: LC-10AD VP, Shimadzu Inc.; HPLC system controller: SCL- 10A VP, Shimadzu Inc; UV-Detector: SPD-IOA VP, Shimadzu Inc; Autosampler: CTC HTS, PAL, Leap Scientific; Mass spectrometer: API 150EX with Turbo Ion Spray source, AB MDS Sciex; Software: Analyst 1.2.
Example 1.1: Preparation of Compounds of Formula (la).
H3R antagonist of Formula (la) can be prepared according to procedures described in WO2008/005338, for example: 4'-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4-sulfonic acid 4-chloro-benzylamide can be prepared according to Example 1.9 (Cmpd 24), WO2008/005338; 4'- [2-((R)-2-Methyl-pyrrolidin- 1 -yl)-ethyl] -biphenyl-4-sulfonic acid amide can be prepared according to Example 1.23 (Cmpd 35), WO2008/005338; 4'-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]- biphenyl-4-sulfonic acid ethylamide can be prepared according to Example 1.21 (Cmpd 37), WO2008/005338; Propionic acid l-{4'-[2-((R)-2-methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4- sulfonyl}-piperidin-4-yl ester can be prepared according to Example 2.27 (Cmpd 53),
WO2008/005338; Propionic acid 2-(l-{4'-[2-((R)-2-methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4- sulfonyl}-piperidin-4-yl)-ethyl ester can be prepared according to Example 2.24 (Cmpd 63), WO2008/005338; and Propionic acid l-{4'-[2-((R)-2-methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-4- sulfonyl}-piperidin-4-ylmethyl ester can be prepared according to Example 2.23 (Cmpd 68), WO2008/005338.
Example 1.2: Preparation of Compounds of Formula (Ila).
H3R antagonist of Formula (Ila) can be prepared according to procedures described in WO2008/048609, for example: (R)-l-[2-(4'-Methanesulfonyl-biphenyl-4-yl)-ethyl]-2-methyl- pyrrolidine can be prepared according to Example 1.13 (Cmpd 1), WO2008/048609; (R)-l-[2-(4'- Ethanesulfonyl-biphenyl-4-yl)-ethyl]-2-methyl-pyrrolidine can be prepared according to Example 1.15 (Cmpd 2), WO2008/048609; (R)-l-{2-[4'-(2-Methoxy-ethanesulfonyl)-biphenyl-4-yl]-ethyl}- 2-methyl-pyrrolidine can be prepared according to Example 1.5 (Cmpd 3), WO2008/048609; (R)- 2-Methyl-l-{2-[4'-(propane-l-sulfonyl)-biphenyl-4-yl]-ethyl}-pyrrolidine can be prepared according to Example 1.6 (Cmpd 5), WO2008/048609; (R)-2-Methyl-l-[2-(4'- phenylmethanesulfonyl-biphenyl-4-yl)-ethyl] -pyrrolidine can be prepared according to Example 1.7 (Cmpd 6), WO2008/048609; 6-{4-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]-phenyl}-l,l-dioxo- ^6-thiochroman-4-one can be prepared according to Example 1.4 (Cmpd 7), WO2008/048609; (R)- 1 - { 2-[4'-(3-Methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl-pyrrolidine can be prepared according to Example 1.8 (Cmpd 8), WO2008/048609; 3-{4'-[2-((R)-2-Methyl- pyrrolidin-l-yl)-ethyl]-biphenyl-4-sulfonyl}-propan-l-ol can be prepared according to Example 1.20 (Cmpd 15), WO2008/048609; and 2-{4'-[2-((R)-2-Methyl-pyrrolidin-l-yl)-ethyl]-biphenyl-3- sulfonylj-ethanol can be prepared according to Example 1.19 (Cmpd 17), WO2008/048609.
Example 1.3: Preparation of Compounds of Formula (Ilia).
H3R antagonist of Formula (Ilia) can be prepared according to procedures described in WO2009/058300, for example: (,S,)-4-((4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4- yl)methyl)oxazolidin-2-one can be prepared according to Example 1.26 (Cmpd 1),
WO2009/058300; (,S,)-4-(4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)oxazolidin-2-one can be prepared according to Example 1.25 (Cmpd 2), WO2009/058300; (R)-4-((4'-(2-((R)-2- methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)methyl)oxazolidin-2-one can be prepared according to Example 1.4 (Cmpd 4), WO2009/058300; (R)-3-methyl-4-(4'-(2-((R)-2-methylpyrrolidin-l- yl)ethyl)biphenyl-4-yl)oxazolidin-2-one can be prepared according to Example 1.5 (Cmpd 5), WO2009/058300; (R)-3-isopropyl-4-(4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4- yl)oxazolidin-2-one can be prepared according to Example 1.7 (Cmpd 7), WO2009/058300; (S)-3- methyl-4-((4'-(2-((R)-2-methylpyrrolidin- 1 -yl)ethyl)biphenyl-4-yl)methyl)oxazolidin-2-one can be prepared according to Example 1.10 (Cmpd 11), WO2009/058300; (¾-3-(2-methoxyethyl)-4-((4'- (2-((R)-2-methylpyrrolidin- 1 -yl)ethyl)biphenyl-4-yl)methyl)oxazolidin-2-one can be prepared according to Example 1.11 (Cmpd 12), WO2009/058300; (,S,)-4-((4'-(2-((R)-2-methylpyrrolidin-l- yl)ethyl)biphenyl-4-yl)methyl)-l,3-oxazinan-2-one can be prepared according to Example 1.15 (Cmpd 13), WO2009/058300; 5-((4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4- yl)oxazolidin-2-one can be prepared according to Example 1.17 (Cmpd 14), WO2009/058300; and (lSr)-3-methyl-4-(4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)oxazolidin-2-one can be prepared according to Example 1.23 (Cmpd 15), WO2009/058300.
Example 1.4: Preparation of Compounds of Formula (IVa).
H3R antagonist of Formula (IVa) can be prepared according to procedures described in WO2009/105206, for example: (R)-3-methoxy-l-(7-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l//)-yl)propan-l-one can be prepared according to Example 1.19 (Cmpd 1), WO2009/105206; (R)-cyclopropyl(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)mefhanone can be prepared according to Example 1.10 (Cmpd 3), WO2009/105206; (R)-cyclopropyl(5 4 2 2-methylpyrrolidin-l-yl)ethyl)phenyl)isoindolin-2- yl)methanone can be prepared according to Example 1.24 (Cmpd 5), WO2009/ 105206; (R)-(4- methoxyphenyl)(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2( \H)- yl)methanone can be prepared according to Example 1.12 (Cmpd 9), WO2009/ 105206; (R)-(5-(4- (2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)isoindolin-2-yl)(tetrahydro-2/i-pyran-4-yl)methanone can be prepared according to Example 1.26 (Cmpd 12), WO2009/105206; (R)-(6-(4-(2-(2- methylpyrrolidin-l-yl)emyl)phenyl)-3,4-dmydroisoquinolin-2(l//)-yl)(pyridin-4-yl)mem can be prepared according to Example 1.14 (Cmpd 14), WO2009/105206; (R)-3-hydroxy-l-(6-(4-(2- (2-memylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dmydroisoquinolin-2(l//)-yl)propan-l-one can be prepared according to Example 1.17 (Cmpd 16), WO2009/105206; and (R)-(6-(4-(2-(2- methylpyrrolidin-l-yl)emyl)phenyl)-3,4-dmydroisoquinolin-2(l//)-yl)(pyridin-2-yl)mem can be prepared according to Example 1.6 (Cmpd 18), WO2009/105206. Example 1.5: Preparation of Compounds of Formula (Va).
Compounds found in Examples 1.5-1 to 1.5-32 refer to the compounds and their corresponding Compound numbers as shown in Table A.
Example 1.5-1: Preparation of Intermediate (/f)-6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l,2,3,4-tetrahydroisoquinoline.
The intermediate (R)-6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4- tetrahydroisoquinoline was prepared in a similar manner as described in WO2009/105206 which is incorporated herein by reference in its entirety.
Step A: Preparation of (/f)-l-(4-Bromophenethyl)-2-methylpyrrolidine.
A 4 L jacketed reactor equipped with a mechanical stirrer, thermocouple, gas inlet, heating/cooling and condenser was charged with 4-bromophenethyl methanesulfonate (199.8 g, 716 mmol), followed by acetonitrile (2.2 L), and the resulting slurry was stirred. Water (270 mL) was then added, followed by gradual addition of potassium carbonate (297.2 g, 2.147 mol). (R)-2- methylpyrrolidine L-tartrate (168.8 g, 717 mmol) was then added, and the reaction mixture was heated at 71 °C overnight. The reaction mixture was cooled and the solvent was removed. The residue was suspended in water (500 mL) and extracted with isopropyl acetate (2 X 400 mL). The organic extracts were combined, rinsed with water (150 mL), dried over sodium sulfate, filtered and concentrated to dryness to provide a golden yellow oil (191 g). This material was combined with 185 g of material which was prepared by the same method and dissolved in isopropyl acetate (2 X 500 mL). The mixture was extracted with 1 N HC1 (2 X 300 mL and 200 mL). The acidic aqueous layer was separated and pH adjusted to 11-12 with 25% NaOH. This was then extracted with isopropyl acetate (2 X 350 mL, washed with water (150 mL) and dried over MgS04 (100 g). Upon filtration and solvent removal, a pale yellow oil was obtained to provide the title compound (337.5 g). LCMS m/z = 268.1 [M+H]+; ¾ NMR (CDC13, 400 MHz) δ ppm 1.16 (d, 7 = 6.2 Hz, 3H), 1.46- 1.55 (m, 1H), 1.71-1.81 (m, 1H), 1.82-1.90 (m, 1H), 1.94-2.01 (m, 1H), 2.24-2.31 (m, 1H), 2.32- 2.39 (m, 1H), 2.41-2.47 (m, 1H), 2.84 (t, 7 = 8.2 Hz, 2H), 3.01-3.08 (m, 1H), 3.26-3.31 (m, 1H), 7.11 (d, 7 = 8.5 Hz, 2H), 7.42 (d, 7 = 8.1 Hz, 2H).
Step B: Preparation of (/f)-4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenylboronic Acid.
A I L 3 -neck flask equipped with mechanical stirrer, thermometer, and addition funnel under N2 was charged with a solution of (R)-l-(4-bromophenethyl)-2-methylpyrrolidine (26.8 g, 100 mmol) in anhydrous THF (250 mL). The reaction mixture was then cooled to an internal temperature of -78 °C. A solution of butyllithium (2.5 M in hexane, 52 mL, 130 mmol) was added dropwise, maintaining an internal temperature < -70 °C. Once addition was complete, stirring was continued an additional 15 min prior to the addition of triisopropyl borate (75 g, 400 mmol), followed by a rinse with 50 mL anhydrous THF while maintaining an internal temperature < -65 °C during addition. The reaction mixture was then allowed to warm to ambient temperature over 1.5 h, and was then quenched by dropwise addition of 2 N HC1 (100 mL). The resulting mixture was stirred overnight, and the solvent volume was reduced to about 150 mL. The resulting suspension was cooled in an ice bath and filtered, rinsing sparingly with cold isopropanol. The filtrate volume was again reduced to 50 mL and the process was repeated. The filter cakes were combined, taken up in boiling isopropanol (250 mL), dissolving most, but not all of the solids. The mixture was then cooled in an ice bath and filtered, then the filtrate was concentrated to half volume and the process was repeated to provide two additional crops. A white solid was obtained as the title compound (23 g). LCMS m/z = 234.3 [M+H]+; ¾ NMR (400 MHz, DMSO- ) δ ppm 1.41 (d, 7= 6.6 Hz, 3H), 1.59-1.68 (m, 1H), 1.89-2.00 (m, 2H), 2.15-2.22 (m, 1H), 3.00-3.07 (m, 2H), 3.11-3.19 (m, 2H), 3.37-3.50 (m, 2H), 3.57-3.65 (m, 1H), 4.80-6.75 (bs, 3 H), 7.27 (d, 7= 7.6 Hz, 2H), 7.76 (d, 7 = 8.2 Hz, 2H).
Step C: Preparation of Intermediate (fl)-6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l,2,3,4-tetrahydroisoquinoline.
To a round -bottom flask was added 6-bromo-l,2,3,4-tetrahydroisoquinoline hydrochloride (2.00 g, 8.05 mmol), (R)-4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenylboronic acid (2.063 g, 8.85 mmol), tetrakis(triphenylphosphine)palladium (0) (0.279 g, 0.241 mmol), benzene (30.00 mL), ethanol (10.00 mL), and 2.0 M aqueous solution of sodium bicarbonate (8.05 mL, 16.09 mmol).
The reaction mixture was refluxed for 6 h. Upon completion, water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na2S04, and concentrated. The residue was taken up in 1 M HC1 solution and washed with ethyl acetate. The aqueous layer was basified with 10% aqueous NaOH to pH~l 1, extracted with ethyl acetate, and concentrated. The residue was purified by silica gel column, eluting with 5-10% 2.0 M ammonia in methanol/DCM to give a yellow solid (1.20 g). LCMS m/z = 321.4 [M+H]+; ¾ NMR (400 MHz, DMSO- ) δ ppm 0.99-1.04 (m, 3H), 1.22-1.33 (m, 1H), 1.59-1.69 (m, 2H), 1.81-1.92 (m, 1H), 2.13 (q, J = 8.67 Hz, 1H), 2.20-2.34 (m, 2H), 2.65-2.83 (m, 5H), 2.94-3.04 (m, 3H), 3.10-3.18 (m, 1H), 3.91 (s, 2H), 7.09 (d, J = 8.08 Hz, 1H), 7.29 (d, J = 8.08 Hz, 2H), 7.33-7.40 (m, 2H), 7.53 (d, J = 8.08 Hz, 2H). Example 1.5-2: Preparation of (/f)-l-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)-2-(l/ -tetrazol-5-yl)ethanone (Compound 1).
(R)-6-(4-(2-(2-Methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline (20.0 mg, 0.062 mmol), 2-(l/Metrazol-5-yl)acetic acid (8.8 mg, 0.069 mmol), DIEA (32.7 μί, 0.187 mmol), and HATU (28.5 mg, 0.075 mmol) were added to a vial with DMF (0.4 mL). The reaction was briefly heated with a heat gun to dissolve all the starting material. Then the reaction was stirred overnight at room temperature. The reaction was diluted with DMSO (0.4 mL) and purified by preparative LC/MS to give the TFA salt of the title compound (24.6 mg). LCMS mJz = 431.2.
Example 1.5-3: Preparation of (/f)-4-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-4-oxobutanoic Acid (Compound 2).
To a mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride (260 mg, 0.661 mmol) and dihydrofuran-2,5-dione (79 mg, 0.793 mmol) in THF (15 mL) was added TEA (0.332 mL, 2.379 mmol). The reaction was stirred at 60 °C overnight. The mixture was filtered. The filtrate was concentrated to give a solid. The solid was trituated in ACN, filtered, and collected to give the neutral form of the title compound. LCMS mJz = 421.6 [M+H]+; ¾ NMR (400 MHz, DMSO- ) δ ppm 1.01 (d, J = 6.00 Hz, 3H), 1.23-1.34 (m, 1H), 1.59-1.70 (m, 2H), 2.81-2.91 (m, 1H), 2.15 (t, /= 8.72, 1H), 2.22-2.35 (m, 2H), 2.46 (t, J = 6.22, 2H), 2.60-2.66 (m, 2H), 2.66-2.85 (m, 3H), 2.90-3.03 (m, 2H), 3.10-3.17 (m, 1H), 3.66-3.74 (m, 2H), 4.62 (s, 1H), 4.69 (s, 1H), 7.26 (t, / = 8.80 Hz, 1H), 7.30 (d, / = 8.13 Hz, 2H), 7.45 (d, / = 6.01 Hz, 2H), 7.55 (d, / = 8.03 Hz, 2H).
Example 1.5-4: Preparation of (/f)-2,2,3,3-tetrafluoro-4-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoic acid (Compound 8).
In a reaction vial was placed (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline dihydrochloride (30 mg, 0.076 mmol), 3,3,4,4-tetrafluorodihydrofuran-2,5- dione (39.4 mg, 0.229 mmol) and sodium bicarbonate (32.0 mg, 0.381 mmol) in THF (1.5 mL). The reaction was stirred at room temperature for 1 h and heated at 60 °C for 30 min. The mixture was filtered and the filtrate was concentrated. The residue was dissolved in 2 M HC1 and purified by HPLC to give the TFA salt of the title compound. LCMS mJz = 493.4 [M+H]+.
Example 1.5-5: Preparation of (/f)-3,3-Dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 3). The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 4,4-dimethyldihydro-2/i-pyran-2,6(3//)-dione. LCMS m/z = 463.6 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 0.97 (s, 3H), 1.02 (s, 3H), 1.15 (d, / = 6.74, 0.3H), 1.35 (d, / = 6.52, 2.7H), 1.58-1.71 (m, IH), 1.90-2.00 (m, 2H), 2.10-2.20 (m, IH), 2.25 (s, IH), 2.28 (s, IH), 2.53 (s, IH), 2.55 (s, IH), 2.81-2.93 (m, 2H), 2.95-3.07 (m, 4H), 3.25-3.35 (m, IH), 3.39-3.40 (m, IH), 3.61-3.71 (m, IH), 3.77-3.82 (m, 2H), 4.72 (s, IH), 4.74 (s, IH), 7.18 (t, J = 8.37 Hz, IH), 7.28 (d, 7 = 8.17 Hz, 2H), 7.39 (d, / = 8.04 Hz, 2H), 7.52 (d, 7 = 8.16 Hz, 2H). Example 1.5-6: Preparation of (/f)-2,2-Dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 4).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 3,3-dimethyldihydro-2/i-pyran-2,6(3H)-dione. LCMS m/z = 463.5 [M+H]+.
Example 1.5-7: Preparation of 3-Methyl-5-(6-(4-(2-((/f)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 5).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 4-methyldihydro-2/i-pyran-2,6(3//)-dione. LCMS m/z = 449.3 [M+H]+.
Example 1.5-8: Preparation of 3-hydroxy-5-(6-(4-(2-((/f)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic acid (Compound 6).
In a reaction vial was placed (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline dihydrochloride (48 mg, 0.122 mmol), 4-(tert- butyldimethylsilyloxy)dihydro-2/i-pyran-2,6(3//)-dione (89 mg, 0.366 mmol), and sodium bicarbonate (51.3 mg, 0.610 mmol) in DMF (2 mL). The reaction was stirred at 60 °C overnight. The mixture was filtered and purified by HPLC to give the TFA salt of the title compound. LCMS m/z = 451.4 [M+H]+.
Example 1.5-9: Preparation of (K)-2-(2-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-2-oxoethylthio)acetic Acid (Compound 7).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and l,4-oxathiane-2,6-dione. LCMS m/z = 453.5 [M+H]+. Example 1.5-10: Preparation of (/f)-2^-Dimethyl-4-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoic Acid (Compound 9).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-4 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline dihydrochloride and 3,3-dimethyldihydrofuran-2,5-dione. LCMS mJz = 449.6 [M+H]+.
Example 1.5-11: Preparation of (K)-2-(2-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)-2-oxoethoxy)acetic Acid (Compound 10).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline dihydrochloride (30 mg, 0.076 mmol), l,4-dioxane-2,6-dione (10.62 mg, 0.092 mmol), and sodium carbonate (28.3 mg, 0.267 mmol) in THF (2 mL) was heated at 60 °C for 30 min. The mixture was then stirred at room temperature overnight. The precipitate was filtered, dissolved in 2 M HCl and purified by HPLC to give the TFA salt of the title compound. LCMS mJz = 437.4 [M+H]+.
Example 1.5-12: Preparation of (/f)-5-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoic Acid (Compound 11).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline dihydrochloride (30 mg, 0.076 mmol), dihydro-2/i-pyran-2,6(3//)-dione (10.44 mg, 0.092 mmol), and TEA (0.038 mL, 0.275 mmol) in THF (2 mL) was heated at 60 °C for 1 h. The mixture was purified by HPLC to give the TFA salt of the title compound. LCMS mJz = 435.6 [M+H]+.
Example 1.5-13: Preparation of (fl)-Methyl 4-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l/ )-yl)-4-oxobutanoate (Compound 12).
To (R)-4-(6-(4-(2-(2-me lpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)- yl)-4-oxobutanoic acid (15 mg, 0.036 mmol) was added HCl (1.25 M in MeOH, 2 mL, 2.500 mmol). The mixture was stirred at 60 °C for 30 min and room temperature for 1 h. The solvent was removed and residue was triturated in acetonitrile three times and dried to give the title compound as the HCl salt. LCMS mJz = 435.4 [M+H]+.
Example 1.5-14: Preparation of (S)-Ethyl 2-amino-5-(6-(4-(2-((fl)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoate (Compound 13).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline dihydrochloride (30 mg, 0.076 mmol), (S)-tert-butyl 2,6-dioxotetrahydro- 2#-pyran-3-ylcarbamate (22.73 mg, 0.099 mmol), and TEA (0.038 mL, 0.275 mmol) in THF (1.5 mL) was heated at 60 °C for 3 h. The resulting mixture was concentrated. The residue was treated with 2 M HCl in EtOH and purified by HPLC to give the TFA salt of the title compound. LCMS mJz = 478.4 [M+H]+.
Example 1.5-15: Preparation of (/f)-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l/ )-yl)(l/ -tetrazol-5-yl)methanone (Compound 14).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tettahydroisoquinoline bis(2,2,2-trifluoroacetate) (30 mg, 0.055 mmol), disodium salt of IH- tetrazole-5-carboxylic acid (8.64 mg, 0.055 mmol), HATU (22.88 mg, 0.060 mmol), and TEA
(0.023 mL, 0.164 mmol) in DMF (1.5 mL) was stirred at 60 °C for 1 h. The mixture was filtered. The filtrate was purified by HPLC to give the TFA salt of the title compound. LCMS mJz = 417.4 [M+H]+. Example 1.5-16: Preparation of (fl)-Ethyl 4-(6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(lH)-yl)-4-oxobutanoate (Compound 15).
To (R)-4-(6-(4-(2-(2-me lpyrrolidin-l-yl)ethyl)phenyl)-3,4-dmydroisoquinolin-2(l//)- yl)-4-oxobutanoic acid (15 mg, 0.036 mmol) was added HCl (2 M in ethanol, 1.783 mL, 3.57 mmol). The solution was stirred for 24 h at room temperature and concentrated. The residue was trituated with ACN to give the title compound. LCMS mJz = 449.6 [M+H]+.
Example 1.5-17: Preparation of (fl)-Methyl 2-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l//)-yl)-2-oxoacetate (Compound 16).
To (R)-6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline (50 mg, 0.156 mmol) in THF (1.5 mL) was added methyl 2-chloro-2-oxoacetate (28.7 mg, 0.234 mmol). The reaction was stirred at room temperature for 1 h. The mixture was concentrated and purified by HPLC to give the TFA salt of the title compound. LCMS m/z = 407.5 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.27 (d, J = 6.87 Hz, 0.3H), 1.46 (d, J = 6.52 Hz, 2.7H), 1.71- 1.85 (m, 1H), 2.02-2.12 (m, 2H), 2.24-2.31 (m, 1H), 2.94-3.02 (m, 2H), 3.05-3.19 (m, 4H), 3.37- 3.48 (m, 1H), 3.50-3.60 (m, 1H), 3.69 (t, J= 5.97, 1H), 3.73-3.81 (m, 1H), 3.83 (t, J= 6.12, 1H),
3.91 (s, 3H), 4.64 (s, 1H), 4.74 (s, 1H), 7.22-7.32 (m, 1H), 7.39 (d, / = 8.19 Hz, 2H), 7.45-7.53 (m, 2H), 7.63 (d, 7 = 8.19 Hz, 2H).
Example 1.5-18: Preparation of (K)-2-(l-(2-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l/ )-yl)-2-oxoethyl)cyclopentyl)acetic Acid (Compound 17).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tettahydroisoquinoline (40 mg, 0.125 mmol) and 8-oxaspiro[4.5]decane-7,9-dione (20.99 mg, 0.125 mmol) in THF (1 mL) was heated at 60 °C overnight. The mixture was added 1 M HCl and purified by HPLC to give the TFA salt of the title compound. LCMS m z = 489.4 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.27 (d, 7 = 6.83 Hz, 0.3H), 1.46 (d, 7 = 6.53 Hz, 2.7H), 1.49-1.69 (m, 4H), 1.70-1.80 (m, 5H), 2.02-2.12 (m, 2H), 2.23-2.33 (m, IH), 2.46 (d, 7 = 12.2 Hz, 2H), 2.71 (d, 7 = 5.53 Hz, 2H), 2.96 (t, J= 6.04 Hz, IH), 3.01 (t, 7 = 5.84 Hz, IH), 3.06-3.20 (m, 4H), 3.38-3.48 (m, IH), 3.53-3.61 (m, IH), 3.74-3.84 (m, IH), 3.85-3.92 (m, 2H), 4.81 (s, IH), 4.83 (s, IH), 7.29 (t, 7 = 7.89, IH), 7.40 (d, 7 = 8.17 Hz, 2H), 7.48-7.53 (m, 2H), 7.64 (d, 7= 8.18 Hz, 2H).
Example 1.5-19: Preparation of (K)-2-(l-(2-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l/ )-yl)-2-oxoethyl)cyclohexyl)acetic Acid (Compound 18).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline (20 mg, 0.062 mmol) and 3-oxaspiro[5.5]undecane-2,4-dione (11.37 mg, 0.062 mmol) in DMF (1 mL) was stirred at room temperature for 3 h. The mixture was purified by HPLC to give the TFA salt of the title compound. LCMS m/z = 503.2 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.28 (d, 7 = 6.87 Hz, 0.3H), 1.29-1.30 (m, IH), 1.48 (d, 7 = 6.52 Hz, 2.7H), 1.49-1.65 (m, 9H), 1.70-1.85 (m, IH), 2.01-2.12 (m, 2H), 2.24-2.32 (m, IH), 2.53 (d, 7 = 11.6 Hz, 2H), 2.65 (d, 7 = 5.28 Hz, 2H), 2.97 (t, J= 6.01 Hz, IH), 3.02 (t, 7 = 5.79 Hz, IH), 3.07-3.18 (m, 4H), 3.37-3.46 (m, IH), 3.50-3.61 (m, IH), 3.75-3.84 (m, IH), 3.90-3.95 (m, 2H), 4.83 (s, IH), 4.88 (s, IH), 7.27-7.33 (m, IH), 7.40 (d, 7= 8.18 Hz, 2H), 7.48-7.54 (m, 2H), 7.64 (d, 7 = 8.20 Hz, 2H).
Example 1.5-20: Preparation of (15^5)-2-(6-(4-(2-((K)-2-Methylpyrrolidin-l-yl)ethyl)phenyl)- l,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic Acid (Compound 19).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline (20 mg, 0.062 mmol) and (3aR,7aR)-hexahydroisobenzofuran-l,3-dione (9.62 mg, 0.062 mmol) in DMF (0.7 mL) was stirred at room temperature for 3 h. The mixture was purified by HPLC to give the TFA salt of the title compound. LCMS m/z = 475.3 [M+H]+.
Example 1.5-21: Preparation of (fl)-Methyl 3-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 3,4-dihydroisoquinolin-2(l//)-yl)-3-oxopropanoate (Compound 20).
A mixture of (R)-6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)- 1 ,2,3,4- tetrahydroisoquinoline dihydrochloride (30 mg, 0.076 mmol), methyl 3-chloro-3-oxopropanoate (11.45 mg, 0.084 mmol), and TEA (0.034 mL, 0.244 mmol) was stirred at room temperature for 3 h. The mixture was added 1 M HCl (1 mL) and purified by HPLC to give the TFA salt of the title compound. LCMS m/z = 421.5 [M+H]+.
Example 1.5-22: Preparation of (fl)-Methyl l-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)- l,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclopropanecarboxylate (Compound 21). To a solution of (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline dihydrochloride (44 mg, 0.112 mmol) and 1-
(methoxycarbonyl)cyclopropanecarboxylic acid (17.73 mg, 0.123 mmol) in DMF (1 mL) were added HATU (46.8 mg, 0.123 mmol) and TEA (0.047 mL, 0.336 mmol). The reaction was stirred at room temperature for 4 h. The mixture was purified by HPLC to give the TFA salt of the title compound. LCMS mJz = 447.4 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.15 (d, J = 6.88 Hz, 0.3H), 1.17-1.22 (m, IH), 1.22-1.27 (m, IH), 1.34 (d, J = 6.44 Hz, 2.7H), 1.34-1.38 (m, 2H), 1.59- 1.71 (m, IH), 1.90-1.99 (m, 2H), 2.11-2.21 (m, IH), 2.77-2.87 (m, 2H), 2.94-3.23 (m, 5H), 3.25- 3.34 (m, IH), 3.59 (s, 3H), 3.63-3.73 (m, 3H), 4.60 (s, 2H), 7.09-7.19 (m, IH), 7.27 (d, / = 8.21 Hz, 2H), 7.34-7.40 (m, 2H), 7.52 (d, / = 8.16 Hz, 2H).
Example 1.5-23: Preparation of (/f)-2-Methyl-l-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-2-(l/ -tetrazol-5-yl)propan-l-one
(Compound 22).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-22 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline dihydrochloride and 2-methyl-2-(l/i-tetrazol-5-yl)propanoic acid. LCMS mJz = 459.4 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.15 (d, / = 6.37 Hz, 0.3H), 1.34 (d, / = 6.50 Hz, 2.7H), 1.58 (s, 6H), 1.60-1.70 (m, IH), 1.90-1.99 (m, 2H), 2.1 1-2.21 (m, IH), 2.93-3.08 (m, 4H), 3.24-3.37 (m, 2H), 3.40-3.49 (m, 2H), 3.60-3.71 (m, 2H), 4.25-4.49 (m, 2H), 6.96 (bs, IH), 7.21-7.27 (m, 2H), 7.27-7.33 (m, IH), 7.47 (d, 7 = 8.11 Hz, 2H).
Example 1.5-24: Preparation of (fl)-Methyl 3,3-dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-5-oxopentanoate (Compound 23).
The HC1 salt of the title compound was prepared in a similar manner to the one described in Example 1.5-13 using (R)-3,3-dimethyl-5-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-5-oxopentanoic acid. LCMS mJz = 477.4 [M+H]+.
Example 1.5-25: Preparation of (15,3tf)-Methyl 3-(6-(4-(2-((tf)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-l^,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylate
(Compound 24).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-22 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline bis(2,2,2-trifluoroacetate) and (lR,3S)-3- (methoxycarbonyl)cyclohexanecarboxylic acid. LCMS mJz = 489.5 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.15 (d, J = 6.86 Hz, 0.3H), 1.17-1.37 (m, 3H), 1.35 (d, J = 6.51 Hz, 2.7H), 1.38- 1.52 (m, IH), 1.59-1.71 (m, 2H), 1.71-1.79 (m, IH), 1.90-1.99 (m, 2H), 2.10-2.20 (m, IH), 2.30- 2.39 (m, IH), 2.50-2.72 (m, IH), 2.73-2.79 (m, IH), 2.82-2.88 (m, IH), 2.94-3.06 (m, 4H), 3.24- 3.35 (m, IH), 3.37-3.48 (m, IH), 3.52 (s, 3H), 3.64-3.74 (m, 3H), 4.58 (s, IH), 4.63 (s, IH), 7.15 (d, 7 = 7.79 Hz, IH), 7.27 (d, 7 = 8.16 Hz, 2H), 7.33-7.40 (m, 2H), 7.51 (d, 7 = 8.16 Hz, 2H). Example 1.5-26: Preparation of (lr,4r)-Methyl 4-(6-(4-(2-((/f)-2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l^,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylate
(Compound 25).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-22 using (R)-6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1,2,3,4- tetrahydroisoquinoline £>w(2,2,2-trifluoroacetate) and (lr,4r)-4-
(methoxycarbonyl)cyclohexanecarboxylic acid. LCMS mJz = 489.5 [M+H]+; H NMR (400 MHz, CD3CN) δ ppm 1.28 (d, 7 = 6.87 Hz, 0.3H), 1.46 (d, 7 = 6.52 Hz, 2.7H), 1.48-1.59 (m, 4H), 1.70- 1.90 (m, 3H), 1.99-2.12 (m, 4H), 2.24-2.41 (m, 2H), 2.66-2.77 (m, IH), 2.84-2.91 (m, IH), 2.96- 3.00 (m, IH), 3.05-3.21 (m, 4H), 3.40-3.49 (m, IH), 3.54-3.62 (m, IH), 3.65 (s, 3H), 3.73-3.83 (m, 3H), 4.70 (s, IH), 4.76 (s, IH), 7.24-7.30 (m, IH), 7.39 (d, 7 = 8.18 Hz, 2H), 7.45-7.51 (m, 2H),
7.64 (d, 7 = 8.18 Hz, 2H).
Example 1.5-27: Preparation of (K)-l-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-1^ ,4- tetrahydroisoquinoline-2-carbonyl)cyclopropanecarboxylic Acid (Compound 26).
To a solution of (R)-methyl l-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-l,2,3,4- tetrahydroisoquinoline-2-carbonyl)cyclopropanecarboxylate 2,2,2-trifluoroacetate (7 mg, 0.012 mmol) in MeOH (1 mL) was added 5 M NaOH (0.062 mL, 0.312 mmol). The reaction was stirred at 60 °C for 4 h. The mixture was concentrated and the residue was dissolved in H20 (2 mL). The mixture was added 1 M HC1 (1 mL) and purified by HPLC. LCMS mJz = 433.2 [M+H]+; ¾ NMR (400 MHz, CD3CN) 5 ppm l. l5 (d, 7 = 6.87 Hz, 0.3H), 1.17-1.24 (m, 2H), 1.34 (d, 7= 6.29 Hz, 2.7H), 1.33-1.38 (m, 2H), 1.60-1.71 (m, IH), 1.90-1.99 (m, 2H), 2.11-2.21 (m, 2H), 2.80-2.87 (m, IH), 2.95-3.07 (m, 4H), 3.25-3.35 (m, IH), 3.40-3.49 (m, IH), 3.58-3.72 (m, 3H), 4.62 (s, 2H), 7.14-7.19 (m, IH), 7.28 (d, 7 = 8.14 Hz, 2H), 7.34-7.39 (m, 2H), 7.52 (d, 7= 8.16 Hz, 2H). Example 1.5-28: Preparation of (lr,4r)-4-(6-(4-(2-((/f)-2-Methylpyrrolidin-l-yl)ethyl)phenyl)- l,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic Acid (Compound 27).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-27 using (lr,4r)-methyl 4-(6-(4-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylate 2,2,2-trifluoroacetate. LCMS mJz = 475.5 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.16 (d, 7 = 6.85 Hz, 0.3H), 1.34 (d, 7 = 6.52 Hz, 2.7H), 1.36-1.47 (m, 4H), 1.59-1.76 (m, 3H), 1.88-1.99 (m, 3H), 2.11-2.21 (m, 2H), 2.55-
2.65 (m, IH), 2.72-2.79 (m, IH), 2.82-2.89 (m, IH), 2.94-3.10 (m, 4H), 3.25-3.36 (m, IH), 3.39- 3.50 (m, 1H), 3.61-3.71 (m, 3H), 4.58 (s, 2H), 4.64 (s, 1H), 7.16 (d, 7 = 7.76 Hz, 1H), 7.28 (d, 7 = 8.15 Hz, 2H), 7.33-7.40 (m, 2H), 7.52 (d, 7 = 8.13 Hz, 2H).
Example 1.5-29: Preparation of (lS tf)-3-(6-(4-(2-((tf)-2-Methylpyrrolidin-l- yl)ethyl)phenyl)-l^,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic Acid (Compound 28).
The TFA salt of the title compound was prepared in a similar manner to the one described in Example 1.5-27 using (1 S, 3R) -methyl 3-(6-(4-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)phenyl)- 1 ,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylate 2,2,2 -trifluoroacetate. LCMS mJz = 475.6 [M+H]+.
Example 1.5-30: Preparation of (/f)-4-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-l-oxo- 3,4-dihydroisoquinolin-2(l//)-yl)-4-oxobutanoic Acid (Compound 29).
Step A: Preparation of (K)-6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-l(2//)-one.
In a microwave reaction vial was placed (R)-4-(2-(2-methylpyrrolidin-l- yl)ethyl)phenylboronic acid hydrochloride (500 mg, 1.855 mmol), 6-bromo-3,4- dihydroisoquinolin-l(2//)-one (419 mg, 1.855 mmol), dichlorobis(phenyldi-feri- butylphosphine)palladium (11.54 mg, 0.019 mmol), and sodium carbonate (393 mg, 3.71 mmol) in a mixture of toluene (10 mL), EtOH (3 mL), and water (2 mL). The reaction was heated under microwave irradiation at 125 °C for 2 h. The mixture was extracted with toluene and 1 M HCl. The aqueous layer was separated and purified by HPLC to give the title compound. LCMS mJz = 335.3 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.15 (d, 7 = 6.86 Hz, 0.3H), 1.34 (d, 7 = 6.53 Hz, 2.7H), 1.59-1.70 (m, 1H), 1.90-1.99 (m, 2H), 2.11-2.21 (m, 1H), 2.42-2.48 (m, 2H), 2.87-2.93 (m, 2H), 2.93-3.06 (m, 4H), 3.39-3.49 (m, 1H), 3.62-3.72 (m, 1H), 6.81 (d, 7 = 8.16 Hz, 1H), 7.26 (d, 7 = 8.22 Hz, 2H), 7.31-7.39 (m, 2H), 7.49 (d, 7 = 8.25 Hz, 2H), 8.19 (s, 1H).
Step B: Preparation of (/f)-4-(6-(4-(2-(2-Methylpyrrolidin-l-yl)ethyl)phenyl)-l-oxo- 3,4-dihydroisoquinolin-2(l//)-yl)-4-oxobutanoic Acid (Compound 29).
To a suspension of NaH (8.03 mg, 0.334 mmol) in THF (1 mL) was added (R)-6-(4-(2-(2- methylpyrrolidin-l-yl)ethyl)phenyl)-3,4-dihydroisoquinolin-l(2//)-one 2,2,2-trifluoroacetate (15 mg, 0.033 mmol). The mixture was stirred at room temperature for 30 min. Dihydrofuran-2,5-dione (16.74 mg, 0.167 mmol) was then added to the mixture. The reaction was stirred at room temperature for 3 h. The mixture was quenched with 1 M HCl and purified by HPLC. LCMS mJz = 435.2 [M+H]+; ¾ NMR (400 MHz, CD3CN) δ ppm 1.15 (d, 7 = 6.85 Hz, 0.3H), 1.35 (d, 7 = 6.53 Hz, 2.7H), 1.59-1.71 (m, 1H), 1.91-2.00 (m, 2H), 2.11-2.21 (m, 1H), 2.42-2.47 (m, 2H), 2.59-2.65 (m, 2H), 2.85-2.91 (m, 2H), 2.95-3.07 (m, 4H), 3.14-3.19 (m, 2H), 3.25-3.35 (m, 1H), 3.39-3.50 (m, 1H), 3.62-3.72 (m, 1H), 7.25-7.32 (m, 3H), 7.38-7.47 (m, 2H), 7.54 (d, 7 = 8.25 Hz, 2H). Example 1.5-31: Preparation of (tf)-Ethyl 2-(l-(2-(6-(4-(2-(2-Methylpyrrolidin-l- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l/ )-yl)-2-oxoethyl)cyclohexyl)acetate (Compound 30).
The HC1 salt of the title compound was prepared in a similar manner to the one described in Example 1.5-13 using (R)-2-(l-(2-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)-2-oxoethyl)cyclohexyl)acetic acid. LCMS m/z = 431.7 [M+H]+.
Example 1.5-32: Preparation of (ltf,2tf)-Methyl 2-(6-(4-(2-((tf)-2-methylpyrrolidin-l- yl)ethyl)phenyl)-l^,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylate
(Compound 31).
To (2R)- 1 -(4-(2-(( lR,2R)-2-carboxycyclohexanecarbonyl)- 1 ,2,3,4-tetrahydroisoquinolin-6- yl)phenethyl)-2-methylpyrrolidinium 2,2,2-trifluoroacetate (2.0 mg, 3.40 μηιοΐ) was added HC1 (1.25 M in methanol, 1.006 mL, 1.257 mmol). The reaction was stirred at 60 °C overnight. The mixture was concentrated and triturated with ACN to give the title compound. LCMS m/z = 489.4 [M+H]+.
Example 2: [3H] N-Alpha-Methyl-Histamine Competitive Histamine H3 Receptor Binding Assay.
The histamine receptor binding assay was conducted using standard laboratory procedures as described below. A crude membrane fraction was prepared from whole rat brain cortex using a polytron to homogenize the tissue followed by differential centrifugation in a HEPES-based buffer containing protease inhibitors. Membranes where frozen at -80 °C until needed. Frozen membranes were thawed and resuspended in ice-cold assay buffer consisting of 50 mM TRIS containing 5 mM EDTA (pH = 7.4). 50 μg of membrane protein was added to each well of a 96-well assay plate along with test compound and [3ITj-Ai-a-methyl-histamine (1 nM final assay concentration). Imetit was used as an assay positive control at varying concentrations. The plate was incubated for 30 min at room temperature. The assay was terminated by rapid filtration through a 96-well glass fiber filtration plate (GF/C) using a cell harvester (Perkin-Elmer). Captured membranes were washed three times with cold assay buffer and plates were dried at 50 °C. 35 L of scintillation cocktail was added to each well and membrane -bound radioactivity was recorded using a TopCount 96-well plate scintillation counter (Perkin-Elmer).
The following table shows the observed activities for certain compounds of the present invention.
Compound Name
Kj Binding Assay (nM) (Cmpd No. in Reference)
Figure imgf000112_0001
Each of the Compounds in Table A and Table B of WO2008/005338 (Formula (la)) had an observed ¾ value in the range of about 260 nM to about 0.5 pM.
Each of the Compounds in Table A and Table B of WO2008/048609 (Formula (Ila)) had an observed ¾ value in the range of about 20 μΜ to about 0.4 pM; Cmpd 37 in WO2008/048609 was not tested in this assay. Each of the Compounds in Table A and Table B of WO2009/058300 (Formula (ma)) had an observed ¾ value in the range of about 205 nM to about 0.8 pM; Cmpd 32 in WO2009/058300 was not tested in this assay.
Each of the Compounds in Table A and Table B of WO2009/105206 (Formulae (IVa) and (IVb)) had an observed ¾ value in the range of about 255 nM to about 0.2 pM; Cmpds 42 and 50 in WO2009/105206 were not tested in this assay.
Example 3: Receptor Binding Assays
Compounds that bind to GPCRs can be identified by their ability to displace a radiolabeled or fluorescently labeled tracer ligand from the receptor. The tracer ligand can be a receptor agonist, inverse agonist or a neutral antagonist. Receptor binding assays can be performed using either whole cells or membrane fractions prepared from such cells.
In a typical radioligand binding assay, cell membranes prepared from cells either endogenously or recombinantly expressing the desired receptor, are prepared and allowed to equilibrate with a mixture of a test ligand and tracer radioligand. Following equilibration, the cell membranes are captured by rapid filtration and washed with cold assay buffer to remove any unbound compounds. A liquid scintillant is typically added to the captured membranes and the samples are then counted on a scintillation counter. Compounds that bind to the receptor and displace the radioligand result in lower counts. For some receptors, the radiolabeled tracer ligand can be replaced by a fluorescently labeled ligand.
Homogeneous binding assays have also been developed for some receptors. These can involve the use of either radioactive tracer ligands (e.g., scintillation proximity assays) or fluorescent tracer ligands (fluorescence polarization binding assays). Example 3.1: Human Histamine H3 Receptor Binding Assay - MDS Pharma Services (Taiwan).
Compounds of the invention were tested for their ability to bind to the human histamine H3-receptor using the MDS Pharma Services (Taiwan) assay, Catalogue No. 239810. Certain compounds of the present invention and their corresponding activity values are shown in following table.
Figure imgf000113_0001
- I l l - 6- { 4- [2-((R)-2-Methyl-pyrrolidin- 1 -yl)-ethyl] -phenyl } - 1 , 1 -dioxo- 1λ6-
2.9 thiochroman-4-one (Cmpd 7, WO2008/048609)
(¾-4-((4X2-((R)-2-methylpyrrolidin- 1 -yl)ethyl)biphenyl-4-
6.9 yl)methyl)oxazolidin-2-one (Cmpd 1, WO2009/058300)
(5)-4-(4'-(2-((R)-2-methylpyrrolidin-l-yl)ethyl)biphenyl-4-yl)oxazolidin-
4.4 2-one (Cmpd 2, WO2009/058300)
(R)-cyclopropyl(5-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)isoindolin- 0.57
2-yl)methanone (Cmpd 5, WO2009/ 105206)
(R)-(6-(4-(2-(2-methylpyrrolidin-l-yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)(pyridin-2-yl)methanone (Cmpd 18, 2.15
WO2009/105206)
Example 3.2: Rat Cortex H3 Receptor Radioligand Binding Assay.
Cell membranes prepared from isolated, Sprague-Dawley rat cortex, which are known to abundantly express the rat H3 receptor, are plated into 96-well microtiter plates at a concentration of 50 μg total membrane protein per well. Test compounds, prepared in assay buffer (50 mM Tris- HC1, pH7.4, with 5 mM EDTA) containing the selective H3 receptor agonist radioligand [3H] N- methylhistamine (final assay concentration 1.25 nM), are added to each well on the plate. Test compound concentrations typically begin at 2 or 10 μΜ (final assay concentration) and 1:5 serial dilutions are prepared in order to generate 10-point dose-response curves for IC50 and ¾ determinations. After a 1 hour, room temperature incubation, membranes are harvested into a PEI- washed filter plate (Whatman GF/C Unifilter) by rapid filtration (Perkin Elmer harvester) and washed three times with ice cold assay buffer (3 x 150 μΕ). Filter plates are then partially dried in a 50°C oven for 1-2 hours. Finally, the plate bottoms are sealed and BetaScint (Perkin Elmer 1205- 440; 25 μΕ per well) is added to each well. Plates are then read on a TopCount (Packard
Instruments). Raw counts are then used to plot dose-response curves for serially diluted test compounds. The resulting IC50 values can be converted to ¾ values using the Cheng-Prusoff equation and the ¾ value for [3H] N-methylhistamine at the rat H3 receptor (0.4 nM).
Example 3.3: Assays for Determination of GPCR Activation or Inhibition.
A variety of assays are available for assessment of activation or inhibition of GPCRs. The following are illustrative; those of ordinary skill in the art are credited with the ability to determine those techniques that are preferentially beneficial for the needs of the artisan.
1. Membrane [35S]GTPyS Binding Assays.
When a G protein-coupled receptor is in its active state, either as a result of agonist ligand binding or constitutive activation, the receptor couples to G proteins, stimulating the release of GDP and subsequent binding of GTP to the G protein alpha subunit. The activated G protein alpha subunit acts as a GTPase and slowly hydrolyzes the GTP back to GDP. The non-hydrolyzable GTP analog, [35S]GTPyS, can be utilized to monitor binding of GTP to membrane-associated G proteins. Typically, test compounds are incubated with receptor-expressing cell membranes in the presence of [ SJGTPyS for 30 to 60 minutes. If the test compound is an agonist or an inverse agonist at the receptor of interest, enhanced or diminished uptake of [35S]GTPyS into the membrane-associated G-proteins will be detected. A neutral antagonist, with no intrinsic efficacy at the receptor, can be detected by its ability to prevent agonist-stimulated [35S]GTPyS exchange. The advantage of using [35S]GTPyS binding to measure activation is that: (a) under appropriate assay conditions, it is generically applicable to all G protein-coupled receptors; (b) it is proximal at the membrane surface making it less likely to pick-up molecules which affect the rest of the G protein mediated intracellular signaling cascade.
Human H3 Receptor GTPyS Assay.
In a typical [35S]GTPyS assay, recombinant human H3 receptor-expressing CHO-K1 cell membranes (90 μg membrane protein per well) are equilibrated in assay buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, 1 mM EDTA) containing test compounds and 10 μΜ GDP for 20 minutes. SPA beads (scintillation microsphere beads) are then added and incubated for 60 minutes at 30°C. The reaction is then initiated by the addition of 0.3 nM
[35S]GTPyS for 30 minutes. Plates are then sealed, centrifuged and counted in a scintillation counter (Packard TopCount).
The recombinantly-expressed histamine H3 receptor is known to be constitutively active. In the above example, test compounds that have either positive (agonist) or negative (inverse agonist) efficacy at the H3 receptor would be detected by their ability to increase or decrease basal levels of [35S]GTPyS binding, respectively. In an alternate configuration, the assay may be modified to include a low dose (typically an EC8o-9o concentration) of a selective H3 receptor agonist such as N-methylhistamine. In this approach, the ability to detect agonists is diminished and the ability to detect inverse agonists is increased. Additionally, antagonists (ligands with no intrinsic receptor efficacy) that block binding of the agonist to the receptor will be detected.
2. cAMP Assays.
GPCRs coupled to either Gs or Gi G-proteins modulate levels of intracellular cAMP and cAMP levels can be determined using a variety of commercially available assay kits. Examples of commonly used cAMP detection assays include FlashPlate® (New England Nuclear), HTRF® (Cisbio), cAMP-Screen® (Applied Biosystems), HitHunter® (Applied Biosystems/DiscoveRx), CatchPoint® (Molecular Devices), AlphaScreen® (Perkin Elmer), GloSensor® (Promega) and numerous ELISA products. Most of these assays rely on the use of an anti-cAMP antibody to detect cAMP.
Homogeneous time-resolved fluorescence assays (HTRF®, Cisbio) detect levels of cAMP in lysed cell preparations using a europium or terbium cryptate-labeled anti-cAMP antibody and fluorophore-labeled cAMP (cAMP-d2). In the absence of exogenous cAMP, the anti-cAMP antibody binds to cAMP-d2. Photoexcitation of the cryptate donor results in a combination of cryptate emission at 620 nm and fluorescence resonance energy transfer (FRET) from the cryptate to the acceptor d2 fluorophore, which then fluoresces at 665 nm. The 620/665 nm emission ratio is monitored. In the presence of exogenous cAMP, which competes with cAMP-d2 for binding to the anti-cAMP antibody, FRET is decreased and the 620/665 nm emission ratio therefore increases, providing a sensitive and accurate means to measure cAMP levels in biological assays.
Human H3 Receptor HTRF cAMP Assay.
Compounds of the present invention were evaluated using the human H3 receptor (H3R) HTRF cAMP assay. In this assay, HEK293 cells expressing the human H3 receptor were suspended in PBS containing 100 μΜ IB MX and plated into 384-well assay plates (Perkin Elmer Proxiplate 384-Plus; 15,000 cells per well; 5 μΕ plating volume) and allowed to equilibrate for an hour. Test compounds were serially diluted in 100% DMSO and then further diluted in PBS containing forskolin (2 μΜ). Test compounds (5 μΕ) were then added to the assay plate and the mixture was incubated for 1 hour. HTRF assay reagents (Cisbio, Dynamic 2 cAMP Kit), cAMP-d2 and cryptate- labeled anti-cAMP antibody, are mixed with cell lysis buffer and added to the assay plate. After 1- hour incubation with these reagents, the assay plate was read on an HTRF-compatible microplate reader (Perkin Elmer En Vision or BMG Pherastar). A cAMP standard curve was included on each assay plate and HTRF emission ratios for test compounds were fit to this curve to generate accurate measures of cAMP levels in each test well. The observed H3R IC50 values in the HTRF cAMP assay for several compounds of the present invention are shown in Table C. Table C
Figure imgf000116_0001
In addition to the compounds and their corresponding H3R IC50 values disclosed in Table C, all other compounds in Table A had observed H3R IC50 values in the HTRF cAMP assay ranging from about 430 pM to about 7 nM.
In an alternate configuration, designed to detect antagonists (ligands with no intrinsic receptor efficacy) that block binding of the agonist to the receptor the assay is modified to include a low dose of histamine (typically 20 nM) in the test compound buffer. The recombinantly-expressed histamine H3 receptor is known to be constitutively active. In the above example, test compounds that have either positive (agonist) or negative (inverse agonist) efficacies are detected by their ability to decrease or increase forskolin stimulated levels of cAMP, respectively. In this configuration, both inverse agonists and antagonists are efficiently detected.
3. Xenopus Melanophore Assays.
Melanophores are skin cells found in lower vertebrates. They contain pigmented organelles termed melanosomes. Melanophores are able to redistribute these melanosomes along a microtubule network upon G-protein coupled receptor (GPCR) activation. The result of this pigment movement is an apparent lightening or darkening of the cells. In melanophores, the decreased levels of intracellular cAMP that result from activation of a Gi-coupled receptor such as the H3 receptor cause melanosomes to migrate to the center of the cell, resulting in a dramatic lightening in color. If cAMP levels are then raised, following activation of a Gs-coupled receptor or addition of an H3 receptor inverse agonist, the melanosomes are re-dispersed and the cells appear dark again. The response of the melanophores takes place within minutes of receptor activation and results in a simple, robust color change. The response can be easily detected using a conventional absorbance microplate reader or a modest video imaging system.
Since the H3 receptor is a constitutively active Gi-coupled receptor, melanophores expressing the H3 receptor will exhibit partial pigment aggregation in the resting state. Stimulation with an H3 receptor agonist or inverse agonist will cause either further pigment aggregation or dispersion, respectively. A neutral antagonist at the H3 receptor would be detected by its ability to inhibit pigment aggregation stimulated by a selective H3 receptor agonist
4. Intracellular Calcium and Inositol Phosphate Assays.
GPCRs coupled to Gq G-proteins regulate the activity of phospholipase C, resulting in the modulation of intracellular inositol phosphates (IP) and diacylglycerol levels. Increased IP levels result in activation of the IP receptor with consequent release of calcium ions into the cytosol. Levels of intracellular IP can be determined in cells loaded with [3H]-myo-inositol, resulting in the production of tritiated IP, which can be detected using standard radiometric techniques. IP levels can also be determined using an HTRF IP-One assay (Cisbio) which relies on an antibody to inositol monophosphate to detect IP.
Cytosolic calcium can be monitored using membrane -permeable dyes that become fluorescent when bound to calcium. The most widely used instrument for conducting intracellular calcium release assays is the Fluorometric Imaging Plate Reader (FLIPR®, Molecular Devices). The FLIPR instrument is able to simultaneously add test compounds to all wells on appropriate microplates and take real-time measurements of the fluorescence of calcium bound dye, allowing accurate measurement of intracellular calcium levels. Similar experiments can be performed with a number of alternate, commercially available instruments or by the imaging of single cells or small numbers of cells with a fluorescence microscope.
Intracellular calcium levels can also be measured in cells engineered to express calcium sensitive proteins such as aequorin. Aequorin is a photoprotein isolated from jellyfish. While the calcium sensitive dyes used in FLIPR experiments require an excitation source in order to fluoresce, aequorin emits light in the presence of calcium without the need for an excitation source.
Receptors that do not normally couple to Gq G-proteins, such as the H3 receptor, can be artificially coupled to the IP/calcium signaling pathway through the use of promiscuous G-proteins (Gi5 and Gi6). These G-proteins signal through the Gq pathway and therefore regulate intracellular calcium release, but are promiscuous in the sense that they can couple to receptors that do not normally interact with Gq proteins. Alternatively, chimeric G-proteins may be used. These chimeric proteins typically utilize a Gq alpha protein in which approximately 5 amino acids at the carboxy- terminus are replaced with the corresponding amino acids from Gi alpha subunits. The resulting chimeric alpha subunit will recognize and be activated by Gi-coupled receptors but will signal though the Gq pathway to release intracellular calcium
In a typical FLIPR assay, cells expressing the H3 receptor and either a promiscuous G- protein (G15 or Gi6) or a chimeric G-protein (Gqi) are suspended in assay buffer and plated into black 384-well assay plates. Calcium dye is then added to the wells and allowed to incubate with the cells for one hour prior to test compound addition on an instrument such as a FLIPR. H3 receptor agonists will stimulate and H3 receptor inverse agonists will inhibit, respectively, calcium release. Antagonists and inverse agonists are typically detected by their ability to block the action of a selective H3 receptor agonist.
5. β-Arrestin Assays.
Activation of a GPCR typically results in receptor phosphorylation via a variety of kinases and then recruitment of β-arrestin from the cytosol. By monitoring the translocation of β-arrestin proteins from the cytosol to GPCRs in the cell membrane or quantitating the amount of receptor- arrestin complex formed in the cell, one can determine the extent of receptor activation. The recruitment of arrestin and the formation of arrestin-GPCR complexes can result from both constitutive receptor activity and the influence of test compounds, with agonists promoting arrestin- receptor complexation.
a. ) Arrestin translocation assays. In a typical arrestin translocation assay, cells expressing the receptor of interest are plated in transparent assay plates and allowed to fully adhere to the bottom of the wells. Test compounds are then added and incubated with the cells for up to an hour. The intracellular location of arrestin may be monitored by the use of cells recombinantly expressing a modified arrestin protein fused to a fluorescent protein such as green fluorescent protein (GFP). Alternatively, cells can be fixed and permeabilized, then treated with a fluorescently labeled anti-β- arrestin antibody.
b. ) Arrestin-receptor interaction assays. The association of β-arrestin with a GPCR can be monitored by tagging both β-arrestin and the receptor of interest with peptides or proteins that can interact to produce a biological readout when the receptor and arrestin are brought into close proximity upon receptor activation. Commercially available examples include Path Hunter® (DiscoveRx), in which the receptor and arrestin are tagged with complementary fragments of β- galactosidase that can combine via enzyme complementation upon receptor activation to produce active luciferase enzyme; and Tango® (Invitrogen), which utilizes an arrestin protein tagged with a protease and the receptor of interest tagged with a transcription factor at the cytoplasmic carboxyl terminus. The transcription factor is linked to the receptor via a protease-sensitive amino acid sequence and upon recruitment of protease-tagged arrestin, the transcription factor is cleaved from the receptor and translocates to the cell nucleus to initiate a transcriptional readout. Another commonly used technique for the measurement of arrestin-GPCR protein-protein interactions is bioluminescence resonance energy transfer (BRET). In this example, both arrestin and the receptor of interest are tagged with fluorescent proteins. One of the proteins is considered a donor while the other is an acceptor. Under conditions of low receptor activation, where arrestin is located primarily in the cytosol and the receptor is located primarily at the cell membrane, photoexcitation of the donor fluorescent protein, tagged to either the receptor or arrestin, results primarily in fluorescence emission from the donor. Upon receptor activation, resulting in close association of the receptor and arrestin, excitation of the donor protein is followed by BRET energy transfer to the acceptor protein and fluorescence emission from the acceptor protein at a longer wavelength that would be expected from the donor protein.
6. Reporter Gene Assays.
Activation of a GPCR typically results in changes in the activities of numerous protein signaling pathways within the cell. Some of these signaling pathways also lead to the modulations of intracellular concentrations of second messenger molecules such as cAMP, inositol phosphates, diacylglycerol and calcium. Many of these signaling pathways can ultimately result in a transcriptional response in the cell nucleus. Reporter gene assays take advantage of this response. Typically, cells are engineered to express a reporter gene product such as β-galactosidase or luciferase with gene expression regulated by a promoter that is sensitive to the type of signaling expected from the receptor of interest. In a typical example, cells expressing a receptor capable of modulating cAMP levels within the cell (Gi or Gs coupled) and a luciferase reporter gene under the control of a cAMP response element (CRE) can be used to determine intracellular cAMP levels. Activation of a Gs-coupled receptor or treatment with forskolin, leading to the production of cAMP, will increase reporter gene expression. Activation of a Gi coupled receptor such as the H3 receptor, leading to reductions in cAMP production, will reduce levels of reporter gene expression. For the constitutively active H3 receptor, agonists or inverse agonists would be detected by their ability to either decrease or increase forskolin-stimulated reporter gene expression, respectively. A neutral antagonist would be detected by its ability to block the actions of a selective H3 agonist.
Example 4: Administration of H3R Antagonists Significantly Inhibits Histamine Induced Itch (Histamine Induced Pruritus Model). H3R antagonists, 6-[(3-cyclobutyl-2,3,4,5-tetrahydro-l/i-3-benzazepin-7-yl)oxy]-Ai- methyl-3-pyridinecarboxamide (Compound B) and 4-(3-(4-(piperidin-l-yl)but-l- ynyl)benzyl)morpholine (Compound C), were dissolved in distilled water and saline, respectively, at the appropriate concentration and 100 μL was administered orally into 8-week-old C57BL 6 female mice. The selective H3R antagonist, (R)-2 -hydroxy- 1 -(6-(4-(2-(2-methylpyrrolidin- 1- yl)ethyl)phenyl)-3,4-dihydroisoquinolin-2(l//)-yl)ethanone (Compound A), was formulated in saline (Figure 5B and Figure 5C) or PBS (Figure 5D). Thirty minutes post-dosing, 25 μL· of 40 mg/mL histamine solution was injected subcutaneously at the rostral part of the back. Mice were video recorded and the number of scratching bouts that occurred between 5 and 25 minutes after histamine injection was counted. Compound B and Compound C were found to strongly inhibit histamine-induced itch after oral administration (Figure 5A). In addition, the selective H3R antagonist, (R)-2-hydroxy- 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2(l//)-yl)efhanone Compound A, inhibited histamine-induced scratching behavior in a dose dependent manner at oral doses of 0.3 mg kg and higher (Figure 5B). In separate experiments, Compound A also inhibited scratching induced by serotonin and compound 48/80 (Figure 5C and Figure 5D). To determine if Compound A is efficacious when applied topically, the test compound was prepared as a 0.2 or 0.6% ethanol suspension and 25 μL· of the suspension was applied topically onto a depilated area of the rostral part of the back, followed by intradermal injection of histamine 30 minutes later as described above. The 0.6% suspension was observed to significantly inhibit scratching behavior (Figure 6).
Example 5: H3R Antagonists Significantly Inhibit Itch in Allergic Models for Pruritus (DNP- Ovalbumin Pruritus Model).
To perform the DNV-ova pruritus model, female C57BL/6 mice (5/group) were sensitized via intraperitoneal administration of the allergen DNP-ova precipitated in aluminum hydroxide. 14 days later, mice were orally dosed with the indicated doses of dexamethasone, diphenhydramine (non-peripherally restricted HI antagonist), naloxone (opioid receptor antagonist), or Compound A 30 minutes prior to subcutaneous DNP-ova (100 μg in PBS) challenge. Scratching bouts were counted from 5 to 25 minutes (over a 20 minute period) post-DNP-ova challenge (Figure 7). Compound A inhibited pruritus in a dose dependent fashion.
To perform the DNFB allergic pruritus model, female C57BL/6 mice (5/group) were sensitized two times/week via epicutaneous administration of the allergen DNFB (0.3% in 4: 1 acetone: olive oil) on the rostral back skin. On the 14th day after initial DNFB administration, mice were challenged again with epicutaneous DNFB; at 5h post-DNFB challenge, mice were dosed with Compound 1 at 1, 3, 10, and 30 mg/kg, IP. Scratching bouts were counted from 30-50 minutes post-compound dosing (Figure 9). Compound 1 inhibited pruritus in a dose dependent fashion. These data demonstrate that H3R antagonists can inhibit pruritus mediated by endogenous pruritogens released from an immune response.
These data demonstrate that H3R antagonists can inhibit pruritus mediated by endogenous pruritogens released from an immune response.
Example 6: Central Administration of a H3R Antagonist Does not Inhibit Histamine-induced Pruritus.
When administered peripherally, centrally penetrant H3R antagonists such as Compound A have many effects in the rodent. These include increasing cognition, and inhibiting HA-induced pruritus. In order to assess whether there was a brain-mediated component to blockade of HA- induced pruritus, Compound A was administered directly into the brain, and effects on both HA- induced pruritus and performance in a cognitive task were measured. Male C57BL/6 mice were implanted with chronically indwelling cannulae aimed at the lateral ventricle. Cannulated mice (6- 7/group) were dosed with either vehicle (PO) and vehicle (ICV), Compound A (10 mg/kg PO) and vehicle (ICV), or vehicle (PO) and Compound A (10μg ICV). Scratching bouts were counted from 5-25 minutes post-histamine challenge. In the Object Recognition task, cannulated mice were allowed to explore an open-field (50 X 37 X 24 cm) for five minute habituation sessions on two consecutive days. On day three (training day), two identical objects were presented near opposite corners of the open-field (10 cm from walls), and mice allowed to explore the objects for 10 min. Immediately after training, mice were removed from the arena and injected ICV with either vehicle or Compound A (10μg). 24h later, subjects were placed back in the open field in which one of the previously presented objects was replaced by a novel object. Subjects were allowed to explore the two objects for a five-minute session during which their behavior was video recorded for subsequent analysis. The difference between exploration times of the novel and familiar objects as a factor of total object exploration time ((N-F)/(N+F)) was taken as a measure of performance (cognitive index). These data demonstrated that an intracerebro ventricular (ICV) dose of
Compound A increased cognitive function (Figure 8A) but was unable to inhibit HA-induced pruritus (Figure 8B). This suggests that brain exposure is not sufficient for activity in the HA- induced pruritus test, and therefore suggests that HA-induced pruritus is peripherally mediated.
Example 7: Histamine-induced Model for Pruritus.
H3R inhibitors, Compound 1 and Compound 3, were dissolved in saline at the appropriate concentration and 100 μL was administered orally (Compound 3) or intraperitoneally (Compound 1) into 8-week-old C57BL/6 female mice. Thirty minutes post-dosing, 25 μL of 40 mg/mL histamine solution was injected subcutaneously at the rostral part of the back. Mice were video recorded and the number of scratching bouts that occurred between 5 and 25 minutes after histamine injection was counted. Both compounds were found to inhibit histamine-induced scratching behavior in mice, see Figure 10 (Compound 1) and Figure 11 (Compound 3).
Example 8. Identification of Peripherally Restricted H3R Antagonists
Identification of peripherally restricted H3R antagonists can be performed, for example, using a caco-2 permeability assay. The Caco-2 cell line was purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and grown with Minimum Essential Medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 1 % nonessential amino acids at 37°C in 5% C02. Multi-well insert systems with an array of 12 or 24 individual inserts were used in the permeability assay. Each multi-well insert unit has two compartments: the top compartment and the bottom compartment. The top compartment is the insert and is commonly referred to as the apical compartment or apical side (A). The bottom compartment is the well of a multi-well plate where the insert is placed and is referred to as the basal compartment or basal side (B). In each insert, Caco-2 cells were seeded on a porous filter membrane. Cell culture medium was changed every two days. Cells were used for the transport experiments between 24 to 26 days after seeding.
The bidirectional transport of H3 compounds (final concentration: 10 μΜ) and the known P-gp substrate 3H-digoxin (-15 nM) across Caco-2 cell monolayers was determined in the absence and presence of the known P-gp inhibitor cyclosporin A (5 μΜ). All samples from the permeability experiments of H3 compounds were mixed with acetonitrile containing internal standard. After protein precipitation by centrifugation, supernatant was analyzed using LC/MS/MS. Samples from 3H-digoxin transport experiment were mixed with liquid scintillation counting cocktail and counted with a liquid scintillation counter. The apparent permeability coefficients (Papp) were calculated using the following equation:
Papp = (AQ/At)/(A x Co)
where AQ/At is the appearance rate (DPM/sec of radiolabeled test compound; μηιοΐ/sec of non- labeled test compound) on the receiver side during the permeation process, A is the surface area of the cell monolayers, and Co is the initial concentration (DPM/mL of radiolabeled test compound; μηιοΙ/mL of non-labeled test compound) on the donor side.
Efflux ratio was defined as the ratio of Papp in the basolateral to apical direction over the
Papp in the apical to basolateral direction.
Example 9: Blockade of RAMH-Induced Drinking Assay.
When administered to rodents, H3 agonists, such as R-a-methyl-histamine (RAMH), induce a drinking response that is sensitive to reversal with an H3 antagonist. Blockade of RAMH- induced drinking can therefore be utilized as an in vivo assay for functional H3 antagonist activity. In this assay, male Sprague Dawley rats (250-350 g) were housed three per cage and maintained under a reverse 12 h light cycle (lights off at 11 : 30 h) . At 10: 30 h on the day of test, rats were individually housed in new cages and food was removed. 120 min later, rats were administered test article (vehicle or H3 antagonist, 0.3 mg kg PO). 30 min later, water was removed, and RAMH (vehicle or RAMH 3 mg kg salt SC) was administered. 10 min after administration of RAMH, weighed water bottles were placed in the cages, and drinking was allowed for 20 min. Water consumption was determined for each animal by weighing each bottle to the nearest 0.1 g. Data is expressed as percentage reduction in water intake according to the following formula:
[l-[(antagonist/RAMH) - (vehicle/RAMH) / (vehicle/RAMH) - (vehicle/vehicle)]]* 100
Figure imgf000123_0001
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptions, or modifications, as come within the scope of the following claims and its equivalents.

Claims

What is claimed is:
1. A method of preparing a pharmaceutical composition comprising formulating an anti- pruritic agent with a pharmaceutically acceptable carrier; wherein said anti-pruritic agent is a H3R antagonist.
2. A method of preparing a pharmaceutical composition comprising a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having contacted in vitro with a host cell or with a membrane of a host cell comprising an activated G protein- coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1 -445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); and determined to inhibit the functionality of the receptor; wherein the ability of said H3R antagonist to inhibit the functionality of the receptor is indicative of said H3R antagonist being an anti-pruritic agent, said method comprising formulating said H3R antagonist having the effect of an anti-pruritic agent with a pharmaceutically acceptable carrier as a pharmaceutical composition.
3. A method of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
(a) determining the ability of a test compound to inhibit the functionality of an activated G protein-coupled receptor in a host cell or with a membrane of a host cell, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1 -445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2; (iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 ;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
wherein the ability of said test compound to inhibit the functionality of the activated G protein-coupled receptor in a host cell or with a membrane of a host cell is indicative of said test compound being an anti-pruritic agent; and
(b) formulating said anti-pruritic agent with a pharmaceutically acceptable carrier as a pharmaceutical composition.
A method of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1 -445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of said test compound to inhibit the functionality of the receptor; wherein the ability of said test compound to inhibit the functionality of the receptor is indicative of said test compound being an anti-pruritic agent; and
(c) formulating said anti-pruritic agent having the effect of a H3R antagonist with a pharmaceutically acceptable carrier as a pharmaceutical composition.
A method of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1 -445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of said test compound to inhibit the functionality of the receptor; wherein the ability of said test compound to inhibit the functionality of the receptor is indicative of said test compound being an anti-pruritic agent; and
(c) admixing said anti-pruritic agent with a pharmaceutically acceptable carrier.
A method of preparing a pharmaceutical composition comprising a H3R antagonist having the effect of an anti-pruritic agent, said H3R antagonist having obtained a decreased response for said H3R antagonist in a pruritus model, wherein the ability of said H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent, said method comprising formulating said H3R antagonist having the effect of an anti-pruritic agent with a pharmaceutically acceptable carrier as a pharmaceutical composition.
A method of preparing a pharmaceutical composition comprising an anti-pruritic agent having the effect of a H3R antagonist, said method comprising:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1 -445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2; (iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 ;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of said test compound to inhibit the functionality of the receptor;
(c) obtaining a decreased response for said test compound in a pruritus model, wherein the ability of said test compound to decrease the response in said pruritus model is indicative of said test compound being an anti-pruritic agent; and
(d) formulating said anti-pruritic agent having the effect of a H3R antagonist with a pharmaceutically acceptable carrier as a pharmaceutical composition.
A method for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane of a host cell comprising an activated G protein-coupled receptor, wherein said G protein-coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1 -445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 ;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of said test compound to inhibit the functionality of the receptor; and
(c) observing a decreased response in a pruritus model test animal previously administered said test compound;
wherein the ability of said test compound to decrease said response in the animal indicative of said test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
A method for identifying an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual, comprising the steps of: (a) contacting a G protein-coupled receptor with an optionally labeled known ligand to the receptor in the presence or absence of a test compound, wherein said G protein- coupled receptor comprises an amino acid sequence selected from the group consisting of:
(i) amino acids 1 -445 of SEQ ID NO:2;
(ii) amino acids 2-445 of SEQ ID NO:2;
(iii) amino acids 2-445 of SEQ ID NO:2, with the proviso that the receptor does not comprise the amino acid sequence of SEQ ID NO:2;
(iv) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide hybridizing under conditions of high stringency to the complement of SEQ ID NO: 1 ;
(v) a variant of SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v);
(b) detecting the complex between said known ligand and said receptor;
(c) determining whether less of said complex is formed in the presence of said test compound than in the absence of said test compound; and
(d) observing a decreased response in a pruritus model test animal previously administered said test compound;
wherein the ability of said test compound to decrease said response in the animal is indicative of said test compound being an anti-pruritic agent useful for the treatment of a condition characterized by itch in an individual.
A method for identifying an anti-pruritic agent for treating a condition characterized by itch in an individual, comprising the steps of:
(a) administering a H3R antagonist to a pruritus model; and
(b) determining whether said H3R antagonist decreases a response in said pruritus model;
wherein the ability of said H3R antagonist to decrease the response in said pruritus model is indicative of said H3R antagonist being an anti-pruritic agent useful for the treatment of said condition characterized by itch in said individual.
11. A pharmaceutical composition prepared by the process according to any one of claims 1 to 7.
A pharmaceutical composition comprising a H3R antagonist and a pharmaceutically acceptable carrier, wherein said pharmaceutical composition is for the use in treating pruritus. A pharmaceutical composition according to claim 12, wherein said H3R antagonist is identified by the method according to any one of claims 8 to 10.
A method for treating pruritus in an individual comprising administering to said individual in need thereof a therapeutically effective amount of a H3R antagonist; or a pharmaceutical composition according to claim 11.
A method according to claim 13, wherein said H3R antagonist is identified by the method according to any one of claims 8 to 10.
Use of a H3R antagonist in the manufacture of a medicament for treating pruritus.
A use according to claim 14, wherein said H3R antagonist is identified by the method according to any one of claims 8 to 10.
A H3R antagonist for use in a method for the treatment of pruritus.
A H3R antagonist according to claim 15, wherein said H3R antagonist is identified by the method according to any one of claims 8 to 10.
A pharmaceutical composition according to claim 12; a method according to claim 13; a use according to claim 14; or a H3R antagonist according to claim 15; wherein said pruritus is associated with a disease or disorder selected from: eczema, atopic eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, irritant dermatitis, xerosis (dry skin), psoriasis, a fungal infection, athlete's foot, a yeast infection, diaper rash, vaginal itch, parasitic infections, parasitic infestations including scabies and lice, lichen planus, lichen simplex, lichen simplex chronicus, lichen sclerosis, itch secondary to medications, senile itch, uremia, idiopathic itch, itch associated with liver cirrhosis, itch associated with inflammation, itch associated with allergies, itch associated with cancer, itch associated with chemotherapy, itch associated with kidney disease, itch associated with haemodialysis, burns, scalds, sunburn, wound healing, itch associated with an insect bite, itch associated with a flea bite, itch associated with an insect sting, itch associated with a mosquito sting, itch associated with a mite bite, urticaria, urticaria caused by a plant, urticaria caused by poison ivy, urticaria caused by stinging nettle, sweat gland abnormalities, bullous pemphigoid, photodermatoses, skin blisters, adult acne, chicken pox, and dermatitis herpetiformis. A method according to any one of claims 1 to 10, 13, and 16 ; pharmaceutical composition according to any one of claims 11, 12, and 16; a use according to claim 14 or 16; or a H3R antagonist according to claim 15 or 16; wherein said anti-pruritic agent or said H3R antagonist is selected from compounds of Formula (la) and
pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000131_0001
(la)
wherein:
R1 and R2 are each selected independently from the group consisting of H, Ci-C6 acyl, Ci-Cs alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C4-alkylenyl, aryloxy-Ci-C4-alkylenyl, heteroaryl-Ci-C4-alkylenyl and heteroaryloxy-Ci-C4-alkylenyl, and each R1 and R2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2- C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, aryl, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo- Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8
dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, heterocyclyl, hydroxyl, thiol, nitro and sulfonamide; wherein each Ci-C8 alkyl may be further substituted with hydroxy;
or
R1 and R2 together with the nitrogen atom to which they are both bonded form a C3-C7 heterocyclyl or a C5-C10 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C1-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6- alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2- C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6
haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; wherein each Ci-C8 alkyl and carboxy may be further substituted with C1-C6 acyloxy, C1-C6 alkoxy, aryl-Ci-C -alkylenyl, or hydroxy;
or R2 is selected independently from the group consisting of H, C1-C6 acyl, Ci-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C4-alkylenyl, aryloxy-Ci-C4-alkylenyl, heteroaryl-Ci-C4-alkylenyl and heteroaryloxy-Ci-C4-alkylenyl, and each R2 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, aryl, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo- Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8
dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, heterocyclyl, hydroxyl, thiol, nitro and sulfonamide;
and
R1 and R12 together with the atoms to which they are both bonded form a C6-C8 heterocyclyl group optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8
alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, aryl, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6
haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, heterocyclyl, hydroxyl, thiol, nitro and sulfonamide;
J is -CH2CH2- or a l,2-C3-C7-cycloalkylenyl group, each optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of Ci-C3 alkyl, Ci-C alkoxy, carboxy, cyano, Ci-C3 haloalkyl, halogen, hydroxyl and oxo;
R3, R4, R5, R6, R7, R10, R11 and R12 are each selected independently from the group consisting of H, Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3 C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
and
R8 and R9 are each selected independently from the group consisting of H, Ci-C alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C7 cycloalkyl, aryl, heterocyclyl, heteroaryl, aryl-Ci-C4-alkylenyl, aryloxy-Ci-C4-alkylenyl, heteroaryl-Ci-C4-alkylenyl and heteroaryloxy-Ci-C4-alkylenyl, and each R8 and R9 is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of Ci-C6 acyl, Ci- C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6- alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2- C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6 haloalkylsulfinyl, Ci-C6 haloalkylsulfonyl, Ci-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
or
R8 and R9 together with the nitrogen atom to which they are both bonded form a C3-C7 heterocyclyl or a C5-Ci0 heterobicyclyl group each optionally substituted with 1, 2, 3, 4, 5 or 6 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6- alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2- C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide; provided that the compound is other than:
N-(3-cyanophenyl)-iV- [2- [4'- [[( 1 , 1 -dimethylethyl) amino] sulfonyl] [1, 1'- biphenyl]-4-yl]ethyl]-glycine methyl ester;
N- [[4'- [2-oxo-2-(phenylamino)ethyl] [1,1 '-biphenyl] -4-yl] sulfonyl] -D-valine 1 , 1 -dimethylethylester;
N- [[4'- [2-0X0-2- [(phenylmethyl)amino] ethyl] [1, 1 '-biphenyl] -4-yl] sulfonyl] -D- valine 1,1 -dimethylethyl ester;
N- [[4'- [2-oxo-2-(phenylamino)ethyl] [1,1 '-biphenyl] -4-yl] sulfonyl] -D-valine; or
N- [[4'- [2-0X0-2- [(phenylmethyl)amino] ethyl] [1, 1 '-biphenyl] -4-yl] sulfonyl] -D- valine.
A method according to any one of claims 1 to 10, 13, and 16 ; pharmaceutical composition according to any one of claims 11, 12, and 16; a use according to claim 14 or 16; or a H3R antagonist according to claim 15 or 16; wherein said anti-pruritic agent or said H3R antagonist is selected from compounds of Formula (Ila) and
pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000134_0001
(Ha)
wherein:
R1 is selected from the group consisting of H, C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6
haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, C3-C7 heterocyclyl, hydroxyl, thiol, nitro, phenyl and sulfonamide, and each is optionally substituted with 1, 2, 3, 4 or 5 substituents selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, C1-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2- C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6- alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2- C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6
haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide; or
R1 together with the W-S02 group and the ring atom to which the W-S02 group is bonded form a C5-C7 heterocyclic ring with Ring A whereby said C5-C7 heterocyclic ring and Ring A share two adjacent ring atoms, and said C5-C7 heterocyclic ring is optionally substituted with 1, 2, 3 or 4 substituents selected independently from the group consisting of Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3- C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro, oxo and sulfonamide;
W is C1-C4 alkylene, C2-C4 alkenylene, C3-C7 cycloalkylene, C3-C7 heterocyclylene or phenylene, each optionally substituted with 1, 2, 3, 4, 5, 6, 7 or 8 substituents selected independently from the group consisting of C1-C3 alkyl, C1-C4 alkoxy, carboxy, cyano, C1-C3 haloalkyl, halogen, hydroxyl and oxo; Ring A is 1,3-phenylene or 1,4-phenylene, each substituted with R , R , R and R15, wherein R12, R13, R14 and R15 are each selected independently from the group consisting of H, Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2- C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide; or
Ring A is a 6-membered heteroarylene or a 5-membered heteroarylene, each optionally substituted with R16, R17 and R18, wherein R16, R17 and R18 are each selected independently from the group consisting of C1-C6 acyl, C1-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8
alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2-C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, Ci-C6 haloalkoxy, Ci-C6 haloalkyl, Ci-C6
haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
R2, R3, R4 and R5 are each selected independently from the group consisting of
H, Ci-C6 acyl, Ci-C6 acyloxy, C2-C8 alkenyl, Ci-C6 alkoxy, Ci-C8 alkyl, Ci-C8 alkylcarboxamide, C2-C8 alkynyl, Ci-C8 alkylsulfonamide, Ci-C8 alkylsulfinyl, Ci-C8 alkylsulfonyl, Ci-C8 alkylthio, Ci-C8 alkylureyl, amino, Ci-C8 alkylamino, C2-C8 dialkylamino, carbo-Ci-C6-alkoxy, carboxamide, carboxy, cyano, C3-C7 cycloalkyl, C2- C8 dialkylcarboxamide, C2-C8 dialkylsulfonamide, halogen, C1-C6 haloalkoxy, C1-C6 haloalkyl, C1-C6 haloalkylsulfinyl, C1-C6 haloalkylsulfonyl, C1-C6 haloalkylthio, hydroxyl, thiol, nitro and sulfonamide;
R6, R7, R8 and R9 are each selected independently from the group consisting of H, C1-C3 alkyl, C1-C4 alkoxy, carboxy, cyano, C1-C3 haloalkyl, halogen and hydroxyl; and
R10 and R11 together with the nitrogen atom to which they are both bonded form 2-methyl-pyrrolidin- 1-yl;
provided:
1) that Ring B and the sulfur of the R1-W-S(0)2- group are not bonded to adjacent ring atoms of Ring A; and 2) if Ring A is 1,3-phenylene or 1,4-phenylene, and W is C3-C7 heterocyclylene, then the ring atom of W that is directly bonded to the sulfur of the R^W-SfO^- group is other than nitrogen. 19. A method according to any one of claims 1 to 10, 13, and 16 ; pharmaceutical
composition according to any one of claims 11, 12, and 16; a use according to claim 14 or 16; or a H3R antagonist according to claim 15 or 16; wherein said anti-pruritic agent or said H3R antagonist is selected from compounds of Formula (Ilia) and
pharmaceutically acceptable salts, solvates, and hydrates thereof:
Figure imgf000136_0001
wherein:
ring A is heterocyclyl optionally substituted with one, two or three substituents selected from Ci-C6 alkyl and oxo; wherein each Ci-C6 alkyl is optionally substituted with a C1-C6 alkoxy substituent;
R1 is H, C1-C6 alkoxy, Ci-C6 alkyl or halogen;
R2 is H, Ci-C6 alkoxy, Ci-C6 alkyl or halogen;
R3 is H, C1-C6 alkoxy, Ci-C6 alkyl or halogen;
R4 is H or Ci-C4 alkyl; and
n is 0, 1 or 2.
20. A method according to any one of claims 1 to 10, 13, and 16 ; pharmaceutical
composition according to any one of claims 11, 12, and 16; a use according to claim 14 or 16; or a H3R antagonist according to claim 15 or 16; wherein said anti-pruritic agent or said H3R antagonist is selected from compounds of Formula (IVa) and
pharmaceutically acc
Figure imgf000136_0002
(IVa)
wherein:
R is H or C1-C4 alkyl;
R2 is H or halogen; R3 is H, C1-C4 alkyl or C3-C6 cycloalkyl, and R4 is H; or R3 and R4 together with the atom to which they are both bonded form a C3-C6 cycloalkyl;
R5 is selected from: C1-C6 alkyl, aryl, C3-C6 cycloalkyl, heteroaryl and heterocyclyl; each of which is optionally substituted with one or more substituents selected from: C1-C6 alkoxy, halogen, heterocyclyl and hydroxyl;
R6, R7 and R8 are each independently selected from: H, C1-C6 alkoxy, C1-C6 alkyl, amino, halogen, heterocyclyl and hydroxyl;
m is 0 or 1 ;
n is 1 or 2; and
V is CH2, O or absent.
A method according to any one of claims 1 to 10, 13, and 16 ; pharmaceutical composition according to any one of claims 11, 12, and 16; a use according to claim 14 or 16; or a H3R antagonist according to claim 15 or 16; wherein said anti-pruritic agent or said H3R antagonist is selected from and the following compounds and pharmaceutically acceptable salts, solvates, and hydrates thereof:
(R)- 1 - { 2-[4'-(3-methoxy-propane- 1 -sulfonyl)-biphenyl-4-yl] -ethyl } -2-methyl- pyrrolidine;
(R)-2-hydroxy- 1 -(6-(4-(2-(2-methylpyrrolidin- 1 -yl)ethyl)phenyl)-3,4- dihydroisoquinolin-2( 1 //)-yl)ethanone;
6-[(3-cyclobutyl-2,3,4,5-tetrahydro-l/i-3-benzazepin-7-yl)oxy]-Ai-methyl-3- pyridinecarboxamide; and
4-(3-(4-(piperidin-l-yl)but-l-ynyl)benzyl)morpholine.
A method according to any one of claims 2 to 5 and 7 to 9, wherein said G protein-coupled receptor comprises the amino acids 1-445 of SEQ ID NO: 2.
A method according to claim 2 to 5, 7, 8, and 22, wherein said host cell comprises an expression vector, the expression vector comprising a polynucleotide encoding said G protein-coupled receptor.
A method according to claim 2 to 5, 7, 8, 22, and 23, wherein said activated G protein- coupled receptor is constitutively active or is activated by a ligand.
A method according to any one of claims 2 to 5, 7, 8, and 22 to 24, wherein said determining the ability of the test compound to inhibit the functionality of the receptor is through the measurement of a level of a second messenger. A method according to claim 25, wherein the second messenger is selected from the group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol 1,4,5-triphosphate (IP3), diacylglycerol (DAG), MAP kinase activity, MAPK/ERK kinase kinase- 1 (MEKK1) activity, and Ca2+.
A method according to claim 26, wherein the second messenger is cAMP and the level of cAMP is decreased.
A method according to any one of claims 2 to 5, 7, 8, and 22 to 24, wherein said determining the ability of the test compound to inhibit the functionality of the receptor is through the use of a Melanophore assay, or through the measurement of GTPyS binding to a membrane comprising said GPCR.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist has an IC50 (H3R) of about 100 nM or less.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist has an IC50 (H3R) of about 10 nM or less.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is a selective H3R antagonist.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist has a selectivity for H3R over H1R, H2R, or H4R of at least about 10 fold. A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is administered orally and has an IC50 (H3R) of about 100 nM or less.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is applied topically.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is applied topically to the site afflicted with itch.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is applied topically and has an IC50 (H3R) of about 100 nM or less.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is an antagonist of the human H3R.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is in an amount sufficient to reduce itch in a human. A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist has a brain to plasma ratio of about 0.5 or less after administration in a pruritus model animal.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist has a brain to plasma ratio of about 0.2 or less after administration in a pruritus model animal.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti -pruritic agent or said H3R antagonist is administered orally.
A method according to any one of claims 1 to 10, 13, and 16 to 28; pharmaceutical composition according to any one of claims 11, 12, and 16 to 28; a use according to any one of claims 14 and 16 to 28; or a H3R antagonist according to any one of claims 15, and 16 to 28; wherein said anti-pruritic agent or said H3R antagonist is peripherally restricted.
A method according to any one of claims 6 to 10, 17 to 21, 39, and 40, wherein said pruritus model is a primate model, a dog model, a guinea pig model, a mouse model, or a cat model.
A method according to any one of claims 6 to 10, 17 to 21, 39, 40, and 43, wherein said pruritus model is selected from the group: pruritogen injection model, passive cutaneous anaphylaxis model, allergic pruritus model, and spontaneous pruritus model.
A method according to any one of claims 6 to 10, 17 to 21, 39, 40, 43, and 44, wherein said pruritus model is selected from the group: histamine induced pruritus model, DNP- Ovalbumin pruritus model, and DNFB pruritus model.
A method according to any one of claims 3 to 5, 7, 8, and 17 to 21, wherein determining the ability of said test compound to inhibit functionality of the receptor comprises comparing the functionality of the receptor in the presence and absence of said test compound and observing a decreased functionality in the presence of said test compound as compared to in the absence of said test compound.
47. A method according to any one of claims 8 to 10 and 17 to 46, further comprising the step of subsequently admixing said anti-pruritic agent with a pharmaceutical carrier to form an anti-pruritic pharmaceutical composition.
48. A method according to claim 47, further comprising the step of formulating said antipruritic pharmaceutical composition into a form suitable for oral application.
49. A method according to claim 47, further comprising the step of formulating said antipruritic pharmaceutical composition into a form suitable for topical application.
PCT/US2013/034925 2012-04-03 2013-04-02 Methods and compounds useful in treating pruritus, and methods for identifying such compounds WO2013151982A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US201261619594P 2012-04-03 2012-04-03
US61/619,594 2012-04-03
US201261682400P 2012-08-13 2012-08-13
US61/682,400 2012-08-13
US201361792503P 2013-03-15 2013-03-15
US61/792,503 2013-03-15

Publications (1)

Publication Number Publication Date
WO2013151982A1 true WO2013151982A1 (en) 2013-10-10

Family

ID=48366558

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/034925 WO2013151982A1 (en) 2012-04-03 2013-04-02 Methods and compounds useful in treating pruritus, and methods for identifying such compounds

Country Status (1)

Country Link
WO (1) WO2013151982A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105640980A (en) * 2016-01-29 2016-06-08 郑锦辉 Medicine for burns and scalds and preparation method thereof
WO2020257753A1 (en) * 2019-06-20 2020-12-24 Yale University Phosphatidylserine decarboxylase inhibitors

Citations (177)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1980A (en) 1841-02-13 Mode oe
US774216A (en) 1903-11-19 1904-11-08 Alexander F Ward Machine for pointing and lapping hoops.
US4166452A (en) 1976-05-03 1979-09-04 Generales Constantine D J Jr Apparatus for testing human responses to stimuli
US4256108A (en) 1977-04-07 1981-03-17 Alza Corporation Microporous-semipermeable laminated osmotic system
US4265874A (en) 1980-04-25 1981-05-05 Alza Corporation Method of delivering drug with aid of effervescent activity generated in environment of use
US4704362A (en) 1977-11-08 1987-11-03 Genentech, Inc. Recombinant cloning vehicle microbial polypeptide expression
WO1992015567A1 (en) 1991-02-27 1992-09-17 Seed Capital Investment (Sci) B.V. Imidazole-derivatives having agonistic or antagonistic activity on the histamine h3-receptor
WO1993012093A1 (en) 1991-12-18 1993-06-24 Schering Corporation Imidazolyl-alkyl-piperazine and -diazepine derivatives as histamine h3 agonists/antagonists
WO1993012108A1 (en) 1991-12-18 1993-06-24 Schering Corporation Imidazolyl or imidazoylalkyl substituted with a four or five membered nitrogen containing heterocyclic ring
WO1993012107A1 (en) 1991-12-18 1993-06-24 Schering Corporation Imidazoylalkyl substituted with a six membered nitrogen containing heterocyclic ring
WO1993020062A1 (en) 1992-04-01 1993-10-14 The University Of Toledo Process for the preparation of 4-pyridyl- and 4-piperidinylimidazoles useful as intermediates for the synthesis of histamine receptor antagonists
WO1993020061A1 (en) 1992-04-01 1993-10-14 The University Of Toledo 4-[4'-piperidinyl or 3'-pirrolidinyl] substituted imidazoles as h3-receptor antagonists and therapeutic uses thereof
WO1995006037A1 (en) 1993-08-27 1995-03-02 Vrije Universiteit New imidazole derivatives having agonistic or antagonistic activity on the histamine h3 receptor
WO1995011894A1 (en) 1993-10-29 1995-05-04 The University Of Toledo Histamine h3-receptor antagonists and therapeutic uses thereof
US5462856A (en) 1990-07-19 1995-10-31 Bunsen Rush Laboratories, Inc. Methods for identifying chemicals that act as agonists or antagonists for receptors and other proteins involved in signal transduction via pathways that utilize G-proteins
WO1996038142A1 (en) 1995-05-30 1996-12-05 Gliatech, Inc. 1h-4(5)-substituted imidazole derivatives
WO1996038141A1 (en) 1995-05-30 1996-12-05 Gliatech, Inc. 2-(1h-4(5)-imidazoyl) cyclopropyl derivatives
WO1996040126A1 (en) 1995-06-07 1996-12-19 Gliatech, Inc. 1h-4(5)-substituted imidazole derivatives
WO1998006394A1 (en) 1996-08-16 1998-02-19 Schering Corporation Treatment of upper airway allergic responses with a combination of histamine receptor antagonists
WO1999006377A1 (en) 1997-08-01 1999-02-11 Gliatech, Inc. Derivatives of 1-((3,3-dimethyl-3-silabutoxy)methyl)-2-(phenylsulfonyl)imidazole
WO1999024421A1 (en) 1997-11-07 1999-05-20 Schering Corporation Imidazoylalkyl substituted with a five, six or seven membered heterocyclic ring containing one nitrogen atom
US5922576A (en) 1998-02-27 1999-07-13 The John Hopkins University Simplified system for generating recombinant adenoviruses
WO1999042458A1 (en) 1998-02-19 1999-08-26 James Black Foundation Limited Histamine h3 receptor ligands
WO2000006254A2 (en) 1998-07-29 2000-02-10 Societe Civile Bioprojet Non-imidazole alkylamines as histamine h3-receptor ligands and their therapeutic applications
WO2000023438A1 (en) 1998-10-16 2000-04-27 Schering Corporation N-(imidazolylalkyl)substituted cyclic amines as histamine-h3 agonists or antagonists
WO2000042023A1 (en) 1999-01-18 2000-07-20 Novo Nordisk A/S Substituted imidazoles, their preparation and use
WO2000053596A2 (en) 1999-03-08 2000-09-14 Schering Corporation Imidazole compounds as histamine h3 ligands
WO2001066534A2 (en) 2000-03-09 2001-09-13 Abbott Laboratories Cyclic and bicyclic diamino histamine-3 receptor antagonists
WO2001068652A1 (en) 2000-03-17 2001-09-20 Novo Nordisk A/S Condensed imidazoles as histamine h3 receptor ligands
WO2001074813A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. METHOD FOR USING 2- OR 3-ARYL SUBSTITUTED IMIDAZO[1,2-a] PYRIDINES AS H3 ANTAGONISTS
WO2001074773A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Phenyl-substituted indoles as histamine h3-receptor antagonists
WO2001074814A1 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Phenyl-substituted indolizine derivatives and their use as histamine h3 ligands
WO2001074815A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Phenyl-substituted imidazopyridines
WO2001074810A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Method for using 2-aryloxyalkylaminobenzoxazoles and 2-aryloxyalkylaminobenzothiazoles as h3 antagonists
WO2002012214A2 (en) 2000-08-08 2002-02-14 Ortho Mcneil Pharmaceutical Inc. Non-imidazole aryloxyalkylamines as h3 receptor ligands
WO2002012224A2 (en) 2000-08-08 2002-02-14 Ortho Mcneil Pharmaceutical, Inc. Bicyclic compounds as h3 receptor ligands
WO2002012190A2 (en) 2000-08-08 2002-02-14 Ortho Mcneil Pharmaceutical, Inc. Non-imidazole aryloxypiperidines as h3 receptor ligands
WO2002024659A2 (en) 2000-09-20 2002-03-28 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antgonists
WO2002024695A2 (en) 2000-09-22 2002-03-28 Ortho Mcneil Pharmaceutical, Inc. Octahydro-indolizines and quinolizines and hexahydro-pyrrolizines
WO2002024657A2 (en) 2000-09-20 2002-03-28 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antagonists
WO2002024658A2 (en) 2000-09-20 2002-03-28 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antagonists
WO2002032893A2 (en) 2000-10-17 2002-04-25 Schering Corporation Piperidine compounds as anti-allergic
WO2002042461A2 (en) 2000-11-27 2002-05-30 Arena Pharmaceuticals, Inc. Endogenous and non-endogenous versions of human g protein-coupled receptors
WO2002044141A2 (en) 2000-09-20 2002-06-06 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antagonists
WO2002056871A2 (en) 2001-01-17 2002-07-25 Pfizer Limited Histamine receptor antagonists
WO2002064212A1 (en) 2001-02-13 2002-08-22 Cornell Research Foundation, Inc. Method and compositions for reducing cardiac dysfunctions with a selective histamine h3 receptor agonist
WO2002072093A2 (en) 2001-02-08 2002-09-19 Schering Corporation Use of dual h3/m2 antagonists with a bipiperidinic structure in the treatment of cognition deficit disorders
WO2002076925A2 (en) 2001-03-23 2002-10-03 Eli Lilly And Company Non-imidazole aryl alkylamines compounds as histamine h3 receptor antagonists, preparation and therapeutic uses
WO2003024928A2 (en) 2001-09-14 2003-03-27 Novo Nordisk A/S Novel aminoazetidine, -pyrrolidine and -piperidine derivatives
WO2003024929A1 (en) 2001-09-14 2003-03-27 Novo Nordisk A/S Substituted piperidines with selective binding to histamine h3-receptor
WO2003031432A1 (en) 2001-10-12 2003-04-17 Novo Nordisk A/S Substituted piperidines and their use for the treatment of diseases related to the histamine h3 receptor
WO2003033488A1 (en) 2001-10-15 2003-04-24 Schering Corporation Synthesis of 4-(piperidyl) (2-pyridyl)methanone-(e)-o-methyloxime and its salts
WO2003040106A1 (en) 2001-11-09 2003-05-15 Novo Nordisk A/S Preparation and use of substituted imidazoles
WO2003042359A2 (en) 2001-11-13 2003-05-22 Merck & Co. Inc. Histamine receptor h3 polynucleotides
WO2003064411A1 (en) 2002-02-01 2003-08-07 Novo Nordisk A/S Amides of aminoalkyl-substituted azetidines, pyrrolidines, piperidines and azepanes
WO2003066604A2 (en) 2002-02-05 2003-08-14 Novo Nordisk A/S Novel aryl- and heteroarylpiperazines
WO2003088967A1 (en) 2002-04-18 2003-10-30 Schering Corporation (1-4-piperidinyl) benzimidazole derivatives useful as histamine h3 antagonists
WO2003099821A1 (en) 2002-05-24 2003-12-04 X-Ceptor Therapeutics, Inc. Azepinoindole and pyridoindole derivatives as pharmaceutical agents
WO2003103669A1 (en) 2002-04-18 2003-12-18 Schering Corporation 1-(4-piperidinyl) benzimidazolones as histamine h3 antagonists
WO2003104235A1 (en) 2002-06-06 2003-12-18 Novo Nordisk A/S Substituted hexahydropyrrolo[1,2-a]pyrazines, octahydropyrido[1,2-a]pyrazines and decahydropyrazino[1,2-a]azepines
WO2004000831A1 (en) 2002-06-24 2003-12-31 Schering Corporation Indole derivatives useful as histamine h3 antagonists
WO2004009015A2 (en) 2002-07-18 2004-01-29 Merck & Co., Inc. Combination therapy for the treatment of obesity
WO2004018432A1 (en) 2002-08-20 2004-03-04 Eli Lilly And Company Substituted azepines as histamine h3 receptor antagonists, preparation and therapeutic uses
WO2004026837A2 (en) 2002-09-18 2004-04-01 Eli Lilly And Company Histamine h3 receptor antagonists, preparaton and therapeutic uses
WO2004035544A1 (en) 2002-10-16 2004-04-29 Glaxo Group Limited Benzo[d]azepine derivatives for the treatment of neurological and psychiatric disorders
WO2004035556A1 (en) 2002-10-16 2004-04-29 Glaxo Group Limited Substituted piperazines, (1,4) diaszepines, and 2,5-diazabicyclo (2.2.1) heptanes as histamine h1 and/or h3 antagonists or histamine h3 reverse antagonists
WO2004037788A1 (en) 2002-10-22 2004-05-06 Glaxo Group Limited Bicyclic benzamide compounds as histamine h3 receptor ligand useful in the treatment of neurological diseases
WO2004037800A1 (en) 2002-10-22 2004-05-06 Glaxo Group Limited Aryloxyalkylamine derivates as h3 receptor ligands
WO2004055026A1 (en) 2002-12-18 2004-07-01 Suven Life Sciences Limited Tetracyclic 3-substituted indoles having serotonin receptor affinity
WO2004054973A2 (en) 2002-12-18 2004-07-01 Novo Nordisk A/S Piperidine and pyrrolidine derivatives as antagonists of histamine h3 receptor
WO2004056821A2 (en) 2002-12-20 2004-07-08 Glaxo Group Limited Quinolizidine derivatives
WO2004056369A1 (en) 2002-12-20 2004-07-08 Glaxo Group Limited Benzo ‘ d!azepine derivatives for the treatment of neurological disorders
WO2004065380A1 (en) 2003-01-14 2004-08-05 Arena Pharmaceuticals Inc. 1,2,3-trisubstituted aryl and heteroaryl derivatives as modulators of metabolism and the prpphylaxis and treatment of disorders related thereto such as diabetes and hyperglycemia
WO2004066960A2 (en) 2003-01-28 2004-08-12 Schering Corporation Combination of h1, h3 and h4 receptor antagonists for treatment of allergic and non-allergic pulmonary inflammation, congestion and allergic rhinitis
WO2004069338A1 (en) 2003-01-31 2004-08-19 Schering Corporation Use of combinations of h1 and h3 histamine receptor antagonists for the preparation of a medicament for the treatment of allergic skin and allergic ocular conditions
WO2004089373A1 (en) 2003-04-10 2004-10-21 Glaxo Group Limited 4- (4-(heterocyclylalkoxy}phenyl)-1-(heterocyclyl-carbonyl)piperidine derivatives and related compounds as histamine h3 antagonists for the treatment of neurological diseases such as alzheimer’s
WO2004101546A1 (en) 2003-04-23 2004-11-25 Glaxo Group Limited Piperazine derivatives and their use for the treatment of neurological and psychiatric diseases
WO2004108675A1 (en) 2003-06-04 2004-12-16 Pfizer Limited 2-amino-pyridine derivatives as beta-2 adrenoreceptor agonists
WO2005009976A1 (en) 2003-07-29 2005-02-03 Novo Nordisk A/S Pyridazinyl- piperazines and their use as histamine h3 receptor ligands
WO2005014579A1 (en) 2003-07-21 2005-02-17 Astrazeneca Ab Imidazol derivatives of piperdine as histamine antagonists
WO2005014571A1 (en) 2003-07-18 2005-02-17 Glaxo Group Limited Substituted piperidines as histamine h3 receptor ligands
WO2005040144A1 (en) 2003-10-15 2005-05-06 Glaxo Group Limited Novel compounds
WO2005053796A1 (en) 2003-12-02 2005-06-16 B & B Beheer Nv Use of d4 and 5-ht2a antagonists, inverse agonists or partial agonists
WO2005056056A2 (en) 2003-12-15 2005-06-23 H. Lundbeck A/S The combination of a serotonin reuptake inhibitor and a histamine 3 receptor antagonist, inverse agonist or partial agonist
WO2005058837A1 (en) 2003-12-17 2005-06-30 Glaxo Group Limited Benzazepine derivatives as histamine h3 antagonists
WO2005082893A2 (en) 2004-02-25 2005-09-09 Eli Lilly And Company Histamine h3 receptor antagonists, preparation and therapeutic uses
WO2005087746A1 (en) 2004-03-12 2005-09-22 Glaxo Group Limited Benzazepine derivatives for the treatment of neurological and psychiatric disorders
WO2005089761A1 (en) 2004-03-17 2005-09-29 Pfizer Limited Combination for treating inflammatory diseases
WO2005097111A2 (en) 2004-03-26 2005-10-20 Merck & Co., Inc. Treatment of tremor with histamine h3 inverse agonists or histamine h3 antagonists
WO2005097740A1 (en) 2004-04-01 2005-10-20 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2005097778A1 (en) 2004-04-08 2005-10-20 Glaxo Group Limited Tetrahydrobenzazepines as histamine h3 receptor ligands
WO2005121080A1 (en) 2004-06-02 2005-12-22 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2005123723A1 (en) 2004-06-18 2005-12-29 Glaxo Group Limited 3-cycloalkylbenzazepines as histamine h3 antagonists
WO2006019833A1 (en) 2004-07-26 2006-02-23 Eli Lilly And Company Oxazole derivatives as histamine h3 receptor agents, preparation and therapeutic uses
WO2006018260A1 (en) 2004-08-16 2006-02-23 Glaxo Group Limited Tetrahydrobenzazepines as antagonists and/or reverse agonists of the histamine h 3 receptor
WO2006023462A1 (en) 2004-08-23 2006-03-02 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2006029906A1 (en) 2004-09-17 2006-03-23 Glaxo Group Limited Methylene dipiperidine derivatives
WO2006040192A1 (en) 2004-10-15 2006-04-20 Glaxo Group Limited Pyrrolidine derivatives as histamine receptors ligands
WO2006044228A1 (en) 2004-10-18 2006-04-27 Eli Lilly And Company Histamine h3 receptor inhibitors, preparation and therapeutic uses
WO2006046131A1 (en) 2004-10-29 2006-05-04 Pfizer Products Inc. Tetralin histamine-3 receptor antagonists
WO2006052608A2 (en) 2004-11-01 2006-05-18 Amylin Pharmaceuticals, Inc. Treatment of obesity and related disorders
WO2006058023A2 (en) 2004-11-23 2006-06-01 Merck & Co., Inc. Treatment of stroke with histamine h3 inverse agonists or histamine h3 antagonists
WO2006061193A1 (en) 2004-12-07 2006-06-15 Glaxo Group Limited Indenyl derivatives and use thereof for the treatment of neurological disorders
WO2006071750A1 (en) 2004-12-23 2006-07-06 Athersys, Inc. 4-piperidinol tertiary amines as histamine 3 receptor inhibitors for the treatment of obesity and other cns disorders
WO2006078775A1 (en) 2005-01-21 2006-07-27 Schering Corporation Imidazole and benzimidazole derivatives useful as histamine h3 antagonists
WO2006097691A1 (en) 2005-03-14 2006-09-21 Glaxo Group Limited Fused thiazole derivatives having affinity for the histamine h3 receptor
WO2006101808A1 (en) 2005-03-17 2006-09-28 Eli Lilly And Company Pyrrolidine derivatives as histamine h3 receptor antagonists
WO2006103537A2 (en) 2005-04-01 2006-10-05 Bioprojet Treatment of epilepsy with non-imidazole alkylamines histamine h3-receptor ligands
WO2006103057A1 (en) 2005-03-31 2006-10-05 Ucb Pharma, S.A. Compounds comprising an oxazoline or thiazoline moiety, processes for making them, and their uses
WO2006103546A2 (en) 2005-04-01 2006-10-05 Bioprojet Treatment of parkinson's disease, obstructive sleep apnea, dementia with lewy bodies, vascular dementia with non-imidazole alkylamines histamine h3-receptor ligands
WO2006124490A2 (en) 2005-05-17 2006-11-23 Schering Corporation Heterocycles as nicotinic acid receptor agonists for the treatment of dyslipidemia
WO2006125665A1 (en) 2005-05-25 2006-11-30 Glaxo Group Limited Substituted piperidine antagonist of hi receptor to be used for the treatment of rhinitis
WO2006136924A1 (en) 2005-06-22 2006-12-28 Pfizer Products Inc. Histamine-3 receptor antagonists
WO2007001975A1 (en) 2005-06-20 2007-01-04 Schering Corporation Piperidine derivatives useful as histamine h3 antagonists
WO2007002057A1 (en) 2005-06-20 2007-01-04 Schering Corporation Carbon-linked substituted piperidines and derivatives thereof useful as histamine h3 antagonists
WO2007005503A1 (en) 2005-07-01 2007-01-11 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2007009741A1 (en) 2005-07-19 2007-01-25 Glaxo Group Limited Piperazinone derivatives useful as histamine h3 receptor antagonists and/or inverse agonists
WO2007009739A1 (en) 2005-07-19 2007-01-25 Glaxo Group Limited Compounds
WO2007025596A1 (en) 2005-07-06 2007-03-08 Glaxo Group Limited Pyrazolo [3 , 4-d] azepine derivatives as histamine h3 antagonists
WO2007048595A1 (en) 2005-10-27 2007-05-03 Ucb Pharma, S.A. Compounds comprising a lactam or a lactam derivative moiety, processes for making them, and their uses
WO2007049123A1 (en) 2005-10-27 2007-05-03 Pfizer Products Inc. Histamine-3 receptor antagonists
WO2007063385A2 (en) 2005-12-01 2007-06-07 Pfizer Products Inc. Spirocyclic amine histamine-3 receptor antagonists
WO2007069053A1 (en) 2005-12-14 2007-06-21 Pfizer Products Inc. Benzimidazole antagonists of the h-3 receptor
WO2007071691A1 (en) 2005-12-20 2007-06-28 Glaxo Group Limited 3- (4-{ [4-(4-{ [3-(3, 3-dimethyl-1-piperidinyl) propyl] 0xy} phenyl) -1-piperidinyl] carbonyl }-1-naphthalenyl) propanoic or propenoic acid as h1 and h3 receptor antagonists for the treatment of inflammatory and/or allergic disorders
WO2007075702A2 (en) 2005-12-21 2007-07-05 Schering Corporation Treatment of nonalcoholic fatty liver disease using cholesterol lowering agents and h3 receptor antagonist/inverse agonist
WO2007076140A2 (en) 2005-12-23 2007-07-05 University Of Cincinnati Treatment methods employing histamine h3 receptor antagonists, including betahistine
WO2007075629A2 (en) 2005-12-21 2007-07-05 Schering Corporation Phenoxypiperidines and analogs thereof useful as histamine h3 antagonists
WO2007082840A1 (en) 2006-01-23 2007-07-26 F. Hoffmann-La Roche Ag Cyclohexyl sulfonamide derivatives having h3 receptor activity
WO2007088450A2 (en) 2006-02-01 2007-08-09 Pfizer Products Inc. Chromane antagonist of the h-3 receptor
WO2007088462A1 (en) 2006-02-01 2007-08-09 Pfizer Products Inc. Spirochromane antagonists of the h-3 receptor
WO2007095039A2 (en) 2006-02-09 2007-08-23 Schering Corporation Pharmaceutical formulations
WO2007099423A1 (en) 2006-03-02 2007-09-07 Pfizer Products Inc. 1-pyrrolidine indane derivatives as histamine-3 receptor antagonists
WO2007105053A2 (en) 2006-03-13 2007-09-20 Pfizer Products Inc. Tetralines antagonists of the h-3 receptor
WO2007122156A1 (en) 2006-04-20 2007-11-01 Glaxo Group Limited 2-substituted 4-benzylphthalazinone derivatives as histamine h1 and h3 antagonists
WO2007127457A2 (en) 2006-04-28 2007-11-08 The Administrators Of The Tulane Educational Fund Ghrelin/growth hormone releasing peptide/growth hormone secretatogue receptor antagonists and uses thereof
WO2007137955A1 (en) 2006-05-30 2007-12-06 F. Hoffmann-La Roche Ag Piperidinyl pyrimidine derivatives
WO2007138431A2 (en) 2006-05-30 2007-12-06 Pfizer Products Inc. Azabicyclic ether histamine-3 antagonists
WO2007137968A1 (en) 2006-05-29 2007-12-06 High Point Pharmaceuticals, Llc 3- (1, 3-benz0di0x0l-5-yl) -6- (4-cyclopropylpiperazin-1-yl) -pyridazine, its salts and solvates and its use as histamine h3 receptor antagonist
WO2008005338A1 (en) 2006-06-29 2008-01-10 Arena Pharmaceuticals, Inc. Modulators of the histamine h3-receptor useful for the treatment of disorders related thereto
WO2008032156A1 (en) 2006-09-08 2008-03-20 Pfizer Products Inc. Diaryl ether derivatives and uses thereof
WO2008045371A2 (en) 2006-10-06 2008-04-17 Wyeth N-substituted-azacyclylamines as histamine-3 antagonists
WO2008048609A1 (en) 2006-10-17 2008-04-24 Arena Pharmaceuticals, Inc. Biphenyl sulfonyl and phenyl-heteroaryl sulfonyl modulators of the histamine h3-receptor useful for the treatment of disorders related thereto
WO2008059335A1 (en) 2006-11-13 2008-05-22 Pfizer Products Inc. Diaryl, dipyridinyl and aryl-pyridinyl derivatives and uses thereof
WO2008115574A1 (en) 2007-03-21 2008-09-25 Reliant Pharmaceuticals, Inc. Cb1 antagonist and a dyslipidemic agent and/or metabolic regulator, and methods of making and using same
WO2008153958A2 (en) 2007-06-08 2008-12-18 Arena Pharmaceuticals, Inc. Crystalline forms of (r)-1-{2-[4'- (3-methoxy-propane-1- sulfonyl)-biphenyl-4-yl]-ethyl}-2-methyl-pyrrolidine, and compositions, and methods related thereto
WO2008154126A1 (en) 2007-06-11 2008-12-18 High Point Pharmaceuticals, Llc New heteocyclic h3 antagonists
WO2009005646A2 (en) 2007-06-28 2009-01-08 Schering Corporation Substituted piperazines as cb1 antagonists
WO2009005671A2 (en) 2007-06-28 2009-01-08 Schering Corporation Substituted piperazines as cb1 antagonists
WO2009005645A1 (en) 2007-06-28 2009-01-08 Schering Corporation Substituted piperazines as cb1 antagonists
WO2009009501A2 (en) 2007-07-06 2009-01-15 Bristol-Myers Squibb Company Non-basic melanin concentrating hormone receptor-1 antagonists and methods
WO2009012252A1 (en) 2007-07-16 2009-01-22 Wyeth Aminoalkylazole derivatives as histamine-3 antagonists
WO2009024823A2 (en) 2007-08-22 2009-02-26 Astrazeneca Ab Oxadiazole derivatives as dgat inhibitors
WO2009030716A1 (en) 2007-09-06 2009-03-12 Glaxo Group Limited Piperazine derivative having affinity for the histamine h3 receptor
WO2009036132A1 (en) 2007-09-11 2009-03-19 Abbott Laboratories Octahydro-pyrrolo[3,4-b]pyrrole n-oxides
WO2009055437A2 (en) 2007-10-22 2009-04-30 Memory Pharmaceuticals Corporation (1,4-diaza-bicyclo[3.2.2]non-6-en-4-yl)-heterocyclyl-methanone ligands for nicotinic acetylcholine receptors, useful for the treatment of disease
WO2009058300A1 (en) 2007-10-30 2009-05-07 Arena Pharmaceuticals, Inc. Biphenyl derivatives as modulators of the histamine-h3 receptor useful for the treatment of disorders related thereto
WO2009063953A1 (en) 2007-11-13 2009-05-22 Taisho Pharmaceutical Co., Ltd. Phenylpyrazole derivatives
WO2009071988A1 (en) 2007-12-07 2009-06-11 Pfizer Inc. Tosylate salt of trans-n-isobutyl-3-fluoro-3- [3-flu0r0-4- (pyrrolidin-1-yl-methyl) -phenyl] cyclobut anecarboxamide
WO2009092764A1 (en) 2008-01-24 2009-07-30 Ucb Pharma, S.A. Compounds comprising a cyclobutoxy group
WO2009097567A1 (en) 2008-01-30 2009-08-06 Cephalon, Inc. Substituted spirocyclic piperidine derivatives as histamine-3 (h3) receptor ligands
WO2009095394A1 (en) 2008-02-01 2009-08-06 Evotec Neurosciences Gmbh Diazepanes as histamine h3 receptor antagonists
WO2009097306A1 (en) 2008-01-30 2009-08-06 Cephalon, Inc. Substituted pyridazine derivatives which have histamine h3 antagonist activity
WO2009105206A1 (en) 2008-02-19 2009-08-27 Arena Pharmaceuticals, Inc. Modulators of the histamine h3 receptor useful for the treatment of disorders related thereto
WO2009121812A1 (en) 2008-03-31 2009-10-08 Evotec Neurosciences Gmbh Tetrahydronaphthyridines and aza derivatives thereof as histamine h3 receptor antagonists
WO2009128907A1 (en) 2008-04-16 2009-10-22 Arena Pharmaceuticals, Inc. Processes useful for the synthesis of (r)-1-{2-[4'-(3-methoxypropane-1-sulfonyl)-biphenyl-4-yl]-ethyl}-2-methyl-pyrrolidine
WO2009135842A1 (en) 2008-05-08 2009-11-12 Evotec Neurosciences Gmbh Azetidines and cyclobutanes as histamine h3 receptor antagonists
WO2009142732A2 (en) 2008-05-20 2009-11-26 Cephalon, Inc. Substituted pyridazinone derivatives as histamine-3 (h3) receptor ligands
WO2009147149A1 (en) 2008-06-06 2009-12-10 Ucb Pharma, S.A. Compounds comprising a cyclobutoxy group
WO2009150101A1 (en) 2008-06-09 2009-12-17 Glaxo Group Limited Pharmaceutical compositions comprising n-[2-((2r)-2-{[4-[(4-chlorophenyl)methyl]-l-oxo-2(ih)-phthalaz inyl]methyl}-l- pyrrolidinyl)ethyl]-4-(methyloxy)butanamide
WO2010000456A1 (en) 2008-07-03 2010-01-07 Janssen Pharmaceutica Nv Substituted 6- (1-piperazinyl) -pyridazines as 5-ht6 receptor antagonists
WO2010007382A1 (en) 2008-07-18 2010-01-21 Takeda Pharmaceutical Company Limited. Benzazepine derivatives and their use as hstamine h3 antagonists
WO2010011657A1 (en) 2008-07-23 2010-01-28 Schering Corporation Bicyclic heterocycle derivatives as histamine h3 receptor antagonists
WO2010011653A1 (en) 2008-07-23 2010-01-28 Schering Corporation Tricyclic heterocycle derivatives as histamine h3 antagonists
WO2010052222A1 (en) 2008-11-07 2010-05-14 Evotec Neurosciences Gmbh (dihydro)naphthyridinone derivatives as histamine h3 receptor antagonists
WO2010086403A1 (en) 2009-02-02 2010-08-05 Evotec Neurosciences Gmbh Azetidines as histamine h3 receptor antagonists
WO2010129242A2 (en) 2009-04-27 2010-11-11 Abbott Laboratories Treatment of osteoarthritis pain
WO2010151611A1 (en) 2009-06-26 2010-12-29 Sanofi-Aventis Novel fumarate salts of a histamine h3 receptor antagonist

Patent Citations (180)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1980A (en) 1841-02-13 Mode oe
US774216A (en) 1903-11-19 1904-11-08 Alexander F Ward Machine for pointing and lapping hoops.
US4166452A (en) 1976-05-03 1979-09-04 Generales Constantine D J Jr Apparatus for testing human responses to stimuli
US4256108A (en) 1977-04-07 1981-03-17 Alza Corporation Microporous-semipermeable laminated osmotic system
US4704362A (en) 1977-11-08 1987-11-03 Genentech, Inc. Recombinant cloning vehicle microbial polypeptide expression
US4265874A (en) 1980-04-25 1981-05-05 Alza Corporation Method of delivering drug with aid of effervescent activity generated in environment of use
US5462856A (en) 1990-07-19 1995-10-31 Bunsen Rush Laboratories, Inc. Methods for identifying chemicals that act as agonists or antagonists for receptors and other proteins involved in signal transduction via pathways that utilize G-proteins
US6051386A (en) 1990-07-19 2000-04-18 Bunsen Rush Laboratories, Inc. Methods for identifying chemicals that act as agonists or antagonists for receptors and other proteins involved in signal transduction via pathways that utilize G-proteins
WO1992015567A1 (en) 1991-02-27 1992-09-17 Seed Capital Investment (Sci) B.V. Imidazole-derivatives having agonistic or antagonistic activity on the histamine h3-receptor
WO1993012108A1 (en) 1991-12-18 1993-06-24 Schering Corporation Imidazolyl or imidazoylalkyl substituted with a four or five membered nitrogen containing heterocyclic ring
WO1993012093A1 (en) 1991-12-18 1993-06-24 Schering Corporation Imidazolyl-alkyl-piperazine and -diazepine derivatives as histamine h3 agonists/antagonists
WO1993012107A1 (en) 1991-12-18 1993-06-24 Schering Corporation Imidazoylalkyl substituted with a six membered nitrogen containing heterocyclic ring
WO1993020062A1 (en) 1992-04-01 1993-10-14 The University Of Toledo Process for the preparation of 4-pyridyl- and 4-piperidinylimidazoles useful as intermediates for the synthesis of histamine receptor antagonists
WO1993020061A1 (en) 1992-04-01 1993-10-14 The University Of Toledo 4-[4'-piperidinyl or 3'-pirrolidinyl] substituted imidazoles as h3-receptor antagonists and therapeutic uses thereof
WO1995006037A1 (en) 1993-08-27 1995-03-02 Vrije Universiteit New imidazole derivatives having agonistic or antagonistic activity on the histamine h3 receptor
WO1995011894A1 (en) 1993-10-29 1995-05-04 The University Of Toledo Histamine h3-receptor antagonists and therapeutic uses thereof
WO1996038142A1 (en) 1995-05-30 1996-12-05 Gliatech, Inc. 1h-4(5)-substituted imidazole derivatives
WO1996038141A1 (en) 1995-05-30 1996-12-05 Gliatech, Inc. 2-(1h-4(5)-imidazoyl) cyclopropyl derivatives
WO1996040126A1 (en) 1995-06-07 1996-12-19 Gliatech, Inc. 1h-4(5)-substituted imidazole derivatives
WO1998006394A1 (en) 1996-08-16 1998-02-19 Schering Corporation Treatment of upper airway allergic responses with a combination of histamine receptor antagonists
WO1999006377A1 (en) 1997-08-01 1999-02-11 Gliatech, Inc. Derivatives of 1-((3,3-dimethyl-3-silabutoxy)methyl)-2-(phenylsulfonyl)imidazole
WO1999024421A1 (en) 1997-11-07 1999-05-20 Schering Corporation Imidazoylalkyl substituted with a five, six or seven membered heterocyclic ring containing one nitrogen atom
WO1999042458A1 (en) 1998-02-19 1999-08-26 James Black Foundation Limited Histamine h3 receptor ligands
US5922576A (en) 1998-02-27 1999-07-13 The John Hopkins University Simplified system for generating recombinant adenoviruses
WO2000006254A2 (en) 1998-07-29 2000-02-10 Societe Civile Bioprojet Non-imidazole alkylamines as histamine h3-receptor ligands and their therapeutic applications
WO2000023438A1 (en) 1998-10-16 2000-04-27 Schering Corporation N-(imidazolylalkyl)substituted cyclic amines as histamine-h3 agonists or antagonists
WO2000042023A1 (en) 1999-01-18 2000-07-20 Novo Nordisk A/S Substituted imidazoles, their preparation and use
WO2000053596A2 (en) 1999-03-08 2000-09-14 Schering Corporation Imidazole compounds as histamine h3 ligands
WO2001066534A2 (en) 2000-03-09 2001-09-13 Abbott Laboratories Cyclic and bicyclic diamino histamine-3 receptor antagonists
WO2001068652A1 (en) 2000-03-17 2001-09-20 Novo Nordisk A/S Condensed imidazoles as histamine h3 receptor ligands
WO2001074815A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Phenyl-substituted imidazopyridines
WO2001074773A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Phenyl-substituted indoles as histamine h3-receptor antagonists
WO2001074814A1 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Phenyl-substituted indolizine derivatives and their use as histamine h3 ligands
WO2001074813A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. METHOD FOR USING 2- OR 3-ARYL SUBSTITUTED IMIDAZO[1,2-a] PYRIDINES AS H3 ANTAGONISTS
WO2001074810A2 (en) 2000-03-31 2001-10-11 Ortho Mcneil Pharmaceutical, Inc. Method for using 2-aryloxyalkylaminobenzoxazoles and 2-aryloxyalkylaminobenzothiazoles as h3 antagonists
WO2002012190A2 (en) 2000-08-08 2002-02-14 Ortho Mcneil Pharmaceutical, Inc. Non-imidazole aryloxypiperidines as h3 receptor ligands
WO2002012224A2 (en) 2000-08-08 2002-02-14 Ortho Mcneil Pharmaceutical, Inc. Bicyclic compounds as h3 receptor ligands
WO2002012214A2 (en) 2000-08-08 2002-02-14 Ortho Mcneil Pharmaceutical Inc. Non-imidazole aryloxyalkylamines as h3 receptor ligands
WO2002024659A2 (en) 2000-09-20 2002-03-28 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antgonists
WO2002024657A2 (en) 2000-09-20 2002-03-28 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antagonists
WO2002024658A2 (en) 2000-09-20 2002-03-28 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antagonists
WO2002044141A2 (en) 2000-09-20 2002-06-06 Schering Corporation Substituted imidazoles as dual histamine h1 and h3 agonists or antagonists
WO2002024695A2 (en) 2000-09-22 2002-03-28 Ortho Mcneil Pharmaceutical, Inc. Octahydro-indolizines and quinolizines and hexahydro-pyrrolizines
WO2002032893A2 (en) 2000-10-17 2002-04-25 Schering Corporation Piperidine compounds as anti-allergic
WO2002042461A2 (en) 2000-11-27 2002-05-30 Arena Pharmaceuticals, Inc. Endogenous and non-endogenous versions of human g protein-coupled receptors
WO2002056871A2 (en) 2001-01-17 2002-07-25 Pfizer Limited Histamine receptor antagonists
WO2002072093A2 (en) 2001-02-08 2002-09-19 Schering Corporation Use of dual h3/m2 antagonists with a bipiperidinic structure in the treatment of cognition deficit disorders
WO2002064212A1 (en) 2001-02-13 2002-08-22 Cornell Research Foundation, Inc. Method and compositions for reducing cardiac dysfunctions with a selective histamine h3 receptor agonist
WO2002076925A2 (en) 2001-03-23 2002-10-03 Eli Lilly And Company Non-imidazole aryl alkylamines compounds as histamine h3 receptor antagonists, preparation and therapeutic uses
WO2003024928A2 (en) 2001-09-14 2003-03-27 Novo Nordisk A/S Novel aminoazetidine, -pyrrolidine and -piperidine derivatives
WO2003024929A1 (en) 2001-09-14 2003-03-27 Novo Nordisk A/S Substituted piperidines with selective binding to histamine h3-receptor
WO2003031432A1 (en) 2001-10-12 2003-04-17 Novo Nordisk A/S Substituted piperidines and their use for the treatment of diseases related to the histamine h3 receptor
WO2003033488A1 (en) 2001-10-15 2003-04-24 Schering Corporation Synthesis of 4-(piperidyl) (2-pyridyl)methanone-(e)-o-methyloxime and its salts
WO2003040106A1 (en) 2001-11-09 2003-05-15 Novo Nordisk A/S Preparation and use of substituted imidazoles
WO2003042359A2 (en) 2001-11-13 2003-05-22 Merck & Co. Inc. Histamine receptor h3 polynucleotides
WO2003064411A1 (en) 2002-02-01 2003-08-07 Novo Nordisk A/S Amides of aminoalkyl-substituted azetidines, pyrrolidines, piperidines and azepanes
WO2003066604A2 (en) 2002-02-05 2003-08-14 Novo Nordisk A/S Novel aryl- and heteroarylpiperazines
WO2003088967A1 (en) 2002-04-18 2003-10-30 Schering Corporation (1-4-piperidinyl) benzimidazole derivatives useful as histamine h3 antagonists
WO2003103669A1 (en) 2002-04-18 2003-12-18 Schering Corporation 1-(4-piperidinyl) benzimidazolones as histamine h3 antagonists
WO2003099821A1 (en) 2002-05-24 2003-12-04 X-Ceptor Therapeutics, Inc. Azepinoindole and pyridoindole derivatives as pharmaceutical agents
WO2003104235A1 (en) 2002-06-06 2003-12-18 Novo Nordisk A/S Substituted hexahydropyrrolo[1,2-a]pyrazines, octahydropyrido[1,2-a]pyrazines and decahydropyrazino[1,2-a]azepines
WO2004000831A1 (en) 2002-06-24 2003-12-31 Schering Corporation Indole derivatives useful as histamine h3 antagonists
WO2004009015A2 (en) 2002-07-18 2004-01-29 Merck & Co., Inc. Combination therapy for the treatment of obesity
WO2004018432A1 (en) 2002-08-20 2004-03-04 Eli Lilly And Company Substituted azepines as histamine h3 receptor antagonists, preparation and therapeutic uses
WO2004026837A2 (en) 2002-09-18 2004-04-01 Eli Lilly And Company Histamine h3 receptor antagonists, preparaton and therapeutic uses
WO2004035544A1 (en) 2002-10-16 2004-04-29 Glaxo Group Limited Benzo[d]azepine derivatives for the treatment of neurological and psychiatric disorders
WO2004035556A1 (en) 2002-10-16 2004-04-29 Glaxo Group Limited Substituted piperazines, (1,4) diaszepines, and 2,5-diazabicyclo (2.2.1) heptanes as histamine h1 and/or h3 antagonists or histamine h3 reverse antagonists
WO2004037788A1 (en) 2002-10-22 2004-05-06 Glaxo Group Limited Bicyclic benzamide compounds as histamine h3 receptor ligand useful in the treatment of neurological diseases
WO2004037800A1 (en) 2002-10-22 2004-05-06 Glaxo Group Limited Aryloxyalkylamine derivates as h3 receptor ligands
WO2004055026A1 (en) 2002-12-18 2004-07-01 Suven Life Sciences Limited Tetracyclic 3-substituted indoles having serotonin receptor affinity
WO2004054973A2 (en) 2002-12-18 2004-07-01 Novo Nordisk A/S Piperidine and pyrrolidine derivatives as antagonists of histamine h3 receptor
WO2004056821A2 (en) 2002-12-20 2004-07-08 Glaxo Group Limited Quinolizidine derivatives
WO2004056369A1 (en) 2002-12-20 2004-07-08 Glaxo Group Limited Benzo ‘ d!azepine derivatives for the treatment of neurological disorders
WO2004065380A1 (en) 2003-01-14 2004-08-05 Arena Pharmaceuticals Inc. 1,2,3-trisubstituted aryl and heteroaryl derivatives as modulators of metabolism and the prpphylaxis and treatment of disorders related thereto such as diabetes and hyperglycemia
WO2004066960A2 (en) 2003-01-28 2004-08-12 Schering Corporation Combination of h1, h3 and h4 receptor antagonists for treatment of allergic and non-allergic pulmonary inflammation, congestion and allergic rhinitis
WO2004069338A1 (en) 2003-01-31 2004-08-19 Schering Corporation Use of combinations of h1 and h3 histamine receptor antagonists for the preparation of a medicament for the treatment of allergic skin and allergic ocular conditions
WO2004089373A1 (en) 2003-04-10 2004-10-21 Glaxo Group Limited 4- (4-(heterocyclylalkoxy}phenyl)-1-(heterocyclyl-carbonyl)piperidine derivatives and related compounds as histamine h3 antagonists for the treatment of neurological diseases such as alzheimer’s
WO2004101546A1 (en) 2003-04-23 2004-11-25 Glaxo Group Limited Piperazine derivatives and their use for the treatment of neurological and psychiatric diseases
WO2004108675A1 (en) 2003-06-04 2004-12-16 Pfizer Limited 2-amino-pyridine derivatives as beta-2 adrenoreceptor agonists
WO2005014571A1 (en) 2003-07-18 2005-02-17 Glaxo Group Limited Substituted piperidines as histamine h3 receptor ligands
WO2005014579A1 (en) 2003-07-21 2005-02-17 Astrazeneca Ab Imidazol derivatives of piperdine as histamine antagonists
WO2005009976A1 (en) 2003-07-29 2005-02-03 Novo Nordisk A/S Pyridazinyl- piperazines and their use as histamine h3 receptor ligands
WO2005040144A1 (en) 2003-10-15 2005-05-06 Glaxo Group Limited Novel compounds
WO2005053796A1 (en) 2003-12-02 2005-06-16 B & B Beheer Nv Use of d4 and 5-ht2a antagonists, inverse agonists or partial agonists
WO2005056056A2 (en) 2003-12-15 2005-06-23 H. Lundbeck A/S The combination of a serotonin reuptake inhibitor and a histamine 3 receptor antagonist, inverse agonist or partial agonist
WO2005058837A1 (en) 2003-12-17 2005-06-30 Glaxo Group Limited Benzazepine derivatives as histamine h3 antagonists
WO2005082893A2 (en) 2004-02-25 2005-09-09 Eli Lilly And Company Histamine h3 receptor antagonists, preparation and therapeutic uses
WO2005087746A1 (en) 2004-03-12 2005-09-22 Glaxo Group Limited Benzazepine derivatives for the treatment of neurological and psychiatric disorders
WO2005089761A1 (en) 2004-03-17 2005-09-29 Pfizer Limited Combination for treating inflammatory diseases
WO2005097111A2 (en) 2004-03-26 2005-10-20 Merck & Co., Inc. Treatment of tremor with histamine h3 inverse agonists or histamine h3 antagonists
WO2005097740A1 (en) 2004-04-01 2005-10-20 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2005097778A1 (en) 2004-04-08 2005-10-20 Glaxo Group Limited Tetrahydrobenzazepines as histamine h3 receptor ligands
WO2005121080A1 (en) 2004-06-02 2005-12-22 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2005123723A1 (en) 2004-06-18 2005-12-29 Glaxo Group Limited 3-cycloalkylbenzazepines as histamine h3 antagonists
WO2006019833A1 (en) 2004-07-26 2006-02-23 Eli Lilly And Company Oxazole derivatives as histamine h3 receptor agents, preparation and therapeutic uses
WO2006018260A1 (en) 2004-08-16 2006-02-23 Glaxo Group Limited Tetrahydrobenzazepines as antagonists and/or reverse agonists of the histamine h 3 receptor
WO2006023462A1 (en) 2004-08-23 2006-03-02 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2006029906A1 (en) 2004-09-17 2006-03-23 Glaxo Group Limited Methylene dipiperidine derivatives
WO2006040192A1 (en) 2004-10-15 2006-04-20 Glaxo Group Limited Pyrrolidine derivatives as histamine receptors ligands
WO2006044228A1 (en) 2004-10-18 2006-04-27 Eli Lilly And Company Histamine h3 receptor inhibitors, preparation and therapeutic uses
WO2006046131A1 (en) 2004-10-29 2006-05-04 Pfizer Products Inc. Tetralin histamine-3 receptor antagonists
WO2006052608A2 (en) 2004-11-01 2006-05-18 Amylin Pharmaceuticals, Inc. Treatment of obesity and related disorders
WO2006058023A2 (en) 2004-11-23 2006-06-01 Merck & Co., Inc. Treatment of stroke with histamine h3 inverse agonists or histamine h3 antagonists
WO2006061193A1 (en) 2004-12-07 2006-06-15 Glaxo Group Limited Indenyl derivatives and use thereof for the treatment of neurological disorders
WO2006071750A1 (en) 2004-12-23 2006-07-06 Athersys, Inc. 4-piperidinol tertiary amines as histamine 3 receptor inhibitors for the treatment of obesity and other cns disorders
WO2006078775A1 (en) 2005-01-21 2006-07-27 Schering Corporation Imidazole and benzimidazole derivatives useful as histamine h3 antagonists
WO2006097691A1 (en) 2005-03-14 2006-09-21 Glaxo Group Limited Fused thiazole derivatives having affinity for the histamine h3 receptor
WO2006101808A1 (en) 2005-03-17 2006-09-28 Eli Lilly And Company Pyrrolidine derivatives as histamine h3 receptor antagonists
WO2006103057A1 (en) 2005-03-31 2006-10-05 Ucb Pharma, S.A. Compounds comprising an oxazoline or thiazoline moiety, processes for making them, and their uses
WO2006103045A1 (en) 2005-03-31 2006-10-05 Ucb Pharma S.A. Compounds comprising an oxazole or thiazole moiety, processes for making them, and their uses
WO2006103537A2 (en) 2005-04-01 2006-10-05 Bioprojet Treatment of epilepsy with non-imidazole alkylamines histamine h3-receptor ligands
WO2006103546A2 (en) 2005-04-01 2006-10-05 Bioprojet Treatment of parkinson's disease, obstructive sleep apnea, dementia with lewy bodies, vascular dementia with non-imidazole alkylamines histamine h3-receptor ligands
WO2006124490A2 (en) 2005-05-17 2006-11-23 Schering Corporation Heterocycles as nicotinic acid receptor agonists for the treatment of dyslipidemia
WO2006125665A1 (en) 2005-05-25 2006-11-30 Glaxo Group Limited Substituted piperidine antagonist of hi receptor to be used for the treatment of rhinitis
WO2007001975A1 (en) 2005-06-20 2007-01-04 Schering Corporation Piperidine derivatives useful as histamine h3 antagonists
WO2007002057A1 (en) 2005-06-20 2007-01-04 Schering Corporation Carbon-linked substituted piperidines and derivatives thereof useful as histamine h3 antagonists
WO2006136924A1 (en) 2005-06-22 2006-12-28 Pfizer Products Inc. Histamine-3 receptor antagonists
WO2007005503A1 (en) 2005-07-01 2007-01-11 Eli Lilly And Company Histamine h3 receptor agents, preparation and therapeutic uses
WO2007025596A1 (en) 2005-07-06 2007-03-08 Glaxo Group Limited Pyrazolo [3 , 4-d] azepine derivatives as histamine h3 antagonists
WO2007009741A1 (en) 2005-07-19 2007-01-25 Glaxo Group Limited Piperazinone derivatives useful as histamine h3 receptor antagonists and/or inverse agonists
WO2007009739A1 (en) 2005-07-19 2007-01-25 Glaxo Group Limited Compounds
WO2007048595A1 (en) 2005-10-27 2007-05-03 Ucb Pharma, S.A. Compounds comprising a lactam or a lactam derivative moiety, processes for making them, and their uses
WO2007049123A1 (en) 2005-10-27 2007-05-03 Pfizer Products Inc. Histamine-3 receptor antagonists
WO2007063385A2 (en) 2005-12-01 2007-06-07 Pfizer Products Inc. Spirocyclic amine histamine-3 receptor antagonists
WO2007069053A1 (en) 2005-12-14 2007-06-21 Pfizer Products Inc. Benzimidazole antagonists of the h-3 receptor
WO2007071691A1 (en) 2005-12-20 2007-06-28 Glaxo Group Limited 3- (4-{ [4-(4-{ [3-(3, 3-dimethyl-1-piperidinyl) propyl] 0xy} phenyl) -1-piperidinyl] carbonyl }-1-naphthalenyl) propanoic or propenoic acid as h1 and h3 receptor antagonists for the treatment of inflammatory and/or allergic disorders
WO2007075702A2 (en) 2005-12-21 2007-07-05 Schering Corporation Treatment of nonalcoholic fatty liver disease using cholesterol lowering agents and h3 receptor antagonist/inverse agonist
WO2007075629A2 (en) 2005-12-21 2007-07-05 Schering Corporation Phenoxypiperidines and analogs thereof useful as histamine h3 antagonists
WO2007076140A2 (en) 2005-12-23 2007-07-05 University Of Cincinnati Treatment methods employing histamine h3 receptor antagonists, including betahistine
WO2007082840A1 (en) 2006-01-23 2007-07-26 F. Hoffmann-La Roche Ag Cyclohexyl sulfonamide derivatives having h3 receptor activity
WO2007088450A2 (en) 2006-02-01 2007-08-09 Pfizer Products Inc. Chromane antagonist of the h-3 receptor
WO2007088462A1 (en) 2006-02-01 2007-08-09 Pfizer Products Inc. Spirochromane antagonists of the h-3 receptor
WO2007095039A2 (en) 2006-02-09 2007-08-23 Schering Corporation Pharmaceutical formulations
WO2007099423A1 (en) 2006-03-02 2007-09-07 Pfizer Products Inc. 1-pyrrolidine indane derivatives as histamine-3 receptor antagonists
WO2007105053A2 (en) 2006-03-13 2007-09-20 Pfizer Products Inc. Tetralines antagonists of the h-3 receptor
WO2007122156A1 (en) 2006-04-20 2007-11-01 Glaxo Group Limited 2-substituted 4-benzylphthalazinone derivatives as histamine h1 and h3 antagonists
WO2007127457A2 (en) 2006-04-28 2007-11-08 The Administrators Of The Tulane Educational Fund Ghrelin/growth hormone releasing peptide/growth hormone secretatogue receptor antagonists and uses thereof
WO2007137968A1 (en) 2006-05-29 2007-12-06 High Point Pharmaceuticals, Llc 3- (1, 3-benz0di0x0l-5-yl) -6- (4-cyclopropylpiperazin-1-yl) -pyridazine, its salts and solvates and its use as histamine h3 receptor antagonist
WO2007137955A1 (en) 2006-05-30 2007-12-06 F. Hoffmann-La Roche Ag Piperidinyl pyrimidine derivatives
WO2007138431A2 (en) 2006-05-30 2007-12-06 Pfizer Products Inc. Azabicyclic ether histamine-3 antagonists
WO2008005338A1 (en) 2006-06-29 2008-01-10 Arena Pharmaceuticals, Inc. Modulators of the histamine h3-receptor useful for the treatment of disorders related thereto
WO2008032156A1 (en) 2006-09-08 2008-03-20 Pfizer Products Inc. Diaryl ether derivatives and uses thereof
WO2008045371A2 (en) 2006-10-06 2008-04-17 Wyeth N-substituted-azacyclylamines as histamine-3 antagonists
WO2008048609A1 (en) 2006-10-17 2008-04-24 Arena Pharmaceuticals, Inc. Biphenyl sulfonyl and phenyl-heteroaryl sulfonyl modulators of the histamine h3-receptor useful for the treatment of disorders related thereto
WO2008059335A1 (en) 2006-11-13 2008-05-22 Pfizer Products Inc. Diaryl, dipyridinyl and aryl-pyridinyl derivatives and uses thereof
WO2008115574A1 (en) 2007-03-21 2008-09-25 Reliant Pharmaceuticals, Inc. Cb1 antagonist and a dyslipidemic agent and/or metabolic regulator, and methods of making and using same
WO2008153958A2 (en) 2007-06-08 2008-12-18 Arena Pharmaceuticals, Inc. Crystalline forms of (r)-1-{2-[4'- (3-methoxy-propane-1- sulfonyl)-biphenyl-4-yl]-ethyl}-2-methyl-pyrrolidine, and compositions, and methods related thereto
WO2008154126A1 (en) 2007-06-11 2008-12-18 High Point Pharmaceuticals, Llc New heteocyclic h3 antagonists
WO2009005646A2 (en) 2007-06-28 2009-01-08 Schering Corporation Substituted piperazines as cb1 antagonists
WO2009005671A2 (en) 2007-06-28 2009-01-08 Schering Corporation Substituted piperazines as cb1 antagonists
WO2009005645A1 (en) 2007-06-28 2009-01-08 Schering Corporation Substituted piperazines as cb1 antagonists
WO2009009501A2 (en) 2007-07-06 2009-01-15 Bristol-Myers Squibb Company Non-basic melanin concentrating hormone receptor-1 antagonists and methods
WO2009012252A1 (en) 2007-07-16 2009-01-22 Wyeth Aminoalkylazole derivatives as histamine-3 antagonists
WO2009024823A2 (en) 2007-08-22 2009-02-26 Astrazeneca Ab Oxadiazole derivatives as dgat inhibitors
WO2009030716A1 (en) 2007-09-06 2009-03-12 Glaxo Group Limited Piperazine derivative having affinity for the histamine h3 receptor
WO2009036132A1 (en) 2007-09-11 2009-03-19 Abbott Laboratories Octahydro-pyrrolo[3,4-b]pyrrole n-oxides
WO2009055437A2 (en) 2007-10-22 2009-04-30 Memory Pharmaceuticals Corporation (1,4-diaza-bicyclo[3.2.2]non-6-en-4-yl)-heterocyclyl-methanone ligands for nicotinic acetylcholine receptors, useful for the treatment of disease
WO2009058300A1 (en) 2007-10-30 2009-05-07 Arena Pharmaceuticals, Inc. Biphenyl derivatives as modulators of the histamine-h3 receptor useful for the treatment of disorders related thereto
WO2009063953A1 (en) 2007-11-13 2009-05-22 Taisho Pharmaceutical Co., Ltd. Phenylpyrazole derivatives
WO2009071988A1 (en) 2007-12-07 2009-06-11 Pfizer Inc. Tosylate salt of trans-n-isobutyl-3-fluoro-3- [3-flu0r0-4- (pyrrolidin-1-yl-methyl) -phenyl] cyclobut anecarboxamide
WO2009092764A1 (en) 2008-01-24 2009-07-30 Ucb Pharma, S.A. Compounds comprising a cyclobutoxy group
WO2009097567A1 (en) 2008-01-30 2009-08-06 Cephalon, Inc. Substituted spirocyclic piperidine derivatives as histamine-3 (h3) receptor ligands
WO2009097306A1 (en) 2008-01-30 2009-08-06 Cephalon, Inc. Substituted pyridazine derivatives which have histamine h3 antagonist activity
WO2009095394A1 (en) 2008-02-01 2009-08-06 Evotec Neurosciences Gmbh Diazepanes as histamine h3 receptor antagonists
US20100331312A1 (en) * 2008-02-19 2010-12-30 Arena Pharmaceuticals, Inc. Modulators of the histamine h3 receptor useful for the treatment of disorders related thereto
WO2009105206A1 (en) 2008-02-19 2009-08-27 Arena Pharmaceuticals, Inc. Modulators of the histamine h3 receptor useful for the treatment of disorders related thereto
WO2009121812A1 (en) 2008-03-31 2009-10-08 Evotec Neurosciences Gmbh Tetrahydronaphthyridines and aza derivatives thereof as histamine h3 receptor antagonists
WO2009128907A1 (en) 2008-04-16 2009-10-22 Arena Pharmaceuticals, Inc. Processes useful for the synthesis of (r)-1-{2-[4'-(3-methoxypropane-1-sulfonyl)-biphenyl-4-yl]-ethyl}-2-methyl-pyrrolidine
WO2009135842A1 (en) 2008-05-08 2009-11-12 Evotec Neurosciences Gmbh Azetidines and cyclobutanes as histamine h3 receptor antagonists
WO2009142732A2 (en) 2008-05-20 2009-11-26 Cephalon, Inc. Substituted pyridazinone derivatives as histamine-3 (h3) receptor ligands
WO2009147149A1 (en) 2008-06-06 2009-12-10 Ucb Pharma, S.A. Compounds comprising a cyclobutoxy group
WO2009150101A1 (en) 2008-06-09 2009-12-17 Glaxo Group Limited Pharmaceutical compositions comprising n-[2-((2r)-2-{[4-[(4-chlorophenyl)methyl]-l-oxo-2(ih)-phthalaz inyl]methyl}-l- pyrrolidinyl)ethyl]-4-(methyloxy)butanamide
WO2010000456A1 (en) 2008-07-03 2010-01-07 Janssen Pharmaceutica Nv Substituted 6- (1-piperazinyl) -pyridazines as 5-ht6 receptor antagonists
WO2010007382A1 (en) 2008-07-18 2010-01-21 Takeda Pharmaceutical Company Limited. Benzazepine derivatives and their use as hstamine h3 antagonists
WO2010011657A1 (en) 2008-07-23 2010-01-28 Schering Corporation Bicyclic heterocycle derivatives as histamine h3 receptor antagonists
WO2010011653A1 (en) 2008-07-23 2010-01-28 Schering Corporation Tricyclic heterocycle derivatives as histamine h3 antagonists
WO2010052222A1 (en) 2008-11-07 2010-05-14 Evotec Neurosciences Gmbh (dihydro)naphthyridinone derivatives as histamine h3 receptor antagonists
WO2010086403A1 (en) 2009-02-02 2010-08-05 Evotec Neurosciences Gmbh Azetidines as histamine h3 receptor antagonists
WO2010129242A2 (en) 2009-04-27 2010-11-11 Abbott Laboratories Treatment of osteoarthritis pain
WO2010151611A1 (en) 2009-06-26 2010-12-29 Sanofi-Aventis Novel fumarate salts of a histamine h3 receptor antagonist

Non-Patent Citations (65)

* Cited by examiner, † Cited by third party
Title
"Bioreversible Carriers in Drug Design", 1987, AMERICAN PHARMACEUTICAL ASSOCIATION AND PERGAMON PRESS
"From Neuron To Brain", 1992, SINAUER ASSOCIATES, INC., article "Indirect Mechanisms of Synaptic Transmission"
"Handbook of Pharmaceutical Excipients", 2003, PHARMACEUTICAL PRESS
"Harry's Cosmeticology", 1982, CHEMICAL PUBLISHING
"Remington: The Science and Practice of Pharmacy", 2000, LIPPINCOTT WILLIAMS & WILLDNS
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING CO.
ACTA VET SCAND., vol. 51, 20 October 2009 (2009-10-20), pages 40
ARRANG ET AL., NATURE, vol. 302, 1983, pages 832 - 7
AUSUBEL ET AL.: "Short Protocols in Molecular Biology", 1995, WILEY & SONS
BENOIST ET AL., NATURE (LONDON, vol. 290, 1981, pages 304 - 310
BERGE ET AL., JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 66, 1977, pages 1 - 19
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BONGERS G; KRUEGER KM; MILLER TR; BARANOWSKI JL; ESTVANDER BR; WITTE DG; STRAKHOVA MI; VAN MEER P; BAKKER RA; COWART MD: "An 80-Amino Acid Deletion in the Third Intracellular Loop of a Naturally Occurring Human Histamine H3 Isoform Confers Pharmacological Differences and Constitutive Activity", J PHARMACOL EXP THER, vol. 323, no. 3, 2007, pages 888 - 98
BR J DERMATOL., vol. 149, no. 1, July 2003 (2003-07-01), pages 17 - 22
BROWN ET AL., PROG. NEUROBIOL., vol. 63, 2001, pages 637 - 672
BRUTLAG ET AL., COMP APP BIOSCI, vol. 6, 1990, pages 237 - 245
CELLO ET AL., SCIENCE, vol. 297, 2002, pages 1016 - 8
CHAN, L.S. ET AL., J. INVEST. DERMATOL., vol. 117, 2001, pages 977 - 83
CHEN, J.; LIU, C.; LOVENBERG, T. W.: "Molecular and pharmacological characterization of the mouse histamine H(3) receptor", EUR J PHARMACOL, vol. 467, no. 1-3, 2003, pages 57 - 65
CLIN EXP ALLERGY., vol. 34, no. 3, March 2004 (2004-03-01), pages 456 - 9
DUNFORD, P.J. ET AL., J. ALLERGY CLIN. IMMUNOL., vol. 119, 2007, pages 176 - 83
EXP DERMATOL., vol. L 1, no. 4, August 2002 (2002-08-01), pages 285 - 91
GRAHAM ET AL., J. GEN VIROL., vol. 36, 1977, pages 59
HAMER ET AL., J. MOL. APPL. GEN., vol. 1, 1982, pages 273 - 288
HARLOW; LANE: "Antibodies. A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
HE TC ET AL., PROC NATL ACAD SCI USA, vol. 95, 1998, pages 2509 - 2514
HOOD ET AL.: "Immunology", 1984, BENJAMIN
HUNKAPILLER; HOOD, NATURE, vol. 323, 1986, pages 15 - 16
HUSTON ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 5879 - 5883
J NEUROSCI., vol. 28, no. 30, 23 July 2008 (2008-07-23), pages 7659 - 69
J. PHARM. EXPER. THERAPEUTICS, vol. 332, no. 1, 2010, pages 164 - 172
JOHNSTON ET AL., PROC. NATL. ACAD. SCI. (USA, vol. 79, 1982, pages 6971 - 6975
JONATHAN A. COVEL ET AL: "Design and Evaluation of Novel Biphenyl Sulfonamide Derivatives with Potent Histamine H 3 Receptor Inverse Agonist Activity", JOURNAL OF MEDICINAL CHEMISTRY, vol. 52, no. 18, 24 September 2009 (2009-09-24), pages 5603 - 5611, XP055075479, ISSN: 0022-2623, DOI: 10.1021/jm900857n *
JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 126, 2006, pages 1705 - 1718
K.J. GUILLORY: "Polymorphism in Pharmaceutical Solids", vol. 95, 1999, MARCEL DEKKER, INC., article "Generation of Polymorphs, Hydrates, Solvates, and Amorphous Solids", pages: 202 - 209
KENALDN, LIFE SCIENCES, vol. 43, 1988, pages 1095 - 1101
LANCET, vol. 361, 2003, pages 690 - 94
LANZAVECCHIA ET AL., EUR. J. IMMUNOL., vol. 17, 1987, pages 105
LEURS ET AL., NAT. REV. DRUG. DISCOV., vol. 4, 2005, pages 107 - 120
LOVENBERG, T.W.; ROLAND, B.L.; WILSON, S.J.; JIANG, X.; PYATI, J.; HUVAR, A.; JACKSON, M.R.; ERLANDER, M.G.: "Cloning and functional expression of the human histamine H3 receptor", MOL. PHARMACOL., vol. 55, no. 6, 1999, pages 1101 - 1107
MATHER ET AL., ANNALS N. Y. ACAD. SCI, vol. 383, 1982, pages 44 - 68
MATHER, BIOL. REPROD., vol. 23, 1980, pages 243 - 251
MCKNIGHT, CELL, vol. 31, 1982, pages 355 - 365
MCLEAN ET AL., MOL. PHARMA. MOL PHARMACOL., vol. 56, 1999, pages 1182 - 91
MILLIGAN ET AL., TRENDS IN PHARMACEUTICAL SCIENCES, vol. 20, 1999, pages 118 - 124
MILLIGAN; REES, TRENDS IN PHARMACEUTICAL SCIENCES, vol. 20, 1999, pages 118 - 24
NEUROPHARMACOLOGY, vol. 56, no. 8, 2009, pages 1131 - 1137
OFFERMANNS ET AL., J BIOL CHEM, vol. 270, 1995, pages 15175 - 15180
OFFERMANNS; SIMON, J BIOL CHEM, vol. 270, 1995, pages 15175 - 80
PASSANI ET AL., TRENDS IN PHARMACOL. SCI., vol. 25, 2004, pages 618 - 625
PASSANI ET AL., TRENDS PHARMACOL. SCI., vol. 25, 2004, pages 618 - 625
PAUS, R. ET AL., J. CLIN. INVEST., vol. 116, 2006, pages 1174 - 1185
RAMSAY ET AL., BR. J. PHARMACOLOGY, 2001, pages 315 - 323
ROSSBACH K ET AL: "Histamine H, Hand Hreceptors are involved in pruritus", NEUROSCIENCE, NEW YORK, NY, US, vol. 190, 1 June 2011 (2011-06-01), pages 89 - 102, XP028274954, ISSN: 0306-4522, [retrieved on 20110613], DOI: 10.1016/J.NEUROSCIENCE.2011.06.002 *
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR
SHIM WON-SIK ET AL: "Histamine-induced itch and its relationship with pain", MOLECULAR PAIN, BIOMED CENTRAL, LONDON, GB, vol. 4, no. 1, 31 July 2008 (2008-07-31), pages 29, XP021045078, ISSN: 1744-8069, DOI: 10.1186/1744-8069-4-29 *
SILVER ET AL., PROC. NATL. ACAD. SCI. (USA, vol. 81, 1984, pages 5951 - 59SS
SUZUKI ET AL., HUM GENE THER, vol. 7, 1996, pages 1883 - 1893
T. HIGUCHI; V. STELLA: "Pro-drugs as Novel Delivery Systems", vol. 14, A.C.S. SYMPOSIUM SERIES
TAKANO, N. ET AL., EUR. PHARMACOL., vol. 471, 2003, pages 223 - 228
UMEUCHI, H ET AL., EUR J PHARM., vol. 518, 2005, pages 133 - 139
WELLENDORPH P; GOODMAN MW; BURSTEIN ES; NASH NR; BRANN MR; WEINER DM: "Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor", NEUROPHARMACOLOGY, vol. 42, no. 7, 2002, pages 929 - 40
WISE ET AL., ANNU REV PHARMACOL TOXICOL, vol. 44, 2004, pages 43 - 66
ZHENG, T. ET AL., J. INVEST. DERMATOL., 16 October 2008 (2008-10-16)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105640980A (en) * 2016-01-29 2016-06-08 郑锦辉 Medicine for burns and scalds and preparation method thereof
WO2020257753A1 (en) * 2019-06-20 2020-12-24 Yale University Phosphatidylserine decarboxylase inhibitors

Similar Documents

Publication Publication Date Title
US11834447B2 (en) Indole derivatives and use thereof
JP6435001B2 (en) Pyrimidine carboxamides as sodium channel blockers
US9745287B2 (en) Pyrimidines and use thereof
US9714252B2 (en) Cyclic sulfonamides as sodium channel blockers
US9120786B2 (en) Triazine carboxamides as sodium channel blockers
US10738026B2 (en) Isoquinoline derivatives and use thereof
CN102153562B (en) Spiro-oxindole compounds and their uses as therapeutic agents
CN105008355B (en) It is used as the 7-azaindole derivatives of kinases inhibitor
EA020777B1 (en) 4-pyrazolyl-n-arylpyrimidin-2-amines, 4-pyrazolyl-n-pyrazolylpyrimidin-2-amines and 4-pyrazolyl-n-pyridylpyrimidin-2-amines as janus kinase inhibitors
AU2017202759A1 (en) Nitrogen-containing aromatic heterocyclic compound
EP2968307A2 (en) Carboxamide derivatives and use thereof
Daval et al. Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors
BR112016010041A2 (en) compound, medication, and, use of the compound or salt
AU2021240162A1 (en) EGFR inhibitor compounds
CA2564139A1 (en) Modulators of human g protein-coupled receptors for the treatment of hyperglycemia and related disorders
CN1917921A (en) Calcitonin gene related peptide receptor antagonists
WO2013151982A1 (en) Methods and compounds useful in treating pruritus, and methods for identifying such compounds
US9365511B2 (en) Biphenyl-ethyl-pyrrolidine derivatives as histamine H3 receptor modulators for the treatment of cognitive disorders
KR20200116115A (en) CXCR7 receptor agonist (3S,4S)-1-cyclopropylmethyl-4-｛[5-(2,4-difluoro-phenyl)-isoxazole-3-carbonyl]-amino｝-piperidine- Crystalline form of 3-carboxylic acid (1-pyrimidin-2-yl-cyclopropyl)-amide
Guo et al. Discovery of BGB-8035, a Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase for B-Cell Malignancies and Autoimmune Diseases
Maheshwari et al. Evaluation of 1, 2, 5-thiadiazoles as modulators of M1/M5 muscarinic receptor subtypes
JP2024500543A (en) Conolidine analogs as selective ACKR3 modulators for the treatment of cancer and cardiovascular diseases
WO2014028322A1 (en) Modulators of the histamine h3 receptor and the treatment of disorders related thereto
Pegoli Dibenzodiazepinone-type mAChR ligands: radio-and fluorescence labeling enable unveiling of dualsteric M2R binding and conjugation to short peptides as an avenue to highly selective M2R ligands

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13721570

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13721570

Country of ref document: EP

Kind code of ref document: A1