WO1993002206A1 - Purification du facteur neurotrophique ciliaire recombine et du facteur neurotrophique ciliaire tronque a terminaison c et procedes de traitement des lesions de nerfs peripheriques - Google Patents

Purification du facteur neurotrophique ciliaire recombine et du facteur neurotrophique ciliaire tronque a terminaison c et procedes de traitement des lesions de nerfs peripheriques Download PDF

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WO1993002206A1
WO1993002206A1 PCT/US1992/006136 US9206136W WO9302206A1 WO 1993002206 A1 WO1993002206 A1 WO 1993002206A1 US 9206136 W US9206136 W US 9206136W WO 9302206 A1 WO9302206 A1 WO 9302206A1
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cntf
protein
recombinant
peripheral nerve
human
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PCT/US1992/006136
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English (en)
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Franklin D. Collins
Deborah Russell
John R. Mcdonald
Erwin Freund
Larry J. Wilhelm
Duane Mach Bonam
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Syntex-Synergen Neuroscience Joint Venture
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Priority to EP92917747A priority Critical patent/EP0596034A4/fr
Priority to JP5503057A priority patent/JPH07500725A/ja
Priority to AU24432/92A priority patent/AU666327B2/en
Publication of WO1993002206A1 publication Critical patent/WO1993002206A1/fr
Priority to NO949194A priority patent/NO940194L/no
Priority to NO940194A priority patent/NO940194D0/no
Priority to FI940302A priority patent/FI940302A0/fi

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to neurotrophic factors and ciliary neurotrophic factor (CNTF) in particular, as well as methods of purifying CNTF and producing recombinant CNTF.
  • CNTF ciliary neurotrophic factor
  • Neurotrophic factors are a class of molecules that promote the survival and functional activity of nerve or glial cells. Evidence exists to suggest that neurotrophic factors will be useful as treatments to prevent nerve or glial cell death or malfunction resulting from the conditions enumerated above. Appel, 1981, Ann. Neuro1oq 10:499.
  • NGF nerve growth factor
  • a complication of the use of neurotrophic factors is their specificity for only those subpopulations of nerve cells which possess the correct membrane receptors. Most nerve cells in the body lack NGF receptors and are apparently unresponsive to this neurotrophic factor. It is, therefore, of critical importance to discover new neurotrophic factors that can support the survival of different types of nerve or glial cells than does NGF. New neurotrophic factors have been searched for by their ability to support the survival in culture of nerve cells that are not responsive to NGF.
  • One widely used screening assay is designed to discover factors that promote the survival of ciliary ganglionic motor neurons that innervate skeletal and smooth muscle. These ciliary ganglionic nerve cells belong to the parasympathetic nervous system and their survival is not supported by NGF.
  • ciliary ganglionic nerve cells have been reported from a variety of tissues and species. Many of these ciliary ganglionic neurotrophic activities have the following similar chemical and biological properties: (1) the activity is present in high concentration in sciatic nerves; (2) the neurotrophic activity survives exposure to the ionic detergent sodium dodecyl sulfate (SDS) and to the reducing agents beta-mecaptoethanol (BME) or dithiothreitol (DTT) during electrophoresis on SDS polyacrylamide reducing gels; and (3) on such gels the activity migrates with an apparent molecular weight between 24-28 kd. Collins, 1985, Developmental Biology. 109:255-258, Manthorpe, et al.. 1986, Brain Research, 367:282-286.
  • SDS ionic detergent sodium dodecyl sulfate
  • BME beta-mecaptoethanol
  • DTT dithiothreitol
  • ciliary neurotrophic factor typically referred to as "ciliary neurotrophic factor” or "CNTF”
  • CNTF ciliary neurotrophic factor
  • the peripheral nervous system consists of those nerve cells that extend axonal processes outside the spinal cord and brain.
  • the principle nerve cell types in the peripheral nervous system are primary motor neurons innervating skeletal muscle and controlling movement, autonomic neurons (both sympathetic and parasympathetic) innervating the cardiovascular system and other internal organs and regulating their function, and sensory neurons innervating sensory receptors throughout the body and conveying sensations including pain and proprioception.
  • peripheral nerve damage Conditions that compromise the survival and proper function of one or more of these types of peripheral nerve cells cause peripheral nerve damage. Such damage may occur through physical injury, which causes the degeneration of the axonal processes of peripheral nerve cells that pass through or near the site of injury. Such damage may also occur because of intentional or accidental exposure to neurotoxins, such as the cancer and AIDS chemotherapeutic agents cisplatinu and dideoxycytidine (ddC) , respectively. Such damage may also occur because of chronic metabolic diseases, such as diabetes or renal dysfunction. Such damage may also occur because of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) , which causes the degeneration of primary motor neurons and consequently motor dysfunction.
  • ALS Amyotrophic Lateral Sclerosis
  • peripheral nerve damage is compromised function and/or survival of peripheral nerve cells and their axonal processes.
  • This invention describes treatments that can support peripheral nerve cells: that is, promote the normal function and survival of peripheral nerve cells against the effects of conditions that typically lead to peripheral nerve damage, or to reverse or minimize the effects of peripheral nerve damage.
  • CNTF human protein ciliary neurotrophic factor
  • CNTF used according to these methods can be effective in preventing or reversing peripheral nerve damage in adult animals.
  • the appropriate route of administration and the appropriate dosing of CNTF needed to treat different forms of peripheral nerve damage are also disclosed. These methods differ among different diseases primarily in the way CNTF is administered and the dosing that is used.
  • An object of this invention is to provide recombinant human ciliary neurotrophic factor protein having an amino acid sequence as in Figure 1 where the amino acids at the C-terminal end are cleaved.
  • Preferred truncated forms of CNTF are truncated by two or six amino acids.
  • the invention also provides for an expression vector comprising a DNA sequence encoding for C- terminal truncated forms for CNTF and their expression in bacterial and preferably E. coli expression systems.
  • this invention provides for recombinant human CNTF substantially free of truncated forms of CNTF.
  • the present invention provides a method for preparing a substantially purified CNTF comprising: (a) applying cell lysate containing soluble CNTF protein to an anion exchange column which reversibly binds CNTF; (b) collecting fractions comprising CNTF by eluting the CNTF protein bound to the anion exchange column with salt; (c) applying the fractions containing CNTF protein to a cation exchange column; (d) collecting fractions comprising CNTF by eluting the CNTF protein with a pH gradient of from about 7 to about 8.5; (e) applying the fractions containing CNTF protein to an anion exchange column; and (f) eluting the substantially purified CNTF protein with a salt gradient.
  • the present invention also includes a method for preventing or treating peripheral nerve damage which comprises administering to a patient in need thereof a therapeutically effective amount of CNTF; the use of a therapeutically effective amount of CNTF for the manufacture of medicament suitable for preventing or treating peripheral nerve damage; and an agent for preventing or treating peripheral nerve damage which comprises a therapeutically effective amount of CNTF.
  • the invention provides methods for administering therapeutically effective amounts of CNTF by therapeutically effective routes of administration in order to prevent and reverse peripheral nerve damage.
  • the invention also demonstrates the adequacy of these methods to prevent or reverse peripheral nerve damage from a variety of insults.
  • Figure 1 shows the DNA and inferred amino acid sequence of human CNTF.
  • the human CNTF coding sequence is interrupted by a single ca. 1.3-kb intron located between amino acids 38 and 39.
  • the splice acceptor/donor sequences at this site are: [GTAAGT...1.3kb...TTTCCTGTATCCTCGGCCAG] .
  • the internal sequence of human CNTF is interrupted by a single ca. 1.3-kb intron located between amino acids 38 and 39.
  • the splice acceptor/donor sequences at this site are: [GTAAGT...1.3kb...TTTCCTGTATCCTCGGCCAG] .
  • Hindlll and Nhel sites used in construction of the expression vector are underlined as are the oligon cleotides used for cloning.
  • Figure 2 shows the inferred amino acid sequences of human, rabbit and rat CNTF.
  • the amino acid sequences are presented in single letter code. Numbers to the right indicate position in the human sequence. Regions in which the sequence is identical in all three species are shaded. Since the inferred rabbit protein is one amino acid shorter than either human or rat CNTF, a gap (indicated by a dash) has been introduced into the rabbit sequence to maximize alignment.
  • Figure 3 shows the SDS-Page analysis of selected fractions eluted from initial Q-Sepharose ion- exchange chromatography column (Step 3) .
  • Cell extract was chromatographed on a column of Q-Sepharose (1.5 X 20 cm) .
  • the chromatogram was developed at 2 ml/min and 2 ml fractions were collected. Selected fractions were subjected to SDS-PAGE and the gels stained with CBB.
  • samples (15 ⁇ l, lanes 1 and 2 or 30 ⁇ l, lanes 3-28) were diluted in SDS-sample buffer (final concentrations: 10% glycerol, 1% DTT, 0.5% SDS, 0.002% bromophenol blue and 25 Mm Tris-HCl, pH 6.8) and boiled for 2 min.
  • Figure 4 shows the Q-Sepharose ion-exchange chromatography (Step 4) .
  • the CNTF pool from the first Q-Sepharose column was dialyzed and chromatographed on a second Q-Sepharose column (1.5 X 15 cm).
  • the chomatogram was developed at 2 ml/min and 2 ml fractions were collected. Selected fractions were prepared for electrophoresis, subjected to SDS-PAGE and the gels silver-stained.
  • the inset shows the protein content of electrophoresed fractions of the CNTF pool (fractions 114-130) .
  • FIG. 5 shows the S-Sepharose ion-exchange chromatography (Step 5) .
  • the CNTF pool from Step 4 was dialyzed and chromatographed on an S-Sepharose column (1 X 10 cm) .
  • the chromatogram was developed at 2 ml/min and 4 ml fractions were collected.
  • Figure 6 shows the Zn 2+ -affinity chromatography (Step 6) .
  • the CNTF pool from Step 5 was dialyzed and chromatrographed on a Zn 2+ -IDA-agarose column (1 X 10 cm) .
  • the chromatogram was developed at
  • FIG 7 shows the RP-HPLC analysis of purified recombinant human CNTF.
  • CNTF, 5 ⁇ g (A) and 50 ⁇ g (B) were applied to SynChrom RP-8 reverse phase HPLC column (250 X 4.6 mm) equilibrated with 0.1% TFA. Protein was eluted with a linear gradient of acetonitrile containing 0.1% TFA (1% acetonitrile/min; flow rate, 1 ml/min) .
  • Figure 8 shows the multiple forms of CNTF.
  • Purified human recombinant CNTF (12 ⁇ g) was subjected to SDS-PAGE with (A) or without (B) prior heating in SDS sample buffer to 100°C for 2 min.
  • the protein was transblotted onto a nitrocellulose membrane and treated with primary antibody (rabbit anti-CNTF) and secondary antibody (goat anti-rabbit IgG-alkaline phosphatase) .
  • Figure 9 shows the u.v.-absorption spectrum of recombinant human CNTF.
  • the u.v. absorption spectrum was recorded on a Beckman DU-50 spectrophotometer and the concentration of CNTF (1.28 mg/ml) determined by amino acid analysis.
  • Figure 10 illustrates the rate of recovery of cutaneous sensation after sciatic nerve crush (on day 0) in adult rats treated with vehicle only or with vehicle containing 0.25mg/kg human recombinant CNTF delivered in daily subcutaneous injections on days -2 to +11.
  • Figure 11 illustrates the rate of recovery of motor function (measured by recovery of the ability to spread toes 1-5) after sciatic nerve crush (on day 0) in adult rats treated with vehicle only or with vehicle containing 0.25mg/kg human recombinant CNTF delivered in daily subcutaneous injections on days -2 to +11.
  • This application includes recombinant methods of production of human ciliary neurotrophic factor
  • CNTF C-terminal truncated CNTF
  • the C-terminal truncated CNTFs are identical to full length human CNTF as shown in figure 1 but are truncated at the C-terminus by either two or six amino acid residues.
  • the C-terminal truncated CNTFs of the present invention preferably are produced during the bacterial expression of human CNTF by the expression of vectors containing the gene coding for CNTF.
  • Such C-terminal truncated CNTFs may also be produced by the expression of vectors containing the gene coding for the C-terminal truncated CNTFs.
  • This invention also includes purification processes for obtaining substantially purified CNTF obtained from recombinant production systems.
  • PCT application WO 90/07341 of Collins et al. PCT/US90/00022
  • the Collins application includes a description of the rabbit and human genes coding for CNTF and the production of recombinant human CNTF from mammalian and bacterial expression systems.
  • the WO 90/07341 application is specifically incorporated herein, in its entirety, by this reference, including without limitation all definitions and experimental procedures.
  • a novel process for the production and purification of recombinant human CNTF is given below in Example l. Further included in this example is a six step process for the purification of recombinant human CNTF comprising: 1. the preparation of cell free extracts;
  • CNTF composition is prepared that contains less than 0.1% non-CNTF proteins.
  • a preferred method of purification of human recombinant CNTF as taught in Example 2 below comprises: (a) applying cell lysate containing soluble CNTF protein to an anion exchange column which reversibly binds CNTF; (b) collecting fractions comprising CNTF by eluting the CNTF protein bound to the anion exchange column with salt; (c) applying the fractions containing CNTF protein to a cation exchange column; (d) collecting fractions comprising CNTF by eluting the CNTF protein with a pH gradient of from about 7 to about 8.5; (e) applying the fractions containing CNTF protein to an anion exchange column; and (f) eluting the substantially purified CNTF protein with a salt gradient.
  • Example 2.below An alternate purification of CNTF is described in Example 2.below, wherein C-terminal truncated CNTFs are isolated and identified.
  • the C- terminal truncated CNTFs are isolated from a bacterial expression of a vector containing the gene coding for full length human CNTF.
  • the C-terminal truncated CNTFs so isolated are identical to human CNTF — as shown in Figure 1 — but are truncated at the c-terminus by either 2 or 6 amino acid residues.
  • Also described are procedures for isolating CNTF from C-terminal truncated CNTFs and substantially purified CNTF which is substantially free from C-terminal truncated CNTFs.
  • C-terminal truncated CNTFs that retain any of the biological activity associated with CNTF. It has also been found that recombinant CNTF produced as described in Example 2 can be further purified by using an additional chromatography step. As described in Example 3 below, columns that have been found effective in lowering the amount of non-CNTF proteins from purified CNTF solution include hydroxy apatite resin, butyl HIC (hydrophobic interaction chromatography) resin and Zn-IMAC (immobilized metal affinity chromatography) resin.
  • the present invention further relates to methods for preventing and treating peripheral nerve damage in patients suffering therefrom.
  • These methods comprise the route of administration of a therapeutically effective amount of a ciliary neurotrophic factor (CNTF) to a patient suffering from peripheral nerve damage or to a patient at risk of suffering peripheral nerve damage.
  • CNTF ciliary neurotrophic factor
  • a disease or medical indication is to be considered to be peripheral nerve damage if the survival or function of peripheral nerve cells and/or their axonal processes is compromised.
  • a patient is at risk of suffering peripheral nerve damage or actually has peripheral nerve damage as the result of one of the following conditions: 1) Physical injury, which causes the degeneration of the axonal processes of peripheral nerve cells that pass through or near the site of injury; 2) Exposure to neurotoxins, such as the cancer and AIDS chemotherapeutic agents cisplatinum and dideoxycytidine (ddC) , respectively; 3) Chronic metabolic diseases, such as diabetes or renal dysfunction; and, 4) Neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) , which causes the degeneration of primary motor neurons and consequently motor dysfunction.
  • ALS Amyotrophic Lateral Sclerosis
  • a non-exclusive list of conditions involving peripheral nerve damage includes Amyotrophic Lateral Sclerosis, Diabetic
  • Peripheral Polyneuropathy Toxic Peripheral Neuropathy caused by the cancer chemotherapeutic agents taxol or cisplatin or vincristine, Toxic Peripheral Neuropathy caused by the AIDS chemotherapeutic agents ddl or ddC, and physical damage to peripheral nerves such as that caused by crush or cut injuries to the arm and hand.
  • peripheral nerve damage includes the ability to reverse permanent peripheral nerve damage and the ability to enhance naturally occurring recovery processes by either speeding up such processes or by effecting a more complete recovery from the peripheral nerve damage.
  • the prevention of peripheral nerve damage includes the ability to totally prevent nerve damage against the effects of conditions that typically lead to peripheral nerve damage, as well as the ability to lessen the extent of peripheral nerve damage associated with such conditions.
  • preferred CNTFs are naturally occurring proteins.
  • the naturally-occurring proteins are preferred in part because they pose a comparatively low risk of producing unforeseen and undesirable physiological side effects in patients treated therewith.
  • Human CNTFs are preferred for use in this invention.
  • non ⁇ human CNTFs are substantially equivalent to human CNTFs and possess equivalent biological activity, they are considered to be within the scope of this invention.
  • a protein is deemed to be "naturally-occurring” if it or a substantially equivalent protein can be found to exist normally in healthy humans.
  • “Naturally- occurring” proteins specifically includes forms of proteins found to exist in healthy humans that are partially truncated at the amino or carboxyl terminus of such proteins or that have amino acids that are deamidated or otherwise chemically modified.
  • “Naturally-occurring” proteins may be obtained by recombinant DNA methods as well as by isolation from cells which ordinarily produce them. “Naturally- occurring” also encompasses proteins that contain or lack an NH 2 -terminal methionyl group as a consequence of expression in E. coli.
  • Substantially equivalent as used throughout the specification and claims is defined to mean possessing a very high degree of amino acid residue homology (See generally M. Dayhoff, Atlas of Protein
  • CNTFs of the present invention are the naturally-occurring proteins that have previously been described in PCT application WO 90/07341 of Collins, et al. entitled "Purified Ciliary Neurotrophic Factor.”
  • CNTF is modified by attachment of one or more polyethylene glycol (PEG) or other repeating polymeric moieties.
  • PEG polyethylene glycol
  • One disclosed method consists of isolating CNTF from various sources, such as peripheral nerve tissues.
  • a second disclosed method involves isolating the genes responsible for coding CNTF, cloning the gene in suitable vectors and cell types, and expressing the gene in order to produce the CNTF.
  • the latter method which is exemplary of recombinant DNA methods in general, is a preferred method of the present invention. Recombinant DNA methods are preferred in part because they are capable of achieving comparatively higher amounts at greater purity.
  • the above described CNTFs are produced by the aforementioned method in "substantially pure” form.
  • substantially pure it is meant that CNTF, in an unmodified form, has a comparatively high specific activity. It is to be recognized, however, that derivatives of CNTF may have different specific activities.
  • a therapeutic composition comprising CNTF is administered in an effective amount to patients suffering from peripheral nerve damage.
  • the method of the present invention could be practiced by administering a therapeutic composition whose active ingredient consists of that portion (or those portions) of CNTF which controls (or control) CNTF neurotrophic function.
  • the therapeutic composition of the present invention is preferably administered parenterally by injection or intrathecally by continuous infusion from an implanted pump.
  • other effective administration forms such as parenteral slow-release formulations, inhalant mists, orally active formulations, or suppositories, are also envisioned.
  • the carrier and the CNTF constitute a physiologically-compatible, slow- release formulation.
  • the primary solvent in such a carrier may be either aqueous or non-aqueous in nature.
  • the carrier may contain other pharmacologically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmacologically-acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the CNTF.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dose or multi- dose form or for intrathecal delivery by continuous or periodic infusion from an implanted pump or intrathecally by periodic injection.
  • the therapeutic composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready to use form or requiring reconsti ution immediately prior to administration.
  • the preferred storage of such formulations is at temperatures at least as low as 4°C and preferably at -70°C. It is also preferred that such formulations containing CNTF are stored and administered at or near physiological pH. It is presently believed that storage and administration in a formulation at a pH below approximately pH 5.5 and above approximately pH 8.0 is undesirable.
  • the manner of parenterally administering the formulations containing CNTF is via a subcutaneous or intramuscular route.
  • a subcutaneous or intramuscular route to achieve the desired dose of CNTF.
  • repeated daily or less frequent subcutaneous or intramuscular injections may be administered. It is believed that the administration of CNTF in daily doses below approximately O.OOl g/kg may not be effective, while the administration of daily doses of greater than lmg/kg have undesirable side effects.
  • a preferred dosage range for the parenteral treatment of peripheral nerve damage is between about 0.01 and 0.25 mg per kg of patient body weight per 24 hours administered in a single dose per 24 hours.
  • the administration of CNTF will begin up to one week before the condition or initiation of events that typically leads to peripheral nerve damage.
  • administration of CNTF will begin up to 1 week before the initiation of treatment with the chemotherapeutic agent and will continue during the period of exposure to the agent.
  • the frequency of dosing will depend on pharmacokinetic parameters of CNTF in the formulation used and will be readily ascertained by one skilled in the art.
  • CNTF may be administered intrathecally into the subarachnoid space of the spinal cord. Administration may be continuous or periodic and may be accomplished by a constant- or programmable-flow implantable pump or by periodic injections.
  • CNTF which is administered in this fashion is encapsulated.
  • the encapsulated CNTF may be formulated with or without those carriers customarily used in the compounding of solid dosage forms.
  • the capsule is designed so that the active portion of the formulation is released at that point in the gastro-intestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional excipients may be included to facilitate absorption of CNTF. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • the specific dose is calculated according to the approximate body weight or surface area of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them without undue experimentation, especially in light of the dosage information and assays disclosed herein. These dosages may be ascertained through use of the established assays for determining dosages utilized in conjunction with appropriate dose-response data.
  • CNTF formulations described herein may be used for veterinary as well as human applications and that the term "patient” should not be construed in a limiting manner. In the case of veterinary applications, the dosage ranges should be the same as specified above.
  • Example 4 describes the application of the present invention to peripheral nerve damage from physical injury to a peripheral nerve, as described herein.
  • the differences, if any, between this treatment and the treatment of patients suffering from other forms of peripheral nerve damage would be readily and routinely identified by one of ordinary skill in the art.
  • the ability to accelerate the recovery of sensory and motor function after physical injury to peripheral nerves by administering CNTF as shown in the following example shows that the administration of CNTF may be equally effective in preventing and treating other forms of peripheral nerve damage, as defined herein.
  • EXAMPLE 1 PURIFICATION OF RECOMBINANT CILIARY NEUROTROPHIC FACTOR Materials and Methods - The purification, cloning and expression of CNTF has been previously described and is incorporated by reference herein, Collins et al.. WO 90/07341. (See also, Collins, et al. , U.S. Patent 5,011,914 and Collins et al.. U.S. Patent 4,997,929). Numbers in brackets refer to references listed below in the section entitled References for Example 1.
  • CNTF-1 TAT/C GTN AAA/G CAT/C CAA/G GG (Tyr-Val- Lys-His-Gln-Gly)
  • CNTF-2 AAT/C AAA/G AAT/C ATT/C/A ATT/C C/TT (Asn-Lys-Asn-Ile-Asn-Leu)
  • CNTF-3a AAA/G TTA/G TGG GGN TTA/G AA
  • CNTF-3b AAA/G TTA/G TGG GGN CTN AA
  • CNTF-3C AAA/G CTN TGG GGN TTA/G AA;
  • Oligonucleotides 1 (sense) and 3a to 3d (anti-sense) were used as primers in PCR with human genomic DNA. Polymerase chain reactions were performed as previously described [1] except that each reaction contained 1.75 mM MgCl 2 , 100 ng of each oligonucleotide, and 0.5 ⁇ g of human genomic DNA prepared from placenta [2]. To identify DNA bands amplified from the CNTF gene, DNA (Southern) blots of the PCR products were probed with 32 P-labeled oligonucleotide 2, which occurs just downstream of oligonucleotide 1 in the rabbit gene [7]. A single ca.
  • the ca. 400-bp fragment amplified from human genomic DNA was labeled with 32 P by random priming and used to screen a human genomic DNA library at high stringency.
  • the human genomic DNA library was constructed by cloning genomic DNA [18], partially digested with Sau3AI, into the BamHI site of Charon 30 [3]. Out of lxlO 6 clones, nine positive clones were isolated. Two of these clones were sequenced and the rest appeared related to these based on DNA (Southern) blot analysis.
  • the sequenced clones contained an open reading frame (Fig. 1) that was 89% identical to the rabbit CNTF coding sequence [7], In addition, each open reading frame contained a segment identical to the fragment amplified from human genomic DNA by PCR.
  • Restriction endonuclease fragments from the human genomic DNA clones corresponded to those observed on DNA (Southern) blot analysis of human genomic DNA, indicating that the clones were representative of the organization of the CNTF gene in genomic DNA.
  • Phagemid 1 contains a single Nhel site at amino acids 22-23 in the human CNTF coding sequence (Fig. 1) . Partially overlapping complementary oligonucleotides,
  • Oligonucleotides 2 and 3 also contain a transnational coupler to promote effective translation in E. coli [6] .
  • Phagemid 2 DNA was then digested with BamHI and Hindlll to release the DNA fragment referred to as CNTF-Synl which contains DNA sequences suitable for expression in E. coli and encoding human CNTF upstream of the Hindlll site (Fig. 1) .
  • a human genomic DNA clone for CNTF in phage Charon 30 was cut with the restriction enzyme Hindlll and a 2.1 kb-fragment, containing the CNTF coding sequences downstream of the Hindlll site (Fig. 1) , was subcloned into Hindlll-cut plasmid pEMBL ⁇ [7].
  • a Spel site was inserted into the 2.1 kb-insert DNA by oligonucleotide directed mutagenesis 13 base pairs downstream of the stop codon ending the CNTF sequence using the synthetic oligonucleotide 4 (5' - ATG TAG CAG TTA GTC ACT AGT CTC TTC CTT GCT - 3') .
  • the mutated plasmid was cut with Hindlll and Spel to release the DNA referred to as CNTF-Syn2.
  • CNTF-Synl and CNTF-Syn2 were ligated at the Hindlll overhangs to produce CNTF-Synl/2, which was subcloned into the BamHI- and Spel-cut phagemid expression vector pJU1003 [8] to produce pJU1003- huCNTF, which was transformed into E. coli strain BL21(DE3) [9].
  • IPTG isopropyl S-D-thio-galactopyranoside
  • CNTF-A Overnight cultures of CNTF-A were prepared in Luria broth [10] supplemented with 10 ⁇ g/ml of tetracycline. These cultures were diluted (1 to 50) with the same medium and grown until the A 600 reached 1.0 (3-4 h) . Expression of CNTF was achieved by adding IPTG to a final concentration of 0.5 mM and incubating for 4 h. Cells were harvested by centrifugation (9,000 X g, 5 min), washed with 50 mM sodium phosphate, pH 8.0, and recentrifuged. Cell pastes were either used immediately or stored frozen at -80°C.
  • Step 1 Preparation of Cell Free Extracts - A cell paste (4-5 g wet weight) was suspended in 3-4 volumes of buffer A (50 mM sodium phosphate, pH 8.0, containing 5 mM EGTA and 5 mM EDTA) and passed through a buffer A (50 mM sodium phosphate, pH 8.0, containing 5 mM EGTA and 5 mM EDTA) and passed through a buffer A (50 mM sodium phosphate, pH 8.0, containing 5 mM EGTA and 5 mM EDTA) and passed through a
  • Step 2 Removal of Nucleic Acids - PEI 5 was added to the supernatant to a final concentration 0.25% (v/v) to facilitate removal of nucleic acids [11]. Without this treatment, the nucleic acid contained in the supernatant would bind to the anion-exchange
  • Step 3 O-Sepharose Ion-exchange Chromatography - Cell extract was loaded onto a column (1.5 X 20 cm) of Q-Sepharose previously equilibrated with buffer A.
  • Step 4 O-Sepharose Ion-exchange Chromatography -
  • the above CNTF pool was loaded onto a column (1.5 X 15 cm) of Q-
  • the CNTF pool was dialyzed twice against 10 volumes of buffer C (5 mM sodium phosphate, pH 7.1, containing 0.1 mM EGTA and 0.1 mM EDTA) .
  • Step 5 S-Sepharose Ion-exchange Chromatography -
  • the above CNTF pool was loaded onto a column (1 X 10 cm) of S-
  • CNTF pool was dialyzed twice against 10 volumes of buffer D (10 mM Hepes, pH 7.5, containing 50 mM NaCl, 0.1 mM EGTA, and 0.1 mM EDTA) .
  • buffer D 10 mM Hepes, pH 7.5, containing 50 mM NaCl, 0.1 mM EGTA, and 0.1 mM EDTA.
  • the CNTF pool was loaded onto a column (I X 10 cm) of Zn 2+ -IDA agarose previously equilibrated with buffer D without the metal ion chelators, EGTA and EDTA. After loading, the column was washed with the same buffer until the A 280 reached baseline. Bound proteins were eluted with a gradient (50 ml) of 0 to 50 mM histidine in buffer D (without chelators) . The final, purified CNTF pool was dialyzed twice against 10 volumes of 10 mM phosphate, pH 8.0, containing 50 mM NaCl, 0.1 mM EGTA and 0.1 mM EDTA and stored at - 80°C.
  • RP-HPLC - TFA and acetonitrile were added to protein samples to final concentrations of 0.1% (v/v) and 5% (v/v) , respectively, prior to injection.
  • RP- HPLC was performed using a 250 X 4.6 mm SynChropak RP-8 column (SynChrom, Inc., Lafayette, IN) with 0.1% aqueous TFA as solvent A and 0.1% TFA in acetonitrile as solvent B.
  • Electrophoresis and Blotting Techniques were performed in 12.5% polyacrylamide slab gels (1.5 mm thick), with a 5% acrylamide stacking gel, in the presence of 0.1% (w/v) SDS at 40 mA, with the discontinuous buffer system of Laemmli [12]. Gels were stained with CBB as described previously [13] or silver-stained using a Rapid-Ag-Stain Kit (ICN Radiochemicals, Irvine, CA) . Gels to be used to separate proteins prior to Western blotting and protein sequencing were pre-electrophoresed for 16 h at 15 mA in the presence of 25 mM thioglycolic acid and 10 mM DTT. This prevents blockage of amino-terminal amino acid groups during electrophoresis of protein samples [14]. Western blotting was preformed as previously described [15] using Immobilon-P (Millipore
  • nitrocellulose membranes were subjected to treatment with antibodies to CNTF, and subsequently, with goat anti-(rabbit IgG) conjugated to alkaline phosphatase (Cappel) .
  • the secondary antibody was detected using a kit with 5- bromo-4-chloro-indo-3-yl phosphate and nitroblue tetrazolium supplied by Promega (Madison, WI) .
  • Bioassays - Bioassays for CNTF activity were performed as described by Lin et al.. [10]. Briefly, the in vitro assay for CNTF activity [17] measures the survival of chick embryo ciliary ganglion (E8) , sympathetic chain (Ell) or dorsal root ganglion (E10) neurons. Two thousand purified neurons were placed into each well of a 96-well dish and serial dilutions of samples to be assayed were added. After 20 h
  • neuronal survival was estimated by the ability of live cells to reduce the vital dye MTT (3-4[,5-dimethylthiazol-2-yl]-2,5- diphenyltetra-zoliu ) (Sigma) .
  • MTT vital dye
  • The' titer of bioactivity in trophic units (TU) per ml was defined as the dilution that gave 50% of the maximal neuronal survival in the MTT assay. For example, if a dilution of 1:1000 was required to give 50% survival, the titer was defined as 1,000 TU/ml.
  • Peptide Mapping and Protein Sequencing - Generation of C-terminal peptides of CNTF was achieved by first digesting the protein with CNBr overnight at room temperature in hexafluoro-acetone hydrate. Peptides were separated on a narrow-bore C8 RP-HPLC column (Brownlee, Inc., Santa Clara, CA) , with 0.085% aqueous TFA as solvent A and 0.085% TFA in 80% acetonitrile as solvent B. The C-terminal peptide was then subdigested with endoproteinase ASP-N [7] and the peptides separated as above. Amino-acid analysis and protein sequencing were carried out as described by Armes and Forney [18] .
  • Human CNTF Gene The genomic DNA sequence and inferred amino acid sequence encoding human CNTF are shown in Figure 1.
  • the human DNA and protein sequences are 89% and 86% identical to the rabbit CNTF [7] and 85% and 83% identical to the rat CNTF [8] DNA and protein sequences, respectively.
  • Alignment of the inferred amino acid sequences of human, rabbit and rat CNTF is shown in Figure 2. Only a single band that hybridized to CNTF-specific probes was observed in DNA (Southern) blots of human genomic DNA digested with various restriction endonucleases (not shown) , consistent with only a single gene in human genomic DNA hybridizing at high stringency.
  • CNTF Purification of recombinant CNTF - Upon induction with IPTG, cultures of the bacterial transformant pJU1003-CNTF-A synthesized recombinant human CNTF. At the end of the culture period, CNTF accounted for approximately 13% of the soluble protein in cell extracts (25 mg/liter/A 60rj unit) as judged by laser densitometer analysis of CBB-stained gels (Fig. 3, lane 1) .
  • the resultant CNTF pool (Fig. 4, inset) was dialyzed into a low ionic strength buffer at pH 7.1 and subjected to cation-exchange chromatography on S- Sepharose. CNTF bound to the resin was eluted by application of a salt gradient as a peak between 125- 250 mM NaCl (Fig. 5).
  • the CNTF pool (Fig. 5, inset) was then subjected to a final affinity chromatography step on a Zn 2+ -IDA-agarose column. CNTF bound to the column, probably via an interaction between zinc and histidine residues, of which CNTF possesses ten per molecule (Table I) .
  • CNTF was eluted from the column by application of a histidine gradient at 30-35 mM histidine (Fig. 6) .
  • CNTF The amino acid composition of human recombinant CNTF, purified as above, corresponded well to the amino acid composition predicted from the human coding sequence (Table I) .
  • peptide map and amino acid sequence analyses of the purified protein indicated only the presence of CNTF sequences.
  • the amino acid sequence of recombinant human CNTF was that expected from the human coding sequence (Fig. 1) , except for the failure to detect an amino-terminal methionine. Amino acid sequence analysis of three different CNTF preparations yielded less than 0.1% of the expected amount of methionine at the amino-terminal position. Removal of the amino- terminal methionine during expression in bacteria is not uncommon for proteins, such as CNTF, in which the amino-terminal methionine is following by an alanine residue [20, 21]. The purity of CNTF was further analyzed by
  • CNTF CNTF. Therefore, the two peaks represent forms of CNTF that differ in an unknown way. These CNTF forms may be a consequence of deamidation.
  • proline isomerization which has been reported for other proteins, including insulin [22, 23, 24] .
  • No CNTF sequence was detected from a strip of immobilon-P excised from between the 46,000 and 23,000 dalton species. This suggests that the detection of any CNTF sequence at the 46,000 dalton level is not a consequence of streaking of native CNTF upon. SDS-PAGE.
  • the 21,000 dalton species may be a carboxyl-terminally truncated form of CNTF, since its amino terminus was intact (too little material was available for sequencing of the carboxyl-terminal peptide) . Based on later densitometry of CBB stained gels, the apparent dimer accounted for approximately 0.01% and the truncated CNTF for approximately 0.1% of the total protein. The 0.1% RP-HPLC peak, discussed above, was tentatively identified as a putative truncated form of CNTF. Loss of the highly charged carboxyl-terminus of CNTF (Fig. 1) would reasonably be expected to alter migration on RP-HPLC.
  • Bioactivity of recombinant human CNTF - There would appear to be no need to perform a refolding step in order to produce biologically active CNTF from the bacterial expression system. Since there is only one cysteine in human CNTF (amino acid 17 in Fig. 1) , there can be no intramolecular disulfide bonds that would need to reform correctly. Also, since CNTF can regain biological activity after exposure to SDS, acetonitrile, and TFA [10], CNTF appears able to spontaneously refold after some forms of denaturation. As anticipated, both crude bacterial lysate and purified recombinant CNTF exhibited CNTF biological activity.
  • the primary structure of human CNTF exhibits no N-linked glycosylation sites (N-X-T/S) (Fig. 1) .
  • the biological activity of bacterially- expressed human CNTF indicates that other forms of glycosylation, even if they occur in vivo, are not essential for the biological activity of CNTF.
  • the percentage of CNTF in crude extract and subsequent pooled fractions was estimated by laser densitometer analysis of CBB, rather than silver stained gels. The percent values are the mean of at least three separate determinations.
  • CG chick embryo E8 ciliary ganglion
  • SC Ell sympathetic chain
  • DRG dorsal root ganglio
  • Typical production scales were 10 or 160 liters.
  • the cells were thawed and water was added to obtain 20% cell solids and the pH was adjusted to 8.2 with 0.5 M Tris-base. Alternatively freshly harvested cells were used. The entire process from performed at 4-
  • the cells were lysed by a continuous homogenizer.
  • the lysate was clarified by centrifugation and diluted with 10-15 volumes of cold water to a conductivity equal to that of the lead column equilibration buffer.
  • Step 1 (CNTF CAPTURE) Q-SEPHAROSE FAST FLOW COLUMN STEP
  • the product was captured on an anion exchange column (2.5 cm diameter by 7.1 cm) with 35 mL bed volume of Q-Sepharose fast flow resin (Pharmacia) , equilibrated with 10 mM Tris-HCl pH 8.1 and 1 mM EDTA.
  • the clarified lysate was pumped at 11 mL per minute through a 3 uM filter and then on the column.
  • the column was washed at the same flowrate with column equilibration buffer until the OD (280 nm) returned to base line (approximately three bed volumes) .
  • CNTF was step eluted from the column at 3.7 mL per minute with 80 mM NaCl prepared in 10 mM Tris-HCl pH 8.2 and 1 mM EDTA. The entire peak was pooled and diluted two fold with water and the pH was adjusted to 7.2 with 0.1 N H 3 P0 4 .
  • Step 2 S-SEPHAROSE FAST FLOW COLUMN STEP (CNTF C- terminal TRUNCATED FORMS I, II AND III SEPARATION)
  • the pooled CNTF was diluted two-fold with cold water and loaded at 4.7 mL/minute onto the cation- exchange (S-Sepharose, Pharmacia) column (2.5 cm diameter and 2,500 mL bed volume) equilibrated with 25 mM NaPO pH 7.1, 25 mM NaCl, and 0.1 mM EDTA.
  • the column was washed at the same flowrate with equilibration buffer until the OD (280 nm) returned to baseline.
  • the CNTF was eluted with a pH gradient going from 7.1 to 8.1. This was accomplished by a 200 mL gradient made up with two buffers.
  • the low pH buffer was 25 mM NaPi pH 7.1, 25 mM NaCl, and 0.1 mM EDTA.
  • the high pH buffer was made up of 25 mM Tris-HCL, pH 8.1, 25 mM NaCl, and 0.1 mM EDTA.
  • the CNTF was gradient eluted at a flowrate of 1.2 mL/minute. Of the three peaks, the last peak (pH 7.8-8.1) was pooled from 0D (280 nm) 0.2 to 0.2.
  • the last peak contains substantially pure CNTF Form III, which contains the full amino acid sequence as set forth in Figure 1.
  • the second peak contains C- terminal truncated CNTF Form II, wherein the last two amino acids of the C-terminal end of the amino sequence as set forth in Figure 1 are cleaved.
  • the first peak contains C-terminal truncated CNTF Form I, wherein the last six amino acids of the C-terminal end of the amino acid sequence as set forth in Figure 1 are cleaved.
  • the CNTF C-terminal truncated forms I, II, and III were also separated using NaCl gradients at pH 7.1 using 50 mM Phosphate buffer.
  • Step 3 Q-SEPHAROSE FAST-FLOW COLUMN STEP
  • the pH of the S-Sepharose pool was adjusted to 8.0 with 0.1 N HCl or NaOH.
  • the dimension of the Q- Sepharose (Pharmacia) column was 2.5 cm in diameter with a bed volume of 20 mL.
  • the resin was equilibrated with 10 mM Tris pH 8.0, 50 mM NaCl.
  • the column was loaded at a flowrate of 3.4 mL/minute and washed with equilibration buffer until the OD (280 nm) returns to baseline.
  • the CNTF was eluted at 1.1 mL/minute with a 200 mL salt gradient composed of 2 buffers.
  • the low salt buffer was 10 mM Tris-HCl pH 8.0 and 50 mM NaCl.
  • the high salt buffer was 10 mM Tris-HCl pH 8.0 and 200 mM NaCl.
  • the pooling of the fractions took place between an OD (280 nm) of 0.3 and 0.9.
  • the present chromatography train is Q S
  • the CNTF at this stage is purified to equal to or greater than 99.9% purity with respect to ECP .E. coli protein) . Furthermore it did pass DNA and endotoxin specifications. These are less than 100 pg DNA per dose and less than 5 E.U. per kg body weight per day, respectively.
  • the amounts of CNTF forms were typically 97, 3 and 0.1% of CNTF C-terminal truncated forms III, II, and I, respectively. Note that III is the full size CNTF.
  • the amount of ECP in the final product was between 50-200 ppm as judged by ELISA to ECP. To further lower the amount of ECP in a range below 25 ppm an additional column was needed.
  • This fourth column in the process flow diagram can be either in between the 2nd ('S') and the third column ('Q') or after the third column.
  • examples are given of the various column resins tried that produced material that had a lower ECP content.
  • the column with ceramic Hydroxy Apatite (HA) resin (AIC) was equilibrated with 5 mM NaPi pH 7.0.
  • the pH of the load (the S column) was adjusted to 7.0 with 0.1 N HCl and water was added to the load until its conductivity was equal to the conductivity of the HA column equilibration buffer.
  • the column was washed with equilibration buffer until the OD (280 nm) returned to baseline and the CNTF was eluted with a 10 bed volume phosphate gradient.
  • the low phosphate buffer was 5 mM NaPi pH 7.0.
  • the high phosphate buffer was 150 mM phosphate pH 7.0. Fractions were pooled between an OD (280 nm) of 0.3 and 1.0 at the leading and at the trailing edge of the peak, respectively.
  • the ECP loan decreased to less than 25 ppm.
  • a spheroidal HA from BDH was also successful in removing ECP but it required a lower phosphate concentration throughout all steps.
  • the HA column can also be used after the third column in which case there is no phosphate present in the final bulk product.
  • Example B Butyl HIC resin.
  • Butyl Toyopearl 650 M (Toso-Haas) is a resin used in hydrophobic interaction chromatography (HIC) .
  • HIC hydrophobic interaction chromatography
  • the NaCl and phosphate concentrations in the load of 200 mg CNTF were adjusted to 300 mM and 20 mM pH 7.5, respectively.
  • the column was loaded at 9.6 mL per minute with CNTF and washed with column equilibration buffer.
  • the CNTF was step eluted with 175 mL of a 20 mM imidazole buffer pH 7.5.
  • the CNTF could be eluted with 20 mM Tris pH 7.5 of 50% (v/v) ethylene glycol in 20 mM phosphate buffer pH 7.5 or water or 20% ethanol or 10% glycerol in 20 mM imidazole pH 7.5.
  • the resin was regenerated with a 6 M urea followed by washing with water and requilibration. Butyl resins with bead sizes of 650 M or 650 S are expected to yield similar results.
  • Example C Zn-IMAC (immobilized metal affinity chromatography) resin.
  • the column had a diameter of 1 cm and was filled with 4 mL chelating Sepharose Fast-Flow from
  • the column was equilibrated with 10 mM Hepes pH 7.5 and 50 mM NaCl followed by a charging step using a solution of 1 mg ZnCl 2 /mL prepared in water.
  • the CNTF was loaded to a capacity of 5 mg/mL resin at 3 mL/minute followed by a wash in column equilibration buffer.
  • the CNTF was eluted at 1 mL/minute with a histidine gradient of 80 mL. The gradient was 0 to 75 mM histidine prepared in column equilibration buffer.
  • the column was regenerated with a solution containing 5 mM EDTA in 10 mM Hepes and 1 M NaCl at a pH of 7.5, followed by a 1 hour soak in 1 M NaOH. Then the column was washed in water and requilibrated followed by a recharge with zinc. The location of this zinc column was tried both after the S column as well as after the Q column.
  • Alternative charging metals are copper, cobalt, and nickel.
  • the fur on the left hind limb was clipped from the thigh and hip regions.
  • the clipped area was cleaned with betadine soap and rinsed with ethanol.
  • Using sterile technique throughout the procedure a 15 mm skin incision was made in the proximal half of the line between trochanter major and knee joint.
  • the vastus lateralis and biceps femoris muscles were separated by blunt dissection and the sciatic nerve exposed where it emerges from under the gluteus maximus and runs over the semi-membranous and semitendinosus muscles.
  • the nerve was elevated and Crile hemostatic forceps placed around the nerve 5 mm distal to the trochanter major. The Crile forceps were closed maximally for 30 sec. The muscles were not reopposed.
  • the skin was closed with wound clips.
  • a single intramuscular injection of penicillin G procaine and penicillin G benzathine in an aqueous suspension was given.
  • the rats are ambulatory 10 to 15 min after the surgery. Because the femoral nerve is intact, the rats are able to bear weight on the lesioned limb.
  • the current was generated by a constant-current generator (53500 Precision Instrument, Stoelting, Wood Dale, IL) and transmitted to the skin by dual stimulating electrodes with poles that are 1/8" apart (Lafayette Instrument, Lafayette, IN) .
  • the current was applied to the plantar surface of the paw immediately distal to digit 5.
  • the lowest current to which the rats responded by withdrawing that limb was determined.
  • a percentage recovery was calculated based on the lowest detectable current. Rats responding to 300 ⁇ A were considered to be 100% recovered, since this was the smallest current level that caused withdrawal reproducibly in normal rats.
  • Motor function The sciatic nerve crush results in denervation of the extensors of the digits in the hindlimb.
  • the digits are hyperflexed and held abnormally close together. This loss of toe spreading was used as an index of motor function after sciatic nerve crush. Toe spreading was measured from footprints made by walking rats. Measurements were compared in CNTF-treated and untreated rats during the course of regeneration of the sciatic nerve.
  • the plantar surfaces of both hindlimbs were pressed against an ink pad that was soaked lightly with black ink.
  • the rat was placed, hindlimbs first, at the entrance of a walkway.
  • the walkway was composed of a three-sided cardboard tunnel with its ground surface removed.
  • the tunnel which served to direct the rat's movements, was placed on a strip of white butcher paper.
  • the rat was allowed to move freely through the tunnel.
  • the object was to obtain at least two paired footprints on the butcher paper while the rat was in a walking mode. Two parameters, the footspread (FS) and the distance between the intermediary toes (ID) , were measured from the footprints.
  • FS footspread
  • ID intermediary toes
  • FS is the linear distance to the nearest millimeter from the medial edge of digit 1 to the lateral edge of digit 5.
  • ID is linear distance to the nearest millimeter between the medial edge of digit 2 and the lateral edge of digit 4.
  • the mean distances for FS and ID were determined for each foot. For each parameter, a ratio was calculated from values determined on the lesioned left foot and non-lesioned right foot.
  • Ratios were calculated daily beginning at day 7 from values determined for the paired lesioned and non- lesioned feet and compared for each parameter between CNTF-treated and untreated groups.
  • C. Administration of CNTF Rats were injected subcutaneously with CNTF at the dorsal midline in the region of the scapulae. The CNTF administered was human recombinant CNTF, produced as described in the Collins et al. patent application described above. Injections were made with an insulin syringe with a built-in 28 gauge needle. Minimal physical restraint was required during the injections. The volume of injected CNTF was 1.0 ml per kg body weight. Control rats were injected with 1.0 ml per kg of buffer vehicle, using the same technique. D. Experimental design.
  • CNTF had no effect on.mortality and no significant effect on body weight at the doses found to accelerate recovery after peripheral nerve damage.
  • CNTF had no abnormalities apparent in the motor or sensory function on the control, unlesioned side. This indicates that CNTF had no obvious effect on sensory or motor function in the absence of nerve injury.
  • CNTF was effective in accelerating recovery from peripheral nerve damage after physical injury in rats when CNTF was administered by daily subcutaneous injection around the time of injury.

Abstract

L'invention concerne la production et la purification du facteur neurotrophique ciliaire recombiné, ainsi que la purification des formes tronquées de terminaison C du facteur neurotrophique ciliaire. Des procédés sont également décrits pour empêcher et traîter les lésions de nerfs périphériques. Le procédé consiste à administrer aux patients en ayant besoin une dose thérapeutiquement efficace de facteur neurothrophique ciliaire. Un procédé préféré de production de facteur neurothrophique ciliaire se fait par la technique de recombinaison de l'ADN.
PCT/US1992/006136 1991-07-23 1992-07-21 Purification du facteur neurotrophique ciliaire recombine et du facteur neurotrophique ciliaire tronque a terminaison c et procedes de traitement des lesions de nerfs peripheriques WO1993002206A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP92917747A EP0596034A4 (fr) 1991-07-23 1992-07-21 Purification du facteur neurotrophique ciliaire recombine et du facteur neurotrophique ciliaire tronque a terminaison c et procedes de traitement des lesions de nerfs peripheriques.
JP5503057A JPH07500725A (ja) 1991-07-23 1992-07-21 組換え毛様体向神経因子およびｃ−末端切断毛様体向神経因子の精製ならびに末梢神経障害の処置方法
AU24432/92A AU666327B2 (en) 1991-07-23 1992-07-21 Purification of recombinant ciliary neurotrophic factor and C-terminal truncated ciliary neurotrophic factor and methods for treating peripheral nerve damage
NO949194A NO940194L (no) 1991-07-23 1994-01-19 Rensing av rekombinant ciliaer neurotrof faktor of C-terminal trunket ciliaer neurotrof faktor og fremgangsmaater for aa behandle periferi-nerve-skade
NO940194A NO940194D0 (no) 1991-07-23 1994-01-19 Rensing av rekombinant ciliaer neurotrof faktor og C-terminal trun kert ciliaer neurotrof faktor og fremgangsmaater for aa behandle periferi-n erveskade
FI940302A FI940302A0 (fi) 1991-07-23 1994-01-21 Siliaarisen neurotrofisen rekombinanttifaktorin ja C-päästä lyhentyneen siliaarisen neurotrofisen faktorin puhdistus ja menetelmät perifeeristen hermovaurioiden hoitamiseksi

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US73553891A 1991-07-23 1991-07-23
US735,538 1991-07-23
US75317691A 1991-08-30 1991-08-30
US753,176 1991-08-30

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Publication number Priority date Publication date Assignee Title
US5846935A (en) * 1992-10-09 1998-12-08 Regeneron Pharmaceuticals, Inc. Modified ciliary neurotrophic factors
US6565869B1 (en) 1996-11-19 2003-05-20 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Use of CNTF (ciliary neurotrophic factor) receptor activators for the treatment of obesity

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EP1013280A4 (fr) * 1998-06-10 2004-04-28 Bml Inc Preparations pour la peau et a usage externe

Citations (3)

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WO1990010647A1 (fr) * 1989-03-15 1990-09-20 The Regents Of The University Of California Facteur neurotrope ciliaire purifie
WO1991004316A2 (fr) * 1989-09-15 1991-04-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Facteur neurotrophique ciliaire
US5011914A (en) * 1989-01-05 1991-04-30 Collins Franklin D Purified ciliary neurotrophic factor

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US501914A (en) * 1893-07-25 Method of building engine-foundations
EP0446931A1 (fr) * 1990-03-14 1991-09-18 FIDIA S.p.A. Facteur neuronotrophique de cil humain, séquence d'ADN encodant le facteur, et production du facteur par technologie ecombinante
JPH07503128A (ja) * 1991-11-11 1995-04-06 フィディーア・ソシエタ・ペル・アチオニ ヒト毛様体ニューロン親和性因子の先端欠失型および突然変異タンパク質型の合成と精製

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US5011914A (en) * 1989-01-05 1991-04-30 Collins Franklin D Purified ciliary neurotrophic factor
WO1990010647A1 (fr) * 1989-03-15 1990-09-20 The Regents Of The University Of California Facteur neurotrope ciliaire purifie
WO1991004316A2 (fr) * 1989-09-15 1991-04-04 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Facteur neurotrophique ciliaire

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5846935A (en) * 1992-10-09 1998-12-08 Regeneron Pharmaceuticals, Inc. Modified ciliary neurotrophic factors
US6565869B1 (en) 1996-11-19 2003-05-20 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Use of CNTF (ciliary neurotrophic factor) receptor activators for the treatment of obesity
US6960558B2 (en) 1996-11-19 2005-11-01 Instituto Di Recerche Di Biologia Molecolare P. Angeletti S.P.A. Method of screening for anti-obesity agents using ciliary neutrophic factor receptor

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HU9400133D0 (en) 1994-05-30
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