WO2011098578A2

WO2011098578A2 - Liposome system for ocular administration

Info

Publication number: WO2011098578A2
Application number: PCT/EP2011/052061
Authority: WO
Inventors: Daniel Bar-Shalom; Simon SKJØDE JENSEN
Original assignee: Bioneer A/S
Priority date: 2010-02-12
Filing date: 2011-02-11
Publication date: 2011-08-18
Also published as: WO2011098578A3

Abstract

This invention concerns a pharmaceutical formulation for ocular or intra-ocular administration providing sustained or delayed release of at least one active ingredient, said pharmaceutical formulation comprising liposomes, said liposomes comprising at least part of said at least one active ingredient, at least part of said liposomes exhibiting a charge increasing the adherence of the charged liposomes to the eye after administration, in particular when said pharmaceutical formulation is for ocular administration, and wherein at least one of the lipids in the liposomes is degradable by sPLA₂ activity, allowing sustained or delayed release of said at least one active ingredient from said liposomes upon contact with tear fluid or with sPLA₂ in the eye or in the vicinity of the eye. The pharmaceutical formulation is useful for ophthalmic indications.

Description

LIPOSOME SYSTEM FOR OCULAR ADMINISTRATION FIELD OF THE INVENTION

The present invention relates to liposomes for ocular or intra-ocular administration, allowing sustained or delayed release of an active pharmaceutical ingredient. BACKGROUND OF THE INVENTION

Delivery of drugs to the eye is difficult since most drugs are washed off by lacrimation, tear dilution and tear turnover. This means that retention of drugs in the eye is limited to short periods, resulting in a fast reduction in eye drug concentration, leading to subsequent reduced therapeutic effect. The tear fluid, which protects the eye from bacteria and which lubricates the eye contains several enzymes that participate in the protection against bacteria, one of them being secretory phospholipase A₂ which in particular has a protective effect against infections with gram negative bacteria. sPLA₂ has enzymatic activity towards phospholipids when these lipids are present in certain liposomes. International patent application publication WO 01/58910 in the name of Liplasome Pharma A/S purports to disclose lipid-based prodrugs, which are turned into active drugs by hydrolysis via the extracellular phospholipase. The disclosed drug delivery system is purportedly particularly useful in the treatment or alleviation of diseases which are characterized by localized activity of extracellular PLA₂ activity. International patent application publication WO 07/107161 in the name of Liplasome Pharma A/S purports to disclose a lipid based drug delivery system comprising lipid derivatives which are substrates for extracellular phospholipase A₂, and lipopolymers and/or glycolipids so as to present hydrophilic chains on the surface of the system.

International patent application publication WO 2009/141450 in the name of Liplasome Pharma A/S purports to disclose cholesterol-free liposomes comprising a therapeutic agent, wherein the liposomes have been stabilized by exposure to divalent cations, in particular Calcium ions. The Liplasome technology was developed for targeted delivery of drugs administered systemically, i.e. the drug or prodrug was supposed to be carried by the blood stream and released or activated at sites with increased levels of extracellular PLA₂ activity, thereby providing targeted delivery to e.g. cancerous tissues. However, the targeting aspect did not live up to expectations, most likely due to degradation of the liplasomes in the systemic circulation.

SUMMARY OF THE INVENTION

The problem of systemic degradation of liposomes i.a. observed when exercising the liplasome technology is eliminated when a liposome is delivered directly to the eye.

Surprisingly, it has been demonstrated that the retention of the sPLA₂ sensitive, charged liposomes is enhanced in the prescence of (human) tear fluid comprising sPLA₂ as shown in the examples below.

Prior art liposomes, such as those described in US 4,804,539, typically include high concentrations of cholesterol (the liposomes in US 4,804,539 e.g. include 20-50% mol/mol of cholesterol), thus apparently rendering tear fluid sPLA₂ ineffective in providing the effects disclosed herein.

According to an aspect, the invention concerns a liposome for ocular or intra-ocular administration, said liposome comprising:

lipids and at least one active ingredient;

said liposome preferably exhibiting a charge increasing the adherence of said liposome to the eye after administration, in particular when said liposome is for ocular administration;

wherein at least one of the lipids in the liposome is degradable by sPLA₂ activity, thereby allowing sustained or delayed release of said at least one active ingredient from said liposome upon contact with tear fluid (e.g. human tear fluid) or sPLA₂ in the eye or in the vicinity of the eye.

Positively charged liposomes exhibit a superior retention to the cornea due to the

electronegative charge of the cornea. Positively charged liposomes therefore seem to be most efficient for ocular drug delivery. sPLA₂ in tear fluid is present in very high amounts and is able to degrade liposomes administered to the eye either when the liposomes are freely present in the tear fluid, or when the liposomes adhere to the negatively charged cornea, where the liposomes are retained due to electrostatic attraction. Positively charged liposomes are attracted very efficiently to the cornea, but are degraded relatively slowly by sPLA₂, because sPLA₂ is most active on negatively charged liposomes, which can be disrupted within minutes causing in the subsequent release of the encapsulated drug.

Conversely, in some pathological situations, electropositively charged areas may occur, and in these areas electronegatively charged lipids or liposomes would consequently target themselves. Hence, such electronegatively charged lipids or liposomes are within the scope of the invention according to this aspect.

According to another aspect, the invention concerns a pharmaceutical formulation comprising the liposome according to the first aspect of the invention. The person skilled in the art is aware of suitable excipients to include in such a

pharmaceutical formulation. However, reference is made to the detailed disclosure below.

According to a third aspect, the invention concerns a lipid based delivery system for ocular or intra-ocular administration, said system providing sustained or delayed release of at least one active ingredient,

said system comprising :

(I) lipids comprising:

(a) an organic radical having a least 2 carbon atoms; and

(b) a hydrophilic moiety; said lipids being a substrate for enzymes in (human) tear fluid or for sPLA₂to the extent that the organic radical can be hydrolytically cleaved off, leading to an intramolecular cyclization reaction; and

(II) at least one active ingredient; said lipids allowing the formation of liposomes, said liposomes comprising at least part of said at least one active ingredient;

at least part of said liposomes exhibiting a charge, which increases the adherence of the charged liposomes to the eye after administration; and

wherein at least one of the lipids in the liposome is degradable by enzymes in (human) tear fluid or by secretory phospholipase, in particular sPLA₂ activity, allowing sustained or delayed release of said at least one active ingredient from said liposomes upon contact with secretory phospholipase, in particular sPLA₂.

The sustained or delayed drug release system of the present invention does not depend on increased levels of sPLA₂ induced by the presence of disease or an abnormal condition. According to a fourth aspect, the invention concerns a method of ocular and/or intra-ocular treatment, comprising administration of liposomes, a pharmaceutical formulation and/or a lipid based delivery system according to the invention.

According to a fifth aspect, the invention concerns the use of liposomes, a pharmaceutical formulation and/or a lipid based delivery system according to the invention, for the manufacture of a medicament for an indication disclosed herein.

LEGENDS TO THE FIGURE

Fig. 1 : Overview of liposomes, liposome size and surface charge. A) List of 5 liposomes composed of l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol (POPG) and l,2-dihexadecanoyl-3-trimethylammonium- propane (DOTAP). Ratio of each component in the liposomes is shown, and their expected sensitivity to PLA₂ mediated hydrolysis shown together with their expected cell binding property. B) Liposome size measured in nanometer (nm) by dynamic light scattering on a ZetaPALS zeta potential analyzer from Brookhaven Instruments. C) Liposome surface charge expressed in mV. Liposome compositions for B and C refer to the numbers from figure 1A. Fig. 2: Maldi-TOF analysis of lipid formulations (liposomes 1-5 from figure 1A) hydrolyzed in human tear fluid dependent on time of incubation. The Maldi-TOF peak intensity of LPC16-Na (m/z=518.4) and POPC-Na (m/z=782.6) were collected and the ratio R= PI_LPci6/(PIpopc X%POPC) was calculated. PI_Lpci6 and PIpopc are the peak intensities of LPC16-Na and POPC-Na and%popc denoted fractional content of POPC in the formulation. Zero hydrolysis corresponds to R=0 whereas full hydrolysis corresponds to a large/infinite R-value.

Fig.3 : Liposome binding to human cells dependent on time of incubation and liposome composition. After the indicated incubation time (minutes) cells were washed three times in cell media and left in fresh media until fluorescence measurement. Total amount of liposome added is seen for t=0. Fluorescence measurement was made by to trace liposome presence through incorporation of 0.2% DOPE-rhodamine into the liposome composition. Fig.4: Liposome binding to human cells dependent on presence of tear fluid or bee venom lipase. A) Liposome association with human cells expressed in relative fluorescence units by measurement of DOPE-rhodamine in the liposome compositions compared to total amount of liposome added. After incubation with cells for 60 min, cells were washed three times in media and remaining liposome (cell associated) was measured by fluorescence intensityof the cells. B) The data expressed as percent fluorescence intensity compared to total amount of liposome added to the cells (data adapted from figure 4A).

Fig.5 : Liposome cytotoxicity was measured on human cells using the formulations from figure 1A. The liposomes were incubated with the cells for 10, 30, 60 or 120 min before washing with cell media. After 24 h the liposome induced cytotoxicity was measured by incubation with XTT for 3 hours, allowing live cells to convert XTT. There was no significant cytotoxicity of the cationic liposomes containing DOTAP compared to neutral or negatively charged liposomes. Increased time of incubation did not induce cytotoxicity.

DETAILED DISCLOSURE OF THE INVENTION Definitions

A "liposome" in the present application and claims denotes an artificial prepared vesicle made of at least one lipid bilayer.

A "hydrophobic" drug in the present context denotes a drug substance, which exhibits a distribution coefficient between 1-octanol and water (measured using the shake flask method at 20°C, 1 bar and pH 7.4) higher than 1, i.e. a log D_octanoi/water higher than 0. Preferred hydrophobic drugs of relevance for the present invention exhibit higher log D_octan0|_/water values, such as values of at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.5, and at least 3.0. log D_octanoi/water is calculated as follows:

An "ointment" may be described as a viscous semisolid preparation used topically on a variety of body surfaces. These include the skin and the mucous membranes of the eye (an eye ointment), vagina, anus, and nose. Hence, these provide optional routes of

administration of liposomes according to an embodiment of the invention.

A "cream" may be described as an emulsion of oil and water (both o/w and w/o) in approximately equal proportions, which penetrates the stratum corneum outer layer of skin well.

A gel normally liquefies upon contact with the skin.

A paste usually combines three agents, i.e. oil, water, and powder; it may be described as an ointment in which a powder is suspended.

When using the terms "substantial"/"substantially" or "essential"/"essentially" herein it is intended that the feature which is described by these terms is present in an amount or has an impact which provides for a technical effect with relevance for the exercise of the presently claimed invention. For instance, a "substantial amount" of a substance in a composition is an amount which provides for a technical effect exhibited by the substance to a degree which provides for a technical effect in terms of the present invention. Likewise, if a composition is indicated as comprising "substantially no" of a particular substance, this means that the composition is allowed to include insignificant amounts of the substance, as long as these amounts do not have any technical impact on the other ingredients in the composition and does not in itself "make a difference" - or put in other words, "substantially no" and "essentially no" means that e.g. trace amounts or effects may be present as long as they do not have an overall technical influence.

Specific embodiments of the invention

Additional embodiments of the present invention are described below. It will be clear for the person skilled in the art, that aspects and/or embodiments of the invention may be combined. For ocular delivery of drugs, positively charged liposomes will likely be degraded by sPLA₂ within 1-12 h, which means that the drug entrapped inside the liposomes will be released over many hours, which allows a much longer presence of the drug in the eye.

It will be understood though that the results reported herein primarily demonstrate that there appears to be a correlation between the sPLA₂ sensitivity of lipids in the liposomes and their ability to adhere to negatively charged cell surfaces. However, it cannot be excluded that the effects disclosed in the Examples, where presence of human tear fluid dramatically increases the cell-association of liposomes of the invention may be due to the influence of other enzymes or substances in human tear-fluid (which is a complex aqueous composition).

However, the results demonstrate that positively charged liposomes containing a balanced mixture of anionic lipids and cationic lipids provide for surprisingly improved results in terms of adhesion to corneal surfaces and sustained release to the eye and that these effects are the consequence of the action of tear fluid on the liposomes.

Liposome structure and characteristics

In some embodiments of the invention at least part of the lipids in the liposomes exhibit a positive charge, particularly after ocular administration. It is contemplated that up to 100% (mol/mol) may be positively charged, but lower amounts are preferred. In some

embodiments at most 90%, such as at most 80%, at most 70%, at most 60%, at most 50% and at most 40%. Especially preferred embodiments entail that at most 30% (mol/mol) lipids exhibit a positive charge. The amount of positively charged lipids is hence at most 30%, at most 29%, at most 28%, at most 27%, at most 26%, at most 25%, at most 24%, at most 23%, at most 22%, at most 21% and at most 20%. On the other hand, the amount of positively charged lipids is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, and at least 20%.

According to an embodiment of the invention the liposome comprises an amount of 10-60%, preferably 20-50% positively charged/cationic lipids (mol/mol), typically selected among 0- 5%, 5%-15%, 10-20%, 20-25%, 25-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% (mol/mol) positively charged/cationic lipids At any rate, irrespective of the exact amount of positively charged lipids, some embodiments of the present invention entail that the liposome exhibits a net positive charge, in particular after administration to the normal human eye. For instance, these embodiments include those where the liposome has a zeta potential in the range from 10-60 mV. The zeta potential is typically at most 55 mV, such as at most 50 mV, or at most 45 mV, e.g. at most 40 mV. Also, the zeta potential is typically at least 15 mV, such as at least 20 mV, or at least 25 mV, e.g. at least 30 mV.

In other embodiments of the invention the lipids in the liposomes are substantially not charged, and in certain embodiments the net charge of the liposomes is essentially neutral. In accordance with the findings herein, partilularly preferred liposomes of the invention are those which exhibit an improved binding to normal corneal tissue and the ability to sustainedly release a (hydrophobic) drug to the eye after having being exposed to human tear fluid . An important feature of the liposomes of the invention is their ability to, when contacted with 50% human tear fluid for 60 minutes, subsequently exhibit at least 20% of maximum possible association with HT1080 cells after one hour of incubation. The maximum possible association is in this context the complete association of all liposomes administered to the eye in a situation where a pharmaceutically relevant and acceptable amount of liposomes are administered, i.e. in a situation where the association to the eye is not inhibited by administration of excessive amounts of liposomes. It is preferred that the liposome, when contacted with 50% human tear fluid for 60 minutes, subsequently exhibits at least 30%, such as between 50 and 60%, of maximum possible association with HT1080 cells after one hour of incubation. When testing this ability, the conditions set forth in Example 5 are used . As is apparent from the examples below, the exact lipid composition of the liposomes of the invention dictates the kintetics of the liposomes' release of drugs to the eye, in particular for hydrophobic drugs. If administering a hydrophobic drug such as a corticosteroid to the eye in a simple aqueous solution, the release will be immediate - on the other hand, use of a lipid- based delivery system such as a liposome will delay or even prevent release, since the drug will remain in the hydrophobic environment of the delivery system. The present invention provides for an intermediate between these two extremes and utilises the ability of tear fluid to degrade certain but not all lipids in e.g . liposomes. So, in some embodiments, the liposome of the invention exhibits a release of hydrophobic active ingredient when said liposome is applied to the normal human eye, which is slower than release from an aqueous solution and faster than from reference liposomes comprising >20% mol/mol cholesterol . In these embodiments the liposome preferably releases hydrophobic active ingredient more slowly than a reference liposome having a net negative charge. The aqueous solution and the reference liposomes in these situations comprise the same amount of hydrophobic active ingredient as does the liposome. Expressed in terms of bioavailability, there will be an increase in bioavailability when using the liposomes of the invention which is at least twice as high as that provided by the reference liposomes. However, higher increases in bioavailability are contemplated, such as at least 2.5, at least 3, and at least 4 times.

The above-described embodiments entail that the liposome preferably comprises lipids that exhibit a negative charge as well as lipids that exhibit a positive charge, in particular after ocular administration - as shown in the examples, POPG and derivatives are interesting anionic/negatively charged lipids in the liposomes. The liposomes may consequently also include electroneutral lipids which are e.g . used to balance the charge of the liposome - for instance, POPC and derivatives are, as seen in the examples, interesting neutral lipids to include.

In general, lipids that are electroneutral and which are useful in the liposomes of the invention comprise head groups selected from phosphatidylcholine and

phosphatidylethanolamine to which are coupled hydrophobic groups such as alkyl chains.

Lipids that are negatively charged and which are useful in the liposomes of the invention comprise head groups selected from phosphatidylserine, phosphatidylglycerol, phosphatidic acid, and phosphatidylinositol, to which are coupled hydrophobic groups such as alkyl chains.

The hydrophobic groups that form part of the lipids used in the liposomes of the invention are typically di-acyl or tri-acyl carbon chains having lengths from 8-24 hydrocarbons, cf. the description of alkyl groups below. The hydrocarbons may be saturated or include one or more double bonds.

The lipids in the liposomes may comprise or constitute phospholipids. The lipids may comprise a group selected from phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA), DPG (bisphosphatidyl glycerol), PEOH (phosphatidyl alcohol) and cholesterol.

As mentioned above, the liposome may comprise a cationic lipid, which is preferably selected from the group consisting of stearylamine (SA), dimethyldioctadecylammonium bromide (DDAB), 36-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), 1,2- ditetradecanoyl-3-trimethylammonium-propane (DMTAP), DOTAP derivatives (1,2- dioctadecanoyl-3-trimethylammonium-propane, l,2-di-(9Z-octadecenoyl)-3- trimethylammonium-propane, l,2-dihexadecanoyl-3-trimethylammonium-propane), DODAP derivatives (l,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane, l,2-ditetradecanoyl-3- dimethylammonium-propane, l,2-dihexadecanoyl-3-dimethylammonium-propane, 1,2- dioctadecanoyl-3-dimethylammonium-propane), l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), dioctadecylamide-glycylspermine, SAINT-2, polycationic lipid 2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), GL67TM, l,2-dioctadecanoyl-sn-glycero-3-ethylphosphocholine (Etyl PC), and 1,2- dioctadecanoyl-sn-glycero-3-phosphoethanolamine (DSPE). Preferred cationic lipids are DOTAP and DOTAP derivatives. Additional examples of cationic lipids and lipid components may be found in or made according to US 4,804,539. According to the present invention, it is preferred that the liposomes comprise less than 20% (mol/mol), such as less than 10%, preferably less than 5%, more preferred less than 2% cholesterol, most preferred substantially no cholesterol. However amounts of cholesterol up to about 10% appear to be acceptable, so amounts in the range between 0 and 10% are also within the invention, such as amounts between 1 and 9%, 2 and 8% and 3 and 7%.

The liposomes of the invention preferably include lipids, which when relevant, contain alkyl chains that are C8-C24, preferably C10-C22, more preferred C12-C20, preferably C14-C18, most preferred C16-C18 saturated chains or unsaturated chains, preferably saturated chains the two latter mentioned species may have chain lengths of C12-C18, and may also include double bonds.

Preferred liposomes of the invention are in the form of Large Unilamellar Vesicle (LUV), meaning that LUVs are preferred components of composition comprising liposomes of the invention.

Typically, the liposome of the present invention has a diameter of 50-10,000 nm, preferably 60-5,000 nm, more preferred 70-1,000 nm, preferably 80-120 nm, more preferred 100-500 nm, preferably 200-7,500 nm, more preferred 300-3,000 nm, preferably 500-2,000 nm, more preferred 1,000-1,500 nm.

In some embodiments, the liposome does not comprise a hydrophilic polymer conventionally used in preparation of liposomes. For instance, it is preferred that the liposome does not comprise a polymer selected among PEG [poly(ethylene glycol)], PAcM [poly(N- acryloylmorpholine)], PVP [poly(vinylpyrrolidone)], PLA [poly(lactide)], PG [poly(glycolide)], POZO [poly(2-methyl-2-oxazoline)], PVA [polyvinyl alcohol)], HPMC

(hydroxypropylmethylcellulose), PEO [poly(ethylene oxide)], chitosan [poly(D-glucosamine)], PAA [poly(aminoacid)], polyHEMA [Poly(2-hydroxyethylmethacrylate)] and co-polymers thereof. In preferred embodiments, the liposome contains essentially no hydrophilic polymer.

In other embodiments, the liposome comprises that at least part of the lipids comprise or is conjugated to a hydrophilic polymer, such as the hydrophilic polymers discussed above, subject to the proviso that the average molecular weight of hydrophilic polymer for the totality of the lipids is less than 2,000 Dalton, preferably less than 1,500 Dalton, more preferred less than 1,000 Dalton, preferably less than 500 Dalton, more preferred less than 300 Dalton, preferably less than 200 Dalton, more preferred less than 100 Dalton.

The liposome according to any one of the preceding claims, wherein at least one lipid is a substrate for enzyme(s) in human tear fluid or for sPLA₂. The liposome of the invention include an active ingredient, typically a drug substance or composition. Typically, the liposome comprises substantially all of said at least one active ingredient. Normally, one of the compartments selected among the interior aqueous compartment, a hydrophobic bilayer, and a polar inter-phase of the inner and outer leaflet of the liposome carry said at least one active ingredient.

The at least one active ingredient is generally selected from drugs or drug composition useful in therapeutic or prophylactic treatment or amelioration of conditions of the eye. The active ingredient may also be one which may be administered to the eye but which exhibits its intended clinical effect in a different anatomical location. The skilled person will generally be knowledgeable about the choice of active ingredient and the correct dosage thereof. Typical drugs are antimicrobial drugs/antibiotics/antiviral drugs as well as immunemodulating or anti-inflammatory drugs as well as drugs that exert their effects on nerve tissue or on vasculature. A special group of active ingredients are relevant in the treatment of glaucoma.

In embodiments, said at least one active ingredient is selected from the group consisting of acetazolamide, acyclovir, indomethacin, verapamil, rapamycin, ascomycin, ciprofloxacin, ofloxacin, fusidin, gentamicin, chloramphenicol, levofloxacin, oxytetracyclin, tobramycin, acyclovir, prednisolon, dexamethason, chloramphenico, brimonidin, brimonidintartrat, dorzolamid, timolol, latanoprost, tetryzolinhydrochlorid, and natriumcromoglicat.

Also the at least one active ingredient is a non-steroidal anti-inflammatory drug (NSAID), preferably selected from the group consisting of ketoprofen, flurbiprofen, ibuprofen, diclofenac, ketorolac, nepafenac, amfenac and suprofen.

In preferred embodiments, the active ingredient is in the form of a hydrophobic active ingredient, such as a hydrophobic drug (substance or composition). Particularly interesting hydrophobic drugs are selected from the group consisting of Diclofenac, Fusidine,

Levocabastin, Indometacin, Latanoprost, Travoprost, Olopatadin, Azelastin, Rimexolon, ketotifen, naphazolin, Bimatoprost, Betaxolol, emedastin, Ketorolac, Resorcinol,

Fluormetolon, Atropine, Apraclonidin, Cyclopentolat, Dexamethasone, Lidocain, Prednisolone, Nepafenac, Timolol, Tropicamid, Brimonidine, Chloramphenicol, Pilocarpine, Vancomycin, Fluconazol, Sulfamethizol, and Moxifloxacin. Pharmaceutical compositions of the invention

As discussed above, the liposomes of the present invention are useful as constituents of pharmaceutical formulations of the invention. Any form of such a formulation conventionally used for pharmaceuticals for direct application to the human or animal eye are contemplated and a general reference to the requirements for and preparation of such formulations can be found in Bartlett & Jaanus: Clinical Ocular Pharmacology, Section 1 (chapters 2-4), 2007, ISBN 10: 0-7506-7576-4, ISBN 13: 978-0-7506-7576-5.

The pharmaceutical formulation of the invention is preferably in the form of a cream, an ointment, a gel, a paste or eye-drops.

It is preferred that the pharmaceutical formulation provides release of said at least one active ingredient during a period of 0.5-24 hours, preferably 1-12, more preferred 2-8, preferably 3-6 hours. As detailed above, the precise kinetics of the liposomes forming part of the pharmaceutical composition may be adjusted to aim precisely at a suitable release profiled. For instance, said pharmaceutical formulation can e.g. comprise a mixture of liposomes of the invention with different release characteristics, whereby the pharmaceutical formulation provides sustained release of said at least one active ingredient.

Therapeutic uses or methods

Also part of the invention is the liposome or the lipid-based drug delivery system or the pharmaceutical composition of the inventnion for use as a pharmaceutical. In particular, these aspects of the invention may be for use in prophylactic or therapeutic treatment or amelioration of an ophthalmic indication, such as post-operative pain or ocular inflammation, such as an ocular inflammation which results from iritis, conjunctivitis, seasonal allergic conjunctivitis, acute and chronic endophthalmitis, anterior uveitis, uveitis associated with systemic diseases, posterior segment uveitis, chorioretinitis, pars planitis, masquerade syndromes including ocular lymphoma, pemphigoid, scleritis, keratitis, severe ocular allergy, corneal abrasion or blood-aqueous barrier disruption. Also, they may be for use in treatment or amelioration of the indication post-operative ocular inflammation, such as inflammation resulting from photorefractive keratectomy, cataract removal surgery, intraocular lens implantation or radial keratotomy. Finally, they may also be for use in treatment or amelioration of an indication selected from the group consisting of fungal infection; microbial infection; inflammatory disease; non-ocular inflammatory disease, such as rheumatoid arthritis (RA); dry eye disease and dry eye condition, such as episodic or chronic dry eye condition and aqueous tear deficiency (ATD); keratoconjunctivitis sicca (KCS), such as Sjogren syndrome (SS), associated KCS and non-SS associated KCS; mucin deficiency;

Stevens-Johnson syndrome; ocular injury such as chemical burn; chronic blepharitis; contact lens intolerance; chronic ocular surface inflammation; inflammation of ocular surface disease; glaucoma, such as open-angle glaucoma, primary open-angle glaucoma (POAG) and exfoliation glaucoma (AG); conjunctival inflammation; and corneal disease, such as allergy, conjunctivitis (Pink Eye), corneal infection, Fuchs' Dystrophy, Herpes Zoster (Shingles), iridocorneal endothelial syndrome, keratoconus, lattice dystrophy, map-dot-fingerprint dystrophy, ocular herpes, and pterygium.

Consequently, the present invention also relates to a method for prophylactic or therapeutic treatment or amelioration of an ocular or intraocular disease or condition, the method comprising administering, ocularly or intraocularly, to a subject in need thereof an effective amount of a liposome, lipid based drug delivery system, or a pharmaceutical composition of the invention; the ocular or intraocular disease or condition is preferably identical to or associated with any one of the indications discussed in the previous paragraph.

All cited references are incorporated in their entirety herein. EXAMPLE 1

Liposome preparation

Unilamellar fully hydrated liposomes were made from l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2- dihexadecanoyl-3-trimethylammonium-propane (DOTAP). As a fluorescence marker to measure presence of liposomes in biological systems, 0.2% DOPE-rhodamine was mixed with the lipids as a tracer. The molar ratios of each lipid in the liposomes are outlined in figure 1A. POPC, POPG and DOTAP were all obtained from Avanti Polar lipids. Briefly, weighed amounts of POPC, POPG and DOTAP were dissolved in chloroform. The solvent was removed by a gentle stream of N₂ and the lipid films were dried overnight under low pressure to remove trace amounts of solvent. Multilamellar vesicles were prepared by dispersing the dried lipids in a buffer solution containing : 150 mM KCL, 10 mM HEPES (pH = 7.5), 1 mM NaN₃, 30 μΜ CaCI₂ and 10 μΜ EDTA. The multilamellar vesicles were extruded ten times through two stacked 100 nm pore size polycarbonate filters as described by Mayer et al., Biochim.

Biophys. Acta, 858, 161-168. EXAMPLE 2

Characterization of liposome size and surface charge dependent on composition

Liposomes prepared as outlined in figure 1A were prepared with the attempt to design liposomes with ability to adhere to the ocular surface of the eye as demonstrated by L. Guo, C.T. Redmann and R. Radhakrishnan, US patent No. 4,804,539, 1986. Charged liposomes with up to 40% positively charged lipids show an increased adherence to ocular tissue, most likely due to presence of the negatively charged glycoprotein mucin, possibly combined with the negative surface charge of mammalian cells. Therefore, we designed liposomes with 10 or 20% net positive charge (formulation 4 and 5 in figure 1A), together with control liposomes with negative or nearly neutral charge (formulation 1-3 in figure 1A) . The liposomes were prepared as described in example 1, and their size measured in nanometer (nm) by dynamic light scattering on a ZetaPALS zeta potential analyzer from Brookhaven Instruments. The liposomes showed sizes between 130-180 nm in diameter (Figure IB) . The surface charge (Zeta potential) of the liposomes, was measured in mV and showed surface charge dependent on lipid composition (figure 1C) . High content of the negatively charged POPG (60 molar percent) in the liposome, showed negative surface charge of -35 mV (figure 1C, composition 1) . High content of the neutral lipid POPC (100 molar percent) in the liposome showed weak negative charge (figure 1C, composition 2) . With increased negative and positively charged lipids with the neutral POPC (molar percent 80: 10: 10), the charged lipids counteracted each other, and resulted in a net weak negative charge (figure 1C, composition 3) . Increased amounts of DOTAP to 20% (molar percent 70: 10 : 20), showed a net positive charge of nearly 30 mV (figure 1C, composition 4), whereas the highest amount of DOTAP at 30 mol% (molar percent 60: 10: 30), showed a net positive charge of 40 mV (figure 1C, composition 5) . Formulation 4 and 5 are therefore expected to be the liposomes with adherence to ocular and cellular surfaces due to their electrostatic attraction.

EXAMPLE 3

Liposome hydrolysis in human tear fluid dependent on lipid composition

Tear fluid derived secretory phospholipase A2 type IIA (sPLA₂) which is present in very high amounts in human tear fluid (ranging from 49.7 μg/ml in men to 55,7 μg/ml in women) (O. Kari, V.V. Aho et al ., Acta Ophthalmologica Scandinavia, 2005, 83, 483-486), was used to analyse liposome degradation. Human tear fluid was collected from the eye of a number of adults, pooled and distributed in tubes and stored frozen at -20 C. The liposomes were incubated in 50% tear fluid and PBS for 1 and 24 h at 37 C, and frozen until further analyses. For analyses of lipid degradation by sPLA₂, the liposome samples were mixed with 2: 9 with DHB matrix in methanol and Maldi-TOF spectra were recorded. The Maldi-TOF peak intensity of (lyso-phosphatidyl choline C16) LPC16-Na (m/z= 518.4) and POPC-Na (m/z=782.6) were collected and the ratio R= PI_LPCI6/(PIPOPC X%POPC) was calculated. PI_LPci6 and PI_POpc are the peak intensities of LPC16-Na and POPC-Na and%_POpc denoted fractional content of POPC in the formulation. Zero hydrolysis of the liposome by sPLA₂ corresponds to R=0 whereas full hydrolysis corresponds to a large/infinite R-value (Fig. 2) . All five formulations can be hydrolyzed by sPLA₂ in tear fluid, and the level of hydrolysis increases from lh to 24h incubation. Formulation 1 shows the largest level of hydrolysis which is expected due to the high POPG content, POPG being a good substrate for sPLA₂. The level of hydrolysis of formulation 2 is low, which corresponds to the fact that POPC is a poor sPLA₂ substrate. When POPG is included in the formulations, sPLA₂ has the ability to hydrolyse the lipids to larger extent as seen for formulation 3, but unexpectedly, the hydrolysis profile increases with increased DOTAP content and net positive surface charge. This is unexpected because sPLA₂ is known to have higher preference for negatively charged membranes. One explanation may be that the very high amounts of sPLA₂ causes hydrolysis of otherwise poor substrates. EXAMPLE 4

Liposome association with cells dependent on lipid composition and charge properties

Liposomes prepared according to the composition set forth in Fig. 1 were added to human cells with negative charge properties similar to the negatively charged corneal surface. The human fibrosarcome cell line HT1080 was used as a model system for a negatively charged biological membrane. HT1080 cells were seeded with 10.000 cells/well, and allowed to grow to confluency over 4 days, where the cell layer was completely dense, mimicking the well differentiated and dense corneal surface. The cells were washed with serum free media 3 times, and serum free media added to the cells in a volume of 25 μΙ/well. 5 μΙ liposome (of a 5 mM stock) was added to each well, and incubated for 10, 30, 60 and 120 min prior to washing in cell media three times. The total absorbance was measured at time of addition of liposome as a measure of total amount of added liposome (Fig. 3, t=0). After 10 min incubation and subsequent washing, formulation 4 was the only one that was associated with the cells, where 20% of total amount added was associated with the cells. Formulation 4 increased cell association to a maximum of 40% after 120 min of incubation. Formulation 5 was associated with the cells to nearly 10% of added liposome after 30 min of incubation, and increased to a maximum of 20% after 60 min of incubation (Fig. 3). It can be concluded that liposomes with a net positive charge most efficiently associates with the cells, whereas negatively charged or neutral liposomes showed no or poor cell association. EXAMPLE 5

Enhancement of liposome cell association in presence of human tear fluid dependent on lipid composition and charge properties

Positively charged liposomes show a strongly enhanced retention on ocular surfaces, associated with enhanced bioavailability and stronger therapeutic effect of the associated drug (L. Guo, C.T. Redmann and R. Radhakrishnan, US patent No. 4,804,539, 1986, and R. M. Hathout et al., AAPS PharmSciTech 2007, 8(1), E1-E12.). For hydrophilic drugs like acetazolamide, which are encapsulated inside the liposome, the release profile, bioavailability and therapeutic effect was improved. However, for lipophilic drugs like e.g. glucocorticoids and prostaglandins, the drugs will remain in the liposome or lipid structures and not become bioavailable to the same extent as hydrophilic drugs. Therefore, a drug-release mechanism is required in order to make lipophilic drugs active for treatment of ocular diseases from the liposomes which adhere to the corneal surface. The combination of cationic liposomes which adheres to the ocular surface with the release mechanism provided by the tear fluid derived sPLA₂ mediated liposome degradation, will provide a drug delivery system which will allow better ocular surface retention, higher ocular bioavailability and increased therapeutic activity of lipophilic drugs in the eye.

The effect of tear fluid sPLA₂ on liposomal association with human cells as model system for ocular surface retention was investigated. Liposomes were incubated without lipase, with 50% tear fluid or with 50% bee venom lipase (figure 4A and B). After 60 min incubation, liposomes were added to the cells for another 60 min, washed 3 times and fluorescence intensity remaining in the cells measured. After addition of liposomes the total fluorescence intensity was measured (figure 4A). Normalized to the total amount of fluorescence intensity, the liposomes without lipase associated with cells similar to what was seen in figure 3, with mainly cationic liposomes associated with the cells (figure 4B, formulation 4 and 5).

Incubated with tear fluid, formulation 4 and 5 showed a dramatic increase in cell associated fluorescence intensity, indicating that these liposomes were further activated by sPLA₂, and showed that 50-60% of total added liposome associated with the cells. Incubation with bee venom lipase did not have this effect. This shows that the increased effect was a specific effect of tear fluid and the human sPLA₂ subtype, and that this effect can not be obtained using other types of sPLA₂ such as the bee venom sPLA₂, which has much broader substrate specificity. None of the tested liposomes showed signs of cytotoxicity towards the cells. There was no change in viability when cells were incubated for 10, 30, 60 or 120 min with liposomes, subsequently washed and cultured for 3 h in XTT to measure cell viability (figure 5). Neither was there any change in viability between incubation with the different liposomes. EXAMPLE 5

Release of lipophilic drugs in cell culture model systems with e.g. glucocorticoids or prostaglandins from cationic liposomes with adherence to ocular or cellular surfaces and sensitivity towards tear fluid sPLA₂ Liposomes are prepared using cationic liposomes with a lipid composition of net 0-30% positive charge, e.g. POPC: POPG: DOTAP compositions of 70: 10: 20, 60: 10: 30, 50: 10:40 or between these ratios. Liposomes are prepared as in EXAMPLE 1, but with addition of 0.1-5% of dexamethansone or prostaglandin or their analogues, and with 0.2% DOPE-rhodamine. The liposomes are added to cultured cells where the cells are seeded in 96 well plates 4 days prior to the experiment with a cell density of 10.000 cells/well in cell culture media containing 10% FCS. Prior to the experiment the cells are washed 3 times with media with no serum. 10 μΙ of serum free media is added to the cells, 15 μΙ of human tear fluid and 5 ul of liposome (5 mM) are added to the cells for a certain time, typically between 5-60 min. The cells are washed three times with media and cell-associated liposomes measured as fluorescence intensity in each well. The cells are assayed for presence of drug in several ways. 1)

Dexamethasone is measured in cell lysate using an ELISA kit against dexamethasone. 2) Dexamethasone or its analogues can be assayed for biological activity where a human cell line or primary cells after 1-24 h of liposome contact and washing are exposed to an immune stimulating reagent like LPS, PMA, TNFa, Poly I:C or another TLR-ligand. After approximately 24 h the conditioned media is analysed for suppression of cytokine secretion compared to non-liposome treated cells, or cells treated with liposomes which do not adhere to the cells, but which contains dexamethasone. For a prostaglandin, a similar approach is used. A biological endpoint can be assayed using agonists or antagonists towards the relevant prostaglandin receptor, and measurement of the prostaglandin itself. This can be done using Mass spectrometry, HPLC, RIA, EIA or ELISA kits.

Example 6

Analyses of retention of liposomes on human eyes

Liposomes are prepared using cationic liposomes with a lipid composition as in EXAMPLE 5. Liposomes are prepared as in EXAMPLE 1, but with addition of fluorophore like rhodamine, fluorescein. This can be l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(DOPE rhodamine) or l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (DOPE-fluorescein) which are both headgroup labelled phospholipids. Alternatively, a fluorophore on the acyl chain can be used like e.g. l-oleoyl-2-{6-[(7-nitro-2-l,3- benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoethanolamine (18: 1-06: 0 NBD PE), where NBD is located at the sn-2 position of the phospholipid. The liposomes are prepared according to GMP in clinical grade quality. The liposomes are added to the ocular surface of healthy volunteers, where the liposome formulation is added to one eye, and a control fluorophore to the other eye. The fluorophores must be the same, added in same amount at the same time. Subsequently, the fluorescence signal is measured over time in both eyes, and the difference in signal intensity is registered. If the liposome formulated fluorophore is present for longer time on the cornea, the liposome carrier system causes a longer retention time on the cornea, and is thus an attractive carrier system for ocular drug delivery. Similar experiments may be carried out on laboratory animals like mice, rats, rabbits, minipigs, guneapigs or other species.

EXAMPLE 7

Analyses of therapeutic effect of glucocorticoid containing liposomes on animal or human eyes

Clinical grade liposomes are prepared according to EXAMPLE 1 and 5. A suitable drug is formulated with the liposome. Suitable drugs are lipophilic drugs belonging to the glucocorticoid class like dexamethasone or its analogues (e.g. fluormetolon, prednisolone or rimexolon). Glucocorticoid containing liposomes are useful for treatment of non-infectious inflammatory conditions in the eye like e.g. iritis, scleritis or episcleritis. Non-liposome formulated glucocorticoids are recommended applied 3-6 times per day, and in severe conditions every hour. The present liposome formulated glucocorticoids will be applied once or twice daily to obtain a similar therapeutic effect. Human volunteers with non-infectious inflammatory conditions in the eye like e.g. iritis, scleritis or episcleritis are included in a clinical test, and treated twice daily with liposomal glucocorticoids. A control group is treated with same amounts of non-liposome formulated glucocorticoids two to three times daily. The two groups are compared for therapeutic effect, and tear fluid is obtained after treatment to measure bioavailability dependent on time after treatment, typically 30, 60, 120, 240 minutes after administration. Similar experiments may be carried out on rabbits or other laboratory animals. EXAMPLE 8

Analyses of therapeutic effect of prostaglandin containing liposomes on animal or human eyes

Liposomes are prepared according to EXAMPLE 1 and 5. Example of another suitable drug group for the preent technology are prostaglandin or its analogues (bimatoprost, tafluprost, latanoprost, travoprost) which are useful for treatment of glaucoma. The prostaglandin of interest is formulated with the lipids in the liposome in clinical grade quality. Human volunteers with glaucoma are allocated to two groups, one treated with liposomal prostaglandins, the other with non-liposomal standard prostaglandin. Both groups are treated once daily in the evening. The two groups are clinically examined for side effects after 1-4 weeks treatment. The liposome treated group are expected to show fewer side effects, either because a lower dose is required, or because additives for the liposomal formulation are different from additives in the free formulation. In particular the presence of

benzalkoniumchloride in the free formulation causes side effects. EXAMPLE 9

Test of release of encapsulated drug in animal model

Liposomes are prepared as described in EXAMPLE 1. The liposomes are added to the cornea of a laboratory animal, typically a mouse, rat, rabbit, dog or pig. Controls for such study is the same free drug at the same dose as used in the encapsulated drug in liposomes. Eye fluid is removed from the animal eye at appropriate time points within the first 24 h after administration, and the amount of drug determined at each time point for each formulation. The release profile which is optimal for the specific drug can then be optimized by adjusting the amount of positive and negative charges in the liposome. A slow release can be prepared using liposomes with 10-50% positive charge, potentially with amounts of cholesterol (0-10 mol%). A faster release profile can be achieved by introducing negatively charged phospholipids in the positively charged liposome (e.g. POPG) and reducing the amount of cholesterol. EXAMPLE 10

Eye drop formulation

An eye formulation may be made as indicated in the patent US 5145680, by interchanging the active ingredient with liposomes of the present invention carrying an active ingredient.

Claims

1. A liposome for ocular or intra-ocular administration, said liposome comprising:

lipids and at least one active ingredient;

wherein at least one of the lipids in the liposome is degradable by sPLA₂ activity, thereby allowing sustained or delayed release of said at least one active ingredient from said liposome upon contact with tear fluid or with sPLA₂ in the eye or in the vicinity of the eye.

2. The liposome according to any claim 1, wherein at least part of the lipids exhibit a positive charge, particularly after ocular administration.

3. The liposome according to claim 2, wherein at most 30% (mol/mol) lipids exhibit a positive charge.

4. The liposome according to claim 1 or 2, which has a net positive charge after administration.

5. The liposome according to claim 4, wherein the liposome has a zeta potential in the range from 10-60 mV.

6. The liposome according to claim 1, wherein the lipids are substantially not charged.

7. The liposome according to any one of the preceding claims, which when contacted with 50% human tear fluid for 60 minutes under the conditions described in Example 5 subsequently exhibits at least 40% of maximum possible association with HT1080 cells after one hour of incubation.

8. The liposome according to claim 7, wherein the liposome when contacted with 50% human tear fluid for 60 minutes subsequently exhibits at least 50%, such as between 50 and 60%, of maximum possible association with HT1080 cells after one hour of incubation.

9. The liposome according to any one of the preceding claims, wherein the release of hydrophobic active ingredient when said liposome is applied to the normal human eye is slower than from an aqueous solution and faster than from reference liposomes comprising >20% mol/mol cholesterol, and where the liposome preferably releases hydrophobic active ingredient more slowly than a reference liposome having a net negative charge, where the aqueous solution and the reference liposomes comprise the same amount of hydrophobic active ingredient as does the liposome.

10. The liposome according to any one of the preceding claims, which comprises lipids that exhibit a negative charge as well as lipids that exhibit a positive charge, in particular after ocular administration.

11. The liposom according to any one of the preceding claims, which also comprise lipids that are electroneutral, in particular after ocular administration.

12. The liposome according to any one of the preceding claims, which comprise

substantially all of said at least one active ingredient.

13. The liposome according to any one of the preceding claims, wherein one of the compartments selected among the interior aqueous compartment, a hydrophobic bilayer, and a polar inter-phase of the inner and outer leaflet of the liposome carry said at least one active ingredient.

14. The liposome according to any one of the preceding claims, wherein said at least one active ingredient is selected from the group consisting of acetazolamide, acyclovir, indomethacin, verapamil, rapamycin, ascomycin, ciprofloxacin, ofloxacin, fusidin, gentamicin, chloramphenicol, levofloxacin, oxytetracyclin, tobramycin, acyclovir, prednisolon,

dexamethason, chloramphenico, brimonidin, brimonidintartrat, dorzolamid, timolol, latanoprost, tetryzolinhydrochlorid, and natriumcromoglicat.

15. The liposome according to any one of the preceding claims, wherein said at least one active ingredient is a non-steroidal anti-inflammatory drug (NSAID), preferably selected from the group consisting of ketoprofen, flurbiprofen, ibuprofen, diclofenac, ketorolac, nepafenac, amfenac and suprofen.

16. The liposome according to any one of the preceding claims, wherein the active ingredient is in the form of a hydrophobic active ingredient, such as a hydrophobic drug.

17. The liposome according to claim 16, wherein the hydrophobic active ingredient is selected from the group consisting of Diclofenac, Fusidine, Levocabastin, Indometacin, Latanoprost, Travoprost, Olopatadin, Azelastin, Rimexolon, ketotifen, naphazolin,

Bimatoprost, Betaxolol, emedastin, Ketorolac, Resorcinol, Fluormetolon, Atropine,

Apraclonidin, Cyclopentolat, Dexamethasone, Lidocain, Prednisolone, Nepafenac, Timolol, Tropicamid, Brimonidine, Chloramphenicol, Pilocarpine, Vancomycin, Fluconazol,

Sulfamethizol, and Moxifloxacin.

18. The liposome according to any of the preceding claims, wherein the lipids comprise or are phospholipids.

19. The liposome according to any of the preceding claims, where the lipid comprises or constitutes phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA), DPG (bisphosphatidyl glycerol), PEOH (phosphatidyl alcohol) and cholesterol.

20. The liposome according to any of the preceding claims, wherein at least part of the lipids is a cationic lipid, preferably selected from the group consisting of stearylamine (SA), dimethyldioctadecylammonium bromide (DDAB), 36-[N-(N',N'-dimethylaminoethane)- carbamoyljcholesterol (DC-Cholesterol), l,2-ditetradecanoyl-3-trimethylammonium-propane (DMTAP), DOTAP derivatives (l,2-dioctadecanoyl-3-trimethylammonium-propane, 1,2-di- (9Z-octadecenoyl)-3-trimethylammonium-propane, l,2-dihexadecanoyl-3- trimethylammonium-propane), DODAP derivatives (l,2-di-(9Z-octadecenoyl)-3- dimethylammonium-propane, l,2-ditetradecanoyl-3-dimethylammonium-propane, 1,2- dihexadecanoyl-3-dimethylammonium-propane, l,2-dioctadecanoyl-3-dimethylammonium- propane), l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), dioctadecylamide- glycylspermine, SAINT-2, polycationic lipid 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), GL67TM, 1,2- dioctadecanoyl-sn-glycero-3-ethylphosphocholine (Etyl PC), and 1,2-dioctadecanoyl-sn- glycero-3-phosphoethanolamine (DSPE).

21. The liposome according to any one of the preceding claims, wherein the liposomes comprise 0-60%, preferably 10-60%, such as 20-50% (mol/mol) cationic lipids.

22. The liposome according to any one of the preceding claims, wherein the liposomes comprise less than 20%, such as less than 10%, preferably less than 5%, more preferred less than 2% cholesterol, most preferred substantially no cholesterol.

23. The liposome according to any one of the preceding claims, wherein lipids that are electroneutral comprise head groups selected from phosphatidylcholine and

phosphatidylethanolamine.

24. The liposome according to any one of the preceding claims, wherein lipids that are negatively charged comprise head groups selected from phosphatidylserine,

phosphatidylglycerol, phosphatidic acid, and phosphatidylinositol.

25. The liposome according to any one of the preceding claims, wherein the alkyl chains of the lipids are C8-C24, preferably C10-C22, more preferred C12-C20, preferably C14-C18, most preferred C16-C18 saturated chains or unsaturated chains, preferably saturated chains.

26. The liposome according to any one of the preceding claims, wherein at least one liposome is a Large Unilamellar Vesicle (LUV).

27. The liposome according to any one of the preceding claims, wherein the liposomes have a diameter of 50-10,000 nm, preferably 60-5,000 nm, more preferred 70-1,000 nm, preferably 80-120 nm, more preferred 100-500 nm, preferably 200-7,500 nm, more preferred 300-3,000 nm, preferably 500-2,000 nm, more preferred 1,000-1,500 nm.

28. The liposome according to any one of the preceding claims, which does not comprise a hydrophilic polymer, such as a polymer selected among PEG [poly(ethylene glycol)], PAcM [poly(N-acryloylmorpholine)], PVP [poly(vinylpyrrolidone)], PLA [poly(lactide)], PG

[poly(glycolide)], POZO [poly(2-methyl-2-oxazoline)], PVA [polyvinyl alcohol)], HPMC (hydroxypropylmethylcellulose), PEO [poly(ethylene oxide)], chitosan [poly(D-glucosamine)], PAA [poly(aminoacid)], polyHEMA [Poly(2-hydroxyethylmethacrylate)] and co-polymers thereof.

29. The liposome according to any one of claims 1-27, wherein at least part of the lipids comprise or is conjugated to a hydrophilic polymer, such as the polymers defined in claim 28, subject to the proviso that the average molecular weight of hydrophilic polymer for the totality of the lipids is less than 2,000 Dalton, preferably less than 1,500 Dalton, more preferred less than 1,000 Dalton, preferably less than 500 Dalton, more preferred less than 300 Dalton, preferably less than 200 Dalton, more preferred less than 100 Dalton.

30. The liposome according to any one of the preceding claims, wherein at least one lipid is a substrate for enzymes in tear fluid, such as human tear fluid, e.g. a substrate for tear fluid sPLA₂.

31. Lipid based drug delivery system for ocular or intra-ocular administration, said system providing sustained or delayed release of at least one active ingredient,

said system comprising : (I) lipids comprising:

(a) an organic radical having a least 2 carbon atoms; and

(b) a hydrophilic moiety; said lipids being a substrate for enzymes in tear fluid or for extracellular sPLA₂ to the extent that the organic radical can be hydrolytically cleaved off, leading to an intramolecular cyclization reaction; and

wherein at least one of the lipids in the liposome is degradable by secretory phospholipase, in particular sPLA₂ activity, allowing sustained or delayed release of said at least one active ingredient from said liposomes upon contact with secretory phospholipase, in particular sPLA₂.

32. A pharmaceutical formulation suitable for ocular or intra-ocular administration and providing sustained or delayed release of at least one active ingredient,

said pharmaceutical formulation comprising liposomes,

said liposomes comprising at least part of said at least one active ingredient, and

at least part of said liposomes are liposomes according to any one of claims 1-30.

33. The pharmaceutical formulation for ocular administration according to claim 32, in the form of a cream, an ointment, a gel, a paste or eye-drops.

34. The pharmaceutical formulation according to claim 32 or 33, providing release of said at least one active ingredient during a period of 0.5-24 hours, preferably 1-12, more preferred 2-8, preferably 3-6 hours.

35. The pharmaceutical formulation according to any one the claims 32-34-, wherein said pharmaceutical formulation comprises a mixture of liposomes with different release characteristics, whereby the pharmaceutical formulation provides sustained release of said at least one active ingredient.

36. The Liposome according to any one of claims 1-30 or the lipid-based drug delivery system according to claim 31 or the pharmaceutical composition according to any one of claims 32-35 for use as a pharmaceutical.

37. The Liposome according to any one of claims 1-30 or the lipid-based drug delivery system according to claim 31 or the pharmaceutical composition according to any one of claims 32-35 for use in treatment or amelioration of an ophthalmic indication, such as postoperative pain or ocular inflammation, such as an ocular inflammation which results from iritis, conjunctivitis, seasonal allergic conjunctivitis, acute and chronic endophthalmitis, anterior uveitis, uveitis associated with systemic diseases, posterior segment uveitis, chorioretinitis, pars planitis, masquerade syndromes including ocular lymphoma, pemphigoid, scleritis, keratitis, severe ocular allergy, corneal abrasion or blood-aqueous barrier disruption.

38. The Liposome according to any one of claims 1-30 or the lipid-based drug delivery system according to claim 31 or the pharmaceutical composition according to any one of claims 32-35 for use in treatment or amelioration of the indication post-operative ocular inflammation, such as inflammation resulting from photorefractive keratectomy, cataract removal surgery, intraocular lens implantation or radial keratotomy.

39. The Liposome according to any one of claims 1-30 or the lipid-based drug delivery system according to claim 31 or the pharmaceutical composition according to any one of claims 32-35 for use in treatment or amelioration of an indication selected from the group consisting of fungal infection; microbial infection; inflammatory disease; non-ocular inflammatory disease, such as rheumatoid arthritis (RA); dry eye disease and dry eye condition, such as episodic or chronic dry eye condition and aqueous tear deficiency (ATD); keratoconjunctivitis sicca (KCS), such as Sjogren syndrome (SS), associated KCS and non-SS associated KCS; mucin deficiency; Stevens-Johnson syndrome; ocular injury such as chemical burn; chronic blepharitis; contact lens intolerance; chronic ocular surface inflammation; inflammation of ocular surface disease; glaucoma, such as open-angle glaucoma, primary open-angle glaucoma (POAG) and exfoliation glaucoma (AG); conjunctival inflammation; and corneal disease, such as allergy, conjunctivitis (Pink Eye), corneal infection, Fuchs' Dystrophy, Herpes Zoster (Shingles), iridocorneal endothelial syndrome, keratoconus, lattice dystrophy, map-dot-fingerprint dystrophy, ocular herpes, and pterygium.

40. A method for prophylactic or therapeutic treatment or amelioration of an ocular or intraocular disease or condition, the method comprising administering, ocularly or

intraocularly, to a subject in need thereof an effective amount of a liposome according to any one of claims 1-30, a lipid based drug delivery system according to claim 31 or a

pharmaceutical composition according to any one of claims 32-35.

41. The method according to claim 40, wherein the ocular or intraocular disease or condition is associated with any one of the indications set forth in claims 36-39.