Bibliographic data
`
`Title:
`
`LIPOSOME SYSTEM FOR OCULAR ADMINISTRATION
`
`Pub/Pat no:
`
`WO201 1008s 78A2
`
`Pub/Issue Date:
`
`2011-08-18
`
`Inventor(s):
`
`Applicant(s):
`
`BAR-SHALOM DANIEL |SKJOEDE JENSEN SIMON
`
`BIONEER AS[DK]| BAR-SHALOM DANTEL[DK]| SKIOEDE
`JENSEN SIMON[DK]
`
`Classification:
`
`ASIK9/00AI
`
`Application nurnber:
`Priority. number:
`
`WO201 1LEP5S2061 2011-02-11
`1J$20100303758P 2010-02-12; DE20100070051 2010-02-13:
`
`Abstract of WO2011098578A2
`
`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. iposoimes, said lipdasomes comprising at least part of said at least. one
`activeingredient, at least part of said liposomes exhibiting a charge increasmg 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 liposomesis
`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 orin the
`vicinity of the eye. The pharmaceutical formulation is useful for ophthalmic indications.
`
`

`

`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`
`18 August 2011 (18.08.2011)
`
`(10) International Publication Number
`WO 2011/098578 A2
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU,ID,IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM,PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(51) International Patent Classification:
`A61K 9/00 (2006.01)
`(21) International Application Number:
`.
`
`PCT/EP2011/052061
`
`(22)
`
`InternationalFiling Date:
`
`11 February 2011 (11.02.2011)
`.
`English
`English
`
`(25) Filing Language:
`(26) Publication Language:
`(30) Priority Data:
`12 February 2010 (12.02.2010)
`61/303,758
`PA 2010 70051 12 February 2010 (12.02.2010)
`
`US (84) Designated States (unless otherwise indicated, for every
`DK
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU,TJ,
`except US):
`all designated States
`(for
`(71) Applicant
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`BIONEER A/S [DK/DK]; Kogle Allé 2, DK-2970 Har-
`EE, ES, FI, FR, GB, GR, HR, HU,IE, IS, IT, LT, LU,
`sholm (DK).
`LV, MC, MK,MT, NL, NO, PL, PT, RO, RS, SE, SL SK,
`Inventors; and
`Inventors/Applicants (for US only): BAR-SHALOM, Heeeeorey Ob OM: GA. GN, GQ.
`Daniel |DK/DK]; Rypeveenget 213, DK-2980 Kokkedal
`>
`>
`»
`NE,
`SN,
`TD,
`TG).
`(DK). SKJGDE JENSEN, Simon [DK/DK]; Kagslokke Published:
`2
`j
`1, DK-2700 Brenshaj (DK).
`— without international search report and to be republished
`(74) Agents: KOEFOED,Peteret al.; Inspicos A/S, P.O. Box
`upon receiptof that report (Rule 48.2(g))
`45, Kogle Allé 2, DK-2970 Harsholm (DK).
`
`(72)
`(75)
`
`(34) Title: LIPOSOME SYSTEM FOR OCULAR ADMINISTRATION
`
`(57) 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 com-
`prising at least part of said at least one active ingredient, at least part of said liposomes exhibiting a charge increasing the adher-
`ence of the charged liposomesto the eye after administration, in particular when said pharmaceutical formulation is for ocular ad-
`ministration, and wherein at least one of the lipids in the liposomes is degradable by sPLA:2activity, allowing sustained or delayed
`release ofsaid at least one active ingredient from said liposomes upon contact with tear fluid or with sPLA: in the eye or in the
`vicinity of the eye. The pharmaceutical formulation is useful for ophthalmic indications.
`
`
`
`
`
`Wo2011/098578A2IMITINMINIIMTNANTESA
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`

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`WO 2011/098578
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`PCT/EP2011/052061
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`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 eyeis 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 A> 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 PLA», 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 Az, 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.
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`The Liplasome technology was developed for targeted delivery of drugs administered
`
`systemically, i.e. the drug or prodrug was supposedto be carried by the blood stream and
`
`released or activated at sites with increased levels of extracellular PLA, 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
`
`10
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`15
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`20
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`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 sPLA> sensitive, charged
`
`liposomes is enhanced in the prescence of (human) tear fluid comprising sPLA, 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 sPLA:2 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 SPLA, in the eye or in the
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`25
`
`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.
`
`sPLA, in tear fluid is present in very high amounts and is able to degrade liposomes
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`30
`
`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
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`WO 2011/098578
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`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
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`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.
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`10
`
`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,
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`15
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`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 sPLAsto the extent
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`20
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`that the organic radical can be hydrolytically cleaved off, leading to an intramolecular
`
`cyclization reaction; and
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`(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;
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`25
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`at least part of said liposomes exhibiting a charge, which increases the adherence of the
`
`charged liposomesto 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 sPLA, activity, allowing sustained or delayed
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`release of said at least one active ingredient from said liposomes upon contact with secretory
`
`phospholipase, in particular sPLA>.
`
`The sustained or delayed drug release system of the present invention does not depend on
`
`increased levels of sPLAz 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.
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`LEGENDS TO THE FIGURE
`
`Fig. 1: Overview of liposomes, liposome size and surface charge. A) List of 5 liposomes
`
`composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-
`
`sn-glycero-3-phosphoglycerol (POPG) and 1,2-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=PIipcie6/(Plpopc
`
`XY%opopc) was calculated. PI_pci1g and Plpopc 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=0O whereas full hydrolysis corresponds to a large/infinite R-value.
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`Fig.3: Liposome binding to human cells dependent on time of incubation and liposome
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`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=O. Fluorescence measurement was made by to trace liposome presence
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`30
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`through incorporation of 0.2% DOPE-rhodamine into the liposome composition.
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`Fig.4: Liposome binding to human cells dependent on presence of tear fluid or bee venom
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`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
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`10
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`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
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`of the cationic liposomes containing DOTAP compared to neutral or negatively charged
`
`liposomes. Increased time of incubation did not induce cytotoxicity.
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`DETAILED DISCLOSURE OF THE INVENTION
`
`15
`
`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, /.e. a log Doctanoiwater higher than O. Preferred
`
`hydrophobic drugs of relevance for the present invention exhibit higher log Dectanoiwater 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 Doctanoiwater is calculated as follows:
`
`20
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`25
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`[drUs Jctanot
`log} ————2“etal_.
`water
`water
`
`
`
`e[drug|" +[drugyinennes
`
`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 membranesof the eye (an
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`WO 2011/098578
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`PCT/EP2011/052061
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`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
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`10
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`intended that the feature which is described by these terms is present in an amountor 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 substancein 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
`15
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`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 notin 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
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`20
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`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.
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`For ocular delivery of drugs, positively charged liposomes will likely be degraded by sPLA>
`
`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 sPLA»2 sensitivity of lipids in the liposomes and their
`
`ability to adhere to negatively charged cell surfaces. However, it cannot be excluded that the
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`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
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`of adhesion to corneal surfaces and sustained release to the eye and that these effects are
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`the consequence of the action of tear fluid on the liposomes.
`
`Liposome structure and characteristics
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`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
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`most 29%, at most 28%, at most 27%, at most 26%, at most 25%, at most 24%, at most
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`23%, at most 22%, at most 21% and at most 20%. On the other hand, the amount of
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`positively chargedlipids 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
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`19%, and at least 20%.
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`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 O-
`
`5%, 5%-15%, 10-20%, 20-25%, 25-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%,
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`80-90%, 90-100% (mol/mol) positively charged/cationic lipids
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`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 wherethe liposome has a zeta potential in the range from 10-60 mV. The zeta
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`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.
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`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, /.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 ofa 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 notall 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
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`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.
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`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
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`35
`
`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
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`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
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`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.
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`As mentioned above, the liposome may comprise a cationic lipid, which is preferably selected
`
`from the group consisting of stearylamine (SA), dimethyldioctadecylammonium bromide
`
`(DDAB), 3B-[N-(N',N’-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), 1,2-
`
`ditetradecanoyl-3-trimethylammonium-propane (DMTAP), DOTAP derivatives (1,2-
`
`dioctadecanoyl-3-trimethylammonium-propane, 1,2-di-(9Z-octadecenoyl)-3-
`
`trimethylammonium-propane, 1,2-dihexadecanoyl-3-trimethylammonium-propane), DODAP
`
`derivatives (1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane, 1,2-ditetradecanoyl-3-
`
`dimethylammonium-propane, 1,2-dihexadecanoyl-3-dimethylammonium-propane, 1,2-
`
`dioctadecanoyl-3-dimethylammonium-propane), 1,2-di-O-octadecenyl-3-trimethylammonium
`
`propane (DOTMA), dioctadecylamide-glycylspermine, SAINT-2, polycationic lipid 2,3-
`
`dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate
`
`(DOSPA), GL67TM, 1,2-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 maybe found in or made according to US 4,804,539.
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`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%
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`cholesterol, most preferred substantially no cholesterol. However amounts of cholesterol up
`
`to about 10% appear to be acceptable, so amounts in the range between O and 10%are also
`
`within the invention, such as amounts between 1 and 9%, 2 and 8% and 3 and 7%.
`
`The liposomesof the invention preferably include lipids, which when relevant, contain alkyl
`
`chains that are C8-C24, preferably CLO-C22, more preferred C12—C20, preferably C14-C18,
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`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
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`double bonds.
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`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.
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`Typically, the liposome of the present invention has a diameter of 50-10,000 nm, preferably
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`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,
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`more preferred 1,000-1,500 nm.
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`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)],
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`POZO [poly(2-methyl-2-oxazoline)], PVA [poly(vinyl alcohol)], HPMC
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`(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 sPLAz>.
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`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.
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`Also the at least one active ingredient is a non-steroidal anti-inflammatory drug (NSAID),
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`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,
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`Levocabastin, Indometacin, Latanoprost, Travoprost, Olopatadin, Azelastin, Rimexolon,
`
`ketotifen, naphazolin, Bimatoprost, Betaxolol, emedastin, Ketorolac, Resorcinol,
`
`Fluormetolon, Atropine, Apraclonidin, Cyclopentolat, Dexamethasone, Lidocain, Prednisolone,
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`Nepafenac, Timolol, Tropicamid, Brimonidine, Chloramphenicol, Pilocarpine, Vancomycin,
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`Fluconazol, Sulfamethizol, and Moxifloxacin.
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`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
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`and a general reference to the requirements for