(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`(19) World Intellectual Property
`aS;
`Organization
`International Bureau
`
`(43) International Publication Date
`28 January 2016 (28.01.2016)
`
`WIPO] PCT
`
`A
`
`(1) International Publication Number
`WO2016/013031 Al
`
`AG, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW. BY.
`BZ, CA, CH, CL, CN, CO. CR. CU, CZ, DE. DK, DM,
`DG, DZ, EC, HE, BG, HS, FL GR, GD, GE, GH, GM, GT,
`HIN, HR TAU, ID, AL, IN, IR, 1S, IPL KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG.
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, GM,
`PA, PE, PG, PH, PL. PT, QA, RG, RS, RU, RW, SA, SC,
`
`SD, SE, SG, SK, SL, $M, ST, SV. SY, TH, TH, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`
`(84) Designated States (unless otherwise indicated, jor every
`kind ofregional protection available), ARIPO BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SU, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, RE, BG, CH, CY, CZ, DE,
`DE, BEES, PLR, GB, GR, AR, HULL TE IS, IT, LT, Lu,
`LV, MC, ME, MT, NL, NO, PL, PT, RO, RS, SE, SI, 8K,
`SM, TR), OAPL CBP, BE, CF, CG, CL CM, GA, GN, GQ,
`GW, KM, ML, MK, NE, SN, TD, TG).
`Declarations under Rule 4.17:
`
`as 0 the identity of the inventor (Rule 4.17(1}}
`
`as to applicant's entitlement to apply fer and be@ granteda
`
`
`patent (Ruie 4.47(tt)
`
`as to the applicant's entitlement to claim the priority ofthe
`earlier application (Rule 4.) 7(ii)
`
`of inventorship (Rule 4.47iv)
`Published:
`
`with international search report (Art. 21(3))
`
`bejore the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48.2h))
`
`SY
`
`International Patent Classification:
`AGLE 9/127 (2006.01)
`AGERE 31/4778 (2006.01)
`A&BLK 9/197 (2006.01)
`
`ZH
`
`lnternational Appication Nuriber:
`
`PCT/INZG 1 5/030073
`
`(223
`
`luternational ilng Date:
`
`Filng Language:
`
`Publication Language:
`
`23 July 2015 (23.07.2015)
`
`English
`
`English
`
`Friovity Data:
`25 duly 2614 (25.07 2014)
`24 12/MUM/2014
`UN
`222/MUM/2015 22 Jannary 2015 (22.61.2015)
`UN
`Applicant: SUN PHARMA ADVANCED RESEARCH
`COMPANYLED. [IN/IN], 17/3, Mahal Industrial Estate,
`Off Mahakali Caves Road, Andheri feast), Maharashtra,
`Munibai 400093 (IN),
`
`inverters: BHOWMICK, Subhas; Sun Pharma Ad-
`vanced Research Company Lid, Nima Compound, Near
`Pratham Vinclave, Tandalja, Road, Baroda 3900020 (TN).
`UMRETHIA, Manish Kumar; Sun Pharma Advanced
`Research Company Lid, Nima Compound, Near Pratham
`
`Enclave, Landala,Road, Baroda 390020 (EN). ua
`Keuntal; Sun Pharma Advanced Research Company Lid,
`Nima Compound, Near Pratham Enclave, Tandalja,Road,
`Raroda 390020 CN). THABYA,
`Jayesh Kumar: Sun
`Pharma Advanced Research Company Lid, Nima Com-
`pound, Near Pratham Enclave, Tandalia,Road, Baroda
`
`390026 CIN). PATER. Nitesh Kumar, Sun Pharma Ad-
`vanced Kesearch Company Lid, Nima Compound, Near
`PrathamEnclave, Tandalia,Road, Baroda 390020 (EN).
`Desigmated States (unless otherwise indicated, for every
`kind of national protection available), AK, AG, AL, AM,
`
`23}
`
`28)
`
`BO)
`
`7H
`
`i723)
`
`
`
`(54) Title: LIPOSOME COMPOSITION AND METHOD OF PREPARING THE LIPOS
`
`OME
`
`o.f
`“SsaobeA.
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`‘gooey
`
`(87) Abstract: The present invention relates lo @ method of loadmyg an ionivable drug having 4 group selected from (Pormule Toor
`Formule U} or pharmaceutically acceptable salts thereof into 4 liposome and the Lposomal composition of dig produced therewith.
`
`=a
`
`e
`‘pooed
`Soe?
`aN
`©FJ
`W
`
`

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`WO IWO16M13031
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`POCP/IN2015/050073
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`LIPOSOME COMPOSITION AND METHOD OF PREPARING THE LIPOSOME
`
`FIELD OF THE INVENTION
`
`5 The present invention relates to a method of loading an tonizable drug having a group selected
`
`from
`
`NO=N
`p HN
`HN.
`WN
`\
`(oy US
`or pharmaceutically acceptable salts thereof into a liposome and the long circulating liposomal
`
`i; O
`
`composition of drug prodaced therewith.
`
`ig BACKGROUNDOF THE INVENTION
`
`Liposomes have been widely studied and used as carriers for a variety of agents such as drugs,
`
`putrients, cHagnostic reagents,
`
`and genetic material. Liposomes are actificiaty prepared
`
`spherical vesicle composed of alamellar phase lipid bilayer. Liposomes or lipid vesicles are
`
`often composed of phosphatidylcholine-enriched phospholipids and mayalso contain mixed lipid
`
`15 chains with surfactant properties such as egg phosphatidy] ethanolamine. Since Hposomes consist
`x
`co
`¥i
`they generally have low togicny and therefore are useful in a
`of nontoxic Hpads,
`pharmacentical appheations. Liposomes are a recognized dnig delivery system
`
`
`improve the therapentic activity and increase
`the safety of a surnber of different pharmacentical
`
`agents. Liposome-encepsulated drugs often have biacistribudons and toxicities which differ
`
`2G greatly frora those of free drug. The use of Hposomes increases the therapeutic index of many
`
`drags, and also offers drag targeting and controlled release. To be useful in medical treatments,
`
`Hposome formulations should have an efficient drug to Hpid ratio, ic. a hinher percentage of
`
`dug entrapment, a practical shelf-life and be capable of reproducible preparation. General
`
`techniques of preparing drug loaded liposomes has been extensively studied in the past. The
`
`25 hpesome formalations for pharmaceutical apphcations can be made etther by combining drag
`
`and hpi before formation of the vesicles Gpassive loading techmanue), ar by “loading” drig unre
`
`hpvesicles after they are formed (active loading technique). There are various methoda by
`
`which liposcmmal vesicles can be prepared, such as mechanical dispersion methods inchicine linid
`
`

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`film hydration, sizing
`
`5
`Using sonication, EXtrusio#n.
`
`ent evaporation methods such as ethanal
`
`mijection, ether injection, reverse phase evaporation, detergent removal methods. For drag
`encapsulation, there is a need to in ase the entrapment efficiency such thar the drig to lipid
`
`
`ratio is as high as possible, while maintaining the original chemical integrity of both drug and
`
`Shpid. Once administration to a patient has occurred, drug release is a factor, Rapid releas
`pharmacenicals fran Hposomes reduce the bie-distribution benefits sought i nogy utilizing
`
`vesicle carriers. Accordingly, efforts to optimize pharniaceutical loading and to reduce the rate af
`
`release of pharmaceuticals from lipid vesicles and make them long circulating in plasma have
`
`comtinued. Purther,
`
`the Hposorne formulations should be stable such that they remain intact
`
`10 during shipping and apon storage for the shelf hfe. At present,
`
`there is no single universal
`
`encapsulation method that offers stable encapsulation of most drugs; each drag requires a
`
`different approach to manage all of its properties.
`
`The prior art does not disclose methods of Joading an ionizable drug having a group selected
`
`from
`
`415
`
`=[N
`ad
`ON
`HN.
`
`or
`
`A
`HNTSN
`pet}
`: se
`@
`
`such as losartan, candesartan or pharmaceutically acceptable salt thereof into Hposomes that have
`
`bigh percentage of drug loading and long blood circulating nature when admunistered
`
`parenterally. While atterapting to make a lrosome composition of these ionizable drags such as
`
`losartan or candesartan or pharmaceutically acceptable salts thereof,
`
`the inventors faced the
`
`20 problem of poor drug loading and for short circulating liposome. Particularly, the inventors
`
`found that the salts of the transition metal ions of strong acid provided liposomes that showed
`4t!
`very poor loading. Surprisingly, and unexpectedly, however, when salts of the transition metal
`
`ions of weak acid were employed, the method provided liposomes with very good, efficient
`
`loading of the drug. Not only the drug loading was satisfactory, the so prepared liposomes were
`
`25 long circulatingin the plasma. This was indeed surprising.
`
`Ped
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`SUMMARY OF THE INVENTION
`
`The present invention provides a method of loading an iomzable drug having a group selected
`
`from
`
`N=
`
`PN
`~4
`
`or
`
`’
`
`Ay
`f°
`
`5 or pharmaceutically acceptable salts thereof into a liposome, said method comprising the steps
`
`of:
`
`a) providing preformed liposomes containing an internal solution comprising a weak
`
`acid salt of a transition metal:
`
`6b) adding the ionizable drug to the external solution, and
`
`ia
`
`c)
`
`incubating the external solution of step “b’ with the preformed liposomes of step
`
`‘a’ for a sufficiertt time to load the drug into the Hposornes.
`
`The present
`
`invention further provides a liposomal cornposition prepared by the method
`
`described above.
`
`BRIEF DESCRIPTIONOF FIGURES
`
`15 Figure 1: represents a graph showing the plasma concentration versus time data of losartan after
`
`intravenous administration of reference solution;
`
`liposomal composition of example 1 and
`
`liposomal composition of coraparative example A according to study provided in exarnple 5.
`
`DETAILED DESCRIPTIONOF THE INVEN TRON
`
`The phrase ‘percentage drug loading’ and ‘percentage drug entrapment has been used
`
`20 interchangeably in the specification. ft refers to the percentage by weight of the total drug which
`
`is loaded or entrapped inside the hposome vesicle.
`
`The
`
`term ‘long circulating’
`
`as used herein means
`
`that
`
`the
`
`liposomes
`
`escape
`
`the
`
`reticulcendothelial system (RES) which is responsible for removal of foreign particles from the
`
`bloodstreamand thus circulates in bloodstream for prolonged period. Long circulating liposomes
`
`25 entrap the drug and increase the mean residence time of the drug in the body as compared to
`
`mean residence time from a non-Hposoral solution composition. Mean residence time is an
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`POCP/IN2015/050073
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`avetage time a drug molecule spends in the body. The iiposome composition of the present
`
`invention increases the mean residence time to more than 6 hours, preferably more than 10
`
`hours.
`
`The phrase ‘internal solution” and the “external soletion” as used herein are related to the
`
`5 intermediate steps while preparing the drug loaded liposomes. The method of the present
`
`invention is based on active drug loading imto the preformed liposomes. The preformed
`
`Hposomes before loading the drug into i contain an internal sohition which is aqueous solution
`
`of weak acid salt of a transition metal ion. The phrase “external solution’ refers to a medium in
`
`which an ionizable drug such as losartan or its pharmaceutically acceptable salt is added, and this
`
`10 external solution is incabated with the preformed Hposome for loading the iomzable drug.
`
`Suitably, the external solution may comprise one or more of a sucrose phosphate buffer, sucrose
`
`histidine buffer, HEPES buffer, sucrose phosphate EDTA buffer, acetate buffer, citrate butter,
`
`sucrose HEPES buffer, HEPES EDTA buffer, HEPES sale, histidine and sucrose.
`
`The present invention provides a method of loading an ionmable drug having a group selected
`
`HNSN
`oe‘i
`
`Yp
`
`G
`
`15 from
`
`NooN
`i,
`\
`LN
`N
`
`or
`
`or pharmaceutically acceptable salts thereof into a liposome, said method comprising steps of:
`"1
`a) providing preformed Hposormes containme an internal solution comprising a weak
`
`acid salt of a transition metal;
`
`20
`
`b) adding the ionizable drug or its pharmaceutically acceptable salt to the external
`
`solution, and
`
`c)
`
`incubating the external solution of step ‘b’ with the preformed liposomes of step
`
`‘a’ for a sufficient time to load the drug ito the hposomes.
`
`The present invention further provides a long circulating iposomal composition produced bythe
`
`25 method of the invention described above. The liposomal composition comprises the ionizable
`
`drug or its pharmaceutically acceptable salt; a weak acid salt of a transition metal cation and
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`vesicie forming lipids selected from one or more phospholipids, one or more steroidal lipid, one
`
`or more polymer derivatised lipid or mixtures thereof.
`
`Suitably, a drig suitable for the method of the present invention is an ionizable drug having a
`
`group selected from
`
`NOONwh
`
`5
`
`or
`
`oO
`
`or pharmaceutically acceptable salts thereof. Suitably, these ionizable drugs are weakly acidic in
`
`nature. The pharmaceutically acceptable salts include, any suitable salt that is pharmaceutically
`
`acceptable. Suitable pharmaceutically acceptable salts mclude, but are not Jumited to alkaline
`
`metal salis ike sodium, potassium etc., alkaline earth metal salts ike magnesium, calcturn etc.,
`
`10 medoxonul, cilexetl, mesylate, hydrochloride, hydrobromide, etc.
`
`Preferably, the drugs having the group
`
`N
`
`soe Re
`‘
`N
`
`;
`H Y
`
`inchide, but are not limited to, candesartan,
`
`losartan,
`
`irbesartan, valsartan, olmesartan, EXP-3174 or their active metabolites, esters or
`
`pharmaceutically acceptable salts thereof.
`
`15 Preferably, the drugs having the group “
`
`include, but are not limited to azilsartan or
`
`their active metabolites, esters or pharmaceutically acceptable salts thereof. For example the drug
`
`is azilsartan medoxomnl potassium.
`
`In preferred erabodiments, the method of the present invention comprises an ionizable drug
`
`selected from losartan, candesartan, EXP-3174, clmesartan or their esters or pharmaceutically
`
`20 acceptable
`
`salts
`
`thereof,
`
`such as candesartan cilexeti!,
`
`losartan potassium, olmesartan
`
`medoxomil. The ionizable drig is present in the liposomal composition in therapeutically active
`
`amcaunts, suitable te achieve the desired therapeutic effect.
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`POCP/IN2015/050073
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`In one specific embodiment, the ionizable drug or active agent is losartan or its pharmaceutically
`
`acceptable salts, having the following strucuure:
`
`primer
`
`In one embodiment, the ionizable drug or active agent is candesartan or its pharmaceutically
`
`5 acceptable salts, having the following structure:
`
`
`
`
`HO
`
`In one embodiment, the ionizable drug is EXP-3174, which is an active metabolite of losartan
`
`and have the following structure:
`
`
`
`6
`
`1G
`
`

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`WO IWO16M13031
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`POCP/IN2015/050073
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`In another embodiment, the ionizable drug or active agent is irbesartan or its pharmaceutically
`
`acceptable salts, having the following strucuure:
`
`\
`
`?= 4 _,
`T “
`Ssea, wofyFyrag ‘awl
`
`~é
`
`
`
`In another embodiment, the ionizable drug or active agent is valsartan or its pharmaceutically
`
`5 acceptable salts, having the following structure:
`Ne,
`.
`-|
`
`(#
`(“
`
`Non
`On_OH
`“ONai pS ae No. ”
`[ po K
`Fegeensy
`a
`Nn, a or
`ro
`a
`a,
`Nene Cy
`
`M
`
`thn
`
`ee
`
`In another embodiment, the ionizable drug or active agent is olmesartan or its pharmaceutically
`
`acceptable salts, having the following structure:
`
`
`
`
`eigBe we
`
`sf
`
`
`
`hy.oereees*y
`
`1G
`
`“ennarart,s $
`
`7,
`
`

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`WO IWO16M13031
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`POCP/IN2015/050073
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`In specific embodiment,
`
`the ionizable drug as above is an Angiotensin I] receptor blocker.
`
`Angiotensin Tl receptor blocker and other RAAS inhibitors are anti-hypertensives and are also
`
`knownto reduce the levels of collagen E and Iand basement membrane collagen PVin various
`
`experimental models of fibrosis and reverse renal and cardiac fibrosis in hypertensive patients.
`
`5 Further, it bas been shown to inhibit collagen | production in tumors. The liposomal composition
`
`prepared by the method of the present invention are suitable for use as a collagen modifying
`
`agent to improve the distribution and/or penetration of chemotherapeutics into solid tumours. [ny
`
`one aspect, the Lposomal composition produced by the methad of the present invention,
`
`are
`
`useful in treatment of cancers/nimours such as breast, pancreatic, colorectal, pancreatic, colon,
`
`10 fung, skin, ovarian, cervix, prostate, gastric, gastrointestinal, stomach, head and neck, kidney,
`
`liver cancer or metastatic lestons of these cancers.
`
`In one aspect, the Npasamal composition
`
`produced by the method of the present invention, when adrainistered m combination with other
`
`anticancer agents, provides an improved method of treating cancers or tumors, such as solid
`
`Rumors.
`
`15 In one specific embodiment, the ionizable drig is losartan or its pharmaceutically acceptable salt,
`
`an angiotensin Hf receptor blocker. Suitable salts include losartan potassiumor losartan sodium.
`
`Anyother salt may be used. As used herein in some embodiments, the term ‘losartan’ is intended
`
`to include losartan or its pharmaceutically acceptable salts. A preferred salt is losartan potassium.
`
`Losartan or its pharmaceutically acceptable salt may be present in the hposomal composition
`
`20 prepared according to the method of the present invention,
`
`in an amount ranging from about
`
`0.0001 @wivto about 10.0 Yow/y, preferably from about 0.1 w/vto about 1.0 % wiv,
`
`The weak acid salt of a transition metal according to the present invention is a salt ofa transition
`
`metal cation with a weak acid anion. The transition metal cation suitable for use in the present
`
`25 invention may be selected from a monovalent, divalent,
`
`trivalent or a tetravalent cation,
`
`preferably a divalent or
`
`trivalent cation, more preferably a divalent cation.
`
`in preferred
`
`embodiments, the transition metal/cation is selected from but not limited to copper (monovalent
`9A
`:
`,
`gmt
`#2
`:
`pRuctey
`ot
`+3,
`te
`gg7t
`t2
`+3
`:
`+25,
`Cu’ or divalent Cu), nickel (Ni), iron CFe’*
`or Fe), cobalt (Co™ or Co), zine (in),
`
`manganese (Min), strontium (Sr°°), molybdenam (Mo} and the like. The weak acid anion
`
`20 forming a salt with transition metal according to the present invention inchide but is not linuted
`
`to acetate, glutamate, ghiconats, tartrate, forrnate, citrate or glycinate. Accordingly, non-limiting
`
`&
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`examples of the weak acid that can form a salt with transition metal
`
`includes acetic acid,
`
`glauconic acid, tartaric acid, glutamic acid, citric acid, formic acid, or glycinic acid.
`
`According to preferred embodiments, the weak acid salt of a transition metal include, but are not
`
`himuted to, copper acetate, copper ghiconate, nickel acetate and nickel glaconate. The weak acid
`
`5 sali of transition metal used in preparing Irposomes of the present invention is used in the internal
`
`sohition at a concentration ranging from about LOO mM to about 600 mM, preferably at a
`
`concentration ranging from about 150 mMto about 300 mM. In specific embodiments,
`
`the
`
`Hposome composition formed by the method of the present invention contains copper acetate in
`
`an amount ranging from about 0.0001 % to about 5.0 % weight by vohime of the Hposomal
`
`10 composition.
`
`Suitably, the liposomal composition prepared according to the method ofthe present invention
`
`is composed of one or more vesicle formmeg lipid, selected froma di-aliphatic chain Upid, such as
`
`phospholipids: dighycerides; di-aliphatic glycolipids; single Hpids such as sphingomyelin or
`
`glycosphingolipid: steroidal lipids; hydrophilic polymer derivatised lipids, or mixtures thereof,
`
`15 Preferably, the vesicle forming Hpid comprises one or more phospholipids, one or more steroidal
`
`lipids, and one or more hydrophilic polymer derivatised lipids.
`
`The one or more phospholipids that may be used in the liposomal composition prepared
`
`according to method of the present invention comprises phospholipids that form bilayer vesicular
`
`stracture. The phospholipids that may be used include, bat are not limited to, phospholpid such
`
`20 as phosphatidyl chaline
`
`(PC); phosphathly]
`
`ethanolamine{PE}. phosphatidyl
`
`serime(PS),
`
`phosphatidylglycerol(PG), phosphatidylonositol (PD, sphingomyelin, phosphatidic acid (PA),
`
`lecithin; phosphatidvicholime lipid derivatives such as dipalmntoylphosphatidyicholine (DPPC),
`
`ege phosphatidyichcohne (2PC), hydrogenated egg phosphatidylcholine (HEPC), partially
`
`hydrogenated egg phosphatidylcholine
`
`(PHEPC), distearyiphosphatidyl]
`
`choline (DSPC),
`
`25 dipalmitoy! phosphatidvi choline (DPPC), soy phosphatidy!] choline (PC), hydrogenated soy
`
`phosphatidyl choline CHSPC),
`
`diarachidoy! phosphatidyl choline, dimyristoyl phosphatidyl
`
`ethanolamine (DMPE}, dipalmitoyl phosphatidyl] ethanolamine (DPPE), distearoy! phosphatidyl
`
`ethanolamine (DSPE), diarachidoy] phosphatidyl] ethanclamine CDAPE)
`
`and dipalmitoyl
`
`phosphatidyl] glycerol COPPG) and the lke. These phospholipids may be fully satarated or
`
`30 partially hydrogenated. They may be naturally occerring or
`
`synthetic. The preferred
`
`9
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`phospholipid is hydrogenated soy phosphatidyl choline (HSPC). The steroidal lipids that may be
`
`used in the Hiposomal composition prepared according to method of the present invention
`
`include, but not limited to cholesteral or its derivatives such as cholesteryl sulfate and its salts
`
`(CS), cholesteryl hemisuccinate and its salts, cholesterol phosphate and its salts, cholesteryl
`
`5 phosphocholine and other hydroxycholesterol or amino cholesterol derivatives, cholesteryl
`
`succinate, cholesteryl] oleate, polyethylene glycol derivatives of cholesterol (cholesierol-PEG),
`
`and coprostancl, cholestanol, chelestane, cholic acid, cartisal, corticasterome, hydrocortisone,
`
`ealciferol and the like. Preferably the steroidal
`
`lipid is cholesterol.
`
`In one embodiment,
`
`cholesterol
`
`is used in an amount such that the liposomes have greater than 20 mole percent
`
`10 cholesterol. Preferably, greater than 30 mole percent cholesterol
`
`is used, more preferably the
`
`cholesterol amount ranges from about 40 mole percent to 60 mole percent. According to one
`
`embodiment,
`
`the liposome prepared according to the method of the present
`
`invention is a
`
`polymerized liposome. Such liposomes can be prepared by incorporating one or rnore lipids that
`
`are derivatized by a polymer, along with one or more phospholipids, and optionally, one or more
`pd wn steroids. The exarnples of the polymers that are used to derivatize the Inpids inchide, but are not
`
`Hraited to, polyethylene glycol (PEG): Poly acrylamide (PAA), which is formed from the acryl
`
`amide subunits ie. readily formed from polymerized acryl amide; Poly Q-alkyl-2-oxozoline)
`
`such as Poly(2-methyl-2-oxozoline}; Poly (amine) acids such as synthetic poly(amino acid}s for
`
`example, poly(ghutamic acid} (PGA); Poly (glycerol) such as hyperbranched PG CHPG) with
`
`20 molar masses ranging from 130 Da to 540; poly (vinylpyrrolidone) and the like. In one preferred
`
`ernbodiment, the hydrophilic polymer e.g., polyethylene glycol (PEG) having a molecular weight
`
`between 1-10 Kdaltons, preferably 2-5 Kdaltons (PEG-derivatized phospholipids} may be used.
`
`Preferably,
`
`the polymer derivatised Hipid that may be used includes a hydrophilic polymer
`
`25 derivatised lipid. Non-limiting examples of hydrophilic polymer derivatised lipid,
`
`include,
`
`polyethylene ghycol conjugated phosphatidyl ethanolamine, (PEG-PE}, methoxy polyethylene
`
`glycol conjugated bydrogenated soy phosphaudylicholine (mPEG-HSPC), methoxy polyethylene
`
`glycol-distearoyiphosphatidy! ethanolamine (mPEG-DSPE). Other hydrophilic polymers which
`
`may
`
`be
`
`suitable
`
`imchides
`
`polylactic
`
`acid,
`
`polyglycolic
`
`achl,
`
`polyvinylpyrrolidone,
`
`30 polymethyloxazoline,
`
`polyethyvloxazoline,
`
`polyhydrox ypropyl
`
`methacrylamide,
`
`polymethacrylamide,
`
`polydimethylacrylamide,
`
`and
`
`derivatized
`
`celluloses,
`
`such
`
`as
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`10
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`WO IWO16M13031
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`POCP/IN2015/050073
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`hydrox ymethyicelulose or hydroxyethylcellose. In preferred embodiments,
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`the hydrophilic
`
`polymer derivatised lipid is methoxy polyethylene glycol-distearoylphosphatidyl ethanolamine
`
`(mPEG-DSPE).
`
`In one specific embodiment, the polymerized lipid may be used in an amount
`
`ranging from 0.01 % to 3.0 %weight by volume, preferably, about 0.1 % to 1.0 %weight by
`
`5 volume.
`
`The one or more vesicle forming lipid, that are used in the liposome prepared according to the
`
`method of the present invention, is present
`
`in an amount such that the molar ratio of ionizable
`
`drag to vesicle forming Upids is im the range of about G.0001:1 to about 0.3:1, preferably from
`
`about 0.01:1 to about O.S:1.
`
`16 In one embodiment, the hposomal composition prepared according to the method of the present
`
`Invention comprises one or more phospholipids, one or more steroidal lipids amd one or more
`
`polymer derivatised lipids. The lipids are present im amounts such that
`
`the molar ratio of
`
`phospholipids: steroidal lipid: polymer derivatised lipid ranges from about 30-75 : 21-60 :
`
`I-
`
`LSpreferably from about 48-55 ; 40-503 2-7. In specific embodiment, the liposomal composition
`
`15 prepared according to the method of the present invention comprises one or more phospholipids,
`
`one or more steroidal lipids and one or more polymer derivatised lipids, wherein the molar ratio
`
`of phospholipids: steroidal lipid: polymer derivatised lipid is about 303 45:5.
`
`in some specific embodiments, the long circulating lyposormal composition prepared according to
`
`the method of the present invention comprises an ionizable drug losartan or candesartan or EXP-
`
`20 3174 or their pharmaceutically acceptable salts, a weak acid salt of a transition metal cation and
`
`vesicle forming lipids cormprising one or more phospholipids, one or more steroidal lipid and one
`
`or more polymer derivatised lipid. Preferably, the lypids are present in amounts such that the
`
`molar ratio of phosphalipids: steroidal pid: polymer derivatised lipid ranges from about 30-75 :
`
`21-603 1-15.
`
`Ne as In one embodiment,
`
`the liposomes prepared by the method of the present
`
`invention are
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`unilamellar liposomes. The vesicles have a mean particle size ranging from about 50 nm to about
`
`200 nm, preferably from about 60 nm to about 120 nm.
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`11
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`In one specific embodiment,
`
`3
`
`a
`the Hposomal composition prepared according te the method ofthe
`
`present invention are long circulating in blood and the drug has a mean residence time of greater
`
`than 8 hours
`
`in one specific embodiment, the hposomal composition prepared according to the method ofthe
`
`5 present invention comprises losartan potassium, vesicle forming lipids including a mixture of
`
`hydrogenated soy phosphatidyl choline CHSPC), cholesterol and methoxy polyethylene glycol-
`
`distearoylphosphatidy! ethanolamine GnPEG-DSPE), and copper acetate which is ased as a weak
`
`acid salt of the transition metal ion. Preferably, the lipids are present in amounts such that the
`
`molar ratio of HSPC : Cholestero: mPEG-DSPE may vary from about 30-75 : 21-60: 1-15. It
`
`10 was found by the inventors that
`
`in one embodiment wherein the vesicle forming lipid is a
`
`mixture of hydrogenated soy phosphatidyl
`
`choline CHSPC);
`
`cholesterol
`
`and methoxy
`
`polyethylene glycol-distearoyiphosphatidyl ethanolamine (mPEG-DSPE},
`
`the method of the
`
`present invention preferably requires use of higher mole percentage of cholestero], preferably
`
`greater than 20 mole percentage to achieve higher drug loading.
`
`15 The inventors while atternpting to encapsulate an ionizable drag such as those defined in the
`
`invention, ito a liposomal vesicle,
`
`faced a problem of poor/non-efficient drug loading
`
`particularly when a strong acid salt of a transition metal such as sulphate, nitrate, phosphate salt
`
`of transition metal was used. For instance, when copper sulphate was used, the percentage drug
`
`loading was veryless, for example, as low as 10 % or even lesser in some cases. In one specific
`
`20 embodiment, wherein copper sulphate was used as gradient to load losartan into lrposomies, the
`
`percentage drug loading observed was only 7.36%. In another specific embodiment, wherein
`
`copper sulphate was used as gradient to load candesartan into liposomes, the percentage drug
`
`loading observed was only 34.31%. Surprisingly, however, when a weak acid salt of a transition
`
`metal, such as acetate, or gluconate salt of a transition metal, for instance copper acetate or
`
`25 copper ghiconate or niclek acetate was used, inventors unexpectedly found that the drug loading
`
`cmd be achieved at levels greater than 80%, even greater than 90%. Further, it was surprisingly
`
`found that such liposomes were long circulating in nature. This observation of efficient drug
`
`loading and long circulating nature of liposome, in fact was indeed surprising and unexpected.
`
`This was all the more unexpected in view of the fact that inventor's pricr attempts of preparing
`
`30 hposomes using alkaline earth metal
`
`ion salts sach as calcium acetate, although resulted in
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`acceptable drug loaded liposomes, these liposomes were not long circulating in nature. This was
`
`evidenced by low mean residence time of losartan in plasma (<2hours) upon parenteral infection
`
`of these liposomes (in-vivo pharmacokimetics study). In contrast,
`
`the Iipasomes prepared by
`
`using a weak acid salt of a transition metal cation, such as for example, weak acid salt of copper,
`
`5 viz. copper acetate, are long circulating in nature, which upon intravenous injection remain and
`
`circulates in plasma for a long period of time. This ts evidenced by longer mean residence time
`
`of losartan in plasma which is more than 6 hours, preferably more than 10 hours, upon
`
`intravenous injection of the liposome prepared by the method according to the present invention.
`
`In one preferred aspect,
`
`the pH of the internal solution is less than the pH of the external
`
`10 sohution. In another preferred aspect, the pH of the internal solution is more than the pH of the
`
`external sohition. The pHofthe internal solution may range from about pH 3.0 to 9.0, preferably
`
`from about 5.0 to 9.0 while the pH of the external solution may range from about 5.0 to 9.0,
`
`preferably from about 6.0 to 7.0.
`
`In one aspect, the method of the present invention does not
`
`necessilate presence of a trans-membrane pH gradient or use of an ionophore for achieving drug
`
`15 loading.
`
`According to one embodiment, the method of the present invention involves following steps of :
`
`preparing an aqueous solution of weak acid salt of transition metal ion, in water for injection.
`
`The weak acid salt of transition metal used in preparing liposomes of the present invention may
`
`be taken in arnounts ranging from about 100 mM to about 600 mM, preferably in an amount
`
`20 ranging from about 150 mM to about 300 mM. Suitably, the weak acid salt of transition metal
`
`helps in active loading of the drug into the Hposomes in higher amounts. The lipids are then
`
`dissolved in a suitable organic solvent such as ethanol! or ether and injecting the lipidic solution
`
`into the agucous solution of weak acid salt of transition metal,
`
`to form liposome suspension
`
`having muldlamellar vesicles. The hposomal vesicles maybe prepared by alternative techniques
`
`25 such as
`
`lipid film formation and hydration. Subjecting the Hposome suspension having
`
`miultilamellar vesicles to particle size reduction by techniques known in the art (such as
`
`extrusion, homogenization etc.) se as to obtain preformed liposomes, which are mostly
`
`unilamellar having a mean particle size, preferably in the range of 56-150 am. Exchanging the
`
`external solution, Le.
`
`the extra-lposomal medium with a suitable solvent/puffer at optinvim
`
`30 condition, by methods such as diafiltration or diahysis or ultrafiltration or tangential flow
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`filtration, causing removal of transition metal cation present in the extra-liposomal environment.
`
`The resulting liposomal composition contained the weak acid salt of transition metal in internal
`
`solution/aqueous medium and solvent/buffer salution in external solution (extra-lposomal
`
`medium). Adding suitable buffer solution/pH stabilizer, so as to maintain desired pH in the
`
`5 external medium. Adding the ionizable drug or its pharmaceutically acceptable salt
`
`to the
`
`external sohition or extra-liposomal medium and incubating the Hposorne containing internal
`
`sohition with the external sohition containing drag for sufficient period of time, usually ranging
`
`from about 5 minutes to 120 minutes or Jonger to effect loading of drug into the liposome. This
`
`is usually carried out at suitable temperature preferably about 25°C to 75°C.. Sterilizing the
`
`10 Hposomal composition by membrane filtration, using a O.2 micron membrane filter.
`
`In one embodiment, while preparing the pre-formed liposomes, the lypesomes may be passively
`
`co-encapsulated with another active agent/drug along with the weak acid salt of the transition
`
`metal. Using this approach,
`
`two or more drugs may be mcorporated into the liposome by
`
`corabining passive and active methods of loading.
`
`15 The method of the present
`
`invention provides high percentage of drug loading or drug
`
`entrapment into the liposomes, preferably greater than 70%, more preferably greater than 80%,
`
`more preferably greater than 90%. In one preferred embodiment, the long circulating Hposomal
`
`coraposition prepared by the method of the present invention encapsulates more than 80% by
`
`weight of the added drug into the liposomes.
`
`20 it is to be understood that