CA3032810A1 - Methods for the preparation of liposomes - Google Patents

Abstract

Provided herein are methods for preparing liposomes and uses thereof. In certain embodiments, liposomes are prepared without using heat, organic solvents, proteins, and/or inorganic salts in the process. In certain embodiments, the liposomal preparation contains one or more active agents. In certain embodiments, the liposomal preparations are used in the treatment of diseases or disorders.

Description

TITLE OF THE INVENTION
METHODS FOR THE PREPARATION OF LIPOSOMES
BACKGROUND
The bioavailability of a pharmaceutical drug depends largely in part on the solubility and stability of the drug. Many methods have been employed to improve bioavailability of a drug, including, but not limited to, pH adjustment, associating the drug in micelles of detergents, solubilization in an organic solvent, complexation with cyclodextrin or other polymers, and encapsulating the drug in a liposome bilayer (Strickley, R.G., Pharmaceutical Research, No. 21, 2004: 201-230). Either the drug itself or the excipients used to solubilize the drug may have side effects such as allergic reaction or hemolysis.
It is known that the solvents (e.g., ethanol, propylene glycol, polyethylene glycol, dimethylacetamide, dimethylsulfoxide ("DMSO")), complexing agents (for example, nicotinamide), and surfactants (for example, sodium oleate) are hemolytic and are therefore undesirable for use in injectable solutions. Other limitations to using organic solvents in injectable products include precipitation, pain, and inflammation upon injection.
Liposomes are microscopic lipid vesicles that are composed of a central aqueous cavity surrounded by a lipid membrane formed by concentric bilayer(s) (lamellas).
Liposomes are able to incorporate hydrophilic substances (in the aqueous interior) or hydrophobic substances (in the lipid membrane). Liposomes can be unilamellar vesicles ("UMV"), having a single lipid bilayer, or multilamellar vesicles ("MLV"), having a series of lipid bilayers (also referred to as "oligolamellar vesicles"). The multilamellar vesicles typically range in size from 0.2 p.m to 10 p.m in diameter. See e.g., WO 98/006882. Although anti-hemolytic measures are commonly taken in formulations, maintaining a sufficient amount of liposome in formulation may not be feasible due to the incompatibility of the liposome with an excipient, or the instability of the liposome in the formulation. Further, reconstituting lyophilized formulations containing hydrophobic drugs is often difficult. Moreover, liposomes are not stable in formulations containing concentrated organic solvents.
Unilamellar vesicles with a diameter of less than 0.2 [tm (e.g. between 0.02 and 0.2[tm) 2325999v1/20112-19 are commonly known as small unilamellar vesicles ("SUV"). Unilamellar vesicles with a diameter greater than 0.45 1.tm (in some cases greater than 1 1.tm) are commonly known as large unilamellar vesicles ("LUV").
The bilayer(s) of liposomes most often comprise phospholipids, but may also comprise lipids including but not limited to fatty acids, fatty acid salts and/or fatty alcohols. The properties of the liposomes depend, among other factors, on the nature of the constituents.
Consequently, if liposomes with certain characteristics are to be obtained, the charge of its polar group and/or the length and the degree of saturation of its fatty acid chains must be taken into account.
In addition, the properties of liposomes may be modified, e.g., to incorporatecholesterol and other lipids into the membrane, change the number of lipidic bilayers, or covalently join natural molecules (e.g., proteins, polysaccharides, glycolipids, antibodies, enzymes) or synthetic molecules (e.g., polyethyl glycol) to the surface. There are numerous combinations of phospholipids, optionally with other lipids or cholesterol, in an aqueous medium to obtain liposomes. Depending on the method of preparation and the lipids used, it is possible to obtain vesicles of different sizes, structures, and properties.
Another important parameter to consider with respect to the formation of liposomes isthe rigidity of the lipidic bilayer. The hydrated lipid that foams part of the bilayer may be in either a liquid-crystalline (fluid) or gel state. As the temperature increases, the gel state is converted into the liquid-crystalline state. This occurs at a temperature known as the transition temperature (Tc), which is specific to each lipid. The Tc is directly proportional to chain length and inversely proportional to the degree of unsaturation of the fatty acids and depends on the nature of the polar group.
Despite this, common methods in the preparation of lipid vesicles, such as liposomes,comprise evaporating an organic solvent in which the lipids are dissolved and then dispersed in an optionally buffered aqueous solution. One exemplary method, known as the Bangham method. was originally described in Bangham et at., I Hot. Biol., 11:238-252 (1965).
Variations of the Bangham method are known by those skilled in the art, some of which are described below.
Hydration of a thin lipidic laver. Starting with the organic solution of the constituentlipids of the bilayer, a lipidic film is prepared through removal of organic solvent, which can be achieved by means of evaporation (e.g., at reduced pressure in a rotary evaporator) or by lyophilization. The dry lipidic film obtained is hydrated by adding an aqueous solution and agitating the mixture at temperatures above the Tc.

2 2325999v1/20112-19 Reverse-phase evaporation. Starting with the organic solution of the constituentlipids of the bilayer, a lipidic film is prepared through removal of the organic solvent. The system is purged with nitrogen and the lipids are re-dissolved in a second organic solution, usually constituted by diethyl ether and/or isopropyl ether. The aqueous phase is added to the redissolved lipids. The system is maintained under continuous nitrogen. A gel is formed by removing the second organic solvent.
Solvent injection. The lipids, dissolved in an organic solvent, are injected slowly intoan aqueous solution. The organic solvent used is often a water-miscible solvent, and the aqueous solution may be warmed.
Additional methods for the preparation of multilamellar vesicles can be found, e.g., in Szoka and Papandjopoulos, Ann. Rev. Biophys. Bioeng., 2: 467-508 (1980), and Dousset and Douste-Blazy, Les Liposomes, Puisieux and Delattre, Editors, Tecniques et Documentation Lavoisier, Paris, pp. 1-73 (1985).
Further, when the incorporation of more than one lipid is desired, the lipids shouldremain homogeneously distributed in the liposomal vesicles. Traditionally, this is achieved by previously dissolving the lipids in an organic solvent and using the resulting organic solvent for preparing the liposomes.
U.S. Pat. No. 4,508,703 describes a method for obtaining powdery mixtures of atleast one amphyphilic lipid and, optionally, at least one component of a hydrophobic or partially hydrophobic nature, a method which includes dissolving the components of the mixture in at least one organic solvent and atomizing the obtained solution into an inert gas. The method permits the preparation of lipidic mixtures which can be easily dispersed in an aqueous medium but does not avoid the use of organic solvents.
WO 92/10166 describes a method for preparing liposomes with an elevated encapsulation capacity. The method permits the use of mixtures of lipids; however, the mixture is obtained by means of previous dissolution of the lipids in an organic solvent and subsequent evaporation. In addition, the contact between the lipids and the aqueous solution of active agent is carried out at a temperature above the Tc.
Moreover, it is reported that, where liposomes are made without using organicsolvents, other manipulations, which may result in formulations with certain unfavorable characteristics, are generally required. For example, U.S. Pat. App. Pub. No. 2008/0274172 describes methods of preparing liposomes containing at least two phospholipids without using organic solvents.
However temperatures above the Tc were used to obtain stable liposomes from aqueous solutions containing inorganic salts.

3 2325999v1/20112-19

4 Consequently, existing methods for preparing liposomes utilize organic solvents,protein, inorganic salts, and/or heat. Due to their toxicity and flammability, organic solvents are undesirable in the preparation of liposomes for pharmaceutical, cosmetic and other uses.
Moreover, the use of organic solvents and proteins has negative repercussions in terms of production costs, safety, work hygiene and the environment. Similarly, the use of heat in the preparation of liposomes is undesirable in terms of production costs, safety, and the environment.
The use of inorganic salts in the preparation of liposomes is undesirable as the introduction of inorganic salts increases the size of the liposome and/or results in a more turbid formulation. See e.g. Castile et at., International Journal of Pharmaceutics, 1999, vol. 188, issue 1, pp. 87-95.
Thus, there is a need for a method for preparing liposomes without the use of undesirable agents and procedures.
SUMMARY OF THE INVENTION
Provided herein are methods for preparing liposomes. In certain embodiments, liposomes are prepared without using heat, organic solvents, proteins, and/or inorganic salts in the process.
In one embodiment, a method is provided for the preparation of liposomes, the method comprising:
(a) combining one or more lipids in an aqueous medium at ambient temperature;
(b) dispersing the lipids in the aqueous medium; and (c) adding one or more sugars to the resulting mixture, thereby footling a solution of liposomes.
In certain embodiments, the liposomal preparation contains one or more activeagents. In certain embodiments, the active agent is a pharmaceutical agent. In certain embodiments, active agents include, but are not limited to lapachone; transferrin; cyclosporine;
colchicine; and combinations of lapachone and transferrin in a single liposome formulation.
In certain embodiments, the one or more active agents are added after the liposome orsolution of liposomes has been prepared. In other embodiments, the one or more active agents are added during formation of the liposome.
Also provided herein are uses of liposomal preparations in the treatment of diseases or disorders. In one embodiment, the liposomal preparation is suitable for parenteral administration to a patient suffering from said disease or disorder. In one embodiment, the patient is a human.

2325999v1/20112-19 DEFINITIONS
As used herein, and unless otherwise specified, "lipid" is understood to be a fatty acid, fatty acid salt, fatty alcohol, or phospholipid. Lipids may also be read to include sterols, including, but not limited to, cholesterol; sphingolipids, including, but not limited to, .. sphingomyelin; glycosphingolipids including, but not limited to, gangliosides, globocides and cerebrosides; and surfactant amines including, but not limited to, stearyl, oleyl and linoleyl amines.
As used herein, and unless otherwise specified, "phospholipid" is understood to be anamphyphilic derivative of glycerol, in which one of its hydroxyl groups is esterified with phosphoric acid and the other two hydroxyl groups are esterified with long-chain fatty acids that can be equal to or different from each other and can be saturated or unsaturated. A neutral phospholipid is generally one in which the other phosphoric acid hydroxyl is esterified by an alcohol substituted by a polar group (usually hydroxyl or amino) and whose net charge is zero.
A phospholipid with a charge is generally one in which the other phosphoric acid hydroxyl is esterified by an alcohol substituted by a polar group and whose net charge is positive or negative.
Examples of phospholipids include, but are not limited to phosphatidic acid ("PA"),phosphatidylcholine ("PC"), phosphatidylglycerol (-PG"), phophatidylethanolamine phophatidylinositol (-Pr), and phosphatidylserine (-PS"). sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-alpha-phosphatidylcholine ("DDPC"), dielaidoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleoylphosphatidylcholine ("DOPC "), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-palmitoy1-2-oleoyl-phosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol ("DAPG"), didecanoyl-L-alpha-phosphatidylglycerol ("DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DWG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoylphosphatidylglycerol ("DPPG"), di stearoylphosphatidylglycerol ("DSPG"), 1-palmitoy1-2-oleoyl-phosphatidylglyeerol ("POPG"), diarachidoylphosphatidylethanolamine ("DAPE"), didecanoyl-L-alpha-phosphatidylethanolamine ("DDPE"), dielaidoylphosphatidylethanolamine ("DEPE"), dilauroylphosphatidylethanol amine ("DLPE"), dilinoleoylphosphatidylethanolamine,

5 2325999v1/20112-19 dimyristoylphosphatidylethanolamine ("DMPE"), dioleoylphosphatidylethanolamine ("DOPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), di stearoylphosphatidyl ethanolamine ("DSPE"), 1 -palmitoyl -2-ol eoyl-phosphatidyl ethanolamine ("POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl-L-alpha-phosphatidylinositol ("DDPI"), dielaidoylphosphatidylinositol (DEN"), dilauroylphosphatidylinositol ('DLPI"), dilinoleoylphosphatidylinositol, dimyristoylphosphatidylinositol ("DMPI"), diolcoylphosphatidylinositol ("DOPI"), dipalmitoylphosphatidylinositol ("DPPI"), distearoylphosphatidylinositol ("DSPI"), 1-palmitoy1-2-oleoyl-phosphatidylinositol ("POPI"), diarachidoylphosphatidylserine ("DAP S"), didecanoyl-L-alpha-phosphati dylserine ('DDP S"), di elaidoylphosphatidyl serine ("DEPS"), dilauroylphosphati dylserine ("DLPS"), dilinoleoylphosphatidylserine, dimyri stoylphosphatidylserine ("DMP S"), di ol eoylphosphatidyl serine ("DOPS"), dipalmitoylphosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DS PS"), 1-palmitoy1-2-olcoyl-phosphatidylserine ("POPS"), diarachidoyl sphingomyelin, didecanoyl sphingomyelin, dielaidoyl sphingomyelin, dilauroyl sphingomyelin, dilinoleoyl sphingomyelin, dimyristoyl sphingomyelin, sphingomyelin, dioleoyl sphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, and 1-palmitoy1-2-ol eoyl-sphingomyelin.
As used herein, and unless otherwise specified, "encapsulate" or "encapsulation" is understood to be the process of incorporating an active agent into liposomes or liposomal vesicles. The encapsulated active agent can remain in the aqueous interior or associate with membranes.
As used herein, and unless otherwise specified, the term "enhance" or "enhancing,"when used in connection with the solubility of a compound, means that the methods provided herein result in the increased solubility of the compound as compared to the solubility of the same compound in water. Specifically, the term "enhance" or "enhancing"
means that, when the methods provided herein are used, the solubility of a compound increases about 20 percent or more, about 40 percent or more, about 60 percent or more, about 80 percent or more, about 100 percent or more, or about 200 percent or more of the solubility of the same compound in a reference solvent. In some embodiments, the reference solvent is water.
As used herein, and unless otherwise specified, the term "hydrophobic compound"means a compound with little or no water solubility. In some embodiments, a

6 2325999v1/20112-19 hydrophobic compound has an intrinsic water solubility (i.e., water solubility of the unionized form) of less than about 20 percent by weight, about 15 percent by weight, about 10 percent by weight, about 5 percent by weight, about 1 percent by weight, about 0.1 percent by weight or about 0.01 percent by weight. In other embodiments, a hydrophobic compound has an intrinsic water solubility of less than about 10 mg/mL, about 7 mg/mL, about 5 mg/mL, about 3 mg/mL, about 1 mg/mL or about 0.1 mg/mL.
As used herein, or unless otherwise specified, the terms "aqueous medium"
or"aqueous media" include any water based medium, e.g., water, saline solution, a sugar solution, a transfusion solution, a buffer, and any other readily available water-based medium. Further, an aqueous medium may contain one or more water soluble organic solvents. In the case of a parenteral solution, an aqueous medium is preferably sterile and suitable for use as a carrier of an active agent. Examples of aqueous media include, but are not limited to, water for injection, saline solution, Ringer's solution, D5W, or other solutions of water-miscible substances such as dextrose and other electrolytes.
As used herein, and unless otherwise specified, the term "fatty acid" means acompound whose structure is a carboxylic group attached to a hydrocarbon chain having one or more carbon atoms. The hydrocarbon chain may be saturated or unsaturated (i.e., alkyl, alkenyl or alkynyl hydrocarbon chains). Also, the hydrocarbon chain may be straight or branched.
Moreover, in some embodiments, hydrogens in the hydrocarbon chain may be substituted.
As used herein, and unless otherwise specified, the term "fatty alcohol" means acompound whose structure is an alcohol group attached to a hydrocarbon chain having one or more carbon atoms. The hydrocarbon chain may be saturated or unsaturated (i.e., alkyl, alkenyl or alkynyl hydrocarbon chains). Also, the hydrocarbon chain may be straight or branched.
Moreover, in some embodiments, hydrogens in the hydrocarbon chain may be substituted.
As used herein, and unless otherwise specified, the term "fatty acid salt"
means acompound formed from a reaction between a fatty acid and an inorganic/organic base. In addition, the term encompasses a compound formed from a reaction between a fatty alcohol and an inorganic/organic acid. Examples of such acids include, but are not limited to, sulfuric and phosphoric acid. The hydrocarbon chain of the fatty acid salt may be saturated or unsaturated (i.e., alkyl, alkenyl or alkynyl hydrocarbon chains). In addition, the hydrocarbon chain may be straight or branched. Moreover, in some embodiments, hydrogens in the

7 2325999v1/20112-19 hydrocarbon chain may he substituted.
As used herein, and unless otherwise specified, the term "substituted" means a groupsubstituted by one or more substituents including, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aroyl, halo, haloalkyl (e.g., trifluoromethyl), substituted or unsubstituted heterocycloalkyl, haloalkoxy (e.g., trifluoromethoxy), hydroxy, alkoxy, cycloalkyloxy, heterocylooxy, oxo, alkanoyl, aryl, substituted aryl, substituted or unsubstituted heteroaryl (e.g., indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like), arylalkyl, alkylaryl, heteroaryl, heteroaryl alkyl, alkylheteroaryl, heterocyclo, aryloxy, alkanoyloxy, amino, alkyl amino, aryl ami no, aryl alkyl amino, cycl o alkyl amino, heterocycloamino, mono- and di-substituted amino, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoyl amino, substituted aryl amino, substituted aralkanoylamino, carb amyl (e.g., CONH2), substituted carb amyl (e.g., CONH-alkyl, CONH-aryl, CONH-arylalkyl or instances where there are two substituents on the nitrogen), carbonyl, alkoxycarbonyl, carboxy, cyano, ester, ether, guanidino, nitro, sulfonyl, alkyl sulfonyl, aryl sulfonyl, aryl alkyl sulfonyl, sulfonami do (e.g..
SO2NH2), substituted sulfonami do, thiol, alkylthio, arylthio, aryl alkylthi o, cycloalkylthio, heterocyclothio, alkylthiono, arylthiono and arylalkvithiono.
As used herein, and unless otherwise specified, the term "alkyl" means a saturated straight chain or branched non cyclic hydrocarbon having 1 20 carbon atoms, preferably 1 10 carbon atoms and most preferably 1 4 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n propyl, n butyl, n pentyl, n hexyl, n heptyl, n octyl, n nonyl and n decyl; while saturated branched alkyls include isopropyl, sec butyl, isobutyl, tert butyl, isopentyl, 2 methylbutyl, 3 methylbutyl, 2 methylpentyl, 3 methylpentyl, 4 methylpentyl, 2 methylhexyl, 3 methylhexyl, 4 methylhexyl, 5 methylhexyl, 2,3 dimethylbutyl, 2,3 dimethylpentyl, 2.4 dimethylpentyl, 2,3 dimethylhexyl, 2,4 dimethylhexyl, 2,5 dimethylhexyl, 2,2 dimethylpentyl, 2,2 dimethylhexyl, 3,3 dimtheylpentyl, 3,3 dimethylhexyl, 4,4 dimethylhexyl, 2 ethylpentyl, 3 ethylpentyl, 2 ethylhexyl, 3 ethylhexyl, 4 ethylhexyl, 2 methyl 2 ethylpentyl, 2 methyl 3 ethylpentyl, 2 methyl 4 ethylpentyl, 2 methyl 2 ethylhexyl, 2 methyl 3 ethylhexyl, 2 methyl 4 ethylhexyl, 2,2 diethylpentyl, 3,3 diethylhexyl, 2,2 diethylhexyl, 3,3 diethylhexyl and the like. An alkyl group can be unsubstituted or substituted.
Unsaturated alkyl groups include alkenyl groups and alkynyl groups, which are discussed

8 2325999v1/20112-19 below.
As used herein, and unless otherwise specified, the term "alkenyl" means a straightchain or branched non-cyclic hydrocarbon having 2-20 carbon atoms, preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, and including at least one carbon-carbon double bond.
Representative straight chain and branched (C2-C10) al kenyl s include -vinyl, -allyl, -1-butenyl, -2-butenyl, -i sob utyl enyl, -1 -p entenyl, -2-p entenyl, -3 -m ethyl-1-butenyl, -2-methyl-2-butenyl, -2,3 -dim ethy1-2-butenyl, -1-hex enyl, -2-hex enyl, -3 -h exenyl, -1-heptenyl, -2-heptenyl, -3 -heptenyl, - 1-octenyl, -2-octenyl, -3 -octenyl, -1 -nonenyl, -2-nonenyl , -3-nonenyl, -1-decenyl, -2-decenyl, -3 - decenyl and the like. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. An alkenyl group can be unsubstituted or substituted.
As used herein, and unless otherwise specified, the term "alkynyl" means a straightchain or branched non-cyclic hydrocarbon having 2-20 carbon atoms, preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, and including at lease one carbon-carbon triple bond. Representative straight chain and branched (C2-C i o)al kynyl s include -acetyl enyl, -propynyl, -1-butynyl, -2-butynyl, -1 -p entynyl, -2-p entynyl . -3-methyl- 1-b utynyl, -4-p entynyl, -1-hexynyl, -2-hexynyl, -5-h exynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl, -2-o ctynyl, -7-octynyl, - 1-nonynyl, -2-nonynyl, -8-nonynyl, -1 -decynyl, -2-d ecynyl , -9-decynyl, and the like. The triple bond of an alkynyl group can be unconjugated or conjugated to another unsaturated group. An alkynyl group can be unsubstituted or substituted.
As used herein, and unless otherwise specified, the term "pharmaceutically acceptablesalt" refers to a salt prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
Suitable pharmaceutically acceptable base addition salts for the compositions provided herein include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable nontoxic acids include, but are not limited to, inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,

9 2325999v1/20112-19 nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids.
Examples of specific salts thus include hydrochloride and inesylate salts. Others are well-known in the art, see e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990) or Remington: The Science and Practice of Pharmacy, 19th cd., Mack Publishing, Easton Pa.
(1995).
As used herein, the term "hydrate" means a compound provided herein, or a saltthereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
As used herein, the term "clathrate" means a compound provided herein, or a saltthereof in the form of a crystal lattice that contains spaces (e.g., channels) that have a guest molecule (e.g., a solvent or water) trapped within.
As used herein, and unless otherwise indicated, the term "prodrug" means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound. Examples of prodrugs include, but arc not limited to, derivatives and metabolites of a compound that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Preferably, prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. Esterifying any of the carboxylic acid moieties present on the molecule conveniently forms the carboxylate esters. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley), and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).
As used herein, and unless otherwise specified, the term "stable," when used inconnection with a formulation, means that the active agent of the formulation, when prepared using the methods provided herein, remains solubilized for a specified amount of time and does not significantly degrade or aggregate or become otherwise modified (e.g., as determined by HPLC).
As used herein, and unless otherwise specified, "temperature below the Tc"
2325999v1/20112-19 isunderstood to be a temperature which is lower than the Tc of the lipid having the lowest Tc, and "temperature greater than the Tc" is understood to be a temperature which is greater than the Tc of the lipid having the highest Tc.
As used herein, and unless otherwise specified, the term "harmful ingredient,"
whenused in connection with pharmaceutical compositions, means an ingredient commonly used in a pharmaceutical composition that may cause clinical side effects such as, but not limited to, hcmolysis, hypersensitive reaction, peripheral neuropathies, and/or decrease in the bioavailability of the active ingredient of the composition. Examples of harmful ingredients include, but are not limited to: toxic solvents, including organic solvents such as ethanol, methanol, 1-propanol, 2-propanol, acetone, acetonitrile, ethyl acetate, methyl acetate, diethyl ether, dimethyl ether, diisopropyl ether, methyl tert-hutyl ether ("MTBE"), tetrahydrofuran ("THF"), di chl orom ethane, chloroform, carbon tetrachloride, 1,2 -di cholroethane, pentane, hexanes, heptane, petroleum ether, dioxane, ethylene glycol, diethylene glycol, diglyme, 1,2-dimethoxyethane, 1-butanol, 2-butanol, 2-butanone, benzene, toluene, dimethylsulfoxide ("DMSO"), dimethylformamide ("DMF-), hexamethyiphosphoramide ("HMPA"), N-methylpyrrolidone, glycerin, nitromethane, triethyl amine, xylenes, CREMOPIIOR EL, and polyethylene glycol ("PEG"); co-detergents or surfactants such as polysorbates (e.g., Tweens) or vitamin E; oils such as Castor oil or corn oil; proteins such as HSA; or any other biologic which is potential source of contamination.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A shows a control elution profile for size exclusion HPLC of adalimumab (HUMIRA) antibody without added liposomes. Figure 1B shows an elution profile for size exclusion HPLC of liposome-encapsulated traztuzumab (HERCEPTIN) antibody.
Figure 1C
shows an elution profile for size exclusion HPLC of liposome-encapsulated adalimumab (HUMIRA) antibody.
DETAILED DESCRIPTION OF THE INVENTION
Provided herein is a method for the preparation of liposomes, the method comprising:
(a) Combining one or more lipids in an aqueous medium at ambient temperature;

2325999v1/20112-19 (b) Dispersing the lipids in the aqueous medium; and (c) Adding one or more sugars to the resulting mixture, thereby forming a suspension of liposomes.
In another embodiment, at step (b), the method further comprises homogenization of the lipids in the aqueous medium.
Some embodiments further comprise a step (d), wherein one or more active agents is added to the solution of liposomes. In one embodiment, the active agent is a hydrophobic drug.
In one embodiment, the active agent is added as a solid. In another embodiment, the active agent is added in an organic solvent. In another embodiment, the active agent is added in organic solvent which further comprises one or more fatty acid salts, fatty acids and/or phospholipids.
In another embodiment, the active agent is added during formation of the liposome.
In certain embodiments, the resulting liposomes are less than about 11.tm in diameter.
In one embodiment, the resulting liposomes are less than about 500 nm in diameter.
In one embodiment, the resulting liposomes are less than about 100 nm in diameter.
In one embodiment, at least one of the lipids is a phospholipid or a mixture of phospholipids. Examples of phospholipids include, but are not limited to, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phophatidylethanolamine ("PE"), phophatidylinositol ("P1"), and phosphatidylserine ("PS"), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl L alpha phosphatidylcholine ( DDPC ), dielaidoylphosphatidylcholine ("DEPC "), dilauroylphosphatidylcholine ("DLPC "), dilinolcoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC
), dioleoylphosphatidylcholine ("DOPC "), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ( DSPC ), 1 palmitoyl 2 oleoyl phosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol ( DAPG ), didecanoyl L alpha phosphatidylglycerol ( DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DMPG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), 1 palmitoyl 2 oleoyl phosphatidylglycerol ("POPG"), diarachidoylphosphatidyl ethanolamine ("DAPE"), didecanoyl L alpha 2325999v1/20112-19 phosphati dyl ethanol amine ("DDPE"), di el ai doylphosphati dyl ethanol amine ("DEPE"), dilauroylphosphatidylethanolamine ("DLPE"), dilinoleoylphosphatidylethanolamine, dimyri stoylphosphati dyl ethanol amine ("DMPE"), diol eoylphosphati dyl ethanol amine ("DOPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), distearoylphosphatidylethanolamine ("DSPE"), 1 palmitoyl 2 oleoyl phosphatidylethanolamine ("POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl L alpha phosphatidylinositol ("DDPI"), dielaidoylphosphatidylinositol ("DEPT"), dilauroylphosphatidylinositol ("DLPI"), dilinoleoylphosphatidylinositol, dimyri stoylphosphatidylinositol ("DMPI"), dioleoylphosphatidylinositol ("DOPI"), dipalmitoylphosphatidylinositol ("DPPI"), distearoylphosphatidylinositol ("DSPI"), 1 palmitoyl 2 oleoyl phosphatidylinositol ("POPI"), diarachidoylphosphatidylserine ("DAPS"), didecanoyl L alpha phosphatidylserine ("DDPS"), di el ai doylphosphati dyl serine ("DEP S"), dil auroylphosphatidylserine ("DLPS"), dilinoleoylphosphatidylserine, dimyri stoylphosphatidylserine ("DMP S"), di ol eoylphosphati dyl serine ("DOPS"), dipalmitoylphosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"), 1 palmitoyl 2 oleoyl phosphatidylserine ("POPS"), diarachidoyl sphingomyelin, didecanoyl sphingomyelin, dielaidoyl sphingomyelin, dilauroyl sphingomyelin, dilinolcoyl sphingomyelin, dimyri stoyl sphingomyelin, sphingomyelin, dioleoyl sphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, and 1 palmitoyl 2 oleoyl sphingomyelin.
The phospholipids provided herein may be chiral or achiral. The chiral phospholipidsprovided herein may be D
or L phospholipids, for example, L a phosphatidylcholine or I, 3 phosphatidylcholinc.
In one embodiment, L a phosphatidylcholine is used in the methods provided herein.
In another embodiment, provided herein is a method for the preparation of liposomes, the method comprising:
(a) combining sodium oleate and a phosphatidylcholine in an aqueous medium at ambient temperature;
(b) dispersing sodium oleate and L a phosphatidylcholine in the aqueous medium; and (c) adding one or more sugars to the resulting mixture, thereby forming a solution of liposomes.
In one embodiment, the aqueous medium contains one or more active agents, 2325999v1/20112-19 orpharmaceutically acceptable salts, hydrates, clathrates or prodrugs thereof Examples of active agents include, but are not limited to, lapachone (13-lapachone), taxanes (including, but not limited to, taxol, 7-epitaxol, 7-acetyl taxol, 10-desacetyltaxol, 10-desacety1-7-epitaxol, 7-xylosyltaxol, 10-desacety1-7-sylosyltaxol, 7-glutaryltaxol, 7-N,N-dimethylglycycltaxol, 7-L-alanyltaxol, taxotere, and mixtures thereof), paclitaxel, colchicine, transferrin, cyclosporines, cyclosporin A, ketoprofen, propofol, acetylsalicylic acid, acetaminophen, amphotericin, digoxin, doxorubicin, daunorubicin, epirubicin, idarubicin, angiogenesis inhibitors (e.g, bevacizumab, ranibizumab, vitaxin, carboxyamidotriazole, combretastatin A-4, fumagillin analogs (e.g., TNP-470), CM101, IFN-u, interleukin-10, interleukin-12, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatin, endostatin, 2-methoxyestradiol, tecogalan, thalidomide, prolactin, linomide, angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor), vinca-alkaloids (e.g., vinblastine, vincristine, vindesin, etoposide, etoposide phosphate, and teniposide), cytarabine, actinomycin, etoposide, bleomycin, gentamycin, cyclophosphamide, methotrexate, streptozotocin, cytosine, 13-D-arabinofuranoside-5'-triphosphate, cytochrome C, cisplatin, N-phosphono-acetyl-L-aspartic acid, 5-fluoroorotic acid, acyclovir, zidovudine, interferons, aminoglycosides, cephalosporins, tetracyclines, propranolol, timolol, labetolol, clonidine, hydralazine, imipramine, amitriptyline, doxepim, pheny loin, diphenhydramine, chlorphenirimine, promethazine, prostaglandins, methotrexate, progesterone, testosterone, estradiol, estrogen, epirubicin, beclomethasone and esters, vitamin E, cortisone, dexamethasone and esters, betamethasone valerete, biphenyl dimethyl dicarboxylic acid, calcitonins, camptothecin, captopril, cephazoline, chloroquinine, chlorothiazole, coagulation factors VIII and IX, d-alpha-tocopherol, dexamethasone, dichlofenac, etoposide, feldene, flubiprofen, 5-fluorouracil, fluoxetine, fusidic acid, gentamycin, glyburide, granisetron, growth hormones, indomethacin, insulin, itraconazole, ketoconazole, methotrexate, metronidazole, minoxidil, mitomycin, nafcillin, naproxen, ondansetron, oxyphenbutazone, parazosin, physostigmine, piroxicam, prednisolone, primaquine, quinine, ramipril, taxotane, tenoxicam, terazosin, triamcinolone, urokinase, opioid analgesics (e.g., alfentanil, anileridine, codiene, diamorphine, fentanyl, hydrocodone, hydromorphonc, mcperidine, morphine, oxycodone, oxymorphone, propoxyphene, sufentanil, pentazocine and nalbuphine), non-steroidal anti-inflammatory drugs (e.g., aspirin, indometacin, ibuprofen, mefenamic acid and phenylbutazone), angiotensin converting enzyme ("ACE") inhibitors (e.g., 2325999v1/20112-19 captoprilpolyene), protein kinase C inhibitors, antibiotics (e.g., imidazole and triazole antibiotics), folic acid, anthracycline antibiotics, anti-sense RNAs, tricathecums, microbial ribosomal-inactivating toxins (e.g., gelonin, abrin, ricin A chain, Pseudomonas exotoxin, diptheria toxin, pokeweed antiviral peptide), pipecolic acid derivatives (e.g., tacrolimus), plant alkaloids, dyes, radioisotope-labeled compounds, radiopaque compounds, radiosensitizers (e.g., 5-chloro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine), fluorescent compounds, mydriatic compounds, bronchodilators, local anesthetics (e.g., dibucaine and chlorpromazine), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-diabetic agents, antiglaucoma agents, vitamins, narcotics, and imaging agents. For additional disclosure of active agents, see Gilman et at., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis and Therapy, Berkow, M. D. et al. (eds.), 17th Ed., Merck Sharp &
Dohme Research Laboratories, Rahway, N.J., 1999; Cecil Textbook of Medicine, 20th Ed., Bennett and Plum (eds.), W.B. Saunders, Philadelphia, 1996.
In one embodiment, the active agent is a hydrophobic compound, or a compound withpoor solubility in water.
In another embodiment, the active agent is a non-hydrophobic compound.
In one embodiment, the active agent is a water soluble membrane impermeant agent such as a peptide, a protein, a nucleic acid, a nucleotide, a nucleoside, a carbohydrate or an analog thereof.
In one embodiment, the aqueous medium does not contain an active agent.
In another embodiment, the resulting solution contains 10% by weight trehalose.
In another embodiment, the active agent is lapachone, or a pharmaceutically acceptable salt, hydrate, clathrate or prodrug thereof.
In another embodiment, the active agent is transferrin, or a pharmaceutically acceptable salt, hydrate, clathrate or prodrug thereof.
In another embodiment, the active agent is cyclosporine, or a pharmaceutically acceptable salt, hydrate, clathrate or prodrug thereof In one embodiment, the active agents are transferrin and lapachone, or pharmaceutically 2325999v1/20112-19 acceptable salts, hydrates, clathrates or prodrugs thereof Examples of sugars that may be used in the methods provided herein include, but are not limited to, sucrose, glucose, fructose, lactose, maltose, mannose, galactose and trehalose.
In one embodiment, the sugar is trehalose.
In one embodiment, the liposomal preparation is suitable for parenteral administration to a patient suffering from one or more diseases or disorders.
In one embodiment, the patient is a human.
In certain embodiments, the sequence of the addition of active agent(s) results in enhanced solubility of the active agent(s). The conventional method of incorporating hydrophobic drug to liposome is by adding the drug to lipid before liposome preparation. See, e.g., Immordino, M.L. et al., Journal of Controlled Release, 2003, 91: 417 429. By the conventional process, the incorporation of the drug is only 0.3 to 0.7mg,/mL.
Provided herein are methods for the preparation of liposomes wherein the addition ofthe active agent(s) after formation of the liposome results in enhanced solubility of the active agent(s). In one embodiment, the solubility of the active agent(s) in liposome is increased by at least about two-fold, five-fold or ten-fold compared to the conventional process. In one embodiment, the solubility of the active agent(s) in liposome is increased to about 5 mg/mL.
In certain embodiments in which the active agent is a hydrophobic drug, the activeagent is added to pre made liposome as a solid or in an organic solvent. In one embodiment, the pre made liposome comprises one or more fatty acid salts, fatty acids and/or phospholipids to increase the solubility of the active agent.
In one embodiment. the sequence of the addition of active agent(s) results in greaterefficiency of incorporation of the active agent(s) into liposome. In certain embodiments, the efficiency of incorporation into liposome is 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%. In certain embodiments, the efficiency of incorporation is 90%, 95%, 98%, 99% or 100%.
Without being limited to a particular theory or mechanism, the increase in incorporation of active agent to pre made liposome (e.g., to a concentration of about 5mg/mL) may be due to increase in surface area of liposome.
Also provided herein is a liposomal composition prepared by a method comprising:
(a) Combining one or more lipids in an aqueous medium at ambient temperature;

2325999v1/20112-19 (b) Dispersing the lipids in the aqueous medium; and (c) Adding one or more sugars to the resulting mixture, thereby forming a solution of liposomes.
In another embodiment, provided herein is a liposomal composition prepared by a method comprising:
(a) Combining one or more lipids in an aqueous medium at ambient temperature;
(b) Dispersing the lipids in the aqueous medium;
(c) Adding one or more sugars to the resulting mixture, thereby forming a solution of liposomes; and (d) Adding an active agent to the solution of liposomes.
In one embodiment, the active agent is a hydrophobic drug.
In one embodiment, the active agent is added as a solid.
In one embodiment, the active agent is added in an organic solvent.
In one embodiment, the active agent in organic solvent further comprises one or more fatty acid salts, fatty acids and phospholipids.
In certain embodiments, the methods provided herein result in stable solutions, compositions or formulations comprising liposomes and one or more active agents. In these embodiments, the active agent(s) remain solubilized for a specified amount of time and do not significantly degrade, aggregate or become otherwise modified (e.g., as determined by HPLC).
In some embodiments, about 70 percent or greater, about 80 percent or greater or about 90 percent or greater of the active agent remains solubilized after a week after dilution with an acceptable diluent at an elevated temperature (e.g., about 35 C. or higher).
In other embodiments, about 70 percent or greater, about 80 percent or greater or about 90 percent or greater of the active agent remains solubilized after a week after dilution with an acceptable diluent at room temperature.
In other embodiments, about 70 percent or greater, about 80 percent or greater or about 90 percent or greater of the active agent remains solubilized after a week at a reduced temperature (e.g., about 10 C. or lower).
In certain embodiments, the methods provided herein result in enhanced solubility of an active agent, as compared to the solubility of the same active agent in an aqueous medium.
Specifically, when the methods provided herein are used, the solubility of the active agent 2325999v1/20112-19 increases about 20 percent or more, about 40 percent or more, about 60 percent or more, about 80 percent or more, about 100 percent or more, or about 200 percent or more of the solubility of the same active agent in a reference solvent. In some embodiments, the reference solvent is water.
Also provided herein is a method of treating a disease or disorder using a liposomal composition provided herein. In some embodiments, the disease or disorder includes, but is not limited to, oncological disorders, proliferative disorders, central nervous system disorders, autoimmune disorders, and inflammatory diseases or disorders. In other embodiments the methods are directed to the treatment of bacterial, viral or fungal infections.
Proliferative disorders (e.g. cancer) that may be treated by the methods provided herein include, but are not limited to, neoplasms, tumors (malignant and benign) and metastases, or any disease or disorder characterized by uncontrolled cell growth. The cancer may be a primary or metastatic cancer. Specific examples of cancers that can be prevented, managed, treated or ameliorated in accordance with the methods of the invention include, but are not limited to, cancer of the head, neck, eye. mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach. prostate, breast, ovaries, kidney, liver, pancreas, and brain.
Additional cancers include, but are not limited to, the following: leukemias (e.g., acute leukemia, acute lymphocytic leukemia), acute myelocytic leukemias (e.g., myeloblastic, prornyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome), chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia), polycythemia vera, lymphomas (e.g., Hodgkin's disease, non Hodgkin's disease), multiple myelomas (e.g., smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma), Waldenstrom's macroglobulinemia, monoclonal gammopathy of undetermined significance. benign monoclonal gammopathy, heavy chain disease, bone and connective tissue sarcomas (e.g., bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma), brain tumors (e.g., glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pincocytoma, pineoblastoma, primary brain 2325999v1/20112-19 lymphoma), breast cancer (e.g., adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer), adrenal cancer (e.g., pheochromocytom and adrenocortical carcinoma), thyroid cancer (e.g., papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer), pancreatic cancer (e.g., insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin secreting tumor, and carcinoid or islet cell tumor), pituitary cancers (e.g., Cushing's disease, prolactin secreting tumor, acromegaly, and diabetes insipius), eye cancers (e.g., ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma), vaginal cancers (e.g., .. squamous cell carcinoma, adenocarcinoma, and melanoma), vulvar cancer (e.g., squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease), cervical cancers (e.g., squamous cell carcinoma, and adenocarcinoma), uterine cancers (e.g., endometrial carcinoma and uterine sarcoma), ovarian cancers (e.g., ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor), esophageal cancers (e.g., squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma), stomach cancers (e.g., adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcotna), colon cancers, rectal cancers, liver cancers (e.g., hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma), cholangiocarcinomas (e.g., pappillary, nodular, and diffuse), lung cancers (e.g., non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer), testicular cancers (e.g.. germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma), penile cancers, oral cancers (e.g., squamous cell carcinoma), basal cancers, salivary gland cancers (e.g., adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma), pharynx cancers (e.g., squamous cell cancer, and verrucous), skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma), kidney cancers (e.g., renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer 2325999v1/20112-19 (renal pelvis and/or uterer)), Wilms' tumor, bladder cancers (e.g., transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma), myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.
Other specific diseases and disorders that may be treated by the methods providedherein include, but are not limited to, the following: allergic disorders, inflammation, asthma, arthritis, encephalitis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, inflammatory osteolysis, chronic or acute obstructive pulmonary disease, chronic or acute pulmonary inflammatory disease, inflammatory bowel disease, Crohn's Disease, gout, Bechet's Disease, Henoch Schonlein purpura ("FISP"), septic shock, sepsis, meningitis, colitis, inflammation due to reperfusion, psoriasis, fibrosis including pulmonary fibrosis, Parkinson's disease, bradykinesia, muscle rigidity, Parkinsonian tremor, Parkinsonian gait, motion freezing, depression;
defective long term memory, Rubinstein Taybi syndrome (RTS), dementia, sleep disorders, insomnia, postural instability, hypokinetic disorders, hyperkinetic disorders, synuclein disorders, multiple system atrophies, striatonigral degeneration, olivopontocerebellar atrophy, Shy Drager syndrome, motor neuron disease with parkinsonian features, Lewy body dementia, Tau pathology disorders, progressive supranculear palsy, corticobasal degeneration, frontotemporal dementia; amyloid pathology disorders, mild cognitive impairment, Alzheimer disease, Alzheimer disease with parkinsonism, Wilson disease, Hallervorden Spatz disease, Chediak Hagashi disease, SCA 3 spinocerebellar ataxia, X linked dystonia parkinsonism, Huntington disease, prion disease, chorea, ballismus, dystonia tremors, Amyotrophic Lateral Sclerosis ("ALS"), CNS trauma, myoclonus, and diseases or disorders associated with undesired immune reaction (e.g., organ rejection associated with an organ transplant).
Viral infections that may be treated by the methods provided herein include, but are not limited to, the following: human immunodeficiency virus ("HIV"), herpes simplex virus type 1, herpes simplex virus type 2, influenza viruses, influenza virus type A, influenza virus type B, parainfluenza virus, human papillomavirus ("HPV"), adenoviruses, rhinoviruses, hepatitis A
2325999v1/20112-19 virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E
virus, dengue fever, yellow fever, West Nile virus, Japanese encephalitis virus, GB virus A, GB
virus B, GB virus C, bovine viral diarrhea virus ( BVDV"), classical swine fever virus (i.e., hog cholera virus), border disease virus, varicella zoster virus, smallpox, measles, rabies virus, arbovirus, cytomegalovirus, .. mumps virus, poliovirus, coxsackie B virus, Epstein Barr virus, rubella virus, parvovirus B19, coronaviruses SARS coronavirus), astrovirus, norovirus, rotavirus, and adenoviruses.
Fungal infections that may be treated by the methods provided herein include, but arenot limited to, aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis, fungal sinusitis, hi stoplasmosi s, hypersensitivity pneumonitis, mucormycosis, paracoccidioidomycosis, sporotrichosis, and Valley Fever.
Bacterial infections that may be treated by the methods provided herein include, butare not limited to, brucellosis, cholera, leprosy, leptospirosis, shigellosis, trench fever, tularemia, Q
fever, Whitmore's disease, yersiniosis, yaws, vibrio vulnificus infections, streptococcus infections, staphylococcus infections and E. coli infections.
The invention also contemplates compositions including liposomes that encapsulate antibodies or other therapeutic proteins and methods of making such liposome-encapsulated antibodies or liposome-encapsulated therapeutic proteins. The inventors have found that immunoglobulins and other therapeutic proteins contain a hydrophobic region or pocket that provides them with an affinity for the hydrophobic core of the lipid bilayer of liposomes.
Surprisingly, antibodies and other therapeutic proteins can be spontaneously incorporated into liposomes if the lyophilized antibody or other protein is exposed to a suspension of pre-formed liposomes in an aqueous medium. Such incorporation does not require, and preferably avoids entirely, the use of detergents, surfactants, and organic solvents, and also does not require homogenization, microfluidization, filtration, or other mechanisms that disrupt the lipid bilayer.
Such incorporation also does not require, and preferably avoids, any covalent or even non-covalent modification of the immunoglobulin or other therapeutic protein prior to or following its incorporation into the liposomes. The immunoglobulin or other therapeutic protein preferably associates with lipid alkyl chains by non-covalent binding, such as by hydrophobic interactions and/or van der Waals interactions, and does not involve the use of derivatized lipids and derivatized proteins containing high affinity matched binding pairs, such as streptavidin and biotin, or the like. The resulting antibody- or protein-containing lipid vesicles can have a molar 2325999v1/20112-19 ratio of lipid (e.g., phospholipid) to protein of 100 or less, 80 or less, 60 or less, 50 or less, 40 or less, or even 30 or less. The yield of protein or antibody incorporation (i.e., encapsulation) is at least about 40%, 50%, 60%, 70%, or 80%. The encapsulated antibody or protein is partially or wholly embedded within the hydrophobic portion of the lipid bilayer, and is not substantially entrapped in the vesicle lumen or covalently bound to lipid molecules, or covalently bound to high affinity matched binding molecules such as streptavidin or biotin. The method of encapsulation is gentle, with retention of biological function, and the liposome-encapsulated antibody or protein can be lyophilized and later reconstituted prior to use, such as administration to a patient.
EXAMPLES
Example 1: Preparation of liposomes with no drug: 6% L-a-phosphatidylcholine (Soy) liposome.
6 g of L-a-phosphatidylcholine (Soy) was dispersed in 100 mL of water using a magnetic .. stirrer at 200 rpm for 10 minutes at ambient temperature. The dispersed liposome (multilayer) was passed through a Microfluidic homogenizer at 15,000 psi. Three cycles of passing resulted in a liposome less than 100 nm in diameter. Trehalose was then added to the liposome to a final concentration of 10 % (w/w). The resulting stable isotonic liposome was either used as liquid or lyophilized.
Example 2: Preparation of liposomes encapsulated with lapachone.
200mg of lapachone and 6 g of L a phosphatidylcholine (Soy) were dispersed in mL of water using a magnetic stirrer at 200 rpm for 10 minutes at ambient temperature. The dispersed liposome (multilayer) was passed through a Microfluidic homogenizer at 15,000 psi.
Three cycles of passing resulted in a liposome encapsulated with 2 mg/mL
lapachone less than 100 nm in diameter. Trehalose was then added to the liposome to a final concentration of 10 %(w/w). The resulting stable isotonic liposome encapsulated with lapachone was either used as liquid or lyophilized.
Example 3: Preparation of liposomes encapsulated and micro emulsified with cyclosporine.
500 mg of cyclosporine in 5 mL Mygliol and 6 g of L a phosphatidylcholine (Soy) were 2325999v1/20112-19 dispersed in 100 mL of water using a magnetic stirrer at 200 rpm for 10 minutes at ambient temperature. The dispersed liposome (multilayer) was passed through Microfluidic homogenizer at 15,000 psi. Three cycles of passing resulted in a liposome encapsulated with 5 mg/mL
cyclosporine less than 100 nm in diameter. Trehalose was then added to the liposome to a final concentration of 10 % (w/w). The resulting stable isotonic liposome formulation encapsulated and micro emulsified with cyclosporine was either used as liquid or lyophilized.
Example 4: Preparation of liposomes encapsulated with transferrin.
200 mg of transferrin and 6 g of L a phosphatidylcholine (Soy) were dispersed in 100 mL
of water using a magnetic stirrer at 200 rpm for 10 minutes at ambient temperature. The dispersed liposome (multilayer) was passed through a Microfluidic homogenizer at 15,000 psi.
Three cycles of passing resulted in a liposome encapsulated with 5 mg/mL
transferrin less than 100nm in diameter. Trehalose was then added to the liposome to a final concentration of 10 %(w/w). The resulting stable isotonic liposome formulation encapsulated with transferrin was either used as liquid or lyophilized.
Example 5: Preparation of liposomes encapsulated with colchicine.
6 mg of sodium oleate, 10 g of trehalose and 6 g L a phosphatidylcholine (Soy) were dispersed in 100 mL of water using a magnetic stirrer at 200 rpm for 10 minutes at ambient temperature. The dispersed liposome (multilayer) was passed 10 times through a Microfluidic homogenizer at 15,000 psi. 1004, of colchicine dissolved in acetone was spiked into 1 mL of the pre-made liposomes and lyophilized. The resulting stable isotonic lyophilized liposome encapsulated with the drug is essentially free from organic solvent after lyophilization. After lyophilization, the product can be reconstituted as a lmg/mL, 2mg/mL, 3mg/mL
or 4mg/mL
.. aqueous solution (e.g., Water For Injection).
Example 6. Antibody Encapsulation in Liposomes.
Many therapeutic proteins, including antibodies, have a hydrophobic portion.
In an embodiment, antibodies, just as other hydrophobic proteins or hydrophobic drugs, can be encapsulated by liposomes by adding the antibodies to preformed liposomes.
Surprisingly, liposomal membranes were found to be capable of encapsulating the hydrophobic portions of 2325999v1/20112-19 antibodies in the alkyl chain portion of the bilayer of the liposome. This is in contrast to previous methods in which proteins including antibodies are conjugated to the lipids by chemical modification, such as via a thiol-maleimide reaction or a peptide reaction. In the present invention, no chemical modification of the therapeutic protein is required, and the protein maintains its natural structure.
HERCEPTIN is indicated for adjuvant treatment of breast cancer which is HER2 overexpressing and node positive or node negative (ER/PR negative or with one high risk feature). HERCEPTIN is provided as a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous administration. Each multi-use vial of HERCEPTIN
contains 440 mg trastuzumab, 400 mg E a,a-1,1-trehalose dihydrate, 9.9 mg L-histidine HC1, 6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP.
Reconstitution of a vial of HERCEPTIN with 20 mL of water for injection yielded a solution containing 21 mg/mL trastuzumab, at a pH of approximately 6Ø An aliquot of the solution containing 1.75 mg trastuzumab was lyophilized in a 3 mL vial, and was reconstituted by adding 200 !IL of 6% soybean phosphatidylcholine (SPC) liposomes (prepared using microfluidization) to the lyophilized antibody at a final concentration of antibody of 8.75 mg/mL
(no microfluidization was performed after addition of the antibody, just gentle mixing). The sample was analyzed by size exclusion HPLC by injecting 104, of sample onto a Tosoh G3000 SWXL gel filtration column using 100 mM sodium sulfate and 100 mM sodium acetate pH 6.0 as mobile phase. A HUMIRA sample without liposomes was used as control (see Fig. 1A). The data indicated that 5.6 mg/mL of trastuzumab was encapsulated in the liposome based on its elution at the void volume (Fig. 1B).
The composition of the liposomal HERCEPTIN is shown in Table 1 below.
.. Table 1. Composition of Liposomal Trastuzumab (HERCEPTIN) (1mL composition) Component mg Soy Phosphatidylcholine 60 Trehalose 109.63 Trastuzumab 8.75 2325999v1/20112-19 Hi stidine HC1 1.97 Hi sti dine 0.13 Polysorbate 20 0.04 HUMIRA is a tumor necrosis factor (TNF) blocker indicated for treatment of:
rheumatoid arthritis (RA), psoriatic arthritis, ankylosing spondylitis, Crohn's Disease and plaque psoriasis. HUMIRA is supplied as a sterile, preservative-free solution of adalimumab .. for subcutaneous administration. The drug product is supplied as either a single-use, prefilled pen (HUMIRA Pen) or as a single-use, 1 mL prefilled glass syringe. The solution of HUMIRA
is clear and colorless, with a pH of about 5.2. Each syringe delivers 0.8 mL
of drug product, containing.40 mg adalimumab, 4.93 mg sodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mg dibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80, and Water for Injection, USP. Sodium hydroxide is added as necessary to adjust the pH.
An aliquot containing 1.75 mg adalimumab was lyophilized in a 3 mL vial, and then reconstituted with 200 !IL of 6% SPC liposome with a final concentration of 8.75 mg/mL
antibody. The sample was analyzed by SEC-HPLC by injecting 104, of sample using Tosoh G3000 SWXL gel filtration column using 100 mM sodium sulfate and 100 mM sodium acetate pH 6.0 as mobile phase. Humira sample was used as control without liposome.
Surprisingly, the data indicated that 5.6 mg/mL of Herceptin was encapsulated in the liposome based on the elution at void volume (Fig. 1C). These data suggest that hydrophobic pockets of Herceptin are lodged between the alkyl groups of phosphatidyl choline of the bilayer without any chemical modification.
The composition of the liposomal HUMIRA is shown in Table 2 below.
Table 2. Composition of Liposomal Adalimumab (HUMIRA) (1mL composition) Component mg Soy Phosphatidylcholine 60 Trehalose 100 Adalimumab 8.75 Sodium chloride 0.216 2325999v1/20112-19 Monobasic sodium phosphate 0.030 Dibasic sodium phosphate 0.053 dihydrate Sodium citrate 0.009 Citric acid monohydrate 0.046 Mannitol 0.420 Similar incorporation of antibody was obtained by adding a liquid (i.e., water-solubilized, not lyophilized) preparation of antibody to an aqueous suspension of liposomes.
All references cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

2325999v1/20112-19

Claims (17)

What is claimed is:

1. A liposome-encapsulated antibody composition, comprising a plurality of antibody molecules non-covalently associated with a hydrophobic membrane region of a plurality of liposomes.

2. The liposome-encapsulated antibody composition of claim 1 which is devoid of covalently conjugated antibody molecules.

3. The liposome-encapsulated antibody composition of claim 1, wherein the molar ratio of lipid to antibody is about 50 or less.

4. The liposome-encapsulated antibody composition of claim 1, wherein the plurality of liposomes are suspended in an aqueous medium.

5. The liposome-encapsulated antibody composition of claim 4, wherein the concentration of antibody in the aqueous medium is at least about 5 mg/mL.

6. The liposome-encapsulated antibody composition of claim 1 which is lyophilized.

7. The liposome-encapsulated antibody composition of claim 1, wherein the plurality of liposomes comprise soybean phosphatidylcholine.

8. The liposome-encapsulated antibody composition of claim 1, wherein the composition comprises trehalose.

9. The liposome-encapsulated antibody composition of claim 1 which is essentially devoid of antibody molecules that are not bound to a hydrophobic membrane region of the plurality of liposomes.

10. The liposome-encapsulated antibody composition of claim 1, wherein the antibody is trastuzumab or adalimumab.

11. A method of preparing a liposome-encapsulated antibody composition, the method comprising the steps of:
(a) providing a lyophilized or solubilized antibody and an aqueous suspension of liposomes;
(b) contacting the antibody with the aqueous suspension of liposomes, whereby the antibody becomes associated with a hydrophobic membrane region of the liposomes.

12. The method of claim 11, further comprising:
(c) separating unbound antibody from liposome-bound antibody.

13. The method of claim 12, further comprising:
(d) lyophilizing the liposome-encapsulated antibody composition.

14. The method of claim 11 wherein step (b) is performed without the further use of homogenization, shear, microfluidization, or filtration after adding the antibody.

15. The method of claim 11, wherein at least 60% of the antibody becomes encapsulated.

16. The method of claim 11, wherein the liposomes comprise soybean phosphatidylcholine and the antibody is trastuzumab or adalimumab.

17. The method of claim 11, wherein the antibody is not exposed to a detergent or an organic solvent, and wherein the antibody is not loaded into the liposomes with the use of an osmotic or pH gradient, or with the use of freeze-thaw of the aqueous suspension of liposomes.