CN109562067A - The method for preparing liposome - Google Patents

Abstract

There is provided herein methods for preparing liposome and application thereof.In certain embodiments, liposome is prepared in the method without the use of heating, organic solvent, protein and/or inorganic salts.In certain embodiments, Liposome Preparation contains one or more activating agents.In certain embodiments, Liposomal formulation is for treating disease or illness.

Description

The method for preparing liposome

Background technique

The bioavilability of pharmacy drug depends greatly on the solubility and stability of drug.It has used and has been permitted Multi-method improves the bioavilability of drug, including but not limited to pH adjusts, by drug in conjunction with the micella of detergent, having Dissolution in solvent, with cyclodextrin or other polymer complex and by drug encapsulation in liposome bilayer (Strickley, R.G.,Pharmaceutical Research,No.21,2004:201-230).Drug itself or figuration for dissolving drug Agent may have side effect, such as allergic reaction or haemolysis.

Known solvent (such as ethyl alcohol, propylene glycol, polyethylene glycol, dimethyl acetamide, dimethyl sulfoxide (" DMSO "), complexing Agent (such as niacinamide) and surfactant (such as enuatrol) are hemolytics, therefore are not suitable for Injectable solution.? Other limitations in injectable product using organic solvent include precipitating, pain and the inflammation when injecting.

Liposome is microcosmic lipid vesicle, is made of the aqueous chamber in center, and the aqueous chamber in center is by by (one layer or more) The lipid film that concentric bilayer (lamella) is formed surrounds.Liposome can mix hydroaropic substance (in aqueous interior) or hydrophobicity object Matter (in lipid film).Liposome can be the monolayer vesicle (" UMV ") with single double-layer of lipoid, or have a series of lipids Double-deck multi-layer vesicles (" MLV ") (also referred to as " few layer vesica ").The diameter of multi-layer vesicles is usually in 0.2 μm to 10 μm of size In range.See, for example, WO 98/006882.Although anti-hemolysis measure is generallyd use in the formulation, due to liposome and figuration The unstability of the incompatibility or liposome of agent in the formulation, it may not be feasible for keeping the liposome of sufficient amount in the formulation 's.In addition, lyophilized preparation of the reconstruct containing hydrophobic drug is often difficult.In addition, liposome is organic molten containing being concentrated It is unstable in the preparation of agent.

Diameter is commonly referred to as small monolayer vesicle (" SUV ") less than the monolayer vesicle of 0.2 μm (such as 0.02-0.2 μm).Diameter Monolayer vesicle greater than 0.45 μm (being greater than 1 μm in some cases) is commonly referred to as big monolayer vesicle (" LUV ").

Double-deck (one layer or more) of liposome includes most commonly phosphatide, but also may include lipid, including but not limited to fatty Acid, fatty acid salt and/or fatty alcohol.Among other factors, the characteristic of liposome depends on the property of composition.Therefore, if wanted The liposome with certain features is obtained, then the charge of its polar group and/or the length of its fatty acid chain must be taken into consideration and is satisfied And degree.

In addition, the characteristic of liposome can be modified, for example, cholesterol and other lipids are mixed in film, change lipid Double-deck quantity or by natural molecule (such as protein, polysaccharide, glycolipid, antibody, enzyme) or synthetic molecules (such as polyethyl Glycol) it is covalently attached to surface.Optionally, there are the combinations of a variety of phosphatide and other lipids or cholesterol, in aqueous Jie Liposome is obtained in matter.Depending on preparation method and lipid used, the vesica of different sizes, structure and characteristic can be obtained.

The rigidity that another important parameter considered is double-layer of lipoid is formed about liposome.Form the water of partial double Closing lipid can be liquid crystal (fluid) state or gel state.As temperature increases, gel state is mesomorphic state.This hair The raw temperature in referred to as phase transition temperature (Tc), is specific to every kind of lipid.Tc is directly proportional to chain length, not with fatty acid Saturation degree is inversely proportional, and depends on the property of polar group.

Nevertheless, the common method for preparing lipid vesicle (such as liposome) includes evaporation organic solvent, wherein lipid Dissolution is then dispersed in organic solvent in optional buffered aqueous solution.A kind of illustrative methods, the referred to as side Bangham Method, it is initially described in Bangham et al., J.Hot.Biol., 11:238-252 (1965).It is known to those skilled in the art The deformation of Bangham method, some of them are described below.

The hydration of thin lipid layer.Since the organic solution of double-deck composition lipid, rouge is prepared by removing organic solvent Plasma membrane, this can be realized by evaporation (such as depressurizing in a rotary evaporator) or by desivac.It is aqueous molten by being added Liquid simultaneously stirs mixture in the temperature higher than Tc, makes obtained dried lipid film hydration.

Reverse phase evaporation.Since the organic solution of double-deck composition lipid, lipid film is prepared by removing organic solvent.With Nitrogen purifies the system, and lipid is re-dissolved in the second organic solution being usually made of ether and/or isopropyl ether.By water It is added in the lipid of redissolution.The system is maintained under continuous nitrogen.Gel is formed by removing the second organic solvent.

Solvent injection.The lipid being dissolved in organic solvent is slowly injected into aqueous solution.Organic solvent used is logical It is often solvent miscible with water, and aqueous solution can be warmed.

It can be in such as Szoka and Papandjopoulos, Ann.Rev.Biophys.Bioeng., 2:467-508 And Dousset and Douste-Blazy, Les Liposomes, Puisieux and Delattre, (1980), It is more that preparation is found in Editors, Tecniques et Documentation Lavoisier, Paris, pp.1-73 (1985) The additional method of layer vesica.

In addition, lipid should equably keep being distributed in liposome vesicle when needing to mix more than one lipid. Traditionally, this is by preparing liposome for Lipid dissolution in advance come real in organic solvent and using resulting organic solvent It is existing.

United States Patent (USP) 4,508,703 describes a kind of at least one both sexes (amphyphilic) lipid and optional of obtaining The method of the mixture of powders of at least one hydrophobicity or partially hydrophobic qualitative composition, this method include by the group of the mixture Divide and is dissolved at least one organic solvent and dissolves into resulting solution mist in inert gas.It is mixed that this method allows to prepare lipid Object is closed, can be easily dispersed in aqueous medium, but do not avoid using organic solvent.

WO 92/10166 describes a kind of method for preparing the liposome with the encapsulating ability improved.This method allows Use lipid mixture；However, obtaining mixture by advance evaporating Lipid dissolution in organic solvent and then.This Outside, the contact between lipid and the aqueous solution of activating agent is carried out in the temperature higher than Tc.

Furthermore, it was reported that in the case where preparing liposome without using organic solvent, it usually needs other operations, this can It can lead to the preparation with certain unfavorable features.Contain for example, U.S. Patent Application Publication No.2008/0274172 describes preparation There is the liposome of at least two phosphatide without the use of the method for organic solvent.However, using the temperature higher than Tc from containing inorganic Stable liposome is obtained in the aqueous solution of salt.

Therefore, the existing method for preparing liposome uses organic solvent, protein, inorganic salts and/or heating.Due to it Toxicity and combustibility, organic solvent preparation for drug, cosmetics and other purposes liposome in be undesirable. In addition, the use of organic solvent and protein has negative repercussion in terms of production cost, safety, work hygiene and environment. It similarly, the use of heating is undesirable in terms of production cost, safety and environment in preparing liposome.Preparing lipid It the use of inorganic salts is undesirable in body, because the introducing of inorganic salts increases the size of liposome and/or causes more muddy Preparation.See, for example, Castile et al., International Journal of Pharmaceutics, 1999, vol.188,issue 1,pp.87-95.Therefore, it is necessary to a kind of liposomes for preparing without the use of undesirable reagent and step Method.

Summary of the invention

There is provided herein the methods for preparing liposome.In certain embodiments, prepare in the method liposome without Use heating, organic solvent, protein and/or inorganic salts.

In one embodiment, a kind of method for preparing liposome is provided, this method includes:

(a) it combines one or more lipids in an aqueous medium in environment temperature；

(b) in an aqueous medium by lipid dispersion；With

(c) one or more sugar are added into gained mixture, to form the solution of liposome.

In certain embodiments, Liposome Preparation contains one or more activating agents.In certain embodiments, living Property agent is medicament.In certain embodiments, activating agent includes but is not limited to lapachol；Transferrins；Cyclosporin；Colchicum Alkali；The combination of lapachol and transferrins in single Liposomal formulation.

In certain embodiments, one or more activating agents are added after preparing liposome or liposome solutions.? In other embodiments, one or more activating agents are added during liposome is formed.

Purposes of the Liposome Preparation in treatment disease or illness is also provided herein.In one embodiment, rouge Plastid prepared product is suitable for carrying out parenteral administration to the patient with the disease or illness.In one embodiment, suffer from Person is people.

Definition

As used herein, unless otherwise indicated, " lipid " is interpreted as fatty acid, fatty acid salt, fatty alcohol or phosphatide.Rouge Matter is also construed as including sterol, including but not limited to cholesterol；Sphingolipid, including but not limited to sphingomyelin；Sugar Sphingolipid, including but not limited to gangliosides, globoside (globocide) and cerebroside；With surfactant amine, packet Include but be not limited to stearmide, oleyl amine and sub- oleamide (linoleyl amine).

As used herein, unless otherwise stated, the both sexes (anamphyphilic) that " phosphatide " is interpreted as glycerol are spread out Biology, wherein one hydroxyl can be by Phosphation, another two hydroxyl by long chain fatty acid, the long chain fatty acids It is being equal to each other or different from each other, and can be saturated or unsaturated.Neutral phospholipid is usually wherein other phosphoric acid The phosphatide that the alcohol esterification and its net charge that hydroxyl is replaced by polar group (usually hydroxyl or amino) are zero.Electrically charged Phosphatide is usually the alcohol esterification that wherein other phosphoric acid hydroxyls are replaced by polar group and its net charge is positive or negative phosphatide.

The example of phosphatide include but is not limited to phosphatidic acid (" PA "), phosphatidyl choline (" PC "), phosphatidyl glycerol (" PG "), Phosphatidyl-ethanolamine, phosphatidylinositols (" Pr ") and phosphatidylserine (" PS ").Sphingomyelin (including cranial nerve sheath marrow Phosphatide), lecithin, lysolecithin, lysophosphatidyl ethanolamine, cerebroside, two peanut phosphatidyl cholines (" DAPC "), Two capryl-L- α-phosphatidyl choline (" DDPC "), two mustard phosphatidyl cholines (" DEPC "), dilauroyl phosphatidyl gallbladder Alkali (" DLPC "), Dlpc lipid, L-Dimyristoylphosphatidylcholine (" DMPC "), dioleoyl phosphatidyl Choline (" DOPC "), Dioctonoyl pnosphotidyl choline (" DPPC "), distearoyl phosphatidylcholine (" DSPC "), 1- palm Acyl group -2- oleolyl phosphatidyl choline (" POPC "), two peanut acyl phospholipids acyl glycerol (" DAPG "), two capryl-L- α phosphatide Acyl glycerol (" DDPG "), two mustard acyl phospholipids acyl glycerol (" DEPG "), dilauroylphosphatidylglycerol (" DLPG "), two sub- oil Acyl phospholipids acyl glycerol, dimyristoylphosphatidylglycerol (" DMPG "), dioleoylphosphatidylglycerol (" DOPG "), two palm fibres Palmitic acid acyl phospholipids acyl glycerol (" DPPG "), distearoylphosphatidylglycerol (" DSPG "), 1- palmityl -2- oleolyl phosphatidyl Acyl glycerol (" POPG "), two peanut acylphosphatidyl ethanolamines (" DAPE "), two capryl-L- α-phosphatidyl-ethanolamine (" DDPE "), di-mustard acyl phosphatidylethanolamine (" DEPE "), dilauroyl phosphatidyl-ethanolamine (" DLPE "), two sub- oleoyls Base phosphatidyl-ethanolamine, two myristoyl phosphatidyl-ethanolamines (" DMPE "), dioleoylphosphatidylethanolamine (" DOPE "), Dipalmitoylphosphatidylethanolamine (" DPPE "), distearoylphosphatidylethanolamine (" DSPE "), 1- palmityl -2- oil Acylphosphatidyl ethanolamine (" POPE "), two peanut acyl phospholipids acyl inositols (" DAPI "), two capryl-L- α-phosphatidylinositols (" DDPI "), two mustard acyl phospholipids acyl inositols (DEPI "), dilauroyl phosphatidylinositols (" DLPI "), two sub-oleoyl phosphorus Acyl inositol, two myristoyl phosphatidylinositols (" DMPI "), dioleoyl phosphatidylinositols (" DOPI "), two palmityls Phosphatidylinositols (" DPPI "), distearyl acyl group phosphatidylinositols (" DSPI "), 1- palmityl -2- oleolyl phosphatidyl inositol (" POPI "), two peanut acyl phospholipids acyl serines (" DAPS "), two capryl-L- α-phosphatidylserine (" DDPS "), two Mustard acyl phospholipids acyl serine (" DEPS "), dilauroyl phosphatidylserine (" DLPS "), two sub-oleoyl phosphatidyl silks Propylhomoserin, two myristoyl phosphatidylserines (" DMPS "), di-oleoyl phosphatidylserine (" DOPS "), two palmityls Phosphatidylserine (" DPPS "), distearyl phosphatidylserine (" DSPS "), 1- palmityl -2- oleolyl phosphatidyl Serine (" POPS "), two peanut acyl group sphingomyelins, two capryl sphingomyelins, two mustard acyl group neurolemma marrow phosphorus Rouge, dilauroyl sphingomyelin, two sub-oleoyl sphingomyelins, two myristoyl sphingomyelins, mind Through sphingo, dioleoyl sphingomyelin, two palmityl sphingomyelins, distearyl acyl group sphingomyelin With 1- palmityl -2- oleoyl sphingomyelin.

As used herein, unless otherwise stated, " encapsulating " is interpreted as incorporating active agents into liposome or liposome Process in vesica.The activating agent of encapsulating can be retained in aqueous interior or in conjunction with film.

As used herein, unless otherwise indicated, when being used in combination when the solubility with compound, term " enhancing " means this The method that text provides causes the solubility of compound with the same compound solubility in water compared to increase.Specifically, term " enhancing " refers to, when using method provided herein, the solubility of compound is more molten in reference solvent than the same compound Xie Du increase about 20% or more, about 40% or more, about 60% or more, about 80% or more, about 100% or more or About 200% or more.It in some embodiments, is water with reference to solvent.

As used herein, unless otherwise indicated, term " hydrophobic compound " refers to that water solubility is very little or none water-soluble The compound of Xie Du.In some embodiments, hydrophobic compound, which has, is less than about 20 weight %, about 15 weight %, about 10 Weight %, about 5 weight %, about 1 weight %, the intrinsic water solubility of about 0.1 weight % or about 0.01 weight % are (i.e. non-ionic The water solubility of form).In other embodiments, hydrophobic compound has less than about 10mg/mL, about 7mg/mL, about The intrinsic water solubility of 5mg/mL, about 3mg/mL, about 1mg/mL or about 0.1mg/mL.

As used herein, or unless otherwise indicated, term " aqueous medium " includes any aqueous medium, such as water, salt are molten Liquid, sugar juice, transfusion solution, buffer and any other aqueous medium being easy to get.In addition, aqueous medium can contain one kind Or a variety of water-miscible organic solvents.In the case where parenteral solution, aqueous medium is preferably sterile and is suitable as living The carrier of property agent.The example of aqueous medium includes but is not limited to water for injection, salting liquid, Ringer's solution, D5W or other water The solution of miscible substance such as glucose and other electrolyte.

As used herein, unless otherwise indicated, term " fatty acid " " refers to that its structure is carboxyl and has one or more The compound of the hydrocarbon chain connection of carbon atom.Hydrocarbon chain can be saturated or unsaturated (i.e. alkyl, alkenyl or alkynyl hydrocarbon chain).And And hydrocarbon chain can be linear chain or branched chain.In addition, in some embodiments, the hydrogen in hydrocarbon chain can be substituted.

As used herein, unless otherwise indicated, term " fatty alcohol " refers to that its structure is alcohol radical and has one or more The compound of the hydrocarbon chain connection of carbon atom.Hydrocarbon chain can be saturated or unsaturated (i.e. alkyl, alkenyl or alkynyl hydrocarbon chain).And And hydrocarbon chain can be linear chain or branched chain.In addition, in some embodiments, the hydrogen in hydrocarbon chain can be substituted.

As used herein, unless otherwise indicated, term " fatty acid " salt " refers to by between fatty acid and inorganic/organic base React the compound formed.In addition, the term includes the compound formed by the reaction between fatty alcohol and inorganic/organic acid. The example of these acid includes but is not limited to sulfuric acid and phosphoric acid.The hydrocarbon chain of fatty acid salt can be saturated or unsaturated (i.e. alkane Base, alkenyl or alkynyl hydrocarbon chain).Moreover, hydrocarbon chain can be linear chain or branched chain.In addition, in some embodiments, in hydrocarbon chain Hydrogen can be substituted.

As used herein, unless otherwise indicated, term " substituted " refers to the group being substituted by one or more substituents, The substituent group includes but is not limited to alkyl, alkenyl, alkynyl, naphthenic base, aroyl, halogenated, halogenated alkyl (such as fluoroform Base), substituted or unsubstituted Heterocyclylalkyl, halogenated alkoxy (such as trifluoromethoxy), hydroxyl, alkoxy, Cycloalkyloxy, heterocylooxy, oxo, alkanoyl, aryl, substituted aryl, substituted or unsubstituted heteroaryl (for example, indyl, imidazole radicals, furyl, thienyl, thiazolyl, pyrrolidinyl, pyridyl group, pyrimidine radicals etc.), aryl alkyl, Alkylaryl, heteroaryl, heteroaryl alkyl, miscellaneous alkyl aryl, heterocycle (heterocyclo), aryloxy group, alkanoyloxy, ammonia Base, arylamino, aryl-alkyl amino, cycloalkyl amino, heterocyclic amino group (heterocycloamino), singly takes alkyl amino The amino in generation and disubstituted amino, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoyl ammonia Base, substituted arylamino, substituted aralkanoylamino, carbamoyl (such as CONH₂), replace ammonia Base formoxyl (such as the case where there are two substituent groups on CONH- alkyl, CONH- aryl, CONH- aryl alkyl or in which nitrogen), Carbonyl, alkoxy carbonyl, carboxyl, cyano, ester, ether, guanidine radicals, nitro, sulfonyl, alkyl sulphonyl, aryl sulfonyl, aryl alkane Base sulfonyl, sulfonamido (such as SO₂NH₂), replace sulfonamido, mercaptan, alkylthio group, arylthio, aromatic alkylthio, The thio carbonyl of cycloalkylthio, heterocyclothio, alkane, the thio carbonyl of virtue and the thio carbonyl of aralkyl (arylalkyithiono).

As used herein, unless otherwise indicated, term " alkyl " refers to 1-20 carbon atom, preferably 1-10 carbon original Son, the saturated straight chain or branched non cyclic hydrocarbon of most preferably 1-4 carbon atom.Representative straight chain saturated alkyl includes methyl, second Base, n-propyl, normal-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and positive decyl；Being saturated branched alkyl includes Isopropyl, sec-butyl, isobutyl group, tert-butyl, isopentyl, 2- methyl butyl, 3- methyl butyl, 2- methyl amyl, 3- methylpent Base, 4- methyl amyl, 2- methylhexyl, 3- methylhexyl, 4- methylhexyl, 5- methylhexyl, 2,3- dimethylbutyl, 2,3- Dimethyl amyl group, 2,4- dimethyl amyl group, 2,3- dimethylhexanyl, 2,4- dimethylhexanyl, 2,5- dimethylhexanyl, 2,2- bis- Methyl amyl, 2,2- dimethylhexanyl, 3,3- dimethyl amyl group, 3,3- dimethylhexanyl, 4,4- dimethylhexanyl, 2- ethyl penta Base, 3- ethylpentyl, 2- ethylhexyl, 3- ethylhexyl, 4- ethylhexyl, 2- methyl -2- ethylpentyl, 2- methyl -3- second Base amyl, 2- methyl -4- ethylpentyl, 2- methyl -2- ethylhexyl, 2- methyl-1,3- ethylhexyl, 2- methyl -4- ethyl Hexyl, 2,2- diethyl amyl group, 3,3- diethylhexyl, 2,2- diethylhexyl, 3,3- diethylhexyl etc..Alkyl can be It is unsubstituted or substituted.Unsaturated alkyl includes alkenyl and alkynyl, is discussed below.

As used herein, unless otherwise indicated, term " alkenyl " refers to 2-20 carbon atom, preferably 2-10 carbon original Son, most preferably 2-6 carbon atom and linear chain or branched chain non-cyclic hydrocarbon including at least one carbon-to-carbon double bond.Representative straight chain It include vinyl, allyl, 1- cyclobutenyl, 2- cyclobutenyl, isobutenyl, 1- pentenyl, 2- penta with branch (C2-C10) alkenyl Alkenyl, 3-methyl-1-butene base, 2- methyl-2-butene base, 2,3- dimethyl -2- cyclobutenyl, 1- hexenyl, 2- hexenyl, 3- Hexenyl, 1- heptenyl, 2- heptenyl, 3- heptenyl, 1- octenyl, 2- octenyl, 3- octenyl, 1- nonenyl, 2- nonene Base, 3- nonenyl, 1- decene base, 2- decene base, 3- decene base etc..The double bond of alkenyl can be it is unconjugated or with another not Saturated group conjugation.Alkenyl can be unsubstituted or substituted.

As used herein, unless otherwise indicated, term " alkynyl " refers to 2-20 carbon atom, preferably 2-10 carbon original Son, most preferably 2-6 carbon atom and linear chain or branched chain non-cyclic hydrocarbon including at least one carbon-carbon triple bond.Representative straight chain It include acetenyl, propinyl, 1- butynyl, 2- butynyl, 1- pentynyl, valerylene base, 3- first with branch (C2-C10) alkynyl Base -1- butynyl, 4- pentynyl, 1- hexin base, 2- hexin base, 5- hexin base, 1- heptynyl, 2- heptynyl, 6- heptynyl, 1- Octynyl, 2- octynyl, 7- octynyl, 1- n-heptylacetylene base, 2- n-heptylacetylene base, 8- n-heptylacetylene base, 1- decynyl, 2- decynyl, 9- decine Base etc..Three keys of alkynyl can be unconjugated or be conjugated with another unsaturated group.Alkynyl can be unsubstituted or take Generation.

As used herein, unless otherwise indicated, term " pharmaceutically acceptable salt " refers to by pharmaceutically acceptable nothing Malicious acid or the salt of alkali preparation, the non-toxic acid or alkali include inorganic bronsted lowry acids and bases bronsted lowry and organic bronsted lowry acids and bases bronsted lowry.For provided herein group The suitable pharmaceutically acceptable base addition salts for closing object include but is not limited to the gold made of aluminium, calcium, lithium, magnesium, potassium, sodium and zinc Belong to salt, or by lysine, N, N'- dibenzyl-ethylenediamin, chloroprocanine, choline, diethanol amine, ethylenediamine, meglumine (N- first Base aminoglucose) and procaine made of organic salt.Suitable non-toxic acid includes but is not limited to inorganic and organic acid, such as second Acid, alginic acid, ortho-aminobenzoic acid, benzene sulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, vinyl sulfonic acid, formic acid, fumaric acid, furancarboxylic acid, Galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, apple Tartaric acid, mandelic acid, methanesulfonic acid, glactaric acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, amber Amber acid, sulfanilic acid, sulfuric acid, tartaric acid and p-methyl benzenesulfonic acid.Specific non-toxic acid includes hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and first Sulfonic acid.Therefore, the example of specific salt includes hydrochloride and inesylate salt.It is other be in the art it is well known, see, for example, 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, term " hydrate " refers to compound or its salt provided herein, further comprises by non- The stoichiometry or non-stoichiometric water that power combines between covalent molecule.

As used herein, term " clathrate compound " refers to the salt of compound or its form crystal lattice provided herein, and it includes will Guest molecule (such as solvent or water) captures in space (for example, channel) wherein.

As used herein, unless otherwise indicated, refer to can be under biological condition (in vitro or in vivo) for term " prodrug " Hydrolysis, oxidation are otherwise reacted to provide the derivative of the compound of reactive compound.The example of prodrug includes but not Be limited to include can biological hydrolysis part (such as can biological hydrolysis amide, can biological hydrolysis ester, can biological hydrolysis amino Formic acid esters, can biological hydrolysis carbonic ester, can biological hydrolysis uride and can biological hydrolysis phosphate analog) compound Derivative and metabolin.Preferably, the prodrug of the compound with carboxyl functional group is the lower alkyl esters of carboxylic acid.Esterification exists It is convenient to form carboxylate in any carboxylic moiety on molecule.Well known method preparation usually can be used in prodrug, such as 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) described in method.

As used herein, unless otherwise indicated, when being used together with preparation, term " stable " refers to using herein Offer method preparation when, the activating agent of preparation measure at the appointed time holding dissolution, and not significantly degradation aggregation or with Other modes modification (for example, being measured by HPLC).

As used herein, unless otherwise stated, being interpreted as " lower than the temperature of Tc " lower than the rouge with minimum Tc The temperature of the Tc of matter, and " temperature higher than Tc " is interpreted as being higher than the temperature of the Tc of the lipid with highest Tc.

As used herein, unless otherwise indicated, when being used together with pharmaceutical composition, term " harmful components " refer to medicine Commonly may cause the ingredient of clinical side effects in compositions, the side effect be such as, but not limited to haemolysis, allergic reaction, The bioavilability of peripheral nerve disease, and/or composition active constituent reduces.The example of harmful components includes but is not limited to: having Malicious solvent, including organic solvent such as ethyl alcohol, methanol, 1- propyl alcohol, 2- propyl alcohol, acetone, acetonitrile, ethyl acetate, methyl acetate, second Ether, dimethyl ether, diisopropyl ether, methyl tertiary butyl ether(MTBE) (" MTBE "), tetrahydrofuran (" THF "), methylene chloride, chloroform, four chlorinations Carbon, 1,2- dichloroethanes, pentane, hexane, heptane, petroleum ether, dioxanes, ethylene glycol, diethylene glycol (DEG), diethylene glycol dimethyl ether, 1,2 2 Ethyl Methyl Ether, n-butyl alcohol, 2- butanol, 2- butanone, benzene, toluene, dimethyl sulfoxide (" DMSO "), dimethylformamide (" DMF "), Hexamethyl phosphoramide (" HMPA "), N-Methyl pyrrolidone, glycerol, nitromethane, triethylamine, dimethylbenzene,EL and polyethylene glycol (" PEG ")；Total detergent or surfactant such as polysorbate are (such as ) or vitamin E Tweens；Oils such as castor oil or corn oil；Protein such as HSA；Or any other is the life of potential pollution source Object preparation.

Detailed description of the invention

Figure 1A shows that the control for being added without the size exclusion HPLC of adalimumab (HUMIRA) antibody of liposome is washed De- curve.Figure 1B shows that the elution of the size exclusion HPLC of liposomal encapsulated Herceptin (HERCEPTIN) antibody is bent Line.Fig. 1 C shows the elution curve of the size exclusion HPLC of liposomal encapsulated adalimumab (HUMIRA) antibody.

Specific embodiment

There is provided herein a kind of methods for preparing liposome, and this method includes:

(a) it combines one or more lipids in an aqueous medium in environment temperature；

(b) in an aqueous medium by lipid dispersion；With

(c) one or more sugar are added into gained mixture, to form the suspension of liposome.

In another embodiment, in step (b), this method also includes the lipid that homogenizes in an aqueous medium.

Some embodiments also include step (d), wherein one or more activating agents are added in liposome solutions.? In one embodiment, activating agent is hydrophobic drug.In one embodiment, activating agent is added with solid.At another In embodiment, activating agent is added in organic solvent.In another embodiment, activating agent is added in organic solvent, The organic solvent also includes one or more fatty acid salts, fatty acid and/or phosphatide.

In another embodiment, activating agent is added during liposome is formed.

In certain embodiments, the diameter of gained liposome is less than about 1 μm.

In one embodiment, the diameter of gained liposome is less than about 500nm.

In one embodiment, the diameter of gained liposome is less than about 100nm.

In one embodiment, at least one lipid is the mixture of phosphatide or phosphatide.The example of phosphatide includes but not It is limited to phosphatidic acid (" PA "), phosphatidyl choline (" PC "), phosphatidyl glycerol (" PG "), phosphatidyl-ethanolamine (" PE "), phosphatidyl Inositol (" PI ") and phosphatidylserine (" PS "), sphingomyelin (including cranial nerve sphingo), lecithin, haemolysis Lecithin, lysophosphatidyl ethanolamine, cerebroside, two peanut phosphatidyl cholines (" DAPC "), two capryl-L- α-phosphatide Phatidylcholine (" DDPC "), two mustard phosphatidyl cholines (" DEPC "), Dilauroyl Phosphatidylcholine (" DLPC "), two sub- oil Phosphatidyl choline, L-Dimyristoylphosphatidylcholine (" DMPC "), dioleyl phosphatidyl choline (" DOPC "), two palm fibres Palmitic acid phosphatidyl choline (" DPPC "), distearoyl phosphatidylcholine (" DSPC "), 1- palmityl -2- oleolyl phosphatidyl Phatidylcholine (" POPC "), two peanut acyl phospholipids acyl glycerol (" DAPG "), two capryl-L- α phosphatidyl glycerols (" DDPG "), two Mustard acyl phospholipids acyl glycerol (" DEPG "), dilauroylphosphatidylglycerol (" DLPG "), two sub-oleoyl phosphatidyl glycerols, two Myristoyl phosphatidyl glycerol (" DMPG "), dioleoylphosphatidylglycerol (" DOPG "), dipalmitoylphosphatidylglycerol (" DPPG "), distearoylphosphatidylglycerol (" DSPG "), 1- palmityl -2- oleolyl phosphatidyl glycerol (" POPG "), Two peanut acylphosphatidyl ethanolamines (" DAPE "), two capryl-L- α-phosphatidyl-ethanolamine (" DDPE "), two mustard acyl phospholipids Acyl ethanol amine (" DEPE "), dilauroyl phosphatidyl-ethanolamine (" DLPE "), two sub-oleoyl phosphatidyl-ethanolamines, two Pork and beans Cool acylphosphatidyl ethanolamine (" DMPE "), dioleoylphosphatidylethanolamine (" DOPE "), two palmityl phosphatidyl ethanols Amine (" DPPE "), distearoylphosphatidylethanolamine (" DSPE "), 1- palmityl -2- oleolyl phosphatidyl ethanol amine (" POPE "), two peanut acyl phospholipids acyl inositols (" DAPI "), two capryl-L- α-phosphatidylinositols (" DDPI "), two mustard acyls Base phosphatidylinositols (DEPI "), dilauroyl phosphatidylinositols (" DLPI "), two sub-oleoyl phosphatidylinositols, two Pork and beans Cool acyl phospholipids acyl inositol (" DMPI "), dioleoyl phosphatidylinositols (" DOPI "), two palmityl phosphatidylinositols (" DPPI "), distearyl acyl group phosphatidylinositols (" DSPI "), 1- palmityl -2- oleolyl phosphatidyl inositol (" POPI "), Two peanut acyl phospholipids acyl serines (" DAPS "), two capryl-L- α-phosphatidylserine (" DDPS "), two mustard acyl phospholipids Acyl serine (" DEPS "), dilauroyl phosphatidylserine (" DLPS "), two sub-oleoyl phosphatidylserines, two Pork and beans Cool acyl phospholipids acyl serine (" DMPS "), di-oleoyl phosphatidylserine (" DOPS "), two palmityl phosphatidyl silk ammonia Acid (" DPPS "), distearyl phosphatidylserine (" DSPS "), 1- palmityl -2- oleolyl phosphatidyl Serine (" POPS "), two peanut acyl group sphingomyelins, two capryl sphingomyelins, two mustard acyl group sphingomyelins, two Lauroyl sphingomyelin, two sub-oleoyl sphingomyelins, two myristoyl sphingomyelins, neurolemma marrow Phosphatide, dioleoyl sphingomyelin, two palmityl sphingomyelins, distearyl acyl group sphingomyelin and 1- palm fibre Palmitic acid acyl group -2- oleoyl sphingomyelin.

Phosphatide provided herein can be chiral or achiral.Chirality phosphatide provided herein can be D or L phosphorus Rouge, for example, L- α-phosphatidyl choline or I, 3 phosphatidyl cholines.

In one embodiment, L- α-phosphatidyl choline is in method provided herein.

In another embodiment, there is provided herein the method for preparing liposome, this method includes:

(a) it combines enuatrol and α-phosphatidyl choline in an aqueous medium in environment temperature；

(b) in an aqueous medium by enuatrol and L- α-phosphatidyl choline dispersion；With

(c) one or more sugar are added into gained mixture, to form the solution of liposome.

In one embodiment, aqueous medium contains one or more activating agents or its pharmaceutically acceptable salt, water Close object, clathrate compound or prodrug.The example of activating agent include but is not limited to lapachol (13- lapachol), taxanes (including but not Be limited to taxol, 7- Epitaxol, 7- acetyl paclitaxel, 10- deacetylate taxol, 10- deacetylate -7- Epitaxol, 7- xylosyl taxol, 10-desacetyl-7-sylosyltaxol, 7- glutarylpaclitaxel, 7-N, N Dimethylglycycltaxol, 7-L- alanylpaclitaxel, taxotere and its mixture), taxol, colchicin, turn Ferritin, cyclosporin, cyclosporin A, Ketoprofen, propofol, acetylsalicylic acid, paracetamol, anphotericin, it is high Pungent, Doxorubicin, daunorubicin, epirubicin, idarubicin, angiogenesis inhibitor (such as bevacizumab, Lucentis, Vitaxin, carboxyltriazole, combretastatin A-4, Fumngillin analog (such as TNP-470), CM101, IFN-u, leucocyte are situated between Plain -10, interleukin 12, platelet factor-4, suramin, SU5416, thrombin-sensitive protein, VEGFR antagonist, blood Pipe growth hormone release inhibiting hormone, Endostatin, 2ME2, for can Garland, Thalidomide, prolactin, linomide, angiogenesis Plain -1, basic fibroblast growth factor, vascular endothelial growth factor), (such as vincaleukoblastinum, Changchun are new for vinca alkaloids Alkali, eldisine, Etoposide, etoposide phosphate and Teniposide), it is cytarabine, D actinomycin D, Etoposide, rich It is bleomycin, gentamicin, cyclophosphamide, methotrexate (MTX), streptozotocin, cytimidine, 13-D- cytarabine -5'- triphosphoric acid, thin Cytochrome C, cis-platinum, N- phosphono-acetyl group-L-Aspartic acid, 5- fluororotic acid, acyclovir, Zidovudine, interferons, Aminoglycoside, cephalosporins, Tetracyclines, Propranolol, timolol, labetalol, clonidine, hydrolazine, the third miaow Piperazine, amitriptyline, doxepin (doxepim), pheny loin, diphenhydramine, chlorphenirimine, fenazil, forefront Parathyrine class, methotrexate (MTX), progesterone, testosterone, estradiol, estrogen, epirubicin, beclomethasone and esters, vitamin E, can Pine, dexamethasone and esters, celestone-V, biphenyl dimethyl dicarboxylic acids, calcitonin, camptothecine, captopril, head Spore oxazoline, chloroquine, diuril azoles, blood coagulation factor VIII and IX, d- alpha-tocopherol, Diclofenac, Etoposide, take dexamethasone Pyridine, Flurbiprofen, fluorouracil, Prozac, Fusidic Acid, gentamicin, glibenclamide, Granisetron, growth hormone, indoles Mei Xin, insulin, Itraconazole, ketoconazole, methotrexate (MTX), metronidazole, minoxidil, mitomycin, naphthlazole, Nabumetone Life, Ondansetron, Oxyphenbutazone, parazosin, eserine, piroxicam, prednisolone, primaquine, quinine, Ramipril, Taxotane, tenoxicam, Terazosin, triamcinolone, urokinase, opium sample antalgesic (such as alfentanil, anileridine, Codeine, fentanyl, hydrocodone, Hydromorphone, pethidine, morphine, Oxycodone, Oxymorphone, propoxyhene, relaxes at diamorphine Fentanyl, pentazocine and Nalbuphine), non-steroidal anti-inflammatory drugs (such as aspirin, Indomethacin, brufen, mefenamic acid and Phenylbutazone), it is Angiotensin-Converting (" ACE ") inhibitor (such as captoprilpolyene), inhibitors of protein kinase C, anti- Raw element (such as imidazoles and triazole antibiotics), folic acid, anthracycline antibiotic, antisense RNA, tricathecums, microorganism ribose Body inactivates toxin (such as gelonin, abrin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin, dyers' grapes Antiviral peptide), piperidines acid derivative (such as tacrolimus), plant alkaloid, dyestuff, radioisotope labeled compound, Radiopaque compound, radiosensitizer (such as the chloro- 2'- BrdU of 5-, the bromo- 2'- BrdU of 5- and the iodo- 2'- deoxidation of 5- Uridine), it is fluorescent chemicals, mydriasis compound, bronchodilator, local anesthetic (such as cincaine and chlorpromazine), anti-true (such as Miconazole, terconazole, econazole, Isoconazole, butaconazole, clotrimazole, Itraconazole, nystatin, naphthalene replace microbial inoculum Fragrant and amphotericin B), antiparasitic, hormone, hormone antagonist, immunomodulator, neurotransmitter antagonists, anti-diabetic Agent, antiglaucoma agent, vitamin, anesthetic and imaging agent.About other disclosures of activating agent, referring to Gilman et al.,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, activating agent is hydrophobic compound, or has the compound of the solubility of difference in water.

In another embodiment, activating agent is non-hydrophobic compound.

In one embodiment, activating agent is impermeable dose of water-solubility membrane, for example, peptide, protein, nucleic acid, nucleotide, Nucleosides, carbohydrate or its analog.

In one embodiment, aqueous medium is free of activating agent.

In another embodiment, acquired solution contains the trehalose of 10 weight %.

In another embodiment, activating agent is lapachol or its pharmaceutically acceptable salt, hydrate, clathrate compound Or prodrug.

In another embodiment, activating agent is transferrins or its pharmaceutically acceptable salt, hydrate, cage type Object or prodrug.

In another embodiment, activating agent is cyclosporin or its pharmaceutically acceptable salt, hydrate, cage type Object or prodrug.

In one embodiment, activating agent is transferrins and lapachol, pharmaceutically acceptable salt, hydrate, Clathrate compound or prodrug.

The example for the sugar that can be used in method provided herein includes but is not limited to sucrose, glucose, fructose, lactose, wheat Bud sugar, mannose, galactolipin and trehalose.

In one embodiment, sugar is trehalose.

In one embodiment, Liposome Preparation be suitable for the patient with one or more diseases or illness into Row parenteral administration.

In one embodiment, patient is people.

In certain embodiments, the addition sequence of (one or more) activating agent causes (one or more) activating agent to enhance Solubility.Conventional method by hydrophobic drug incorporation liposome is that drug is added in lipid before liposome preparation.Ginseng See, such as Immordino, M.L.et al., Journal of Controlled Release, 2003,91:417 429.It is logical Conventional method is crossed, the incorporation of drug is only 0.3 to 0.7mg/mL.

There is provided herein the methods for preparing liposome, wherein (one or more) activating agent is added after forming liposome to be caused The solubility of (one or more) activating agent enhancing.In one embodiment, compared with conventional method, in liposome (one Or a variety of) at least about 2 times, 5 times or 10 times of the increase of activating agent solubility.In one embodiment, in liposome (one or It is a variety of) activating agent solubility increases to about 5mg/mL.

In certain embodiments that wherein activating agent is hydrophobic drug, prefabricated liposome is added with solid in activating agent In or be added organic solvent in.In one embodiment, prefabricated liposome includes one or more fatty acid salts, fatty acid And/or phosphatide is to increase the solubility of activating agent.

In one embodiment.The sequence that (one or more) activating agent is added causes to mix (one or more) activating agent Liposome it is more efficient.In certain embodiments, mix liposome efficiency be 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%.In certain embodiments, doping efficiency 90%, 95%, 98%, 99% or 100%.

It is not limited by specific theory or mechanism, incorporates active agents into the increase of prefabricated liposome (for example, increasing to concentration It is about 5mg/mL) it may be the increase long-pending due to surface of liposome.

The liposome composition being prepared by the following method is also provided herein, the method includes:

(a) it combines one or more lipids in an aqueous medium in environment temperature；

(b) in an aqueous medium by lipid dispersion；With

(c) one or more sugar are added into gained mixture, to form liposome solutions.

In another embodiment, there is provided herein the liposome composition being prepared by the following method, the methods Include:

(a) it combines one or more lipids in an aqueous medium in environment temperature；

(b) in an aqueous medium by lipid dispersion；

(c) one or more sugar are added into gained mixture, to form liposome solutions；With

(d) activating agent is added into liposome solutions.

In one embodiment, activating agent is hydrophobic drug.

In one embodiment, activating agent is added with solid.

In one embodiment, activating agent is added in organic solvent.

In one embodiment, the activating agent in organic solvent also include one or more fatty acid salts, fatty acid and Phosphatide.

In certain embodiments, method provided herein generates the stabilization comprising liposome and one or more activating agents Solution, composition or preparation.In these embodiments, (one or more) activating agent keeps dissolving in specific time quantum, and And it does not degrade significantly, assemble or becomes otherwise to modify (for example, measuring by HPLC).

In some embodiments, in the acceptable dilution dilution agent of the temperature of raising (for example, about 35 DEG C or higher) After a week, about 70% or more, about 80% or more, about 90% or more activating agent keeps dissolution.

In other embodiments, in room temperature with acceptable dilution dilution agent after a week, about 70% or more, about 80% Or more, about 90% or more activating agent keep dissolution.

In other embodiments, reduced temperature (for example, about 10 DEG C or lower) after a week, about 70% or more, About 80% or more, about 90% or more activating agent keeps dissolution.

In certain embodiments, compared with the solubility of identical activating agent in an aqueous medium, side provided herein Method causes the solubility of activating agent to enhance.Specifically, when using method provided herein, the solubility ratio of activating agent mutually assimilates Close solubility increase about 20% or more, about 40% or more, about 60% or more, about 80% of the object in reference solvent or more It is more, about 100% or more or about 200% or more.It in some embodiments, is water with reference to solvent.

The method using liposome composition provided herein treatment disease or illness is also provided herein.In some implementations In scheme, the disease or illness include but is not limited to tumor disease, proliferative diseases, central nervous system disease, itself exempt from Epidemic disease and inflammatory disease or illness.In other embodiments, this method is related to the treatment of bacterium, virus or fungal infection.

The proliferative disorders (such as cancer) that can be treated by method provided herein include but is not limited to onch- tumor (pernicious and benign) and transfer, or any disease or illness characterized by the growth of uncontrolled cell.Cancer may be former Hair property or metastatic cancer.Can prevent according to the method for the present invention, manage, treating or the specific example of improved cancer include but It is not limited to head, neck, eyes, oral cavity, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovum The cancer of nest, kidney, liver, pancreas and brain.Other cancers include but is not limited to following: leukaemia (such as acute leukemia, Acute lymphoblastic leukemia), (such as myeloblast, promyelocyte, Myelomonocyte, monokaryon are thin for acute myelocytic leukemia Born of the same parents, erythroleukemia and myelodysplastic syndrome), chronic leukemia it is (such as chronic myelocytic (granulocyte) leukaemia, chronic Lymphocytic leukemia, hairy cell leukemia), polycythemia vera, lymthoma (such as Hodgkin's disease, non-Hodgkin's Disease), (such as depression type multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, thick liquid cell are white for Huppert's disease Blood disease, solitary plasmacytoma and extramedullary plasmacytoma), Walden Si Telunshi macroglobulinemia, not qualitative monoclonal Gamma-globulin mass formed by blood stasis, benign monoclonal gammopathy, heavy chain disease, bone and connective tissue sarcoma (such as osteoma, osteosarcoma, Chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, the fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, blood vessel meat Tumor (angiosarcoma), fibrosarcoma, Kaposi sarcoma, leiomyosarcoma, embryonal-cell lipoma, lymphangioendothelial sarcoma, neurinoma, cross Line muscle tumor, synovial sarcoma), brain tumor (such as glioma, astrocytoma, brain stem glioma, ependymoma, few Prominent glioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pincocytoma, Pinealoblastoma, primary brain lymthoma), breast cancer (such as gland cancer, leaflet (cellule) cancer, intraductal carcinoma, mammary gland marrow sample Cancer, mucus breast cancer, tubular breast cancer, mamillary breast cancer, osteitis deformans and inflammatory breast cancer), kidney Upper gland cancer (such as pheochromocytoma and adrenocortical carcinoma), thyroid cancer (such as mamillary or thyroid follicular cancer, first shape Gland cephaloma and anaplastic thyroid carcinoma), cancer of pancreas (such as insulinoma, gastrinoma, glucagonoma of pancreas, Vipoma (vipoma), Somatostatin Secretion tumour, class cancer or islet-cell tumour), hypophysis cancer (such as Cushing's disease, Prolactin tumour, Acromegalia and diabetes insipius), cancer eye (such as ophthalmomelanoma such as iris melanoma, choroidal melanoma and ciliary body Melanoma, retinoblastoma), carcinoma of vagina (such as squamous cell carcinoma, gland cancer and melanoma), carcinoma of vulva (such as squamous cell carcinoma, Melanoma, gland cancer, basal-cell carcinoma, sarcoma and osteitis deformans, cervical carcinoma (such as squamous cell carcinoma and gland cancer), uterine cancer are (such as Carcinoma of endometrium and sarcoma of uterus), oophoroma (such as epithelial ovarian cancer, borderline tumor, germ cell tumors and mesenchymoma), the cancer of the esophagus (such as squamous cell carcinoma, gland cancer, adenoid cystic carcinoma, mucoepidermoid carcinoma, gland carcinoma squamosum, sarcoma, melanoma, plasmacytoma, excipuliform Cancer and oat cell (cellule) cancer), gastric cancer (such as gland cancer, fungi sample growth (polyp), ulcer, superficial diffusion, diffusivity expand Dissipate, malignant lymphoma, embryonal-cell lipoma, fibrosarcoma and carcinosarcotna), colon and rectum carcinoma, liver cancer (such as liver cell Cancer and hepatoblastoma, gallbladder cancer such as gland cancer), cholangiocarcinoma cells (such as mamillary, nodositas and diffusivity), lung cancer it is (such as non-small Cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), gland cancer, large cell carcinoma and Small Cell Lung Cancer), carcinoma of testis (such as gonioma, Seminoma, denaturation, classical (typical case), sperm mother cell, nonseminoma, embryonal carcinoma, teratoma, choriocarcinoma (yolk sac tumor), prostate cancer are such as, but not limited to gland cancer, leiomyosarcoma and rhabdomyosarcoma), carcinoma of penis, carcinoma of mouth (such as squamous cell carcinoma), substrate cancer, salivary-gland carcinoma (such as gland cancer, mucoepidermoid carcinoma and adenoid cystic carcinoma), pharynx cancer (such as Squamous cell carcinoma and verrucous carcinoma), cutaneum carcinoma (such as basal-cell carcinoma, squamous cell carcinoma and melanoma, shallow spread melanocyte Tumor, nodular melanoma, lentigo maligna melanoma, acra lentigo melanoma), kidney (such as clear-cell carcinoma, gland cancer, Hypernephroma, fibrosarcoma, transitional cell carcinoma (renal plevis and/or ureter)), the nephroblastoma, bladder cancer (such as migratory cell Cancer, squamous cell carcinoma, gland cancer, carcinosarcoma), myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, mesothelium Tumor, synovialoma, hemangioblastoma, epithelioma, cystadenocarcinoma, bronchiogenic cancer, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma and cream Head gland cancer, follicular lymphoma, the cancer with p53 mutation, mammary gland, prostate and ovarian hormone dependent tumors, precancerosis Become such as familial adenomatous polyposis and myelodysplastic syndrome.

It can include but is not limited to following by other disease specifics and illness that method provided herein is treated: anaphylaxis disease It is disease, inflammation, asthma, arthritis, encephalitis, rheumatic 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 disease, Heng Nuo- Perhaps inflammation caused by Lan Shi purpura (" FISP "), septic shock, pyemia, encephalomyelitis, colitis, Reperfu- sion, psoriasis, Fibrosis includes lung fibrosis, Parkinson's disease, bradykinesia, flesh is stiff, parkinsonian tremor, Parkinson's disease gait, moves Freeze, depression；Defective long-term memory, Rubinstein Taybi syndrome (RTS), dementia, sleep disturbance, insomnia, appearance Gesture is unstable, dyskinesia, move obstacle, synapse nucleoprotein obstacle, multi-system atrophy, striatum substantia nigra degeneration, oliva brain more Bridge cerebellar atrophy, Shy Drager syndrome, parkinsonian features motor neuron disease, Lewy body dementia, Tau Pathological barrier, Stein-leventhal syndrome, corticobasal degeneration, Frontotemporal dementia；Amyloid protein pathology obstacle, mild cognitive impairment, Ah Alzheimer's disease, Alzheimer disease are with Parkinson's disease, Wilson disease, Hallervorden Spatz disease, Chediak Hagashi disease, 3 spinocebellar ataxia of SCA, the chain myodystony Parkinson's disease of X, Huntington disease, prion disease, Chorea, ballism (ballismus), myodystony tremble, amyotrophic lateral sclerosis (" ALS "), CNS wound, myoclonia With disease relevant to undesirable immune response or illness (for example, organ rejection relevant with organ transplant).

It can include but is not limited to following by the virus infection that method provided herein is treated: human immunodeficiency virus (" HIV "), herpes simplex types 1 virus, herpes simplex types 2 virus, influenza virus, influenza A virus, influenza B virus, pair Influenza virus, human papilloma virus (" HPV "), adenovirus, rhinovirus, hepatitis A virus, hepatitis type 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), edge disease disease Poison, varicellazoster virus, smallpox, morbilli, hydrophobin, arboviruse, cytomegalovirus, mumps virus, spinal cord Poliovirus, Coxsackie B virus, Epstein epstein-Barr virus, rubella virus, assays for parvovirus B 19, coronavirus severe acute respiratory syndrome coronavirus Virus), astrovirus, norovirus, rotavirus and adenovirus.

It can include but is not limited to aspergillosis, blastomycosis, sorosphere by the fungal infection that method provided herein is treated Bacterium disease, cryptococcosis, fungal rhinosinusitis, histoplasmosis, hylactic pneumonia, mucormycosis, paracoccidioidomycosis, spore Silk bacterium disease and paddy pyreticosis.

Can include but is not limited to by the bacterium infection that method provided herein is treated brucellosis, cholera, leprosy, Leptospirosis, shigellosis, trench fever, tularemia, Q heat, Whitmore's disease, Yersiniosis, yaws, wound Hurt vibrio infection, streptococcal infection, staphy lococcus infection and coli-infection.

The invention further relates to the composition including liposome, the liposomal encapsulated antibody or other therapeutic protein, And the method for preparing these liposomal encapsulated antibody or liposomal encapsulated therapeutic protein.Inventor, which has found, to exempt from Epidemic disease globulin and other therapeutic protein contain hydrophobic region or pocket, and the hydrophobic region or pocket, which are provided for, to be directed to The affinity of the hydrophobic core of liposome lipid bilayer., it is surprising that if the antibody of freeze-drying or the exposure of other protein In preformed liposome suspension in an aqueous medium, then antibody and other therapeutic protein can spontaneously mix rouge In plastid.This incorporation does not need, and preferably completely avoids using detergent, surfactant and organic solvent, and It does not need to homogenize, Micro Fluid, filtering or other mechanism for destroying double-layer of lipoid.It is this before or after mixing liposome Incorporation does not need yet, and immunoglobulin or any covalent or even non-covalent of other therapeutic protein is preferably avoided to repair Decorations.Immunoglobulin or other therapeutic protein preferably pass through Non-covalent binding (such as by hydrophobic interaction and/or model The interaction of moral China) in conjunction with lipid alkyl chain, and be not related to using the derivatization rouge containing high-affinity matching combination pair Matter and derivatization albumen matter, such as streptavidin and biotin etc..Obtain containing antibody or containing the lipid of protein Vesica can have 100 or lower, 80 or lower, 60 or lower, 50 or lower, 40 or lower or even 30 or lower rouge The molar ratio of matter (for example, phosphatide) and protein.Protein or the yield of antibody incorporation (encapsulate) be at least about 40%, 50%, 60%, 70% or 80%.The antibody or protein portion of encapsulating are all embedded in the hydrophobic part of double-layer of lipoid, And do not fall into substantially in vesica chamber or with lipid molecular covalent bond, or be covalently bound to the matched combination of high-affinity point Son such as streptavidin or biotin.The method of encapsulating be it is mild, retain biological function, and liposome packet The antibody or protein of envelope can be frozen the dry doubling then reconstruct before use (such as being administered to patient).

Embodiment

Embodiment 1: the not preparation of the liposome of drug containing: 6%L- α-phosphatidyl choline (soybean) liposome.

Using magnetic stirring apparatus in environment temperature with 200rpm progress 10 minutes L- α-phosphatidyl cholines (soybean) by 6g It is dispersed in 100mL water.The liposome (multilayer) of dispersion is set to pass through Microfluidic homogenizer in 15,000psi.Lead to three times Crossing circulation causes liposomal diameter to be less than 100nm.Then trehalose is added in liposome to final concentration of 10% (w/w).It will The stable isotonic liposome of gained is used with liquid or lyophilized form.

Embodiment 2: with the preparation for the liposome that lapachol is encapsulated.

10 minutes are carried out by 200mg lapachol and 6g L- α-phosphatide with 200rpm in environment temperature using magnetic stirring apparatus Phatidylcholine (soybean) is dispersed in 100 μ l.The liposome (multilayer) of dispersion is set to pass through Microfluidic homogeneous in 15,000psi Device.Cause to be less than 100nm with the liposomal diameter that 2mg/mL lapachol is encapsulated by circulation three times.Then rouge is added in trehalose To final concentration of 10% (w/w) in plastid.By the stable isotonic liposome encapsulated with lapachol of gained with liquid or freeze-drying shape Formula uses.

Embodiment 3: with the preparation of cyclosporin encapsulating and the liposome of microemulsified.

10 minutes are carried out by the 500mg ring in 5mL Mygliol with 200rpm in environment temperature using magnetic stirring apparatus Spore rhzomorph and 6g L- α-phosphatidyl choline (soybean) are dispersed in 100mL water.Make the liposome (multilayer) of dispersion 15, 000psi passes through Microfluidic homogenizer.The liposomal diameter encapsulated with 5mg/mL cyclosporin is led to by circulation three times Less than 100nm.Then trehalose is added in liposome to final concentration of 10% (w/w).By gained it is stable use cyclosporin It encapsulates and the isotonic Liposomal formulation of microemulsified is used with liquid or lyophilized form.

Embodiment 4: with the preparation for the liposome that transferrins is encapsulated.

10 minutes are carried out by 200mg transferrins and 6g L- α-phosphorus with 200rpm in environment temperature using magnetic stirring apparatus Phosphatidylcholine (soybean) is dispersed in 100mL water.The liposome (multilayer) of dispersion is set to pass through Microfluidic in 15,000psi Homogenizer.Cause to be less than 100nm with the liposomal diameter that 5mg/mL transferrins is encapsulated by circulation three times.Then by trehalose It is added in liposome to final concentration of 10% (w/w).By gained it is stable with transferrins encapsulate isotonic Liposomal formulation with Liquid or lyophilized form use.

Embodiment 5: with the preparation for the liposome that colchicin is encapsulated.

10 minutes are carried out by 6mg enuatrol, 10g trehalose and 6g with 200rpm in environment temperature using magnetic stirring apparatus L- α-phosphatidyl choline (soybean) is dispersed in 100mL water.Pass through the liposome (multilayer) of dispersion in 15,000psi Microfluidic homogenizer 10 times.In the 100 μ L colchicins incorporation prefabricated liposome of 1mL being dissolved in acetone and freeze It is dry.After freeze-drying, gained is stable to be substantially free of an organic solvent with the isotonic freeze-dried lipidosome of drug encapsulation.After freeze-drying, product 1mg/mL, 2mg/mL, 3mg/mL or 4mg/mL aqueous solution (such as water for injection) can be reconstructed into.

Embodiment 6: the antibody encapsulating in liposome.

Many therapeutic proteins, including antibody have hydrophobic part.In one embodiment, antibody, such as other Hydrophobic protein or hydrophobic drug are the same, can be and being added to antibody in preformed liposome by liposome Encapsulating.Surprisingly, it has been found that the hydrophobic part of antibody can be encapsulated in the alkyl chain portion of liposome bilayer by liposome membrane In point.(such as reacted by mercaptan-maleimide or peptide reacts) is (including anti-by protein with previously passed chemical modification for this Body) with lipid conjugation method be contrasted.In the present invention, it does not need to be chemically modified therapeutic protein, and Protein keeps its natural structure.

HERCEPTIN be suitable for breast cancer adjuvant treatment, the breast cancer be HER2 be overexpressed and lymph node positive or Lymph Node-negative (ER/PR is negative or has a high risk feature).HERCEPTIN with it is sterile, white to light yellow, without anti- The freeze-dried powder form of rotten agent provides, for intravenously applying.Each of HERCEPTIN is used for multiple times bottle and contains 440mg song Trastuzumab, 400mg α, α -1,1- trehalose dihydrate close object, 9.9mg L-Histidine HCl, 6.4mg L-Histidine and 1.8mg Polysorbate20, USP.

A bottle HERCEPTIN is reconstructed with 20mL water for injection, obtains the solution containing 21mg/mL Herceptin, pH About 6.0.The aliquot of solution containing 1.75mg Herceptin is lyophilized in 3mL bottle, and by by 200 μ L's 6% soy phosphatidylcholine (SPC) liposome (being prepared using Micro Fluid) is added in lyophilized antibodies to reconstruct, and is finally resisted Bulk concentration is 8.75mg/mL (being added after antibody without Micro Fluid, need to only be gently mixed).Sample is analyzed by size exclusion HPLC Product use 100mM sodium sulphate and 100mM acetic acid by the way that 10 μ L samples are injected on Tosoh G3000SWXL solvent resistant column Sodium pH 6.0 is used as mobile phase.HUMIRA sample without liposome is used as control (referring to Figure 1A).Statistics indicate that being based on it In the elution of voidage, the Herceptin of 5.6mg/mL is encapsulated in liposome (Figure 1B).

The composition of liposome HERCEPTIN is shown in table 1 below.

The composition (1mL composition) of 1. liposome Herceptin (HERCEPTIN) of table

Component	mg
		Soy phosphatidylcholine	60
Trehalose	109.63
		Herceptin	8.75
Histidine HCl	1.97
		Histidine	0.13
Polysorbate20	0.04

HUMIRA is a kind of tumor necrosis factor (TNF) blocking agent, is suitable for treatment: rheumatic arthritis (RA), silver bits Sick arthritis, ankylosing spondylitis, Crohn's disease and plaque psoriasis.HUMIRA is with sterile, preservative free A Damu Monoclonal antibody solution provides, and is used for subcutaneous administration.The drug is with disposable pre-filled pen (HUMIRA Pen) or with disposable The 1mL prefilled glass syringe used provides.HUMIRA solution is limpid colourless, and pH value is about 5.2.Each syringe delivering 0.8mL drug products, containing 40mg adalimumab, 4.93mg sodium chloride, 0.69mg sodium dihydrogen phosphate dihydrate, 1.22mg disodium phosphate dihydrate, 0.24mg sodium citrate, 1.04mg citric acid monohydrate close object, 9.6mg mannitol, 0.8mg polysorbate80 and water for injection, USP.Sodium hydroxide is added as needed to adjust pH.

Aliquot containing 1.75mg adalimumab is lyophilized in 3mL bottle, then with 200 μ L 6%SPC rouge Plastid is reconstructed to the final concentration of 8.75mg/mL of antibody.Sample is analyzed by SEC-HPLC, by injecting 10 μ L samples, is used Tosoh G3000SWXL solvent resistant column uses 100mM sodium sulphate and 100mM sodium acetate pH 6.0 as mobile phase.Humira Sample is used as the control of not liposome., it is surprising that statistics indicate that, based on the elution in voidage, by 5.6mg/ MLHerceptin is encapsulated in liposome (Fig. 1 C).These are statistics indicate that the hydrophobic pocket of Herceptin is embedded in double-deck phosphatide Without any chemical modification between the alkyl of phatidylcholine.

The composition of liposome HUMIRA is shown in the following table 2.

The composition (1mL composition) of 2. liposome adalimumab (HUMIRA) of table

Component	mg
		Soy phosphatidylcholine	60
Trehalose	100
		Adalimumab	8.75
Sodium chloride	0.216
		Sodium dihydrogen phosphate	0.030
Disodium phosphate dihydrate	0.053
		Sodium citrate	0.009
Citric acid monohydrate closes object	0.046
		Mannitol	0.420

By liquid (that is, water-soluble, be not lyophilized) prepared product that antibody is added in the aqueous suspension to liposome To obtain similar antibody incorporation.

All references cited herein is incorporated herein in its entirety by reference, and for all purposes, Degree is as clearly and individually pointed out that each individual publication, patent or patent application are incorporated herein by reference in their entirety All purposes.

Claims (17)

1. a kind of liposomal encapsulated antibody compositions, it includes the hydrophobic film region Non-covalent bindings with multiple liposomes Multiple antibody molecules.

2. the liposomal encapsulated antibody compositions of claim 1, without the antibody molecule being covalently conjugated.

3. the liposomal encapsulated antibody compositions of claim 1, wherein the molar ratio of lipid and antibody is about 50 or lower.

4. the liposomal encapsulated antibody compositions of claim 1, wherein the multiple liposome suspends in an aqueous medium.

5. the liposomal encapsulated antibody compositions of claim 4, wherein the concentration of antibody is at least about in the aqueous medium 5mg/mL。

6. the liposomal encapsulated antibody compositions of claim 1 are freeze-dryings.

7. the liposomal encapsulated antibody compositions of claim 1, wherein the multiple liposome includes soy phosphatidylcholine.

8. the liposomal encapsulated antibody compositions of claim 1, wherein the composition includes trehalose.

9. the liposomal encapsulated antibody compositions of claim 1, substantially free of the hydrophobic film not with multiple liposomes The antibody molecule that region combines.

10. the liposomal encapsulated antibody compositions of claim 1, wherein the antibody is that Herceptin or A Damu are mono- It is anti-.

11. a kind of method for preparing liposomal encapsulated antibody compositions, the method comprise the steps of:

(a) antibody of freeze-drying or dissolution and the aqueous suspension of liposome are provided；

(b) contact the antibody and the aqueous suspension of the liposome, to make the hydrophobicity of the antibody and liposome Diaphragm area combines.

12. the method for claim 11, also includes:

(c) antibody of the unbonded antibody in conjunction with liposome is separated.

13. the method for claim 12, also includes:

(d) the liposomal encapsulated antibody compositions are lyophilized.

14. the method for claim 11, wherein after antibody is added, do not further use homogenize, shear, Micro Fluid or mistake Step (b) is carried out in the case where filter.

15. the method for claim 11, wherein at least 60% antibody is encapsulated.

16. the method for claim 11, wherein the liposome includes soy phosphatidylcholine, and the antibody is toltrazuril Monoclonal antibody or adalimumab.

17. the method for claim 11, wherein the antibody is not exposed to detergent or organic solvent, and wherein without using infiltration Thoroughly or pH gradient or the antibody is loaded into liposome without using by the aqueous suspension freeze thawing of liposome.