HK1044291A - Liposome preparations - Google Patents

Description

Liposome formulations

Technical Field

The present invention relates to a pharmaceutical liposome preparation containing, as an active ingredient, a 2-pipecolic acid derivative, in particular a macrolide compound, such as a tricyclic compound called tacrolimus (FK 506) or a pharmaceutically acceptable salt thereof, which has recently been receiving particular attention for its excellent immunosuppressive activity. More particularly, the present invention relates to a liposome preparation which contains the above-mentioned active ingredient stably encapsulated in liposomes and thus can be stably dissolved in various media such as physiological saline, glucose solution for injection, water or body fluid, and thus can be used for various administration methods including injection methods such as intravenous injection, intramuscular injection, and local injection for intra-articular use, and the like, topical administration methods such as application to the skin, instillation into the eyes, nasal administration, and inhalation administration, and oral administration methods and rectal administration methods. In particular, the present invention relates to a liposome preparation characterized by being free of cholesterol and having a rapid effect on diseases such as cerebral ischemia by using a rapid bolus injection.

Background

As a liposome preparation containing tacrolimus, a liposome prepared by incorporating a stabilizer such as cholesterol into phospholipid as a main component forming the liposome is known (WO 93/08802). With this structure, a liquid preparation can be made from tacrolimus, which is slightly soluble in water. Even when the preparation is brought into contact with body fluids, crystallization of the active ingredient does not occur, and therefore the preparation has excellent bioavailability and is stable. Thus, such preparations may take any dosage forms typified by injection, eye drops, nasal administration, inhalant, transdermal absorbent, topical injection, and the like. Further, it is also possible to enhance the concentrated migration of tacrolimus to a site where the migration of tacrolimus is particularly desired and to suppress the migration to a site where the migration of tacrolimus is not necessarily desired. It is known that excellent effects such as improvement in drug efficacy, reduction in side effects, and persistence of drug efficacy are obtained as a result.

The liposome preparation has excellent effect on treating cerebral ischemic diseases such as cerebral infarction. However, since the liposome membrane is too stable, the liposome preparation cannot exhibit sufficiently rapid action as an anticoagulant, fibrinolytic agent and cerebral vasodilator for the treatment of cerebral infarction. Therefore, there is a need to develop a drug having an excellent rapid action capable of treating an emergency such as cerebral infarction.

It is an object of the present invention to improve the above-mentioned problems, thereby providing a liposome preparation containing a 2-piperidinoic acid derivative having an excellent rapid action.

Disclosure of Invention

The present invention relates to a liposome preparation containing, as an active ingredient, a 2-pipecolic acid derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof encapsulated in a liposome.

The 2-pipecolic acid derivatives in the present invention mean that they have a common activity of having affinity with FKBP-type immunophilins and inhibiting peptidyl-coa isomerase and/or rotamase enzyme activity, and have a common chemical structure belonging to 2-pipecolic acid derivatives.

Specific examples of the 2-pipecolic acid derivative include macrolide compounds such as tricyclic compounds of the following general formula (I):wherein adjacent pairs of R¹And R²、R³And R⁴And R⁵And R⁶Independently are:

(a) two adjacent hydrogen atoms; or

(b) Another bond may be formed between the carbon atoms to which they are attached; and

R²or may be an alkyl group;

R⁷is hydrogen, hydroxy, protected hydroxy, or alkoxy, or with R¹Together form an oxo group;

R⁸and R⁹Independently a hydrogen atom or a hydroxyl group;

R¹⁰is a hydrogen atom, an alkyl group substituted with one or more hydroxyl groups, an alkenyl group substituted with one or more hydroxyl groups, or an alkyl group substituted with an oxo group;

x is an oxo group, a state where a hydrogen atom and a hydroxyl group are bonded to one carbon atom at the same time, a state where two hydrogen atoms are bonded to one carbon atom, or a group represented by the formula-CH₂One group represented by O-;

y is an oxo group, a state in which a hydrogen atom and a hydroxyl group are bonded to one carbon atom, a state in which two hydrogen atoms are bonded to one carbon atom, or a group of the formula ═ N-NR¹¹R¹²OR N-OR¹³A group represented by;

R¹¹and R¹²Each independently represents a hydrogen atom, an alkyl group, an aryl group or a tosyl group;

R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²²and R²³Independently represents a hydrogen atom or an alkyl group;

R²⁴is an optionally substituted heterocyclic group which may contain one or more heteroatoms in the ring; and

n represents an integer of 1 or 2.

In addition to the above definitions Y, R¹⁰And R²³Together with the carbon atom to which they are attached, may represent a saturated or unsaturated 5-or 6-membered heterocyclic ring containing nitrogen, sulphur and/or oxygen atoms, which may be substituted by one or more of the following groups: alkyl, hydroxy, alkoxy, benzyl, of the formula-CH₂Se(C₆H₅) A group, and an alkyl group substituted with one or more hydroxyl groups.

The definitions used in the above general formula (I) and specific and preferred examples thereof will be explained and set forth in detail.

Unless otherwise indicated, the term "lower" refers to groups having 1 to 6 carbon atoms.

Preferred examples of the alkyl moiety of "alkyl" and "alkoxy" include straight or branched chain aliphatic hydrocarbon groups, for example, lower alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, neopentyl and hexyl.

Preferred examples of "alkenyl" include straight or branched chain hydrocarbon groups having one double bond, for example, lower alkenyl groups such as vinyl, propenyl (e.g., allyl), butenyl, methylpropenyl, pentenyl and hexenyl.

Preferred examples of "aryl" include phenyl, tolyl, xylyl, cumenyl, mesityl, and naphthyl.

Preferred protecting groups in "protected hydroxy" and the later-mentioned "protected amino" include 1- (lower alkylthio) (lower) alkyl, such as lower alkylthiomethyl (e.g., methylthiomethyl, ethylthiomethyl, propylthiomethyl, isopropylthiomethyl, butylthiomethyl, isobutylthiomethyl, hexylthiomethyl, etc.), more preferably C₁-C₄Alkylthiomethyl, most preferably methylthiomethyl;

trisubstituted silyl groups, e.g. tri (lower) alkylsilyl (e.g. trimethylsilyl, triethylsilyl, tributylsilyl, tert-butyldimethylsilyl, tri-tert-butylsilyl, etc.) or lower alkyldiarylsilyl (e.g. methyldiphenylsilyl, ethylsilylA methyldiphenylsilyl group, a propyldiphenylsilyl group, a tert-butyldiphenylsilyl group, etc.), and more preferably a tris (C)₁-C₄) Alkylsilyl and C₁-C₄Alkyldiphenylsilyl groups, most preferably tert-butyldimethylsilyl and tert-butyldiphenylsilyl; and

acyl groups, for example aliphatic acyl groups, aromatic acyl groups or aliphatic acyl groups substituted by aryl groups, derived from carboxylic, sulfonic or carbamic acids.

Examples of the aliphatic acyl group include lower alkanoyl groups optionally having one or more suitable substituent(s) (e.g., carboxyl group), such as formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, valeryl group, isovaleryl group, pivaloyl group, hexanoyl group, carboxyacetyl group, carboxypropionyl group, carboxybutyryl group, carboxyhexanoyl group and the like;

cyclo (lower) alkoxy (lower) alkanoyl optionally having one or more suitable substituent(s) (e.g., lower alkyl), such as cyclopropyloxy acetyl, cyclobutyloxypropyl, cycloheptyloxybutyryl, menthoxyacetyl, menthoxypropionyl, menthoxybutyryl, menthoxypentanoyl, menthoxyphexanoyl and the like;

a camphorsulfonyl group; or

Lower alkylcarbamoyl having one or more suitable substituent(s) (e.g., carboxy or protected carboxy), for example, carboxy (lower) alkylcarbamoyl (e.g., carboxymethylcarbamoyl, carboxyethylcarbamoyl, carboxypropylcarbamoyl, carboxybutylcarbamoyl, carboxypentylcarbamoyl, carboxyhexylcarbamoyl and the like), tri (lower) alkylsilyl (lower) alkoxycarbonyl (lower) alkylcarbamoyl (e.g., trimethylsilylmethoxycarbonylethylcarbamoyl, trimethylsilylethoxycarbonylpropylcarbamoyl, triethylsilylethoxycarbonylpropylcarbamoyl, t-butyldimethylsilylethoxycarbonylpropylcarbamoyl, trimethylsilylpropyloxycarbonylbarbamoyl and the like) and the like.

Examples of the aromatic acyl group include an aroyl group optionally having one or more suitable substituents such as a nitro group, for example, a benzoyl group, a toluoyl group, a ditoluoyl group, a naphthoyl group, a nitrobenzoyl group, a dinitrobenzoyl group, a nitronaphthoyl group and the like; and

arenesulfonyl optionally substituted with one or more suitable substituents (e.g., halogen), for example, benzenesulfonyl, toluenesulfonyl, xylenesulfonyl, naphthalenesulfonyl, fluorobenzenesulfonyl, chlorobenzenesulfonyl, bromobenzenesulfonyl, sulfophenylsulfonyl, and the like.

Examples of the aliphatic acyl group substituted with the aryl group include aryl (lower) alkanoyl optionally having one or more suitable substituent(s) (e.g., lower alkoxy or trihalo (lower) alkyl), such as phenylacetyl, phenylpropionyl, phenylbutyryl, ethyltrifluoromethyl-2-methoxy-2-phenylacetyl, 2-ethyl-2-trifluoromethyl-2-phenylacetyl, 2-trifluoromethyl-2-propoxy-2-phenylacetyl and the like.

More preferred acyl group among the above-mentioned acyl groups is C optionally having a carboxyl group₁-C₄Alkanoyl with two (C) in the cycloalkyl moiety₁-C₄) Ring of alkyl (C)₅-C₆) Alkoxy (C)₁-C₄) Alkanoyl, camphorsulfonyl, carboxy- (C)₁-C₄) Alkylcarbamoyl, tri (C)₁-C₄) Alkylsilyl (C)₁-C₄) Alkoxycarbonyl radical (C)₁-C₄) Alkylcarbamoyl, benzoyl optionally having one or two nitro groups, phenylsulfonyl with halogen, or with C₁-C₄Alkoxy and trihalo (C)₁-C₄) Phenyl (C) of an alkyl group₁-C₄) An alkanoyl group. Among these, most preferable are acetyl, carboxypropionyl, menthoxyacetyl, camphorsulfonyl, benzoyl, nitrobenzoyl, dinitrobenzoyl, iodobenzenesulfonyl and 2-trifluoromethyl-2-methoxy-2-phenylacetyl.

"saturated or unsaturated 5-or 6-membered heterocyclic ring containing nitrogen, sulfur and/or oxygen atoms" includes pyrrolyl and tetrahydrofuryl.

R²⁴Is an optionally substituted heterocyclic ring which may contain one or more heteroatoms, preferably R²⁴Can be a ring (C)_5-7) Alkyl, the following are examples thereof:

(a)3, 4-dioxocyclohexyl;

(b)3-R²⁰-4-R²¹a cyclohexyl group;

wherein R is²⁰Is hydroxy, alkoxy, oxo or-OCH₂OCH₂CH₂OCH₃Group, R²¹Is hydroxy, -OCN, alkoxy, heteroaryloxy which may be substituted by suitable substituents, 1-or 2-tetrazolyl, -OCH₂OCH₂CH₂OCH₃Radical, protected hydroxy, chlorine, bromine, iodine, aminooxalyloxy, azido, p-tolyloxysulfanyloxy or R²⁵R²⁶CHCOO- (wherein R is²⁵Is optionally protected hydroxy or protected amino, R²⁶Is hydrogen or methyl), or R²⁰And R²¹One oxygen atom bonded to each other in an epoxy ring; or

(c) Cyclopentyl substituted with: methoxymethyl, optionally protected hydroxymethyl, acyloxymethyl (wherein the acyl moiety optionally contains a quaternisable dimethylamino group, or an esterifiable carboxyl group), one or more amino and/or hydroxyl groups which may be protected, or aminooxaloxymethyl.

A preferred example is 2-formylcyclopentyl.

The "heteroaryl group which may be substituted by suitable substituents" moiety of "heteroaryloxy group which may be substituted by suitable substituents" may be R for ｃA compound of formulｃA I of EP-A-532,088¹Those listed, preferably 1-hydroxyethylindol-5-yl, the content of which is incorporated by reference in the present application.

The tricyclic compound (1) and pharmaceutically acceptable salts thereof used in the present invention are known to have excellent immunosuppressive activity, antimicrobial activity and other pharmacological activities, thus, it is useful for the treatment or prevention of rejection due to organ or tissue transplantation, graft-versus-host disease, autoimmune disease and infectious disease, the preparation thereof is also known from [ EP-A-018416 2, EP-A-032304 2, EP-A-423714, EP-A-427680, EP-A-465426, EP-A-480623, EP-A-532088, EP-A-532089, EP-A-569337, EP-A-6285, WO89/05303, WO93/05058, WO96/31514, WO91/13889, WO91/19495, WO93/04680, WO93/5059 and the like ], the contents of which are incorporated herein by reference.

In particular, the compounds designated as tacrolimus, FR 900520 (ascomycin), FR 900523 and FR 900525 are obtained from Streptomyces species such as Streptomyces tsukubaensis No.9993[ stored at national institute of bioscience and Industrial science and technology, human institute of technology and Industrial science and technology (institute of fermentation, former Industrial science and technology), 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, Collection date 10/5/1984, accession number FERM BP-927]Or Streptomyces hygroscopicus Ornithogalum subspecies No.7238[ preserved in national institute of bioscience and Industrial science and technology human technology (original institute of Industrial science and technology fermentation), 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, preservation date 1/12 in 1985, preservation number FERM BP-928][EP-A-0184162]. Tacrolimus of the following chemical formula is a representative compound.Chemical name: 17-allyl-1, 14-dihydroxy-12- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylethenyl]-23, 25-dimethoxy-13, 19, 21, 27-tetramethyl-11, 28-dioxa-4-azatricyclo [22.3.1.0^4，9]Dioctadecyl-18-ene-2, 3, 10, 16-tetraone.

Preferred examples of such tricyclic compounds (I) are those in which each adjacent pair of radicals R³And R⁴And/or R⁵And R⁶Independently of each other, form another bond between the carbon atoms to which they are attached;

R⁸and R²³Each independently is a hydrogen atom;

R⁹is a hydroxyl group;

R¹⁰is methyl, ethyl, propyl or allyl;

x is an oxo group, a state where a hydrogen atom and a hydroxyl group are bonded to one carbon atom, a state where two hydrogen atoms are bonded to one carbon atom, or an oxo group;

y is oxo;

R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹and R²²Each is methyl;

R²⁴is a 3-R²⁰-4-R²¹Cyclohexyl radical, wherein R²⁰Is hydroxy, alkoxy, oxo or-OCH₂OCH₂CH₂OCH₃Group, R²¹Is hydroxy, -OCN, alkoxy, heteroaryloxy which may be substituted by suitable substituents, 1-or 2-tetrazolyl, -OCH₂OCH₂CH₂OCH₃Radical, protected hydroxy, chlorine, bromine, iodine, aminooxalyloxy, azido, p-tolyloxysulfanyloxy or R²⁵R²⁶CHCOO-wherein R²⁵Is optionally protected hydroxy or protected amino, R²⁶Is hydrogen or methyl, or

R²⁰And R²¹Together form one oxygen atom in the epoxy ring;

n is an integer of 1 or 2.

In addition to tacrolimus, the most preferred tricyclic compounds (I) are ascomycin derivatives such as halogenated ascomycin (ASM 981) disclosed in example 66 ｃA of EP-A-427680 (e.g. 33-epi-chloro-33-desoxyascomycin), 3 2-O- (1-hydroxyethylindol-5-yl) ascomycin (L-73 2, 531) disclosed in EP-A-532088, 3 2- (1H-tetrazol-1-yl) ascomycin (ABT 281) disclosed in WO93/04680, and the like.

As another preferred example of the macrolide compound, rapamycin [ the Merck Index (12 th edition), No.8288]And derivatives thereofA compound (I) is provided. Preferred examples of derivatives are 0-substituted derivatives in which the hydroxy group in position 40 of formula A shown on page 1 of WO 95/16691 is replaced by-OR₁In place of, wherein R₁Is hydroxyalkyl, hydroxyalkoxyalkyl, amidoalkyl or aminoalkyl; for example, 40-0- (2-hydroxy) ethyl rapamycin, 40-0- (3-hydroxy) propyl rapamycin, 40-0- [2- (2-hydroxy) ethoxy]Ethyl rapamycin and 40-0- (2-acetamidoethyl) rapamycin.

These 0-substituted derivatives can be prepared by reacting rapamycin (or dihydro or deoxo-rapamycin) with an organic group attached to a leaving group (e.g., RX, where R is the organic group intended as the 0-substituent, such as an alkyl, allyl, or benzyl moiety, and X is a leaving group, such as CCl₃C (NH) O or CF₃SO₃) Under appropriate reaction conditions.

The reaction conditions may be acidic or neutral, for example where X is CCl₃C (NH) O in the presence of an acid such as trifluoromethanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid or their respective pyridinium or substituted pyridinium salt, or when X is CF₃SO₃In the presence of a base such as pyridine, substituted pyridine, diisopropylethylamine or pentamethylpiperidine.

Most preferred is 40-0- (2-hydroxy) ethyl rapamycin, disclosed in WO94/09010, the contents of which are incorporated herein by reference.

Tricyclic compound (I) and rapamycin and its derivatives have similar basic structures, i.e., tricyclic macrolide structures, but at least one similar biological property (e.g., immunosuppressive activity).

The pharmaceutically acceptable salts of the tricyclic compound (I), rapamycin and a derivative thereof may be conventional non-toxic and pharmaceutically acceptable salts, for example, salts with inorganic or organic bases, particularly, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, ammonium salt and amine salts such as triethylamine salt and N-benzyl-N-methylamine salt.

With respect to the 2-pipecolic acid derivative and the macrolide compound used in the present invention, it is clear that conformational isomers and one or more pairs of stereoisomeric forms, such as optical isomers due to asymmetric carbon atoms and geometric isomers due to double bonds, may exist, and these conformational isomers and isomers are also included in the scope of the present invention. In addition, the 2-pipecolic acid derivative and the macrolide compound may be in the form of a solvate, which is included in the scope of the present invention. The solvate preferably includes, for example, a hydrate and an ethanolate.

Further, examples of the 2-pipecolic acid derivative which can be an object of the present invention include the following compounds:

(1) the following Way-124466 compound (Ocain et al, Biochemical and Biophysical Research Communications, 192(3), 1993) synthesized by reacting 4-phenyl-1, 2, 4-triazoline-3, 5-dione with rapamycin;

(2) 2-piperidinoic acid derivative compounds called RAP-Pa (Charkraborty et al, Chemistry and Biology, 3.1995, 2: 157-161);

(3) the following 2-piperidinoic acid derivative compounds (Ikeda et al, J.Am.chem.Soc.)1994, 116, 4143-4144);

(4) wang et al, Bioorganic and medicinal Chemistry Letters, 4(9), 1161-1166, 1994, in particular 2-pipecolic acid derivative compounds disclosed as compounds 2a-2 d;

(5) the following 2-piperidinoic acid derivatives (Birkenshaw et al, Bioorganic and Medicinal Chemistry Letters, 4(21), 2501-2506, 1994);

(6) holt et al, journal of the american society of chemistry (j.am. chem.soc.), 1993, 115, 9925-9938, particularly the 2-pipecolic acid derivative compounds disclosed as compounds 4-14;

(7) 2-piperidinoic acid derivative compounds disclosed in Caffer et al, Bioorganic and medicinal Chemistry Letters, 4(21), 2507-2510, 1994;

(8) 2-pipecolic acid derivatives as disclosed by Teague et al in Bioorganic and medicinal chemistry letters, 3(10), 1947-1950, 1993;

(9) yamashita et al, Bioorganic and Medicinal chemistry letters, 4(2), 325-328, 1994, in particular the 2-pipecolic acid derivative compounds disclosed as compounds 11, 12 and 19;

(10) holt et al, Bioorganic and Medicinal Chemistry Letters, 4(2), 315-320, 1994, in particular the 2-pipecolic acid derivative compounds disclosed as compounds 3-21 and 23-24;

(11) holt et al, Bioorganic and Medicinal Chemistry Letters, 3(10), 1977-1980, 1993, especially the 2-piperidinoic acid derivatives disclosed as compounds 3-15;

(12) hauske et al, journal of medicinal chemistry (j.med. chem.), 1992, 35, 4284-4296, particularly the 2-pipecolic acid derivative compounds disclosed as compounds 6, 9-10, 21-24, 26, 28, 31-32, and 52-55;

(13) 2-pipecolic acid derivatives as disclosed by Teague et al in Bioorganic and Medicinal chemistry letters, 4(13), 1581-1584, 1994; and

(14) stocks et al, Bioorganic and Medicinal chemistry letters, 4(12), 1457-1460, 1994, and in particular the 2-pipecolic acid derivative compounds disclosed as compounds 2, 15-17.

The liposome preparation of the present invention mainly comprises the above-mentioned 2-pipecolic acid derivative encapsulated in a liposome, and other conditions such as the structure, composition, preparation method and size of the liposome, and the type of the compound which can be used in combination with the liposome are not specifically limited unless these conditions adversely affect the rapid action of the drug and these conditions protect the 2-pipecolic acid stably encapsulated in the liposome. Thus, the structure of the liposome may be a Large Unilamellar Vesicle (LUV), a multilamellar vesicle (MLV) or a Small Unilamellar Vesicle (SUV). Thus, the particle size may be 200-1000nm for LUV, 400-3500nm for MLV, and 20-50nm for SUV. Preferably, less SUV accumulates in the reticuloendothelial system (RES).

As the liposome constituting the liposome structure, phospholipid and nitro lipid are used. In general, phospholipids are preferred. Examples thereof include natural phospholipids such as egg yolk lecithin (phosphatidylcholine), soybean lecithin, lysolecithin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, cardiolipin, plasmalogen and the like, or hydrogenated products which can be obtained from the phospholipids by a conventional method; and synthetic phospholipids such as dicetyl phosphate, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, tung oil acyl phosphatidylcholine, tung oil acyl phosphatidylethanolamine, tung oil acyl phosphatidylserine, etc., more preferable examples thereof include lecithin, and most preferable is egg yolk lecithin.

Lipids including these phospholipids may be used alone. Or two or more of them may be used in combination. In this case, the electronegative groups in the phospholipid acyl groups of the lipid and the electropositive groups in the ectopic groups (ethanolamine, choline, etc.) bound thereto are electrically balanced, and thus the whole molecule is electrically neutral. For example, lecithin, sphingomyelin, phosphatidylethanolamine, distearoylphosphatidylcholine, and the like are often used alone. Conversely, if the ectopic group (e.g., serine, glycerol, inositol, etc.) bound to the phosphatidyl group (electronegative group) within the lipid is electrically neutral, the lipid as a whole is electronegative, e.g., phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, etc., or an electronegative lipid such as phosphatidic acid or dihexadecyl phosphate, etc., can be used independently as a lipid in the present invention, but is preferably used in combination with, for example, the neutral ester described above. Among them, phosphatidic acid and dicetyl phosphate do not act as main phospholipids in liposome formation, but are known to act as liposome-forming additives.

In view of stability and handling properties of the liposome formulation of the present invention, it is preferable to use additives such as excipients and/or stabilizers.

Preferred stabilizers for use in the present invention include, for example, stearylamine, alpha-tocopherol, gangliosides, acid glycolipid sulfatides; an acidic glycolipid and a glycolipid with a sulfate group.

Excipients preferably used in the present invention include, for example, pharmaceutically acceptable solid sugars, such as monosaccharides (e.g., glucose, galactose, sorbitol, xylitol, mannitol), disaccharides (e.g., sucrose, lactose, maltose, trehalose), with lactose and maltose being more preferred, and maltose being most preferred.

In the case of liposomes prepared using cholesterol, it is difficult to obtain the same level of rapid action as required in the present invention, and cholesterol is likely to have an adverse effect on cerebral infarction in general. Therefore, cholesterol is not used in the present invention.

Alpha-tocopherol can also be used, with antioxidant functionality expected.

The weight ratio of the 2-pipecolic acid derivative to the lipid forming the liposomal structure is not particularly limited as long as it can produce a liposomal preparation having the desired fast-dissolving property, but is generally in the range of 1: 1 to 1: 500, more preferably 1: 5 to 1: 100, and most preferably 1: 10 to 1: 40.

In view of stability and handling properties of the liposome formulation of the present invention, it is preferable to use an excipient. The amount of excipient is not particularly limited, but the ratio of the 2-pipecolic acid derivative to the stabilizer is in the range of 1: 10 to 1: 1000, more preferably 1: 20 to 1: 500, and most preferably 1: 100 to 1: 400.

The amount of the stabilizer to be added for stabilizing the liposome is not particularly limited, but the weight ratio of the stabilizer to the liposome-forming lipid is preferably 0.01 to 5%, more preferably 0.05 to 2%, most preferably 0.1 to 1%.

In view of storage stability, the liposome preparation of the present invention is preferably provided in the form of a solvent-free dry product (e.g., a freeze-dried product), more preferably in the form of a solid preparation, which is dispersed in a pharmaceutically acceptable sugar as an excipient, particularly a liposome preparation comprising a 2-pipecolic acid derivative or a pharmaceutically acceptable salt thereof. In use, the liposome preparation is re-dispersed in a solvent (e.g., distilled water for injection) and then injected, for example, intravenously. In the case of treating cerebral ischemic diseases such as cerebral infarction using the preparation of the present invention, intravenous bolus administration or bolus administration is most preferable. After completion of the redispersion, the concentration of the 2-pipecolic acid derivative is preferably adjusted to 0.01 to 100mg/mL, more preferably 0.05 to 50mg/mL, most preferably 0.1 to 10 mg/mL.

On the other hand, the dose of the 2-pipecolic acid derivative to be administered using the liposome preparation of the present invention is appropriately adjusted depending on the kind of the derivative, the age of the patient and the condition of the disease to be treated. In the case where the 2-pipecolic acid derivative is the tricyclic compound (I), the liposome preparation is preferably administered in a daily dose of about 0.01 to 1000mg, preferably 0.1 to 500mg, more preferably 0.5 to 100 mg.

There is no particular limitation on the particle size of the liposome, as long as the size is within a range that is pharmaceutically acceptable and can exert a rapid action. The size of the liposomes is preferably 1-200nm, more preferably 10-100nm, most preferably 20-80 nm.

If necessary, a filter may be used when the liposome preparation re-dispersed in distilled water for injection is loaded into the syringe. In this case, conventional filters can be used, and a preferable example thereof is a filter having a pore size of 0.2 μm, 0.22 μm, 0.45 μm, 0.8 μm or 5 μm, which is sterile, pyrogen-free and has low protein-binding properties (Millipore SLGV025LS, SLHVM25LS, SLAA025LS, SLSV025LS, etc.).

The liposome preparation of the present invention can be prepared by the same procedure as in the following examples.

Examples

The embodiments of the present invention are for illustration, but the present invention is not limited by the following embodiments, and modifications may be made without departing from the meaning of the present invention described hereinbefore and hereinafter, and such modifications are also included in the technical scope of the present invention.

The following examples of the preparation of the compounds of the present invention will be described in detail.

(1) Preparative example 1 (ethanol injection method): purified egg yolk lecithin (20g) and tacrolimus (1g) were dissolved in ethanol (200mL), and the resulting solution was injected and dispersed into a solution additionally obtained by dissolving lactose (200g) in water for injection (1800mL) with stirring by a homogenizer. After the dispersion was filtered through a 0.2 μm polycarbonate filter, ethanol was removed from the filtered dispersion by evaporation under reduced pressure, and water for injection was added to the dispersion to make the total volume 2000 mL. The resulting dispersion was filtered through a 0.2 μm polycarbonate filter, and a vial was filled with each 10mL of the dispersion, followed by freeze-drying. The resulting freeze-dried liposome preparation was redispersed in 9mL of water for injection to give a liposome dispersion containing 0.5mg/mL of tacrolimus and having an average particle diameter of 61.5nm (measured by dynamic light scattering method, model C370 manufactured by NICOMECO. Co.).

(2) Preparative example 2 (extrusion method): a solution obtained by dissolving purified egg yolk lecithin (5g) and tacrolimus (0.25g) in ethanol (100mL) was vacuum dried to form a film. The film was roughly dispersed in a 10% maltose aqueous solution (500mL) using a vortex mixer. The dispersion was filtered through 400nm, 200nm, 100nm and 50nm polycarbonate filters in that order. Each 10mL of the filtrate was filled into a vial, and then lyophilized. The resulting freeze-dried liposome preparation was redispersed in 9mL of water for injection to give a liposome dispersion containing 0.5mg/mL of tacrolimus and having an average particle diameter of 110nm (measured by dynamic light scattering method, type C370 manufactured by NICOMCol.).

(3) Preparative example 3 (high pressure emulsion process): a solution obtained by dissolving purified egg yolk lecithin (20g) and tacrolimus (1g) in ethanol (10mL) was vacuum dried to form a film. The film was roughly dispersed in 10% maltose water solution (2000mL) using a magnetic stirrer. The dispersion was treated with a high pressure emulsifier manufactured by Nanomizer co. The resulting liposome emulsion was filled into one vial per 10mL and then freeze-dried. The resulting freeze-dried liposome preparation was redispersed in 9mL of water for injection to give a liposome dispersion containing 0.5mg/mL of tacrolimus and having an average particle diameter of 80nm (measured by dynamic light scattering method, type C370 manufactured by NICOMP Co.).

(4) Preparation of example 4

In the same manner as in preparation example 1, lyophilized liposomes were obtained from the following formulation, and a liposome dispersion was prepared with an appropriate amount of water for injection.

Tacrolimus 3mg

Purified egg yolk lecithin 100mg

Lactose monohydrate 1000mg

Constitutes 1103mg

(5) Preparation of example 5

In the same manner as in production example 3, lyophilized liposome preparations were obtained from the following formulation, and liposome preparations 1) and 2) were obtained with an appropriate amount of water for injection

1) Tacrolimus 3mg

Purified egg yolk lecithin 100mg

Alpha-fertility 0.3mg

Maltose 1000mg

1103.3mg2) Tacrolimus 5mg

Purified egg yolk lecithin 100mg

Alpha-tocopherol 0.3mg

Maltose 1000mg

Make up 1105.3mg

The experimental results are explained below.

Normal mice were anesthetized with trifluorobromochloroethane. A catheter (PE50) was inserted into the femoral vein for administration. The rats were housed in stationary cages. The drug obtained in preparation example 3 was administered within about 30 seconds after awakening. Blood samples were collected from abdominal aorta of mice under anesthesia with trifluorobromochloroethane and ice-cooled, and then whole blood (required amount about 1 ml) was collected. At the time of blood collection, 20. mu.L (about 1000u/mL) of heparin was added per 1mL of whole blood. The blood was stored frozen until the concentration was determined. As for the brain tissue, the brain was removed after cutting the head, blood adhering to the periphery of the brain was washed off and wiped with filter paper, and then the wet weight of the brain was weighed. Brains were placed in centrifuge tubes and stored frozen prior to assay. The cerebral cortex was homogenized with distilled water 9 times the wet weight of the brain on the day of submitting the samples. The concentration of the drug in the sample was determined by an enzyme immunoassay described in unexamined patent publication (Kokai) No. 1-92659. It is clear from the results that sufficient concentrations are reached in the brain or blood in a short time, exerting a sufficiently rapid action. Fig. 1 shows the test results: a, the concentration of tacrolimus-containing liposome formulation in the brain; b, the above concentration in blood.

Brief Description of Drawings

Fig. 1 shows a: concentration of tacrolimus-containing liposome preparation in brain, and B: concentration in blood.

Best mode for carrying out the invention

The present invention relates to a liposome preparation containing, as an active ingredient, a 2-pipecolic acid derivative represented by a macrolide compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof, which is encapsulated in a liposome.

A compound containing 17-allyl-1, 14-dihydroxy-12- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl group of the formula (I)]-23, 25-dimethoxy-13, 19, 21, 27-tetramethyl-11, 28-dioxa-4-azatricyclo [22.3.1.0^4，9]Particularly preferred are liposomal formulations of dioctadecyl-18-ene-2, 3, 10, 16-tetraone or a pharmaceutically acceptable salt thereof as an active ingredient. In a preferred construction, lecithin is used primarily as the liposome-forming lipid, and cholesterol is not included in the formulation as a stabilizer.

Particularly preferred is a form of a solid preparation in which a liposome preparation is dispersed in a pharmaceutically acceptable saccharide, the liposome preparation is composed of liposomes containing lecithin as a main component and containing no cholesterol, and a 2-pipecolic acid derivative or a pharmaceutically acceptable salt thereof is encapsulated in the liposomes.

INDUSTRIAL APPLICABILITY

According to the present invention, since liposomes are easily disintegrated compared to conventional liposome preparations containing cholesterol, it can be expected that administration by bolus injection will give more excellent rapid action. In addition, the preparation of the present invention does not contain a surfactant, so it is possible to obtain excellent effects without any influence on the circulatory organs.

Therefore, the liposome preparation of the present invention is particularly useful for the treatment and prevention of diseases in which a rapid onset of drug action is desired, for example, cerebral ischemic diseases (e.g., head injury, intracerebral hemorrhage (e.g., subarachnoid hemorrhage, intracerebral hemorrhage), cerebral infarction, cerebral thrombosis, cerebral embolism, cardiac arrest, stroke, Transient Ischemic Attack (TIA) hypertensive encephalopathy).

Based on the pharmacological effects of the 2-pipecolic acid derivative as an active ingredient, particularly the tricyclic compound (I), the liposome preparation of the present invention is useful for the treatment and prevention of the following diseases and symptoms:

rejection of organ or tissue (e.g., heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, intervertebral disc, trachea, myoblast, cartilage, etc.) transplantation;

graft versus host response after bone marrow transplantation;

autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, hashimoto's goiter, multiple sclerosis, myasthenia gravis, type I diabetes, and the like;

infections caused by pathogenic microorganisms (e.g., aspergillus fumigatus, fusarium oxysporum, trichophyton asteroides, etc.);

inflammatory or hyperproliferative skin diseases or skin conditions of immunomodulating diseases (e.g., psoriasis, atopic dermatitis, contact dermatitis, eczematoid dermatitis, seborrheic dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, epidermal eosinophilia, lupus erythematosus, acne, and alopecia areata);

autoimmune diseases of the eye (e.g., keratoconjunctivitis, vernal conjunctivitis, uveitis associated with behcet's disease, keratitis, herpetic keratitis, keratoconus keratitis, corneal epithelial dystrophy, corneal leukoplakia, ocular pemphigus, Mooren's ulcer, scleritis, graves ' eye disease, Vogt-Koyanagi-Harada syndrome, keratoconjunctivitis sicca (dry eye), small blisters, iridocyclitis, sarcoidosis, endocrine eye disease, etc.);

reversible obstructive airways diseases [ asthma (such as bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or intractable asthma (such as late asthma and airway hyperreactivity), bronchitis and the like ];

mucosal or vascular inflammation (e.g., gastric ulcer, ischemic or thrombotic vascular injury, ischemic bowel disease, inflammatory bowel disease, necrotizing enterocolitis, intestinal injury associated with thermal burns, leukotriene B4-mediated disease, etc.);

enteritis/allergy (e.g., celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, segmental enteritis, and ulcerative colitis);

food-related allergic diseases that show symptoms away from the gastrointestinal tract (e.g., migraine, rhinitis, and eczema);

renal diseases (e.g., interstitial nephritis, goodpasture's syndrome, hemolytic uremic syndrome, diabetic nephropathy) and nephrotic syndrome (e.g., glomerulonephritis);

neurological diseases (e.g., polymyositis, guillain-barre syndrome, meniere's disease, polyneuritis, mononeuritis, cerebral infarction, presenile dementia, parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), and radiculopathy;

endocrinopathies (e.g., hyperthyroidism and basedorf disease);

hematologic disorders (e.g., pure red cell aplasia, aplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, and failure to erythropoiesis);

bone diseases (e.g., osteoporosis);

respiratory diseases (e.g., sarcoidosis, pulmonary fibrosis, and idiopathic interstitial pneumonia);

skin diseases (e.g., dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, photosensitizing diseases, and cutaneous T-cell lymphoma);

circulatory diseases (e.g., arteriosclerosis, atherosclerosis, aortic inflammation syndrome, polyarteritis nodosa, and non-inflammatory cardiomyopathy);

collagen diseases (e.g., scleroderma, wegener's granulomatosis, and schungren's syndrome);

obesity;

eosinophilic fasciitis;

periodontal disease (e.g., damage to the gums, periodontal tissue, alveolar bone, or cementum);

male pattern alopecia, senile alopecia;

hereditary muscular dystrophy;

pyoderma and west zeri syndrome;

diseases associated with chromosomal abnormalities (e.g., down syndrome);

adrenal insufficiency;

active oxygen modulated diseases [ e.g., organ injury (e.g., ischemic circulatory disorder of organs (e.g., heart, liver, kidney, digestive tract, etc.) associated with preservation, transplantation, or ischemic diseases (e.g., thrombosis, myocardial infarction));

intestinal diseases (e.g., endotoxic shock, pseudomembranous colitis, and drug-or radiation-induced colitis);

renal diseases (e.g., ischemic acute renal insufficiency, chronic renal failure);

pulmonary diseases (e.g., poisoning by pulmonary oxygen or drugs (e.g., prednisone, bleomycin, etc.), lung cancer and emphysema);

eye diseases (e.g., cataract, hemochromatosis (siderophy of the eyeball), retinitis, pigmentary spot disease, age spots, vitreous scarring, corneal alkali burn);

dermatitis (e.g., erythema multiforme, linear immunoglobulin a bullous dermatitis, cement dermatitis);

and other diseases (e.g., gingivitis, periodontitis, sepsis, pancreatitis, and diseases due to environmental pollution (e.g., air pollution), age, carcinogens, cancer metastasis, and baropathy);

by histamine release or leukotriene C₄Release-induced disease;

restenosis of the coronary arteries following angioplasty and prevention of postoperative adhesions;

autoimmune diseases and inflammation (e.g., primary mucosal edema, autoimmune atrophic gastritis, premature menopause, male infertility, juvenile diabetes, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, lens-induced uveitis, idiopathic leukopenia, active chronic hepatitis, idiopathic cirrhosis, discoid lupus erythematosus, autoimmune orchitis, arthritis (e.g., osteoarthritis), or polychondritis);

viral (HIV) infection, aids;

allergic conjunctivitis; and

hypertrophic scars and keloids due to trauma, burns or surgery.

Furthermore, macrolide compounds such as the tricyclic compound (I) have liver regeneration activity and/or activity of stimulating hepatocyte hypertrophy and hyperplasia. Accordingly, the pharmaceutical liposome preparation of the present invention can be used to enhance the therapeutic and/or prophylactic effects of liver diseases [ for example, immunogenic diseases (e.g., chronic autoimmune liver diseases such as autoimmune liver diseases, primary biliary cirrhosis or sclerosing cholangitis), partial hepatectomy, acute hepatic necrosis (e.g., necrosis caused by toxins, hepatitis viruses, shock or hypoxia), hepatitis b, non-a non-b hepatitis, cirrhosis, and liver failure (e.g., fulminant hepatitis, delayed hepatitis, and "chronic to acute" liver failure (acute liver failure in the state of chronic liver diseases)) ].

Further, the liposome composition of the present invention can be also used for enhancing the effect of preventing and/or treating various diseases due to the useful pharmacological activities of the tricyclic macrolide, for example, activity of potentiating chemotherapeutic action, cytomegalovirus infection activity, anti-inflammatory activity, inhibitory activity against peptidyl-co-alanine isomerase or rotamase, antimalarial activity, antitumor activity, etc.

Claims (11)

1. A liposome preparation containing a 2-piperidinoic acid derivative, which comprises a 2-piperidinoic acid derivative or a pharmaceutically acceptable salt thereof encapsulated in a liposome.

2. The liposome preparation as defined in claim 1, wherein lecithin is used mainly as the liposome forming lipid.

3. The liposome formulation as defined in claim 2, wherein the liposomes are substantially free of cholesterol.

4. The liposome preparation as defined in claim 1, wherein the 2-pipecolic acid derivative is a macrolide compound of the following general formula (1) or a pharmaceutically acceptable salt thereof:wherein adjacent pairs of R¹And R²、R³And R⁴And R⁵And R⁶Independently are:

(a) two adjacent hydrogen atoms; or

(b) Another bond may be formed between the carbon atoms to which they are attached; and

R²or may be an alkyl group;

R⁷is hydrogen, hydroxy, protected hydroxy, or alkoxy, or with R¹Together form an oxo group;

R⁸and R⁹Independently a hydrogen atom or a hydroxyl group;

R¹⁰is a hydrogen atom, an alkyl group substituted with one or more hydroxyl groups, an alkenyl group substituted with one or more hydroxyl groups, or an alkyl group substituted with an oxo group;

x is an oxo group, a state where a hydrogen atom and a hydroxyl group are bonded to one carbon atom at the same time, a state where two hydrogen atoms are bonded to one carbon atom, or a group represented by the formula-CH₂One group represented by O-;

y is an oxo group, a state in which a hydrogen atom and a hydroxyl group are bonded to one carbon atom, a state in which two hydrogen atoms are bonded to one carbon atom, or a group of the formula ═ N-NR¹¹R¹²OR N-OR¹³A group represented by;

R¹¹and R¹²Each independently represents a hydrogen atom, an alkyl group, an aryl group or a tosyl group;

R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²²and R²³Independently represents a hydrogen atom or an alkyl group;

R²⁴is an optionally substituted heterocyclic group which may contain one or more heteroatoms in the ring; and

n represents an integer of 1 or 2;

provided that, in addition to the above definitions, Y, R¹⁰And R²³Together with the carbon atom to which they are attached, may represent a saturated or unsaturated 5-or 6-membered heterocyclic ring containing nitrogen, sulphur and/or oxygen atoms, which may be substituted by one or more of the following groups: alkyl, hydroxy, alkoxy, benzyl, formula-CH₂Se(C₆H₅) And alkyl substituted with one or more hydroxyl groups.

5. The liposome preparation as defined in claim 4, wherein the macrolide compound (1) is 17-allyl-1, 14-dihydroxy-12- [2- (4-hydroxy-3-methoxycyclohexyl) -1-methylvinyl ] -23, 25-dimethoxy-13, 19, 21, 27-tetramethyl-11, 28-dioxa-4-azatricyclo [22, 3, 1, 04, 9] octacosa-18-en-2, 3, 10, 16-tetraone or a hydride thereof.

6. A solid formulation wherein the liposome formulation as defined in any one of claims 1 to 5 is dispersed in a pharmaceutically acceptable sugar.

7. A solid preparation comprising a liposome preparation in a pharmaceutically acceptable saccharide, wherein the liposome preparation consists of liposomes containing lecithin as a main component and containing no cholesterol, and a 2-pipecolic acid derivative or a pharmaceutically acceptable salt thereof is encapsulated in the liposomes.

8. The liposome preparation as defined in claim 7, wherein the mass ratio between the solid 2-pipecolic acid derivative or the pharmaceutically acceptable salt thereof, lecithin and the pharmaceutically acceptable saccharide is 1: 1 to 500: 10 to 1000.

9. The solid preparation as defined in claim 7 or 8, wherein the saccharide is lactose or maltose.

10. The liposomal formulation as defined in claim 6 which is a freeze-dried product.

11. The liposomal formulation as defined in claim 1 which is dispersed in distilled water for injection.