WO2000025747A1

WO2000025747A1 - Liposome with lowered toxicity

Info

Publication number: WO2000025747A1
Application number: PCT/JP1999/005976
Authority: WO
Inventors: Yuji Kasuya; Junichi Okada
Original assignee: Sankyo Company, Limited
Priority date: 1998-10-29
Filing date: 1999-10-28
Publication date: 2000-05-11
Also published as: AU6366599A

Abstract

Object: a liposome having a lowered toxicity while sustaining the capability of being retained in the blood. Means for solution: a liposome having a lowered toxicity while sustaining the capability of being retained in the blood, wherein a positively charged compound is employed as a constituent of a liposome modified with a negatively charged nonionic water-soluble polymer to thereby lessen the negatively charged state.

Description

Ming ma Ribosome with reduced toxicity [Technical field]

In physiologic pH aqueous media, add a positively charged compound as a component to ribosomes modified with a negatively charged nonionic water-soluble polymer to reduce the negatively charged state? Also, the present invention relates to a liposome modified with a nonionic water-soluble polymer, the toxicity of which is reduced by making it non-charged.

[Background technology]

Drug carriers such as liposomes are often used to maintain drug concentration in the living body, to ensure the retention of drugs in the blood, or to target drugs to disease sites. .

The retention of ribosomes in the blood can be enhanced by imparting a hydrated structure to the surface of the microparticles by modification with a nonionic water-soluble polymer such as polyethylene glycol (hereinafter referred to as PEG). (See K. Maruyama et al., Biochim. Biophys. Acta, 44-49 (1992)). Therefore, it has been practiced to improve the blood retention of a drug by using a liposome modified with a nonionic water-soluble polymer as a drug carrier.

Thus, liposomes with high blood retention modified by nonionic water-soluble polymers are widely recognized as useful in the field of biomedicine (A. Rollland, eds.

See "Pharmaceutical particulate carriers: therapeutic applicationsj, Marcel Hyekker Inc. (1993) / D. Lasic and F. Martin (eds.)," Stealth liposomesj, CRC Press (1995).).

However, the nonionic water-soluble polymer used for the modification has toxicity such as hemolysis (see Japanese Patent Application Laid-Open No. 7-173079). (DDLasic, "Liposomes: from physics to applicationsj, Elsevier Science Publishers, pp.305-307 (1993) reference. ).

Furthermore, a ribosome composed of a negatively charged lipid, a positively charged lipid, and a neutral lipid modified with a nonionic water-soluble polymer has already been disclosed (see Japanese Patent Application Laid-Open No. 10-506395). However, the charge state and toxicity of the liposome have not been clarified.

[Disclosure of the Invention]

(Problems to be solved by the invention)

The present inventors have found that ribosomes themselves modified by nonionic water-soluble polymers have toxicity. Therefore, it was thought that the usefulness of the liposome would be enhanced by reducing the toxicity of the liposome.

(Means for solving the problem)

The present inventors have formulated a negatively charged non-ionic water-soluble polymer-modified liposome in a physiological pH aqueous medium, and further blended a positively charged compound as a component to form a negatively charged liposome. We have found that reducing the state or making it uncharged reduces toxicity, and has solved the above-mentioned problems.

That is, the present invention further incorporates a positively charged compound as a component into a liposome modified with a nonionic water-soluble polymer that is negatively charged in an aqueous medium at physiological pH, It relates to a liposome that has been made less charged or uncharged.

In the present invention, a positively charged compound is a compound having a positive charge greater than the negative charge in an aqueous medium having a physiological pH, that is, about pH 6.5 to 7. Examples of the positively charged compound used in the present invention include a positively charged lipid, a cationic surfactant and a cationic water-soluble polymer. One or more of these can be used. Here, as a positively charged lipid, for example, ① Alkylamines such as stearylamine;

② 3- / 3 -— [N- (Ν ', N'-dimethylaminoethane) dirubamoyl] Cholesterol-like amine derivatives of cholesterol;

③ Ν- α - trimethyl § emissions ammonio acetyl didodecyl over D- Gurutame such as single Tok port lie de Ν- α _- trimethylammonioacetyl di (C 1 0 to 2 0 alkyl or alkenyl carbon atoms) one D- Glutamate chlorides;

④ Ν— [1— (2,3-Dioleyloxy) propyl] Ν, Ν, — 'N—like trimethylammonium chloride Ν— [1— (2,3—di (carbon Alkyl or alkenyl) oxy) propyl] — 0, Ν, Ν-trimethylammonium chloride;

And positively charged lipids such as

Here, examples of the alkyl group include a pentyl, hexyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, hexacosyl, octacosinole, and triacontacil group. Is mentioned.

These alkyl groups are preferably alkyl groups having 5 to 30 carbon atoms, and more preferably alkyl groups having 10 to 20 carbon atoms.

Examples of the above "alkyl or alkenyl having 10 to 20 carbon atoms" include, for example, decyl, pendecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, decenyl, decynyl, pendecinyl. , Dodecyl or tridecynyl groups. Of these positively charged lipids, preferred are alkylamine, Ν-α-trimethylammonioacetyldi (alkyl or alkenyl having 10 to 20 carbon atoms) -D-daltamitol, and more preferred. Examples include Ν-α-trimethylammonio-ioacetyldidodecyl-1-D-glutamate. In the present invention, in particular, cationic surfactants and cationic water-soluble polymers can also be used as positively charged compounds. As the cationic surfactant, those known to those skilled in the art (M. Rosen (author), Tsubone and Sakamoto (translation) “Surfactants and interfacial phenomena”, pp.16-20, Frederance Journal Inc. (1995) See, for example).

this! : As a cationic surfactant, for example,

① Long-chain alkylamine represented by the following formula or its salt

R ¹

R ² -N

R ³

[In the formula, at least one of R ^{1 to} R ³ represents an alkyl group, and the other represents an organic group. ], For example, stearylamine or dimethylstearylamine and the like;

② Long-chain alkyl or long-chain aralkyl quaternary ammonium salt represented by the following formula

R ¹

^{^{^{R 2 - N + - R 4}}} · X - R 3

[In the formula, at least one of R ^{1 to} R ⁴ represents an alkyl group or an aralkyl group, and the other represents an organic group. X represents a monovalent negative ion. And, for example, trimethyl stearyl ammonium chloride, benzalkonium chloride or benzedemidine chloride;

③ N-long chain alkyl nitrogen-containing aromatic quaternary ammonium salt

: Here, N-long-chain alkyl means that an alkyl group is bonded to a nitrogen atom contained in an aromatic ring to form an ammonium ion, which forms a pair with a monovalent negative ion. ], For example, N-stearylpyridinium chloride, etc .;

ポリ Polyoxyethylene-added long-chain alkylamine represented by the following formula or a salt thereof R ⁵ -N [(CH ₂ CH ₂₀ ) _n -H] ₂

[Wherein, R ⁵ represents an alkyl group, and n represents an integer of 1 to 20]. ], For example, N, N-dipolyoxyethylene stearylamine and the like;

ポリ Polyoxyethylene-added long-chain alkyl quaternary ammonium salt represented by the following formula R ⁶ -N ⁺ -[(CH ₂ CH ₂₀ ) _m -H] ₂ · X-

R ⁷ [In the formula, at least one of R ⁶ and R ⁷ represents an alkyl group, and the other represents an organic group. X— represents a monovalent negative ion. ], For example, N, N-dipolyoxyethylene mono-N-methylstearylamine or the like;

長 Long-chain alkylamine oxide represented by Koji formula

R ⁸

^{^{R 9 - 1 N + -.}} 0- H +

R ¹⁰

[In the formula, at least ^{one of} R ^{8 to} R ¹ ° represents an alkyl group, and the other represents an organic group. Examples thereof include N, N-dimethylstearylamine oxide and the like. Examples of the alkyl groups (1) to (4) include, for example, pentyl, hexyl, octyl, nonyl, decyl, dodecyl, tetradecinole, hexadecyl, octadecyl, icosinore, docosi Λ ^, tetracosinole, hexacosinole, octocosinole, and tria. And a contacyl group.

Examples of the aralkyl group include a benzyl group and a phenethyl group, and preferably a benzyl group.

The alkyl moiety of these aralkyl groups is preferably an alkyl group having 1 to 10 carbon atoms, and more preferably an alkyl group having 1 to 5 carbon atoms.

Examples of the monovalent negative ion include a halogen ion, preferably a chloride ion, a bromine ion, and an iodine ion, and more preferably a chloride ion. Of these cationic surfactants, preferred are long-chain alkylamines or salts thereof, long-chain alkyl or long-chain aralkyl quaternary ammonium salts, polyoxyethylene-added long-chain alkylamines or salts thereof, and polyoxyethylene-added length. Chain alkyl quaternary ammonium salts and long-chain alkylamine oxides, more preferably long-chain alkylamines or salts thereof, long-chain alkyls or long-chain alkylamines. Quaternary ammonium salts and polyoxyethylene-added long-chain alkylamines or salts thereof. Particularly preferred are long-chain alkylamines or salts thereof. '' These cationic surfactants disrupt ribosomes at high concentrations, but in small amounts can be incorporated into ribosomes and become constituents (Urbaneja et al., Biochem. J. 270, 305-308 (1990)). .). Therefore, either an amount of cationic surfactant that does not inhibit liposome formation or an amount of cationic surfactant that does not destroy the formed liposome is added. By adding a cationic surfactant to the liposome dispersion and adsorbing it on the ribosome surface, it becomes one of the constituent components of the ribosome, and the negatively charged state of the polymer-modified ribosome can be reduced. is there. Examples of the cationic water-soluble polymer used in the present invention include cationic water-soluble polymers known to those skilled in the art (see G. Allen et al. (Eds.), "Comprehensive polymer sciencej vol. 6, Pergamon Press (1989)). Examples of such a cationic water-soluble polymer include, for example,

① Polyamines such as polyvinylamine and polyallylamine, poly-N, N-dimethylaminoethyl acrylate, poly-N, N-dimethylaminoethyl methacrylate, poly-N, N-N-ethylaminoethyl acrylate And cationic monomer units such as polyacrylates such as poly (N-N, N-dimethylaminoethyl methacrylate);

Vinylol alcohol, methylvinyl alcohol, vinylolpyrrolidone, vinyloxazolidone, biermethyloxazolidone, 2-vinylpyridine, 4-vinylpyridine, N-vinylsuccinimide, N-vierformamide, N-vinyl-N-methylformamide, N-vinylacetamide, N-butyl-N-methylacetamide, 2-hydroxyethyl methacrylate, atarylamide, methacrylamide, N, N-dimethylacrylamide, N-iso-propyl oleamide, diacetone acrylamide, methyl acrylamide, acryloyl morpholine, acryloyl pyrrolidine, a Cryloylbiperidine, styrene, chloromethylstyrene, bromomethylstyrene, vinyl acetate, methylmethacrylate, butyrylacrylate, methylcyanoacrylate, ethylcyanoacrylate, n-propylcyanoacrylate, n-propylcyanoacrylate, iso-Procyl cyanoacrylate, n-butyl cyanoacrylate, iso-butyl cyanoacrylate, tert; -butyl cyanoacrylate, dalicydyl methacrylate, ethyl vinyl ether, n-propyl At least one non-ionic compound having a double bond, such as bier ether, iso-propyl vinyl ether, n-butyl vinyl ether, iso-butyl vinyl alcohol, tert-butyl vinyl alcohol, etc. Cationic water-soluble composed of one unit of nonionic monomer Cycloalkenyl-based synthetic polymer;

② Cationic water-soluble polyamino acids such as polylysine and polyarginine;

③ Cationic water-soluble synthetic polypeptide composed of different monomer units selected from monomer units of cationic amino acids such as lysine and arginine;

カチオン One unit of cationic amino acid monomer such as lysine and arginine and glycine, alanine, norin, leucine, isoleucine, proline, feninolealanine, serine, threonine, asparagine, glutamin A cationic water-soluble synthetic polypeptide composed of at least one monomer unit selected from monomer units of a nonionic amino acid such as

多 Polysaccharides such as chitosan, agglutinin, acoyutase, nitrite reductase, asparaginase, acetylcholinesterase, adenosine deaminase, α-amylase, 3-amylase, albumin, insulin, and elastase , Elastin a, calcitonin, glucagon, concanavalin 8, cytochrome c, DNA polymerase, transferrin, troponigi, hemoglobin, lactoferrin, lysozyme, immunoglobulin G and other proteins; Cationic water-soluble natural polymers such as;

カチオン a cationic water-soluble modified natural polymer such as getylaminoethyl dextran; 高分子 a polymer selected from the cationic water-soluble polymers ① to ⑥, and the above-mentioned cationic water-soluble which is different from the polymer A cationic water-soluble polymer having, as a structural unit, a polymer selected from polymers;

高分子 A polymer selected from the cationic water-soluble polymers ① to ⑦ A block copolymer or a graft copolymer containing an on-soluble water-soluble polymer as a constituent unit;

プ A block composed of a polymer selected from the cationic water-soluble polymers 1) to 4) and a monomer unit which is a constituent unit of a water-insoluble polymer such as ethylene, propylene or dimethylsiloxane. Polymer or graft copolymer (Whether the copolymer is water-soluble or water-insoluble depends on the ratio and / or arrangement of each monomer unit in the polymer, The cationic polymers that can be used in the present invention are those having a ratio and / or sequence that renders them water-soluble :);

And the like. Among these cationic water-soluble polymers, preferably, a cationic water-soluble vinyl-based synthetic polymer, a cationic water-soluble polyamino acid, a cationic water-soluble synthetic polypeptide, a cationic water-soluble natural polymer or Examples include cationic water-soluble modified natural polymers, more preferably cationic water-soluble vinyl-based synthetic polymers, cationic water-soluble polyamino acids or cationic water-soluble synthetic polypeptides, and particularly preferably cationic. Water-soluble vinyl synthetic polymers. Unlike the adsorption of low molecular weight compounds, the adsorption of these cationic water-soluble polymers is stable and irreversible. The adsorption of high molecules on solid surfaces is stable (G. Allen et al. (Eds.), “Comprehensive polymer science. 2, pp. 733-754, Pergamon Press (1989).) Cationic water-soluble polymers therefore reduce the negatively charged state of the liposome by adsorbing to negatively charged ribosomes. In the ribosome for the purpose of the present invention, the above positively charged compound is mixed with a liposome modified with a nonionic water-soluble polymer which is negatively charged in an aqueous medium having a physiological pH. It is a ribosome that has a reduced negative charge or is uncharged in an aqueous medium at physiological pH.

Here, the aqueous medium is i) water,

ii) buffer,

iii) Water and Z or buffer comprises 5 0 wt ^_0/0 or more, ethanol, medium and water-soluble organic solvent other components such as Jimechirua cell Toami de,

iv) a medium in which an electrolyte and a water-soluble substance such as sugar or saccharide are dissolved in any of the media of i) to iii);

Means either.

Positively charged and positively charged means that the surface zeta potential is positive.Negatively charged and negatively charged means that the surface zeta potential is negative. Means that the zeta potential on the surface is zero. The liposome modified with the nonionic water-soluble polymer in the present invention,

A) Lipid

and,

B) Non-ionic water-soluble polymer bound by a force via --CO (CH ₂ ) ₂ CO-- group or --CO-- group, or by a direct dehydration condensation reaction (hereinafter referred to as "modification").

Holland 小, Pharmaceutical particulate carriers: therapeutic applications, Marcel Dekker Inc. (1993) / D. Lasic and F. .Martin (eds.), Rstealth HposomesJ, CRC Press (1995).)

The above lipids include neutral lipids and negatively charged lipids, which are generally used as components of liposomes.

Here, neutral lipid means a lipid having the same number of positive and negative charges in an aqueous medium at physiological pH, for example,

① Phosphatidic acid derivatives such as dipalmitoylphosphatidylcholine, phosphatidylethanolamine, etc .;

② glycolipids such as digalactosyl glyceride and galactosyl glyceride; ③ Sphingocin derivatives such as sphingomyelins;

ス Cholesterols such as cholesterol, enolegosterone, lanostero, etc .;

Of these neutral lipids, phosphatidic acid derivatives, glycolipids or sterols are preferred,

More preferred are phosphatidic acid derivatives and sterols,

Even more preferably a phosphatidic acid derivative,

Particularly preferred is di (alkanoyl or alkenoyl having 10 to 22 carbon atoms) phosphatidylcholine,

Optimally, dipalmitoyl phosphatidylcholine.

Here, the "alkanoyl or alkenoinole having 10 to 22 carbon atoms" group includes, for example, desilyl, pendecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadesilyl, heptadecyl, octadecyl, nonadecyl, Icosyl, henicosinole, docosyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, icosenyl or docosenyl group.

Di (alkanoyl or alkenol having 10 to 22 carbon atoms) is a carboxylic acid having an alkyl group and a no or alkenyl group having 10 to 22 carbon atoms in two hydroxyl groups of phosphatidylcholine, respectively. It means that it has an ester bond.

Sterols such as cholesterol are themselves used as constituents of liposomes, but can be used as needed in addition to other neutral lipids. By negatively charged lipid is meant a lipid that has more negative charge than positively charged in an aqueous medium at physiological pH, e.g.,

① Phosphatidylglycerols such as dipalmitoylphosphatidylglycerol;

② phosphatidylserines such as dipalmitoylphosphatidylserine;

③ phosphatidylinositol such as dipalmitoyl phosphatidylinositol Le;

And the like.

Of these negatively charged lipids, phosphatidyl glycerols are preferred, and dipalmitoyl phosphatidyl glycerol is particularly preferred. Examples of the lipid modified with the nonionic water-soluble polymer include, for example,

① i) N- (nonionic water-soluble polymer) represented by the following formula: succinylphosphatidylethanolamine

(Non-ionic water-soluble polymer) -COCH ₂ CH ₂ CO-NH-PE

[Wherein,--NH-PE represents an atomic group in which one hydrogen atom has been eliminated from the nitrogen atom of phosphatidylethanolamine. For example, N-monomethoxypolyethylene glycol-succinylphosphatidylethanolamine,

i)) Ν-(nonionic water-soluble polymer) carbonylphosphatidylethanolamine

(Nonionic water-soluble polymer) -CO-NH-PE

[Wherein, Η Η Ρ Ε represents an atomic group in which one hydrogen atom has been eliminated from the nitrogen atom of phosphatidylethanolamine. And, for example, Ν-monomethoxypolyethylene glycol carbonyl phosphatidyl ethanoamine,

Negatively charged lipids modified with a nonionic water-soluble polymer such as;

② Cholesteryl ether represented by the following formula (nonionic water-soluble polymer)

(Nonionic water-soluble polymer) -O-C h

[In the formula, -O-Ch represents that the hydroxyl group of cholesterol and the nonionic water-soluble polymer are bonded by ether. And a neutral lipid modified with a nonionic water-soluble polymer such as polyoxyethylene cholesteryl ether (see H. Ishiwata et al., Chem. Pharm. Bull. 43, 1005-1011 (1995)). ;

③ N— (Nonionic water-soluble polymer) represented by the following formula: Succinyl 2,3-dialkanoyloxypropylamine

(Non-ionic water-soluble _{_{polymer) -COCH 2 CH 2 CO-NHCH}} 2 CH in _{(0-COR) CH 2 0} -COR [ wherein, R represents an alkyl group of 1 0 to 2 0 carbon atoms. ], For example N-Mono Positively charged lipids modified with a nonionic water-soluble polymer such as methoxypolyethylene glycol sacshell-1,2,3-distear yloxypropylamine;

And the like. Among the lipids modified with these nonionic water-soluble polymers,

Preferably, N- (nonionic water-soluble polymer) succinyldi (alkanol or alkenoyl having 10 to 22 carbon atoms) phosphatidylethanolamine or N- (nonionic water-soluble polymer) carbonyldi (carbon An alkanol or an alkenol of the formulas 10 to 22) phosphatidylethanolamine,

More preferably, N— (non-ionic water-soluble polymer) succinyl distearoylphosphatidylethanolamine or N— (non-ionic water-soluble polymer) carboxy-norestea-leuinolephosphatidinole It is an ethanol monoamine,

Still more preferably, N— (nonionic water-soluble polymer) succinyl distearoyl phosphatidylethanolamine,

Particularly preferred is N-monomethoxypolyethylene glycol succinyl disteayl phosphatidylethanolamine.

Here, the “alkanoyl or alkenoinole group having 10 to 22 carbon atoms” has the same meaning as described above.

Di (alkanoyl or alkenoyl having 10 to 22 carbon atoms) refers to a carboxylic acid having an alkyl group having 10 to 22 carbon atoms and a Z or alkenyl group at two hydroxyl groups of phosphatidylethanolamine, respectively. It means that the acid has an ester bond. The above-mentioned "nonionic water-soluble polymer" refers to a polymer having no dissociating group except an end in an aqueous medium such as water or a buffer solution, or a polymer in which the end of the polymer is an alkoxy. It is.

Examples of such nonionic water-soluble polymers include, for example,

① Vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2-bulpyridine, 4-bulpyridine 、 -Vinyl succinimide, Ν-vinyl formamide, Ν-vier-Ν-methylformamide, Ν-vieracetamide, Ν-vininole-Ν-methyl-acetamide, 2-hydroxyhexylmethacrylate Rate, acrylamide, methacrylamide, Ν, Ν-dimethylatarylamide, N-iso-propylacrylamide, diacetoneacrylamide, methylacrylamide, atariloylmorpholinamide Acryloylpyrrolidine, acryloyl olepiperidine, styrene, chloromethylstyrene, bromomethylstyrene, biel acetate, methyl methacrylate, butyl acrylate, methyl lucyano acrylate, ethyl cyanoacrylate , N-propyl cyanoacrylate, iso-propyl cyanoacrylate , N-butyl cyanoacrylate, iso-butyl cyanoacrylate, tert-butyl cyanoacrylate, glycidyl methacrylate, ethynole vinyl ether, n-propyl vinyl ether, iso-propyl vinyl Nonionic vinyl polymer or its high molecule containing one monomer unit such as ether, n-butyl vinylinole, iso-butynole vinyl ether, and tert-butinole vinylinole A polymer having a terminal alkoxylated;

(2) Any one monomer selected from amino acids such as glycine, alanine, noculin, leucine, isoleucine, proline, phenylalanine, serine, threonine, asparagine, and glutamine. A nonionic polyamic acid having a unit as a constituent component or a macromolecule in which a terminal of the macromolecule is alkoxylated;

③ One or more monomer units selected from amino acids such as glycine, alanine, norin, leucine, isoleucine, proline, phenylalanine, serine, threonine, asparagine, and glutamine. A nonionic synthetic polypeptide or a polymer in which the terminal of the polymer is alkoxylated;

非 a nonionic polyester comprising a monomer unit selected from glycolic acid and lactic acid as a component, or a polymer in which the terminal of the polymer is alkoxylated;

非 A nonionic polyether containing a monomer unit selected from glycols such as methylene glycol, ethylene glycol, n-propylene glycol, iso-propylene glycol, and hydroxypropylene glycol, or A polymer in which the terminal of the polymer is alkoxylated;

非 Non-ionic natural substances such as sugars such as dextran, pectin, and pullulan A molecule or a polymer in which the terminal of the polymer is alkoxylated;

非 Nonionic modified natural polymers such as celluloses such as methylcellulose and hydroxypropinoresenorelose, or macromolecules in which the terminals of the polymers are alkoxylated;

プ a block polymer or a graft copolymer having two or more kinds of polymers different from the above-mentioned ① to 構成 as constituent units, or a copolymer in which the terminal of the copolymer is alkoxylated; it can. Of these non-ionic water-soluble polymers, non-ionic polyesters, non-ionic polyesters, non-ionic polyamino acids or non-ionic synthetic polypeptides or those having a non-alkoxy terminal are preferably alkoxylated. More preferably a nonionic polyether or nonionic polyester or a polymer in which the terminal of these polymers is alkoxylated, and still more preferably a nonionic polyether or It is a non-ionic monoalkoxypolyester, particularly preferably polyethylene glycol or monomethoxypolyethylene daryl, and most preferably monomethoxypolyethylene glycol.

The average molecular weight of these nonionic water-soluble polymers is preferably from 1000 to 1200, more preferably from 1000 to 500. Examples of the aqueous phase constituting the inside of the ribosome include an aqueous sodium chloride solution, various commonly used buffers, an aqueous glucose solution, and an aqueous trehalose solution. In addition, lipids having various carbon numbers and / or degrees of unsaturation, such as egg yolk lecithin and soybean lecithin, which are usually available in a mixed state, should be used without separating and purifying them into single components. Can be. Further, if necessary, additives such as α-tocopherol can be used as lipid constituents for the purpose of antioxidant action and the like. Among the above lipids, various lipid types and mixtures are considered, taking into account the morphological stability of ribosomes during storage and in vivo, substance permeability, inclusion properties of the target drug, and pharmacokinetic properties of liposomes. The ratio is selected. The diameter of the target liposome in the present invention varies depending on the type of size adjustment method, but the lower limit of the liposome that can be produced is about 20 nm (DD 丄 asic, “liposomes: from basic to applicationsj, See Elsevier Science Publishers, pp. 3-7 (1993).) The lower limit of the liposome size is not subject to any other special restrictions in the present invention: On the other hand, the upper limit can be administered intravascularly. Emulsification In accordance with the standards for injectables (see the 13th Revised Japanese Pharmacopoeia, The Japanese Compendium, pp. 11 (1996)), it must be 7 μm or less. Alternatively, in consideration of the accumulation property on a diseased tissue such as a tumor or inflammation, the volume average particle diameter is preferably from 50 to 400 nm, more preferably from 50 to 100 nm. desirable.

Here, the volume average particle diameter means the average value of the particle diameter calculated from the average volume of a plurality of particles, and a method known to those skilled in the art using a particle diameter measuring device (for example, RRC ew ^ “Liposomes : a practical approach, pp. 154-160, see IRL Press (1989).) The zeta potential, or charge state, on the surface of the fine particles is determined by the charge density, if well known to those skilled in the art. And the type and amount of electrolyte adsorbed from the medium (see B. Jirgensons and ME Stramanis, A short textbook of colloid chemistry (Second revised ed.), Pergamon Press (1962)). Therefore, one of the factors that makes the liposome modified with a nonionic water-soluble polymer negative in a physiological PH aqueous medium is that the charge density defined by the following equation is negative. Can be mentioned:

Charge density = [(moles of positive charge / moles of negative charge) / (moles of total lipid)] 100 (%). As described above, the components that supply a negative charge to the liposome include negatively charged lipids such as phosphatidylglycerols, phosphatidylserines, and phosphatidylinositols, and N-monomethoxypolyethylene glycol succinyl. Di (alkanoyl or alkenoyl having 10 to 22 carbon atoms) phosphatidylethanolanolamine, N-monomethoxypolyethyleneglycoloxycarbonyldi (alkanoyl or alkenoyl having 10 to 22 carbon atoms) non-phosphatidylethanolamine Negatively charged lipids modified with a zwitterionic water-soluble polymer can be mentioned.

On the other hand, when the charge density is close to 0 and the electrolyte adsorption does not strongly depend on the electrostatic interaction, the phenomenon of preferential adsorption of the anion is generally observed (B. Jirgensons and See ME Stramanis (Author), Bunichi Tamami (translation), "Colloid Chemistry", Baifukan (1967).) From the above, in the present invention, a liposome modified with a water-soluble polymer is “in a negative charge state” in an aqueous medium at physiological pH, which means that the liposome is substantially the following (a) to (a) (c) means any of the above cases.

(a) A negatively charged lipid modified with a nonionic water-soluble polymer is used as a component of a negatively charged liposome, and the negatively charged lipid is modified with a nonionic water-soluble polymer. The charge density becomes negative, and the zeta potential on the liposome surface becomes negative;

(b) A positively charged or neutral lipid modified with a nonionic water-soluble polymer is used as a component of a liposome, where a positively charged lipid modified with a nonionic water-soluble polymer or Negatively charged lipids are contained in excess of other positively charged compounds, resulting in a negative charge density of the liposome modified with the nonionic water-soluble polymer, and a decrease in the surface of the liposome. One potential becomes negative,

(c) The charge density of the liposome modified with the nonionic water-soluble polymer is close to 0, and preferential adsorption of anions from the aqueous medium occurs, and the zeo-electrode on the liposome surface The position becomes negative.

In the present invention, the ribosome modified with a negatively charged nonionic water-soluble polymer in any of these cases is also intended, and by adding a positively charged compound thereto, the `` negative This reduces the state of charge.

Here, that the negatively charged state of the ribosome for the purpose of the present invention is small means that the zeta potential on the liposome surface is preferably from 150 mV to 0 mV, more preferably from 1 mV to 0 mV. It means 30 mV or more and O mV or less, particularly preferably 20 mV or more and O mV or less. these

(1) A positively charged compound is added as a component to a liposome modified with a nonionic water-soluble polymer that is negatively charged in an aqueous medium at physiological pH to reduce the negative charge state. Or a non-charged liposome, preferably

(2) The ribosome according to (1), comprising as components positively charged compounds, lipids, and lipids modified with a nonionic water-soluble polymer.

(3) The liposome according to (1) or (2), which contains as components positively charged compounds, neutral lipids, negatively charged lipids, and negatively charged lipids modified with a nonionic water-soluble polymer. One,

(4) The positively charged compound is one or more selected from a positively charged lipid, a cationic surfactant and a cationic water-soluble polymer, and the positively charged compound is one selected from (1) to (3). Ribosome,

(5) The liposome according to any one of (1) to (4), wherein the positively charged compound is a positively charged lipid.

(6) The positively charged lipid is alkylamine, 3 — — [N— (Ν ', Ν'-dimethylaminoethane) phorbamoyl] cholesterol, Ν—α-trimethylammonio diacetyldi (10 to 20 carbon alkyl or Alkenyl) D—Glutamine Toride or または — [1— (2,3-Dioleyloxy) propyl] One, two or more selected from trimethyl, ammonium, and trimethylammonium chloride The ribosome according to (4) or (5),

(7) The ribosome according to any one of (4) to (6), wherein the positively charged lipid is Ν- _α -trimethylammonioacetyldidodecyl-D-daltamite.

(8) The liposome according to (1), wherein the positively charged compound is a cationic surfactant selected from (1) to (4). (9) The cationic surfactant is a long-chain alkylamine or a salt thereof, a long-chain alkyl or aralkyl quaternary ammonium salt, a polyoxyethylene-added long-chain alkylamine or a salt thereof, or a polyoxyethylene addition. Long-chain alkyl quaternary ammo

(4) The ribosome according to (4) or (8), wherein the ribosome is at least one member selected from the group consisting of a dimethyl salt and a long-chain alkylamine oxide.

(10) The cationic surfactant is one or two selected from a long-chain alkylamine or a salt thereof, a long-chain alkyl or long-chain alkenyl quaternary ammonium salt and a polyoxeti'len-added long-chain alkylamine or a salt thereof. More than species

(4) liposomes as described in (8) or (9),

(11) The ribosome according to any one of (4) and (8) to (10), wherein the cationic surfactant is a long-chain alkylamine or a salt thereof.

(1 2) the ribosome according to one selected from (1) to (4), wherein the positively charged compound is a cationic water-soluble polymer;

(13) The cationic water-soluble polymer is a cationic water-soluble synthetic vinyl polymer, a cationic water-soluble polyamino acid, a cationic water-soluble synthetic polypeptide, a cationic water-soluble natural polymer, and a cationic water-soluble polymer. The liposome according to (4) or (12), which is one or more selected from water-soluble modified natural polymers.

(14) The cationic water-soluble polymer is one or more selected from a cationic water-soluble bubble-based synthetic polymer, a cationic water-soluble polyamino acid, and a cationic water-soluble synthetic polypeptide. (4), (12) or (13) liposome,

(15) The liposome according to any one of (4) and (13) to (14), wherein the cationic water-soluble polymer is a cationic water-soluble vinyl-based synthetic polymer. ,

(16) The neutral lipid is one or more selected from phosphatidylcholine, phosphatidylethanolamine, digalactosylglyceride, galactosylglyceride, sphingomyelin, cholesterol, ergosterol and lanosterol ( 3) the ribosome described,

(17) The neutral lipid according to (3) or (16), wherein the neutral lipid is phosphatidylcholine. Ribosome,

(18) the liposome according to (3), (16) or (17), wherein the neutral lipid is dipalmitoyl phosphatidylcholine;

(1) The liposome according to (1), wherein the neutral lipid is phosphatidylcholine and cholesterol, wherein the liposome is selected from (3) and (16) to (18).

(20) The liposome according to (1), wherein the neutral lipid is dipalmitoyl phosphatidyl choline and cholesterol, and is selected from (3) and (16) to (19).

(21) Negatively charged lipid modified with nonionic water-soluble polymer is N— (nonionic water-soluble polymer) Succinylphosphatidylethanolamine and Z or N— (nonionic water-soluble polymer) The ribosome according to (3), which is carbonylphosphatidylethanolamine;

(22) Negatively charged lipid modified with non-ionic water-soluble polymer is N- (non-ionic water-soluble polymer) succinyl distearoyl phosphatidylethanolamine and or N- (non-ionic water-soluble polymer). Molecule) carbonyl distearoylphosphatidylethanolamine (3) or (21)

(23) The negatively charged lipid modified with a nonionic water-soluble polymer is N- (nonionic water-soluble polymer) succinyl distearoylphosphatidylethanolamine (3), (21) or ( 22 2) the ribosome according to,

(24) Nonionic water-soluble polymer is a nonionic bullet polymer, nonionic polyamino acid, nonionic synthetic polypeptide, nonionic polyester, nonionic polyether, nonionic (1) to (3) and (1) to (3) and one or more types selected from ionic natural polymers or nonionic modified natural polymers, or polymers in which the terminals of these polymers are alkoxylated. (22) The ribosome according to one selected from (23),

(25) Nonionic water-soluble polymer is nonionic vinyl polymer, nonionic polyamino acid, nonionic synthetic polypeptide, nonionic polyester or nonionic polyester Alternatively, the ends of these polymers The ribosome according to any one of (1) to (3) and (22) to (24), which is one or more selected from alkoxylated polymers,

(2) The nonionic water-soluble polymer is one or two selected from nonionic polyesters and nonionic monoalkoxypolyesters (1) to (3) and (22) The ribosome of one of the ribosomes selected from (25) to (25),

(27) Non-ionic water-soluble polymer is monomethoxy polyethylene glycol

(1) the ribosome according to one selected from (3) and (22) to (26),

(28) The average molecular weight of the nonionic water-soluble polymer is from 100 to 1200

The ribosome according to any one of (1) to (3) and (22) to (27),

(29) The average molecular weight of the nonionic water-soluble polymer is from 100 to 500

(1) the ribosome according to one selected from (3) and (22) to (28),

(30) The ribosome according to (3), wherein the negatively charged lipid is one or more selected from phosphatidylglycerol, phosphatidylserine, or phosphatidylinositol.

(31) The ribosome according to (3) or (30), wherein the negatively charged lipid is dipalmitoyl phosphatidylglycerol.

(32) The liposome according to one of (1) to (31), which has a volume average particle diameter of 20 nm to 7 μm,

(33) the ribosome according to one selected from (1) to (32), wherein the volume average particle diameter is 50 nm to 400 nm;

(34) The liposome according to any one of (1) to (33), wherein the volume average particle diameter is 50 nm to 100 nm. Negatively charged liposomes modified with positively charged compounds with nonionic water-soluble polymers By reducing the negatively charged state of the physiological pH in an aqueous medium or making it uncharged, the ribosome of the present invention is produced as follows. The lipids used in the present invention are commercially available (Nippon Oil & Fats Co., Ltd., etc.), or these reagents are extracted and purified from living bodies such as plant seeds, pulp, animals, fish, etc. by methods well known to those skilled in the art. (See, for example, Niro Shiokawa, "Kirkusuma-ichi Kagaku Encyclopedia", Maruzen Co., Ltd., pp.573-575 (1988).)

The cationic surfactant used in the present invention is commercially available (Nippon Oil & Fats Co., Ltd.) or an organic synthesis method well known to those skilled in the art (for example, SHPine, “Organic Chemistrv fifth editi.onJ, For example, long-chain alkylamines can be prepared by a nucleophilic substitution reaction of a long-chain alkyl halide with ammonia. (See McGraw-Hill International Editions (1987).)

The cationic water-soluble polymer used in the present invention is commercially available (Sigma Chemical Company, etc.).

In addition, for the cationic water-soluble vinyl-based synthetic polymer, a general polymer synthesis method (for example, G. Allen et al. (Eds.), “Comprehensive See Pergamon Press (1989), polymer sciencej vols.3, 4. The polymer can be produced by the addition polymerization method, respectively, and the cationic polyamino acid-polypeptide is also nonionic water-soluble as described later. Similar to the method for producing a polymer, a general peptide synthesis method (for example, M. Bodanszky, "Principles of peptide synthesisj, Springer-Verlag (1984) ZM.Bodanszky et al.," The practice of peptide synthesisj, Springer-Verlag ( 1984).), Respectively.

The nonionic water-soluble polymer used in the present invention is commercially available (eg, Sigma Chemical Co.).

For nonionic water-soluble vinyl polymers, nonionic water-soluble polyesters and nonionic water-soluble polyesters, general polymer synthesis methods (for example, G. Allen et al. (Ife), According to "Comprehensive polymer sciencej vols. 3-5, Pergamon Press (1989)"), each is produced by an addition polymerization method and a polycondensation method, respectively. be able to. For example, polyvinyl alcohol, a type of vinyl polymer, can be obtained by addition polymerization of vinyl acetate using a polymerization initiator such as benzoyl peroxide and deacetylation under alkaline conditions. Can be. Polyglycolic acid, which is a kind of polyester, can be polycondensed by heating glycolic acid under reduced pressure. Polymethylene glycol which is a kind of polyether, i.e., a polymer having methylene dallycol as a monomer unit, can be obtained by ring-opening polymerization of cyclic formal using a polymerization initiator such as trifluoromethansulfonic acid. Can be.

In addition, nonionic water-soluble polyamino acids and nonionic water-soluble synthetic polypeptides can be prepared by a general peptide synthesis method (for example, M. Bodanszky, ^ Principles of peptide synthesis J, Springer-Verlag (1984) Z iv. See Bodanszky et al., “The practice of peptide synthesis”, Springer-Verlag (1984).) For example, polyglycine, which is a polyamino acid, can be produced by converting glycine into N-carboxyanhydride by the action of phosgene, and then reacting with a polymerization initiator. Synthetic polypeptides generally involve a process of dehydrating and condensing a C-terminally protected amino acid or peptide with an N-terminally protected amino acid or peptide, followed by deprotection of the N-terminal. It can be manufactured by repeating. In addition, the graft copolymer and the block copolymer can be produced using a polymer reaction or the like according to the method described in G. Allen et al. (徧 “Comprehensive polymer sciencej vol. 6, Pergamon Press (1989)”). The lipid modified with the nonionic water-soluble polymer used in the present invention can be prepared by an organic synthesis method known to those skilled in the art (for example, see SHPine, “Organic Chemistry fifth edition”, McGraw-Hill International Editions (1987)). For example, N-monomethoxypolyethyleneglycolsuccinyl sphatidylethanolamine can be produced by bonding succinic anhydride to a hydroxyl group at one end of monomethoxypolyethylene daricol. To form a carboxyl group and an amino group of phosphatidylethanolamine by dehydration condensation. It can be.

Using these raw materials, methods known to those skilled in the art (DD （asic, liposomes: from basic to applications ", Elsevier Science Publishers pp. 63-107 (1993). ) To produce ribosomes and adjust the size. That is, using the lipid selected above and a sodium chloride aqueous solution, a buffer solution or a glucose aqueous solution, etc., by a thin film method, a reverse phase evaporation method, an ethanol injection method, an ether injection method, a dehydration-rehydration method, or the like. It can be manufactured and its size can be adjusted by a method such as an ultrasonic irradiation method, an ultrasonic irradiation method after freeze-thawing, an extrusion method, a French press method or a homogenization method. Also in the present invention, liposomes are produced according to these methods, and the size of the ribosome is adjusted as necessary.

In addition, since both cationic surfactants and cationic water-soluble polymers are water-soluble, liposomes are added to a dispersion of liposomes modified with a nonionic water-soluble polymer prepared in advance. Components. The method of adding these cationic compounds is an effective method when the drug to be included in the ribosome is cationic. That is, the incorporation efficiency of a charged drug into the liposome (the ratio of the amount of the drug included to the prescribed amount of the drug) can be increased by using lipids having the opposite sign to the drug as components of the ribosome. Yes (see DD 丄 asic, “liposomes: from oasic to applications”, Elsevier science Publishers, pp. 307-317 (1993)). Drugs adsorbed on the outer surface of ribosomes are easily desorbed. The charge state of the resulting drug-containing liposomes is governed by the charge state of the lipids that make up the ribosome, and is likely to be negative, thus efficiently containing the cationic drug and reducing the negative charge state. Although it is generally difficult to prepare a liposome containing a cationic drug efficiently using a negatively charged lipid as a component, a cationic surfactant or cationic water-soluble By adding a molecule, it is possible to efficiently incorporate a cationic drug and to reduce the negatively charged state of the ribosome, and to incorporate the drug into the ribosome according to a method well known to those skilled in the art. (See DD 丄 asic, “liposomes: from basic to applications,” Elsevier Science Publishers, pp. 307-317 (1993).) For example, a drug with high lipophilicity considers the drug as a component of lipids. By mixing with other lipids to produce liposomes, Drugs with high water solubility can be incorporated into liposomes by dissolving them in the aqueous phase to produce liposomes.

When actually manufacturing a liposome modified with a non-ionic polymer containing a drug, the degree of charge of the liposome modified with the non-ionic polymer is used. Is determined by the nature of the drug (eg, toxicity, charge state, etc.). A drug composed of a liposome modified with a nonionic polymer containing a drug has a liposome-constituting lipid concentration of 1 to 300 mM, usually calculated from the composition at the time of formulation. If necessary, dilute using a buffer, physiological saline or an aqueous solution of saccharides or salts, or concentrate by centrifugation, and add a total volume of 1 to 100 m1 to the human vein. Administer by intravenous injection or 100 ml to 1000 ml by intravenous infusion.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between the zeta potential and the charge density of the ribosome prepared in Example 1 in PBS (pH 7.4).

[Fig. 2] Relationship between maximum dose (MTD) of liposomes produced in Example 1 for CDF1 mice and zeta potential (zetapotentia1) in PBS (pH 7.4) (each The number on the plot indicates the number of the prescription example, and the plots of prescription examples 2 to 5 indicate that the MTD is greater than 5 mgZkg.)

[Figure 3] Changes in plasma liposome residual amount after intravenous administration of the liposomes prepared in Example 1 and Comparative Example 1 to Wistar rat (mouth: liposomes of Formulation Example 11; : Ribosome of Formulation Example 1, Δ: Ribosome of Formulation Example 3.)

[Best Mode for Carrying Out the Invention]

Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, and Test Examples, but the present invention is not limited thereto.

Cholesterol is a product of Tokyo Chemical Industry Co., Ltd., and the other lipids are products of Kawahara Yuka Co., Ltd. [Example 1: Production of liposome modified with nonionic water-soluble polymer having different zeta potential] Dipalmitoylphosphatidylcholine (dipalmitoy phosphatidylcholine), a neutral lipid Hereafter referred to as DPPC), cholesterol, negatively charged lipid dipalmitoyl-phosphatidylglycerol (hereinafter referred to as DPPG), and positively charged compound N-α-trimethylammonioacetylzide Decyl-D-glutamate slide (Ν · α-trimethylammonioacetyldidodecyl-D-glutamate chloride ^, referred to as TMA), polyethylene glycol, a negatively charged lipid modified with a nonionic water-soluble polymer N-monomethoxypolyethylene glycol resin with a molecular weight of about 200,000 Di Noree data Roh one Honoré A Mi emissions (N- monomethoxypoly (ethylene glycol) succinyldistearoylphosphatidylethanol- amine 0 Kawahara Yuka KK. Hereinafter referred to PEG-DSPE.), A fluorescent labeling agent ribosomes 1, .gamma. Dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine perchlorate (1, l'-dioctadecylindo-3,3,3', 3'-tetramethylindocarbocyanine perchlorate; product of Lambda Probes & Diagnostics. hereinafter, D i I and one Rere. E. Claassen, J. Immunol. methods 147, 231-240 (1992) reference.) and 1 0 wt ⁰ / o preparative Reharosu solution using, Bangham's method (Mol. According to Biol. 8, 660-668 (1964), ribosomes were produced as follows.

That is, a predetermined amount of the above lipids and the like were weighed in an eggplant-shaped flask, and dissolved by adding 5 mL of a black hole form. Next, a thin layer of lipid was formed on the inner wall of the flask by distilling off the form of the mouth under reduced pressure. A predetermined amount of a 10% by weight aqueous solution of trebrose was added to the lipid thin layer, and the mixture was shaken with a vortex mixer to obtain a crude ribosome dispersion having a lipid concentration of 100 mM. Table 1 shows the composition ratio of lipids in the formulation examples.

【table 1】 *

DPPC Cholesterol DPPG TMA GP EG-D SPE Formulation example 1 5 5 4 0 0 0 5

Formulation example 2 5 3 4 0 0 2 5

Formulation example 3 5 0 4 0 0 5 5

Formulation example 4 4 5 40 0 1 0 5

Formulation example 5 3 5 40 0 20 5

Formulation example 6 3 0 40 2 5 0 5

Formulation example 7 2 5 40 2 5 5 5

Formulation example 8 2 0 40 2 5 1 0 5

Prescription example 9 5 0 4 0 5 0 5

Formulation example 1 0 4 5 4 0 1 0 0 5

* The numerical value of each lipid represents the prescribed μπιο1 number. In addition, in all ribosome treatments, the labeling agent DiI was formulated at 0.2 mol% based on the total number of moles of lipid. Next, the volume average particles of ribosomes were determined by an ultrasonic irradiation method (output: 25 W, irradiation time: 5 minutes to 1 hour) using an ultrasonic crusher (Model 7600, manufactured by Seiko Denshi Kogyo KK). The diameter was adjusted so as to be 100 nm or less, which has high blood retention. Here, the volume average particle diameter and the zeta potential were measured using a particle size measuring device (Nicomp Particle Sizer Model 370, manufactured by Nicomp Particle Sizing Systems) and a zeta potential measuring device (ZEM5000, manufactured by Malvern). It measured using each. The sign and magnitude of the zeta potential of the fine particles can be determined by a method known to those skilled in the art (for example, J. Hunter,

"Otsu eta potential in colloid sciencej, pp.125-178, see Academic Press (1981)."

The volume average particle size of these ribosomes, and 25, phosphate buffered saline (comprising 20 mM sodium dihydrogen phosphate and 135 mM sodium chloride, 1N sodium hydroxide The zeta potential in the solution adjusted to pH 7.4 with an aqueous solution of chromium (hereinafter referred to as PBS) is expressed by the following formula: charge density = [(** moles of positive charge -1 ** moles of negative charge) ** Total lipid moles]

X 1 0 0 (%) Both are shown in Table 2 together with the charge densities calculated from the values determined from the lipid loadings during the liposome preparation.

[Table 2]

Charge density Zeta potential Volume average particle size (mo1%) (mV) nm

Formula 1 1 5-2 0 3 9 7 7 ± 4 4 7 Formula 2 2 3 1 1 4 8 8 2 8 ± 3 7 7 Formula 3 0 1 1 3 9 7 8 4 ± 3 2 8 Formula 4 -5 1 1 2 2 9 7 9 ± 4 1 2 Prescription example 5 + 1 5 1 6 0 9 4 8 ± 3 6 3 Prescription example 6 1 3 0-2 7 3 9 8 2 ± 3 5 4 Prescription example Ί- 2 5-2 5 9 9 7 2 ± 3 2 3 Prescription example 8 1 2 0-2 2 7 9 7 1 ± 4 2 5 Prescription example 9 1 1 0 1 2 1 7 9 7 0 ± 3 3 8 Prescription example 1 0-1 5-2 3 9 9 0 3 ± 3 1 2 Volume average particle size ± expressed as the distribution width of the particle size (standard deviation: SD) _c Thus, all liposomes have physiological pH. It was confirmed that the sample was not positively charged in the aqueous medium. Next, using the data in Table 2, the relationship between zeta potential and charge density is shown in FIG. In this way, the charge density of liposomes modified with a nonionic water-soluble polymer (hereinafter referred to as polymer-modified liposomes) and polymer-modified liposomes containing a positively charged compound and PBS The i ^: is closely related to the zeta potential of the liposome, and the zeta potential of the polymer-modified liposome could be controlled by the type of lipid and the composition ratio of the lipid. That is, it was shown that the negatively charged state of the liposome can be brought close to 0 by including the positively charged compound in the polymer-modified liposome. Furthermore, not only when the polymer-modified ribosome has an excess of negative charge, but also when the positive and negative charge densities are equal, It was shown that, even if it had an excess, it was still negatively charged in an aqueous medium at physiological pH. This fact means that the present invention is applied even when the sign of the charge density calculated from the composition ratio at the time of ribosome production is not negative. The volume-average particle diameter of the polymer-modified liposome prepared with the lipid composition shown in Table 1 and the polymer-modified liposome to which the positively charged compound was added was 100 nm or less, and the retention in blood I was able to adjust the size to be excellent.

[Comparative Example 1] Production of ribosome not modified with nonionic water-soluble polymer In the same manner as in Example 1, ribosome not modified with nonionic water-soluble polymer was used according to the formulation shown in Table 3. Hereinafter, this is referred to as a polymer-unmodified ribosome.) Table 4 shows the charge density of the liposome, the zeta potential in PBS and the value of the volume average particle diameter. [Table 3]

* * * * D P P C Cholesterol D P P G T A G P E G-D S P

E_

Prescription example 1 1 7 0 3_0 0 0 0

**** The numerical value of each lipid represents the prescribed μmo 1 number. In addition, the labeling agent Di I was formulated at 0.2 mol% based on the total number of moles of lipid.

[Table 4]

Charge density Zeta potential ***** Volume average particle diameter (m o 1%) (m V) (nm) Formulation example 1 1 0 1 2. 0 9 5.3 ± 3 2. — 8

Volume average particle diameter It is expressed as the distribution width (standard deviation; SD) of soil particle diameter. ;: Test example 1: Toxicity study Disperse the ribosome dispersion prepared in Example 1 in predetermined volumes of 0.1 to 2.5 ml (every 0.3 ml from 0.1 to 1.0 ml, and 1.0 ml to 2 0.5 ml up to 5 ml) was intravenously administered to a CDF1 mouse (manufactured by Nippon Chara Luzriva Co., Ltd.) weighing 20 to 30 g. By observing the survival of the mice for 1 hour after the administration, the maximum ribosome administration volume, that is, the largest administration volume among the administration volumes in which the mice survived, was determined.

The maximum tolerated dose (MTD) is given by the following formula: MTD (g / kg) = [maximum dose (mL) X lipid concentration (gZmL)] / mouse body weight (kg)

It was calculated from:

The MTD thus determined is shown in FIG. 2 as a function of the zeta potential in PBS. In this figure, the numbers attached to the plots indicate the prescription example numbers, and the plots with the vertical axis scaled out indicate that the MTD is larger than 5. S gZkg.

The MTD of the liposome prepared in Example 1 increased as the negative zeta potential of the liposome in PBS approached zero. That is, the toxicity of the polymer-modified liposome was reduced by adding a positively charged compound to the polymer-modified ribosome and bringing the negative zeta potential of the physiological pH in an aqueous medium close to zero.

[Test Example 2] Examination of blood retentivity Formulation Example 1 prepared in Example 1 and Comparative Example 1 and diluted with a 150 mM sodium chloride aqueous solution at a total lipid concentration of 16.7 mM each 3 or 11 liposomal dispersion was added to a Wistar rat (manufactured by Jishikiken Co., Ltd.) weighing 315 to 385 g with a total lipid dose of 47.7 μmo 1 _g. Was administered from the tail vein such that After a predetermined time of 1 to 24 hours, about 500 μl of blood was collected from the tail vein, and plasma was obtained by centrifugation (1 000 g × 5 minutes). The plasma 1 0 mu 1 to 1 50 mM of chloride isocyanatomethyl Li © anhydrous solution 40 mu 1 was added an additional 1 weight ^_0/0 polyethylene glycol one mono -4 Okuchirufue two Rue one ether of (Tokyo Chemical Industry Co., Ltd.. ) Containing PBS 1 Diluted in ml. Using a spectrofluorometer (650-40 type, manufactured by Hitachi, Ltd.), the fluorescence intensity of this plasma dilution (excitation wavelength: 550 nm, fluorescence wavelength: 565 nm) ₀ ). Using this fluorescence intensity and the fluorescence intensity of a 100-fold diluted solution of the administered ribosome preparation in PBS containing 1% by weight of poly (ethylene glycol) -mono- (4-octyl) phenyl ether, Of 5 weight. / ₀ , the volume fraction of plasma in blood is 60% by weight. / ₀ , and the amount of ribosome remaining in the blood was determined. Figure 3 shows the change in the residual amount of liposomes in the blood.

In this figure, the mouth indicates the polymer-unmodified ribosome prepared in Formulation Example 1, 〇 indicates the polymer-modified liposome manufactured in Formulation Example 1, and Δ indicates the high-molecular-weight liposome containing the positively charged compound manufactured in Formulation Example 3. 1 shows a molecularly modified ribosome. From FIG. 3, it can be seen that the liposome of Formulation Example 3 had an excellent retention in blood equivalent to the liposome of Formulation Example 1.

Also, the ribosome of Formulation Example 11 was inferior in blood retention despite having the same volume average particle size as the ribosomes of Formulation Examples 1 and 3.

From this test result, it was clarified that even when a positively charged compound was included as a component, the effect of improving the blood retention by the nonionic water-soluble polymer that modifies liposomes was maintained.

[Possibility of industrial use]

According to the present invention, a liposome modified with a nonionic water-soluble polymer having high blood retention and significantly reduced toxicity as compared with the conventional one was obtained. The liposome for the purpose of the present invention makes it possible to increase the dose of the contained drug by enhancing the safety of the liposome modified with the nonionic water-soluble polymer, and to increase the therapeutic effect of the drug. Highly useful in enhancing.

Claims

The scope of the claims

For ribosomes modified with a nonionic water-soluble polymer that is negatively charged in an aqueous medium at physiological pH, compounded with a positively charged compound as a constituent IS, to reduce or not negatively charged state Libosome charged.