JPH11302199A - Drug carrier composed of graft copolymer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a graft copolymer useful for forming a fine-grain molecular aggregate of drug carrier which shows excellent drug retentivity in blood or the like by composing of a specific main chain and a specific side chain to make negatively charged in a specific medium. SOLUTION: This graft copolymer is obtained by composing of (A) a polymeric main chain negatively charged at physiological pH in an aqueous medium and (B) a nonionic polymeric side chain to make negatively charged at physiological pH in an aqueous medium. The physiological pH means pH 6.5-7.5. The aqueous medium is either medium consisting of (i) water, (ii) a buffer solution or (iii) at least 50 wt.% of the component i and/or the component ii and a water-soluble organic solvent (e.g. ethanol) or medium obtained by dissolving a water-soluble substance, e.g. an electrolyte, a saccharide or the like, into (iv) some of the components i-iii. As the polymer composing the component A, a vinylic synthetic polymer, a synthetic polyamino acid, a synthetic polypeptide or the like is illustrated, and as the polymer composing the component B, a polyether, a natural polymer or the like is illustrated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an aqueous medium having a physiological pH comprising a polymer main chain and a nonionic polymer side chain which are negatively charged in an aqueous medium having a physiological pH. Graft copolymer to be charged, and fine particle molecule aggregate formed by forming a core material, which is a positively charged material in an aqueous medium at physiological pH, through an electrostatic bond and a shell material which is such a graft copolymer. The present invention relates to a body and a drug carrier in which a drug is included in such a fine particle aggregate.

[0002]

2. Description of the Related Art In order to maintain a drug concentration in a living body and to maintain the blood retention of a drug, a drug carrier composed of a polymer or a liposome is often used. (A. Rolland
(Ed.), “Pharmaceutical particulate carriers: th
erapeutic applications ”, Marcel Dekker Inc. (19
93) / D. Lasic and F. Martin (eds.), “Stealth liposo
mes ", CRC Press (1995)).

[0003] The retention of a drug carrier in blood depends on the liver, spleen,
It is increased by avoiding the capture of the carrier by phagocytic cells present in the lung and the like (DDLasic, "Liposomes:
from physics to applications ", Elsevier Scienc
e Publishers, pp. 261-471 (1993)).

According to previous studies, it is known that the ease of capturing a drug carrier by phagocytic cells can be controlled by improving the physicochemical properties of the carrier, that is, its size, surface potential, surface structure and the like. (L.Illum et al.,
Biomaterials 8, 113-117 (1987) / Y. Ishikawa et al.
J. Biomaterials Sci. Polymer Ed. 1, 53-60 (1991)
/ H. Kawaguchi et al., Biomaterials 7, 61-66 (1986)
/ Y. Tabata et al., J. Colloid Interface Sci. 127, 13
2-140 (1988) / CJvan Oss et al., “Phagocytic eng
alfment and cell adhesiveness '', Marcel Dekker In
c. (1975) / S. Yasukawa et al., J. Microencapsulation 7,
179-184 (1990)), and in particular, modifying the carrier with a water-soluble polymer has been considered effective for this purpose.

The effect of modifying a drug carrier with a water-soluble polymer is affected by the amount of modification of the surface of the drug carrier with the water-soluble polymer. ), The retention of the liposome in the blood increases with the modification density of the PEG, and the liposome exhibits good retention in the blood at a modification density of 5 mol% or more (Maruyama et al., Biochim. Biophys. Ac.
ta 1128 44-49 (1992)). However, P of liposomes
It has been pointed out that when the EG modification density exceeds 5 mol%, the stability of the modified liposome deteriorates (Bedu-Add et al.).
Pharm.Res.13,710-717 (1996) / Bedu-Add et al.Pharm.Res.1
3,718-724 (1996)).

On the other hand, by using PEG having reactivity, the modification density of PEG becomes 11 mo.
It discloses that 1% of the liposome was obtained, and the retention in blood was higher than that of the unmodified liposome (WO90 / 04384).
No.). However, reactive PEG is susceptible to degradation in an aqueous medium and is chemically unstable. Therefore, in the method of Fischer et al., The conditions for modifying liposomes are extremely strictly limited, and the modified liposomes are industrially produced. Not suitable for

That is, when a liposome modified with PEG is used, it is not easy to obtain a drug carrier which satisfies both aspects of stability and retention in blood and is easily industrially manufactured. From such a viewpoint, a new type of drug carrier satisfying the above two aspects and easy to manufacture has been desired.

[0008]

DISCLOSURE OF THE INVENTION The present inventors have developed an aqueous medium having a physiological pH comprising a polymer main chain and a nonionic polymer side chain which are negatively charged in an aqueous medium having a physiological pH. The graft copolymer, which is negatively charged in the medium, and the core material, which is positively charged in an aqueous medium at physiological pH, is coated by such a graft copolymer as an outer material via electrostatic bonding. By producing a fine particle molecule aggregate formed by the above, when using such a fine particle molecular aggregate as a drug carrier, it has been found that the drug has an excellent effect on the blood retention and stability of the drug, the present invention completed.

[0009]

SUMMARY OF THE INVENTION The present invention relates to (1) a linear or branched polymer main chain and a nonionic linear or branched negatively charged main chain in an aqueous medium at physiological pH. Graft copolymer composed of a polymer side chain which is negatively charged in an aqueous medium having a physiological pH, (2) a polymer having a polymer main chain which is negatively charged in an aqueous medium having a physiological pH (3) a polymer whose main chain is negatively charged in an aqueous medium having a physiological pH, and a nonionic polymer (1) to (3), wherein the graft copolymer according to (1) or (2), wherein the polymer is a structural unit; and (4) the polymer main chain contains polyvinyl. The graft copolymer according to any one of (1) to (5), wherein the polymer main chain comprises methyl vinyl ether and The graft copolymer according to any one of (1) to (4), which is a copolymer of maleic acid, and (6) a structural unit in which a polymer whose side chain is a nonionic polymer is a structural unit. The graft copolymer according to any one of (1) to (5),
(7) The graft copolymer according to any one of (1) to (6), wherein the polymer side chain contains a polyether, and (8) the polymer side chain is a polyethylene glycol.
(1) The graft copolymer according to any one of (1) to (7), wherein (9) the side chain of the polymer is monomethoxypolyethylene glycol. Characterized in that there is
(1) The graft copolymer according to any one of (1) to (8), (10) a core substance which is a substance positively charged in an aqueous medium having a physiological pH, via electrostatic bonding, (10) The fine particle molecule aggregate formed by being coated with the shell material as the graft copolymer according to any one of (9) to (9), and (11) the core material is a polymer. Wherein the (12) core material is a liposome, and (13) the core material is di (C _10-18).
Alkanoyl) phosphatidylcholine, as well as, in addition, stearylamine, 3-β- [N- (N ′,
N'-Dimethylaminoethane) carbamoyl] cholesterol and N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride, one or more components selected from the group consisting of Characterized in that it is a liposome composed of
(10) or the fine particle molecule aggregate according to (12), (1)
4) The core substance is dipalmitoyl phosphatidylcholine,
And additionally, stearylamine, 3-β-
[N- (N ′, N′-dimethylaminoethane) carbamoyl] cholesterol and any one selected from the group consisting of N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride (10), (12) or (13), wherein the core material is dipalmitoyl phosphatidylcholine; and (15) the core substance is dipalmitoylphosphatidylcholine, which is a liposome composed of two or more components. 3-β- [N- (N ′, N ′
-Dimethylaminoethane) carbamoyl] cholestero-
Characterized by a liposome composed of
(10), (12), (13) or (14), the fine particle molecule aggregate according to (14), wherein the core substance is dipalmitoyl phosphatidylcholine and N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride. Characterized in that it is a liposome composed of (1)
(0), (12), (13) or (14), wherein (17) the core substance is a liposome to which cholesterol is added as a component. (12), (13), (14), (1
(5) or (16), the fine particle molecule aggregate according to (18),
(10) A drug carrier in which a drug is included in the fine particle molecule aggregate according to any one of (10) to (17), (19) the drug is a fat-soluble compound, (18)
(20) The drug carrier according to (18), wherein the drug is a compound that is negatively charged in an aqueous medium having a physiological pH. The drug carrier according to (18), which is a compound positively charged in an aqueous medium having a pH.

In the present invention, the physiological pH means pH
6.5 to pH 7.5.

The aqueous medium is a medium containing water, a buffer, 50% by weight or more of water and / or a buffer and containing a water-soluble organic solvent such as ethanol or dimethylacetamide as another component. A medium in which a water-soluble substance such as an electrolyte and / or a saccharide is dissolved in a medium.

The term "negatively charged" means a charged state in which the number of negative charges in a molecule is larger than the number of positive charges, and the term "positively charged" means a charged state in which the number of positive charges in the molecule is larger than the number of negative charges. .

[0013] These charged states are measured by using a zeta potential meter or the like, and by a method known to those skilled in the art (J. Hunter, "Zeta potenti").
al in colloid science '', Academic Press (1981) / D.
Glick (Ed.) `` Methods of biochemical analysis '', vo
l.32, John Wiley & Sons, p215-278 (1987). ) Can be calculated by measuring the zeta potential or electrophoretic mobility of the molecule.

Non-ionic means that it has no dissociating group except for the terminal of the polymer chain in an aqueous medium.

A polymer is a polymer having a molecular weight of 1,000 to 100.
0000. A polymer refers to a compound having a repeating structure of a basic unit, a copolymer refers to a polymer having two or more types of constituent components as a basic unit, and a graft copolymer includes a main chain. Polymer
It means a branched copolymer in which a polymer constituting a side chain different from the polymer is covalently bonded.

[0016] The liposome refers to a vesicle composed of a lipid membrane, the structure and function of which are well known in the art.

Fine particles mean particles having a diameter of 1 nm to 1 mm in an aqueous medium or in a dry state as measured by a dynamic light scattering method or the like.
It means particles that are between 0 nm and 1 μm.

The term "molecular assembly" means an aggregate formed by forming a plurality of molecules through electrostatic bonding. Lipid solubility is defined by a method known to those skilled in the art (A. Martin et al., “Physical Pharmacy (3rd.e.
d.) ", see Lea & Febiger, PP272-313 (1983))
The measured partition coefficient in the n-octanol / aqueous medium system is 2 when the aqueous medium phase has a pH of 3 or more and 11 or less.
It means the above, preferably 5 or more.

The polymer constituting the linear or branched polymer main chain is selected from the following substance groups (A) to (G).

That is, (A) a vinyl synthetic polymer which can be negatively charged in an aqueous medium at physiological pH,
A synthetic polyamino acid that can be negatively charged in an aqueous medium of H;
(C) a synthetic polypeptide that can be negatively charged in an aqueous medium at physiological pH, (D) a synthetic polyester that can be negatively charged in an aqueous medium at physiological pH, and (E) an aqueous medium at physiological pH. A negatively chargeable natural polymer, (F) a modified natural polymer that can be negatively charged in an aqueous medium at physiological pH, (G)
A polymer selected from the group of (A) to (F) and, in addition, another polymer selected from the group of (A) to (F) different from the polymer or a nonionic polymer A block copolymer or a graft copolymer having another polymer as a constituent unit.

Here, the block copolymer means a linear copolymer in which two or more kinds of the same basic units are continuously covalently bonded, and two or more kinds of the same basic units are further covalently bonded.

The vinyl synthetic polymer (A) which can be negatively charged in an aqueous medium having a physiological pH includes polymaleic acid, polyfumaric acid, polyitaconic acid, polyitaconic acid monomethyl ester, polycrotonic acid, polycinnamic acid, In addition to polymers such as polystyrene sulfonic acid, polyacrylic acid, polymethacrylic acid, and poly-2-acrylamido-2-methylpropane sulfonic acid, any one of the following structural units selected from the group (a): , And at least one other constituent unit selected from the following groups (a) and (b).

(A) Structural units that can be negatively charged in an aqueous medium at physiological pH maleic acid, fumaric acid, itaconic acid, itaconic acid monomethyl ester, crotonic acid, cinnamic acid, styrene sulfonic acid, acrylic acid, methacrylic acid Acid, 2-acrylamide-2
-Methylpropanesulfonic acid.

(B) Nonionic constitutional units vinyl alcohol, methyl vinyl ether, vinylpyrrolidone, vinyloxazolidone, vinylmethyloxazolidone, 2-vinylpyridine, 4-vinylpyridine, N-vinylsuccinimide, N-vinylformamide, N-vinylformamide Vinyl-N
-Methylformamide, N-vinylacetamide, N-vinyl-N-methylacetamide, 2-hydroxyethylmethacrylate, acrylamide, methacrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, diacetoneacrylamide, methylolacrylamide, acryloylmorpholine , Acryloylpyrrolidine, acryloylpiperidine, styrene, chloromethylstyrene, bromomethylstyrene, vinyl acetate, methyl methacrylate, butyl acrylate, methyl cyanoacrylate, ethyl cyanoacrylate, n-propyl cyanoacrylate, iso-propyl cyanoacrylate,
n-butyl cyanoacrylate, iso-butyl cyanoacrylate, tert-butyl cyanoacrylate, glycidyl methacrylate, ethyl vinyl ether, n-propyl vinyl ether, iso-propyl vinyl ether n-butyl vinyl ether, iso-butyl vinyl ether, tert-butyl vinyl ether.

The synthetic polyamino acid which can be negatively charged in an aqueous medium having a physiological pH of the above (B) means a polymer in which one kind of amino acid such as polyglutamic acid or polyaspartic acid is polymerized via a peptide bond. I do.

The synthetic polypeptide (C) capable of being negatively charged in an aqueous medium having a physiological pH includes any one of the following structural units selected from the group (c): It means a polymer or the like comprising at least one other structural unit selected from the group (d).

(C) Anionic structural units such as glutamic acid and aspartic acid.

(D) Nonionic structural units such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, serine, threonine, asparagine and glutamine.

The synthetic polyester (D) which can be negatively charged in an aqueous medium having a physiological pH is a polymer such as polymalic acid or polytartronic acid, or any one selected from the following group (e): A polymer or the like consisting of one structural unit and at least one other structural unit selected from the group of (e) and (f).

(E) Anionic structural units such as malic acid and tartronic acid.

(F) Nonionic structural units such as glycolic acid and lactic acid.

The natural polymer (E) which can be negatively charged in an aqueous medium having a physiological pH means a polymer selected from the following groups (g) to (i).

(G) polysaccharides such as hyaluronic acid, chondroitin sulfate, heparan sulfate, alginic acid and pectic acid; (h) nitrite reductase, asparaginase, acetylcholinesterase, adenosine deaminase;
α-amylase, β-amylase, albumin, insulin, elastin a, concanavalin A, transferrin, pepsin and other proteins; (i) nucleic acids such as DNA, RNA and oligonucleotides.

The modified natural polymer (F) which can be negatively charged in an aqueous medium having a physiological pH means polymers such as carboxymethylcellulose, dextran sulfate and alkali-treated gelatin.

The polymer constituting the side chain of the graft copolymer means a substance selected from the following groups (H) to (O).

(H) a nonionic vinyl polymer,
(I) Nonionic synthetic polyamino acid (J) Nonionic synthetic polypeptide, (K) Nonionic synthetic polyester, (L) Nonionic polyether (M) Nonionic natural polymer , (N) a nonionic modified natural polymer,
(O) A block copolymer or a graft copolymer comprising two or more polymers selected from the group consisting of (H) to (N) as constituent units.

The nonionic vinyl polymer of the above (H) includes vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2-vinyl pyridine, 4-vinyl pyridine, N-vinyl succin Imide, N-vinylformamide,
N-vinyl-N-methylformamide, N-vinylacetamide, N-vinyl-N-methylacetamide, 2-hydroxyethyl methacrylate, acrylamide, methacrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, diacetone acrylamide, Methylol acrylamide, acryloyl morpholine, acryloyl pyrrolidine, acryloyl piperidine, styrene, chloromethyl styrene, bromomethyl styrene, vinyl acetate, methyl methacrylate, butyl acrylate, methyl cyanoacrylate, ethyl cyanoacrylate, n-
Propyl cyanoacrylate, iso-propyl cyanoacrylate, n-butyl cyanoacrylate, iso-butyl cyanoacrylate, tert-butyl cyanoacrylate,
Glycidyl methacrylate, ethyl vinyl ether, n-
It means a polymer composed of structural units selected from propyl vinyl ether, iso-propyl vinyl ether, n-butyl vinyl ether, iso-butyl vinyl ether, tert-butyl vinyl ether and the like.

The nonionic synthetic polyamino acid of the above (I) is any one of structural units selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, serine, threonine, asparagine, glutamine and the like. Means a polymer consisting of

The nonionic synthetic polypeptide (J) comprises a plurality of structural units selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, serine, threonine, asparagine, glutamine and the like. It means a polymer.

The nonionic synthetic polyester (K) means a polymer having glycolic acid and / or lactic acid as a constitutional unit.

The nonionic polyether (L) means a polymer comprising a structural unit selected from methylene glycol, ethylene glycol, n-propylene glycol, iso-propylene glycol, hydroxypropylene glycol and the like.

The nonionic natural polymer (M) means a polymer such as dextran, pectin, pullulan and the like.

The nonionic modified natural polymer (N) means a polymer such as methylcellulose and hydroxypropylcellulose.

Examples of the core substance include the following substances (P) and (Q).

(P) a liposome capable of being positively charged in an aqueous medium having a physiological pH; (Q) a polymer capable of being positively charged in an aqueous medium having a physiological pH; The liposomes that can be used are stearylamine (stearyamine).
lamine. Hereinafter, it is referred to as SA. ), 3-β- [N- (N
', N'-dimethylaminoethane) carbamoyl] cholesterol and / or N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride (N-
α-trimethylammonioacetyldidodecyl-D-glutamate chl
oride. Hereinafter, it is referred to as TMAG. ) And neutral lipids such as phosphatidylcholine. Cholesterol may be blended as a liposome stabilizer.

Each of the phospholipids such as phosphatidylcholine has two saturated or unsaturated fatty acid ester chains, and the fatty acid (alkanoyl group or alkenoyl group) having 10 to 18 carbon atoms is used as a liposome.
It can be used as a system component. Examples of the “C _10-18 alkanoyl or alkenoyl” group include decyl, undecyl, lauroyl, tridecyl, myristoyl, pentadecyl, palmitoyl, heptadecyl, stearoyl, and oleoyl groups.

The polymer which can be positively charged in the aqueous medium having a physiological pH of the above (Q) includes the following (j) to (n)
Means a polymer selected from the group of

(J) a vinyl polymer which can be positively charged in an aqueous medium; (k) a synthetic polyamino acid which can be positively charged in an aqueous medium; and (1) a synthetic polypeptide which can be positively charged in an aqueous medium. , (M) a natural polymer that can be positively charged in an aqueous medium, (n) a modified natural polymer that can be positively charged in an aqueous medium, and (o) (j) to (n). A block copolymer comprising, as constituent units, a polymer and, in addition, a polymer selected from the group of (a) to (n) different from the polymer or another nonionic polymer. Graft copolymer.

The vinyl polymer which can be positively charged in an aqueous medium in the above (j) includes polyvinylamine, polyN, N-dimethylaminoethyl acrylate, polyN,
N-dimethylaminoethyl methacrylate, poly N, N
-A polymer such as diethylaminoethyl acrylate, poly N, N-dimethylaminoethyl methacrylate, or polyallylamine, or any one of structural units selected from the following group (i), and (i) and (ii) And a polymer comprising at least one other structural unit selected from the group

(I) Structural units capable of being positively charged in an aqueous medium having a pH of 3, vinylamine, N, N-dimethylaminoethyl acrylate, N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl acrylate, N, N-dimethylaminoethyl methacrylate, allylamine.

(Ii) Nonionic constitutional units, vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2-
Vinylpyridine, 4-vinylpyridine, N-vinylsuccinimide, N-vinylformamide, N-vinyl-N-methylformamide, N-vinylacetamide, N-vinyl-N-methylacetamide, 2-hydroxyethyl methacrylate,
Acrylamide, methacrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, diacetoneacrylamide, methylolacrylamide, acryloylmorpholine, acryloylpyrrolidine, acryloylpiperidine, styrene, chloromethylstyrene, bromomethylstyrene, vinyl acetate, vinyl acetate, methyl methacrylate, butyl Acrylate, methyl cyanoacrylate,
Ethyl cyanoacrylate, n-propyl cyanoacrylate, iso-propyl cyanoacrylate, n-butyl cyanoacrylate, iso-butyl cyanoacrylate, tert
-Butyl cyanoacrylate, glycidyl methacrylate, ethyl vinyl ether, n-propyl vinyl ether, iso-propyl vinyl ether, n-butyl vinyl ether, iso-butyl vinyl ether, tert-butyl vinyl ether.

The polyamino acid capable of being positively charged in an aqueous medium in the above (k) means a polymer in which a kind of basic amino acid such as polylysine and polyarginine is polymerized via a peptide bond.

The synthetic polypeptide (1) capable of being positively charged in an aqueous medium includes any one of the following structural units selected from the group consisting of (iii) and (iii) and (iv): And at least one other structural unit selected from the group of

(Iii) Lysine and arginine, which are positively chargeable in pH 3 aqueous medium.

(Iv) Nonionic constitutional unit, glycine,
Alanine, valine, leucine, isoleucine, proline, phenylalanine, serine, threonine, asparagine, glutamine.

The natural polymer (m) capable of being positively charged in an aqueous medium means a polymer selected from the following group (v) or (vi).

(V) polysaccharides such as chitosan, (vi) agglutinin, aconitase, nitrite reductase, asparaginase, acetylcholinesterase, adenosine deaminase, α-amylase, β-amylase, albumin, insulin, elastase, elastin a, calcitonin, Proteins such as glucagon, concanavalin A, cytochrome c, DNA polymerase, transferrin, troponin, hemoglobin, lactoferrin, lysozyme, and immunoglobulin G.

The modified natural polymer (n) capable of being positively charged in an aqueous medium means a polymer such as diethylaminoethyldextran.

The fat-soluble compounds include the following.

Indapamide, cyclozapine, clofazimine, lisinopril, simvastatin, lovastatin,
Naphazoline, pergolide, amiodarone, iodoxamic acid, bupivacaine, diclofenac sodium,
2'-cyano-2'-deoxy--N ⁴ - palmitoyl -
1-β-D-arabinofuranosylcytosine (Patent No. 2
569251), N- [1- (4-methoxyphenyl) -1-methylethyl] -3-oxo-4-aza-5
α-Androst-1-ene-17β-carboxamide (Japanese Patent No. 2097842).

The compound capable of being negatively charged in the aqueous medium means a compound having the following property (p) or (q).

(P) Methods known to those skilled in the art (A. Martin et al.,
"Physical Pharmacy (3rd ed.)", Lea & Febiger
Pp.187-221 (1983). P) measured according to
An acidic compound having a _Ka1 value of 11 or less, and (q) an amphoteric compound having an isoelectric point lower than 11.

Examples of the compound having these properties include the following.

Acetylsalicylic acid, ascorbic acid, barbital, barbituric acid, benzoic acid, benzylpenicillin, penicillin V, pentobarbital, phenobarbital, phenytoin, salicylic acid, sulfadiazine, sulfamerazine, sulfapyridine, sulfathiazole, sulfisoxa Zole, tetracycline, naproxen, nicotinic acid, DNA, oligonucleotide, RNA, 5′-O- {3,4-di (benzyloxy) benzyl} thymidine-3′-O-phosphoryl- (3 ′ → 5 ′)- Guanosine-3′-O-phosphoryl- (3 ′ → 5 ′)-adenosine-3′-O-phosphoryl- (3 ′ → 5 ′)-{3′-O- (2-hydroxyethyl) phosphoryl} guanosine ( JP-A-7-
No. 87982), asparaginase, acetylcholinesterase, adenosine deaminase, insulin, calcitonin, glucagon.

The compound capable of being positively charged in an aqueous medium means a compound having any of the following properties (r) and (s).

(R) Methods known to those skilled in the art (A. Martin et al.,
"Physical Pharmacy (3rd ed.)", Lea & Febiger
Pp.187-221 (1983). ) Measured according to the
A basic compound having a pK _a1 value of the conjugate acid of the compound in an aqueous medium of more than 3, and (s) an amphoteric compound having an isoelectric point of more than 3.

The compounds having these properties include, for example, the following.

Apomorphine, atropine, caffeine,
Cocaine, codeine, ephedrine, epinephrine, erythromycin, morphine, nalorphine, papaverine, physostigmine, pilocarpine, procaine, quinacrine, quinine, reserpine, scopolamine, strychnine, theobromine, theophylline, tolbutamide, lincomycin, lincomycin, clozapin clozapin, clozapinene , Graphenin,
Naphazoline, pergolide, sotalol, vinblastine, amiodarone, methoxamine, mexletin, bupicaine, nizatidine, verapamil, bromazepam, trazodone, pentazocine, doxorubicin, nimustine, 2'-cyano-2'-deoxy-1-β-D-arabinofuranosyl Cytosine (JP-A-4-235182)
No.), asparaginase, acetylcholinesterase, adenosine deaminase, insulin, calcitonin, glucagon, cytochrome c, immunoglobulin G.

[0069]

BEST MODE FOR CARRYING OUT THE INVENTION The polymers (A) to (O) may be purchased from Sigma Chemical Co. or the like, or may be purchased from G. Allen et al. (Eds.), “Comprehensive polymer science” vol.
It can be produced according to the method described in Pergamon Press (1989).

The linear or branched polymer main chain capable of being negatively charged in an aqueous medium and the nonionic linear or branched polymer side chain described in the above (1). The following method can be mentioned as an example of a method for producing a graft copolymer composed of:

Since the polymer constituting the main chain has an acidic functional group such as a carboxyl group, a sulfonic acid group, or a sulfuric acid monoester, a method known to those skilled in the art (for example, S.I.
H.Pine, Organic Chemistry fifth edition (McGraw-H
ill International Editions) (1987). ), These acidic functional groups are activated with an acid halide or the like to become reactive groups. The polymer constituting the side chain needs to have a functional group having a reactivity with the reactive group on the main chain. Examples of the functional group include an amino group or a hydroxyl group. . Then, the main chain polymer and the side chain polymer can be bonded by an amide bond, an ester bond, or the like. These polymers having an acidic functional group used as a main chain or polymers having an amino group or a hydroxyl group used as a side chain are purchased from Sigma Chemical Co., a share water polymer company, or the like. it can.
Polymers having these functional groups are described in G. Allen et al. (Eds.), “Comprehensive polymer science” vol.
It can be produced according to the method described in Pergamon Press (1989).

When a polymer containing an acid anhydride such as maleic anhydride as a component is used as a precursor of a main chain or side chain polymer, a polymer containing the acid anhydride as a component is used. After producing a molecule and using the acid anhydride as a reactive group to produce a graft copolymer as described above, the unreacted acid anhydride is converted into an acid by hydrolysis, and the graft copolymer having an acidic functional group. Can be obtained.

The terminal of the graft copolymer has various ionic or nonionic functional groups. For example, a graft copolymer in which the terminal of polyethylene glycol constituting a side chain is methoxylated (a graft copolymer having a nonionic functional group), a terminal of a polyamino acid constituting a side chain may be Examples include a graft copolymer having an amino group or a carboxyl group (a graft copolymer having an ionic functional group).

In the compound (SA), each of the constituents of the liposome positively charged in the aqueous medium in (P)
3-β- [N- (N ′, N′-dimethylaminoethane)
Carbamoyl] cholesterol, TMAG, phosphatidylcholine or cholesterol) can be purchased from NOF Corporation. These compounds can also be prepared by methods known to those skilled in the art (eg, SHPine, “Organi
c Chemistry fifth edition '', McGraw-Hill Interna
National Editions (1987)).

Using these compounds as starting materials, DDLa
sic, `` Liposomes: from physicsto applications
", Elsevier Science Publishers, pp. 1-171 (1993)
According to the method described in (1), positively charged liposomes can be produced in an aqueous medium.

The positively-chargeable vinyl polymer, the positively-chargeable polyamino acid, the positively-chargeable polypeptide, and the positively-chargeable vinyl polymer contained in the above (Q) (j) to (n) Natural polymers and positively charged modified natural polymers can be purchased from Sigma Chemical Company or the like.

Further, a positively charged vinyl polymer is described in G.
Allen et al. (Eds.), "Comprehensive polymer science" v
ol. 6, Pergamon Press (1989).
Positively charged polyamino acids and positively charged polypeptides are described in M. Bodanszky, "Principles of peptide synt
hesis ", Springer-Verlag (1984) /M.Bodanszky
Et al., "The practice of peptide synthesis", Spring
Each can be produced according to the method described in er-Verlag (1984).

In constructing a fine particle molecular assembly in which the core material is covered with the outer shell material, a negatively charged outer shell material is selected in an aqueous medium in which the core material to be used is positively charged. The aqueous medium has a pH of 3 to 11, an osmotic pressure of 0 to 800 mOsm, and an electrolyte concentration of 0 to 4.
A material having a core material of positive charge and a shell material of negative charge is used.

In the aqueous medium selected in this manner, the core substance and the outer shell substance were added at final concentrations of 0.1 to 500 mg / mL and 0.1 to 100 mg / mL, respectively.
g / mL, by finally mixing at a mixing ratio at which the fine particle molecule aggregate is negatively charged at physiological pH,
A fine particle molecular assembly is manufactured.

Furthermore, a drug carrier can be produced by incorporating a drug into the fine particle molecular assembly, but the production method varies depending on the physicochemical properties of the drug.

First, when the drug to be included is a fat-soluble compound, a liposome (DDLasic, “Liposomes: from phys
icsto applications '', Elsevier Science Publishers
Pp. 261-471 (1993). ) Or after incorporating the drug in a core material composed of a polymer, the core material and the outer shell material are mixed under the same conditions as in the case of producing the above-mentioned drug-free fine particle molecule assembly. Then, a drug carrier containing the drug is produced.

In order to include a drug which can be negatively charged in an aqueous medium, first, a core substance which can be positively charged in an aqueous medium in which the drug is negatively charged is selected. The aqueous medium has a pH of 3 to 11 and an osmotic pressure of 0 to 800.
An aqueous medium is used in which the drug is negatively charged and the core substance is positively charged at an mOsm and electrolyte concentration in the range of 0 to 150 mM.

The drug is mixed with the core substance or a component of the core substance at a mixing ratio at which the positive charge of the core substance becomes excessive, and the size is adjusted as required, whereby the size of the core substance is adjusted. A core material containing the drug in advance is produced.

Thereafter, the final concentrations of the core substance and the shell substance were 0.1 to 500 mg / mL and 0.1, respectively.
The core material and the shell material are mixed under the same conditions as in the case where no drug is contained within the range of 100 to 100 mg / mL to produce a drug carrier containing the drug.

A drug carrier containing a drug which can be positively charged in an aqueous medium can be obtained by combining a fine particle molecule assembly previously produced under the condition where no drug is contained with the drug with p
H is in the range of 3 to 11, the pH range is such that the drug is positively charged and the fine particle molecular assembly is negatively charged, the osmotic pressure is 0 to 800 mOsm, and the electrolyte concentration is 0 to 15
In an aqueous medium of 0 mM, it can be produced by mixing the finally produced drug carrier at a mixing ratio that is negatively charged at physiological pH.

As described above, the graft copolymer of the present invention is coated on a drug or a core substance containing the drug, forms a drug carrier, and is administered to warm-blooded animals including humans.

The administration forms include intravenous, intraarterial,
In addition to subcutaneous, intradermal, and intramuscular injections, local injection into diseased tissues and the like can be mentioned. The dosage for adults is appropriately adjusted depending on the drug involved and the disease to be treated.

The injectable compositions are provided in unit dose ampoules or in multi-dose containers. The composition comprises a tonicity agent,
It may contain additives such as stabilizers, bases and thickeners. It is usually used as a suspension or solution formulation using sterile water or an aqueous liquid containing an organic solvent such as ethanol as a dispersion medium or solvent, or provided with the medium removed by freeze-drying or the like. It is used by suspending or dissolving using the above-mentioned dispersion medium or solvent.

[0089]

The present invention will be described below in more detail with reference to Examples, Comparative Examples and Test Examples. However, these do not limit the present invention.

The compounds A and B used in this example are as follows.

Compound A: 5′-O- {3,4-di (benzyloxy) benzyl} thymidine-3′-O-phosphoryl- (3 ′ → 5 ′)-guanosine-3′-O-phosphoryl- ( 3 '→ 5')-guanosine-3'-O-
Phosphoryl- (3 ′ → 5 ′)-guanosine-3′-
O-phosphoryl- (3 ′ → 5 ′)-adenosine-
3'-O-phosphoryl- (3 '→ 5')-{3'-
O- (2-hydroxyethyl) phosphoryl guanosine (see JP-A-7-87982).

[0092] Compound B: 2'-cyano-2'-deoxy--N ⁴ - palmitoyl -1-beta-D-arabinofuranosyl cytosine (see JP-A-4-235182).

Example 1 Production of a liposome as a core substance

Bangham et al. (J. Mol. Biol. 8, 660-668).
(1964). ) Using the lipids having the composition ratios shown in Tables 1 and 2 and dipalmitoylphosphatidylcholine (hereinafter referred to as DPP).
C. ) And cholesterol, stearylamine (hereinafter referred to as SA) or N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride (N-α-trimethylammoni).
oacetyldodecyl-D-glutamate chloride. Hereinafter, TMA
G. ) Were prepared respectively. These liposomes are hereinafter referred to as SA liposome and TMAG liposome, respectively.

[0095]

Table 1 DPPC 44 (μmol) Cholesterol 40 (μmol) SA 16 (μmol) Add 150 mM aqueous sodium chloride solution to these and add 2 mL
And

[0096]

Table 2 DPPC 44 (μmol) Cholesterol 40 (μmol) TMAG 16 (μmol) ─────────────────────────────── Add 150 mM aqueous sodium chloride solution to these and add 2 mL
And Next, the method known to those skilled in the art (RRC New Edition,
"Liposomes. A Practical Approach", IRL Press,
pp.33-104 (1989). ), An extruder (Liposofast) equipped with a polycarbonate membrane filter (Nomura Microscience) having a pore size of 100 nm.
The size of the liposome was adjusted by an extrusion method using -Basic, manufactured by AVESTIN, or an ultrasonic irradiation method using an ultrasonic crushing apparatus (Model 7600, Seiko Denshi Kogyo).

The volume average particle size of these liposomes and phosphate buffered saline at 25 ° C. (consisting of 20 mM sodium dihydrogen phosphate and 135 mM sodium chloride, adjusted to pH 7.4 with a 1N aqueous sodium hydroxide solution) (Hereinafter referred to as “PBS”), the zeta potential was measured using a Nicomp Particle Sizer Mode.
l370, manufactured by Nicomp Particle Sizing Systems) and a zeta potential meter (ZEM5000, manufactured by Malvern). Table 3 shows the results.

[0098]

[Table 3] 種類 Types of liposomes Size adjustment method Volume average particle size Zeta potential (nm) (mV) ＳＡ SA liposome extrusion method 131.3 33. 0 Ultrasonic irradiation method 57.9 31.0 ＴＭ TMAG extrusion method 106.8 13.9 Liposomes Ultrasonic irradiation method 60.7 10.4 All the liposomes were fine particles and were positively bound in the aqueous medium, and it was confirmed that the liposomes satisfied the conditions as the core substance.

Example 2 Production of a graft copolymer as a shell material.

2 g of a copolymer of methylvinyl ether and maleic anhydride having a molecular weight of about 23,000 (this molecular weight is converted to a degree of polymerization of 147) (manufactured by IPC Japan)
And 8.1 g of monomethoxypolyethylene glycol having an amino group at one end having a linear molecular weight of about 5000 (this molecular weight is converted to a degree of polymerization of 112) (Kawahara Yuka Co., Ltd.) , 100 mL of a mixture of acetone and water (weight ratio of acetone to water 2: 1). The reaction system was stirred at room temperature for 1 hour and further at 60 ° C. for 1 hour to react the amino group at the end of polyethylene glycol with the carboxyl anhydride group in the copolymer.

Evaporator (rotation speed: 100 rpm,
This reaction solution was concentrated under reduced pressure to 60 mL using a temperature of 40 ° C. and a degree of reduced pressure of 700 mmHg. The pH was adjusted to 5.0 using a 1N aqueous sodium hydroxide solution and 1N hydrochloric acid, and the mixture was stirred at 40 ° C. for 24 hours to hydrolyze unreacted carboxyl groups.

This solution was dialyzed against distilled water using a cellulose dialysis membrane having a molecular weight cut off of about 12,000.

Evaporator (rotation speed: 100 rpm,
The solution after dialysis was concentrated under reduced pressure to about 20 mL using a temperature of 40 ° C. and a degree of reduced pressure of 700 mmHg.
mL and 300 mL of ether were added, and the mixture was left under ice cooling for 15 hours to obtain a precipitate as a powder. In addition, 10
Dissolve in 0 mL of distilled water and filter using No. 2 filter paper (Advantech Toyo Co., Ltd.).
After concentration under reduced pressure to mL, ethanol and ether were added to obtain a precipitate. Using a vacuum pump, the obtained powder was dried in a desiccator under reduced pressure for 24 hours, and a graft copolymer (monomethoxypolyethyl) of methyl vinyl ether grafted with monomethoxy polyethylene glycol and maleic acid was dried.
eneglycol-grafted poly (methylvinylether-co-maleic
acid): MPEG-PMVMA).

The ratio of maleyl groups bonded with monomethoxypolyethylene glycol to all maleyl groups of the graft copolymer of methyl vinyl ether and maleic anhydride, ie, the grafting ratio, was determined by the elemental analysis of MPEG-PMVMA (Table 4) and the composition. From the equation (Equation 1), it was determined as follows:

[0105]

Table 4 C (%) H (%) N (%) 52.8 8.28 <0.3 * ０．３ ─ * Indicates that it is below the detection limit.

(C ₇ H ₁₀ O ₅ ) _{(1-a) n} + [C ₁₀ H ₁₇ O ₅ N + (C ₂ H ₄ O) _m ] _an (Formula 1) [In the formula 1, m is monomethoxy It shows the degree of polymerization of polyethylene glycol, n shows the degree of polymerization of the copolymer of methyl vinyl ether and maleic anhydride used, and a shows the degree of grafting. ] Formula 1 is expanded by element, and m = 1
Substituting 12 and n = 147 gives MPEG-PMV
The following formula was obtained as the composition formula of MA.

C _{1029 + 33369a} H _{1470 + 66885a} N _147a O _{735 + 16464a} (Formula 2) From this, the molecular weight of MPEG-PMVMA is (102
9 + 33369a) × 12.0 + (1470 + 6688)
5a) × 1.0 + 147a × 14.0 + (735 + 16
464a) x 16.0 = 25578 + 732795a.

From Table 4 and Equation 2, the following Equations 3 and 4 hold.

[0109] Table 5 shows the respective grafting rates calculated from Equations 3 and 4, and their average values.

[0110]

[Table 5] Thus, the grafting ratio of the graft copolymer obtained in this example was calculated to be 9.5%.

[Example 3] Production of a fine particle molecular assembly using MPEG-PMVMA as an outer shell material and SA liposome as a core material.

The lipid concentration prepared in Example 1 was 50 mM.
In 200 μL of the SA liposome dispersion and 10 mL of a 150 mM aqueous sodium chloride solution adjusted to pH 7 with a 10 mM aqueous sodium hydroxide solution, the MP prepared in Example 2 was added.
A solution in which EG-PMVMA was dissolved at a concentration of 5 mg / mL was mixed at room temperature to obtain a dispersion of fine particle molecule aggregates.

SA liposome 2 used as a core material
The zeta potential in PBS at 5 ° C. was 33 mV as shown in Example 1, but it was -18 mV when mixed with MPEG-PMVMA used as an outer substance. This result indicates that the outer shell material was negatively charged at the pH at which the mixing was performed, and the positively charged core material was covered by the negatively charged outer shell material through electrostatic coupling. This means that an aggregate of fine particle molecules negatively charged at physiological pH was generated.

The volume average particle diameter of the core substance was 131 nm as shown in Example 1, but the MPEG-P
Increased to 230 nm by mixing with MVMA. This also suggested that a fine particle molecule aggregate was formed between the core material and the outer shell material. In addition, it was found that the thus-obtained fine particle molecule aggregate had a size excellent in blood retention when administered into a blood vessel of a living body as described above.

Example 4 Production of a molecular assembly of fine particles using MPEG-PMVMA as a shell material and TMAG liposome as a core material.

The TMAG liposome prepared in Example 1 was used as a core substance instead of SA liposome, and a fine particle molecular assembly (hereinafter referred to as MPEG-P) was prepared in the same manner as in Example 3.
MVMA-TMAG. ) Was prepared. As a result of measuring the zeta potential, the zeta potential of the core substance was 14 m.
V, whereas PEG-
Since it became -13 mV after mixing with PMVMA, the TMAG liposome was converted to excess negatively charged PEG.
-It was found that a fine particle molecular aggregate coated with PMVMA was formed. Regarding the volume average particle diameter, the core substance was 107 nm, but became 203 nm by mixing with the outer substance, so that a fine particle molecule aggregate was formed between the core substance and the outer substance. It was suggested. In addition, it was found that the thus-obtained fine particle molecule aggregate had a size excellent in blood retention when administered into a blood vessel of a living body as described above.

[Example 5] Production of a fine particle molecular assembly using MPEG-PMVMA as a shell material and poly-L-lysine as a core material.

Since the side chain amino group has a pKa value of 10 to 11, poly L-lysine (p) which is one of polyamino acids which can be positively charged at least in a pH range near neutrality is used.
oly (L-lysine). Hereinafter, a PLL is used. )
Was used as the core material.

PLL hydrochloride (average molecular weight 47400, Si
gma. ) At a concentration of 0 or 20 mg / mL and adjusted to pH 7 with 1N aqueous sodium hydroxide solution.
M-sodium chloride solution and MPEG-
PMVMA at a concentration of 0 or 100 mg / mL, adjusted to pH 7 with a 1N aqueous sodium hydroxide solution
M sodium chloride aqueous solution was dissolved in a volume ratio of 1: 1.
To obtain a dispersion liquid of fine particle molecular aggregates, and the volume average particle diameter was measured. Table 6 shows the results.

[0120]

[Table 6] PLL final concentration MPEG-PMVMA final concentration Volume average particle size (mg / mL) (mg / mL) (nm) {0 50 9.0 10 0 1.1 1.1 50 50 12.2} ──────── Thus, either PLL or MPEG-PMVMA monomer, or PLL or MPEG-PMVMA
The volume average particle diameter of each of the aggregates of any of the MAs is 1.1 n.
m and 9.0 nm, but when both were mixed, a larger volume average particle diameter was observed.
From this, it was found that the PLL and MPEG-PMVMA formed a fine particle molecular aggregate.

[Example 6] Production of a drug carrier in which the core substance is an SA liposome containing compound A and the outer shell substance is MPEG-PMVMA.

A fine particle molecular assembly containing compound A, which is a drug that can be negatively charged in an aqueous medium, was produced as follows.

First, liposomes were produced according to the method described in Example 1 using lipids and aqueous solutions having the composition ratios shown in Table 7, and the size was adjusted by the extrusion method. Thus, SA liposomes containing Compound A in advance were obtained.

[0124]

Table 7 DPPC 440 (μmol) Cholesterol 400 (μmol) SA 160 (μmol) ５ 5 (w / w)% containing 1 mg / mL of compound A
Add 10 mL of aqueous glucose solution and add 5 (W / W)
% Glucose was added to make 20 mL.

Next, the SA liposome was used as a core substance, and the concentration of MPEG-PMVMA, which was a negatively chargeable outer shell substance, was set to 1 mg / mL, and a dispersion of a drug carrier was produced according to the method described in Example 3. did. The volume average particle diameter of the carrier was 100.7 nm, and as described above, it was clarified that the carrier had a size excellent in blood retention when administered into a blood vessel of a living body. The ratio of the amount of the drug in the drug carrier to the amount of the drug used in manufacturing the drug carrier (hereinafter, referred to as inclusion rate) was determined as follows.

The drug carrier was precipitated by ultracentrifugation (150,000 g × 20 minutes) of 1 mL of the drug carrier dispersion, the supernatant was removed, and the precipitate was added with a 150 mM aqueous sodium chloride solution to make 1 mL. From 200μ
L and take a 0.01 N sodium hydroxide aqueous solution 200
μL, methanol 400 μL and chloroform 800
The compound A was extracted by adding μL and shaking using a vortex mixer (room temperature, 5 minutes, shaking width 4 cm, 2000 rpm). The supernatant after centrifugation (1000 g × 5 minutes) to remove insoluble components was analyzed by reversed-phase high-performance liquid chromatography under the conditions shown in Table 8 to calculate the amount of Compound A in the drug carrier. Then, the inclusion rate was determined from this.

[0127]

[Table 8] Measuring machine LC-10A (manufactured by Shimadzu Corporation) カ ラ ム Column YMC -A312 (YMC) カ ラ ム Column temperature 40 ℃ ──────検 出 Detection wavelength 260nm ──────────────────── ───────────── Mobile phase Mixed solution of acetonitrile and 0.1 M aqueous solution of triethylammonium acetate 3/7 (volume ratio) ──────────────── Flow rate 1mL / min. ───────────────────────────────── Injection volume 10μL ────────────── ─────────────────── Holding time 8.6 min ────────────────────────── ─────── As a result, the inclusion rate of compound A in the fine particle molecular aggregate was 6
It was 5.4%.

[Example 7] Production of a drug carrier containing doxorubicin in a fine particle molecular assembly using SA liposome as a core substance and MPEG-PMVMA as an outer shell substance.

A drug carrier containing doxorubicin, a drug capable of being positively charged in an aqueous medium, was produced as follows.

First, SA liposomes were produced according to the method described in Example 1 using lipids and aqueous media having the composition ratios shown in Table 9, and the size was adjusted by ultrasonic irradiation.

[0131]

Table 9 DPPC 44 (μmol) Cholesterol 40 (μmol) SA 16 (μmol) ────────────────────────────── These 5% (w / w)% aqueous glucose solution
L.

By further diluting this with 5% glucose, the total lipid concentration calculated from the formulation at the time of manufacture was 1%.
A dispersion of the fine particle molecule aggregate in mM was produced. 1 mL of a dispersion of the fine particle molecular assembly and 1 m of a 5% aqueous glucose solution in which 1 mg / mL of doxorubicin hydrochloride was dissolved
And shaking (25 ° C., 10 minutes, shaking width 7).
cm, 50 rpm) to incorporate doxorubicin to prepare a drug carrier dispersion. To this dispersion was added 2 mL of a 150 mM aqueous sodium chloride solution to prepare a solution for determining the coverage. Of these, 10 μL was used as a sample for measuring the total amount of doxorubicin contained in the solution for determining the coverage. Further, 1 mL of the solution for determining the inclusion ratio was subjected to ultracentrifugation (150,000 g × 20 minutes), and 10 μL of the supernatant obtained by precipitating the drug carrier was obtained.
This was used as a sample for measuring the concentration of doxorubicin not included in the aggregate of fine particle molecules. This sample 1
To 0 μL, 75 μL of distilled water, 150 μL of ethanol, and 75 μL of tertiary butyl alcohol were added to make a clear solution, and a microplate reader (Titertec Multiskan) was used.
PLUS, Flow Labolatories), the concentration of doxorubicin was measured by measuring the absorbance at 492 nm, and the coverage was determined according to equation 5.

[0133] As a result, the doxorubicin inclusion rate in the fine particle molecular assembly was 19.5%.

As a control, when the dispersion of the aggregate of fine particle molecules was diluted and a 150 mM aqueous sodium chloride solution was used as the solvent for doxorubicin, the doxorubicin inclusion rate was reduced to 5.6%.

As described above, since the doxorubicin inclusion rate was reduced in the presence of the electrolyte,
That is, when 5% glucose was used as the diluent for the fine particle dispersion and the solvent for doxorubicin, it was found that the electrostatic interaction between the negative charge of the fine particle molecular assembly and the positive charge of doxorubicin effectively acted on inclusion of doxorubicin. Show.

Example 8 Preparation of a drug carrier in which the core substance is TMAG containing compound B and the outer substance is MPEG-PMVMA.

Compound B, which is a fat-soluble drug, was incorporated into TMAG liposome, which is a positively chargeable core substance, using lipids and aqueous media having the constitutional ratios shown in Table 10 and described in Example 1. According to the method, a dispersion liquid of TMAG liposome containing compound B was prepared, and the size was adjusted by ultrasonic irradiation.

[0138]

Table 10 DPPC 44 (μmol) Cholesterol 40 (μmol) TMAG 16 (μmol) Compound B 10 (mg) ─────────────────────────に Add 5% (w / w)% aqueous glucose solution to these and add 2%
mL.

Next, the liposome was used as the core substance, and the MPEG-PMVMA concentration of the outer shell substance was set to 1 mg / mL.
According to the method described in Example 4, a dispersion of the drug carrier was produced. The volume average particle diameter of the drug carrier containing Compound B was 72.3 nm, and as described above, it was found that the drug carrier had a size excellent in blood retention when administered to the blood of a living body.

Further, the obtained drug carrier dispersion was diluted 100-fold with physiological saline, and 1 mL of the resultant was ultracentrifuged (150,000 g × 20 minutes), and the supernatant was discarded.
A 150 mM aqueous sodium chloride solution was added to the obtained precipitate to make 1 mL. Take 100 μL therefrom and add 1 M acetate buffer (pH 4, prepared with acetic acid and sodium acetate) 100 μL
L and 800 μL of methanol, and shake using a vortex mixer (room temperature, 5 minutes, shake width 4c).
m, 2000 rpm) and then centrifugation (1000 g × 5 minutes) to remove insoluble components.
Then, the supernatant was analyzed by reversed-phase high performance liquid chromatography under the conditions shown in Table 11, and the amount of Compound B in the drug carrier was calculated, and the content was determined therefrom.

[0141]

[Table 11] LC-10A (manufactured by Shimadzu Corporation) カ ラ ム Column YMC-A312 (YMC) ────────────────────────────────── Column temperature 40 ℃ ────検 出 Detection wavelength 247nm ─────────────────移動 Mobile phase Mixture of acetonitrile and distilled water (volume ratio: 3/7) ──────────────── Flow rate 1mL / min. ────────────────────────────────── Injection volume 20μL ───────────── ───────────────────── Holding time 8min ────────────────────────── ──────── As a result, the inclusion ratio of compound B in the fine particle molecular aggregate was 114%, and it was found that the total amount of compound B was included in the fine particle molecular aggregate.

Comparative Example 1 General PEG-modified liposomes
Preparation of a System A general liposome was produced as a PEG-modified fine particle as follows. The PEG-modified liposome used here is
Distearoyl-N- (monomethoxypolyethyleneglycolsuccinyl) phosphatidylethanolamine (molecular weight of PEG moiety: about 2000, Kawahara Yuka Co., Ltd., hereinafter referred to as MPEG2000-DSPE) is used together with other lipids such as dipalmitoyl phosphatidylcholine. , Table 12 to Table 14 (MPEG2000-
Composition ratio of DSPE Table 12: 5 mol% Table 13:10
(Table 14: 20 mol%), according to the production method described in Example 1, and the size was adjusted by an extrusion method. In the extrusion, a filter having a pore size of 1 μm was used.

[0143]

Table 12 DPPC 55 (μmol) MPEG2000-DSPE 5 (μmol) Cholesterol 40 (μmol) １５０ Add 150mM sodium chloride aqueous solution to these and add 2mL
And

[0144]

Table 13 DPPC 50 (μmol) MPEG2000-DSPE 10 (μmol) Cholesterol 40 (μmol) １５０ Add 150mM sodium chloride aqueous solution to these and add 1mL
And

[0145]

Table 14 DPPC 40 (μmol) MPEG2000-DSPE 20 (μmol) Cholesterol 40 (μmol) １５０ Add 150mM sodium chloride aqueous solution to these and add 1mL
And

[Test Example 1] Evaluation of degree of PEG modification of drug carrier and PEG-modified liposome.

The degree of PEG modification of the drug carrier containing Compound A prepared in Example 6 and the PEG prepared in Comparative Example 1
The following experiment was performed to compare the degree of PEG modification of the modified liposome.

The degree of PEG modification of the drug carrier was evaluated based on the partition coefficient of the liposome to a two-phase system consisting of PEG and dextran (see Japanese Patent Application Laid-Open No. 4-501117). In this two-phase system, the higher the partition coefficient to the PEG phase, the higher the hydrophilicity of the liposome surface and the higher the degree of PEG modification. 1 ml of the drug carrier dispersion obtained in Example 6 was subjected to ultracentrifugation (150,000 g × 20 minutes).
Then, the supernatant was removed and redispersed with a 150 m sodium chloride aqueous solution to make 1 ml again, thereby removing the drug A which was not included. To 1 ml of this dispersion, 13.3 (w / v)% P
EG6000 in PBS and 13.3 (w / v)%
Dextran T-500 (Pharmacia) PB
1.5 ml of each S solution was added, and the mixture was allowed to stand at room temperature for 30 minutes to separate into two layers. In order to measure the concentration of Compound A contained in each layer, the drug carrier is precipitated by ultracentrifugation of each layer, a 0.01 N aqueous sodium hydroxide solution, methanol and chloroform are added, and the mixture is shaken (vortexing) Mixer: room temperature, 5 minutes,
Compound A was extracted by shaking 4 cm, 2000 rpm. To remove insoluble components, centrifuge (100
(0 g × 5 minutes), the supernatant was analyzed by reverse-phase high performance liquid chromatography under the conditions shown in Table 15, and the concentration of Compound A was measured.

[0149]

[Table 15] Measuring machine LC-10A (manufactured by Shimadzu Corporation) カ ラ ム Column YMC -A312 (YMC) カ ラ ム Column temperature 40 ℃ ──────検 出 Detection wavelength 260nm ──────────────────── ───────────── Mobile phase Mixture of acetonitrile and 0.1 M aqueous solution of triethylammonium acetate 3: 7 (volume ratio) ─────────────── ────────────────── Flow rate 1ml / min ──────────────────────────── ───── Injection volume 10μL ────── ─────────────────────────── Holding time 8.6 min ──────────────────分配 When the partition coefficient of the drug carrier was calculated as “concentration of compound A in upper layer / concentration of compound A in lower layer”, the value was 76%. As described above, extremely high hydrophilicity was exhibited. Further, the degree of PEG modification of the PEG-modified liposome produced in Comparative Example 1 was evaluated by the distribution method described above. In this case, upper and lower 2
The partition coefficient between the layers was calculated from the turbidity of each layer (wavelength 550 nm, optical path length 0.1 cm). As a result, the partition coefficient of each liposome was 14, and the PEG-D
Even when the composition ratio of SPE is 10 mol% or 20 mol%, the partition coefficient is the same as that of 5 mol%. Therefore, in the ordinary method for producing PEG-modified liposomes, the limit of the degree of PEG modification is about 5 mol%, and the partition coefficient is as follows. It turned out that about 14 is the limit.

[Test Example 2] Changes in blood concentration of Compound A when intravenous administration of a drug carrier whose core substance is an SA liposome containing Compound A and whose outer shell substance is MPEG-PMVMA.

The blood retention of the drug carrier containing compound A produced in Example 6 was examined as follows. Compound A
At a concentration of 0.27 mg / mL and a total lipid concentration of 38.
A 5 mM aggregate of microparticles was administered from a tail vein to Wistar rats (body weight 430 to 490 g) so that the dose of Compound A was 0.5 mg / kg.
Approximately 500 μL from the tail vein after a predetermined time of 0 to 25 hours
Was collected, and plasma was obtained by centrifugation (1000 g × 5 minutes). The plasma was analyzed by reverse-phase liquid chromatography under the conditions described in Test Example 1 to determine the concentration of Compound A in the plasma.

FIG. 1 shows changes in the blood concentration of Compound A. Compound A calculated from the blood concentration transition thus determined
Had a blood half-life of 14.54 hours.

Test Example 3 Changes in blood concentration of Compound A administered intravenously as an aqueous solution.

150 containing 0.5 mg / mL of compound A
mM Aqueous Sodium Chloride was added to Compound A at a dose of 0.
After administration at a concentration of 5 mg / kg, the results of examining the plasma concentration of Compound A under the same conditions as in Test Example 2 are shown in FIG. The blood half-life of Compound A calculated from this change in blood concentration was 2.27 hours.

As described above, it was found that when Compound A was administered in the form of a solution, the retention in blood was remarkably inferior to the case where Compound A was administered after being included in the aggregate of fine particle molecules.

[Test Example 4] Plasma stability of fine particle molecular aggregate and general PEG-modified liposome.

The following experiment was performed to compare the stability of the fine particle molecule aggregate of the formulation of Table 1 prepared in Example 1 and the general PEG-modified liposome of the formulation of Table 13 prepared in Comparative Example 1. went. The fine particle molecular assembly and the liposome
0.1 ml of each plasma was collected from a rat.
Add to 4 ml, shake at 37 ° C for 4 days, and shake (general shaker: 37 ° C, 4 days, shaking width 7 cm, 50 rpm)
m). Turbidity of each plasma immediately after the start of shaking and after the end of shaking (wavelength 550 nm, optical path length 0.1 cm)
Table 16 shows the measurement results.

[0158]

[Table 16] Immediately after starting shaking After shaking for 4 days ────────────────────────────────── Fine particle molecular assembly Body 0.140 0.145 ────────────────────────────────── General PEG-modified liposome 0 0.324 0.538 ────────────────────────────────── Thus, the fine particle molecular aggregate was added. The turbidity of the plasma hardly changed, indicating that the particulate molecular aggregate maintained its morphology. On the other hand, general PEG
The turbidity of the plasma to which the modified liposome was added was remarkably increased by shaking, suggesting that the PEG-modified liposome had undergone a morphological change such as aggregation.

[0159]

According to the present invention, a linear or branched polymer main chain and a nonionic linear or branched polymer side chain which are negatively charged in an aqueous medium at physiological pH are constituted. The graft material that is negatively charged in the medium and the material that is positively charged in the medium that is the core material are coated with the core material by such a graft copolymer as an outer material through electrostatic bonding. Physiological p formed by
Obtaining a negatively charged fine particle molecule aggregate in an aqueous medium of H;
A drug carrier containing a drug therein was manufactured.

The aggregate of fine particles constituting the drug carrier of the present invention is easy to produce, has a high degree of modification of a water-soluble polymer, has good retention in blood, and exhibits excellent stability in plasma. And compounds having various physicochemical properties can be included, and have high versatility as functional fine particles useful pharmaceutically and biochemically.

[Brief description of the drawings]

FIG. 1 is a transition diagram of the concentration of plasma compound A after administration of compound A to rats. (■, molecular assembly; ●, solution)

Claims (21)

[Claims] 1. A physiological solution comprising a linear or branched polymer main chain negatively charged in an aqueous medium at physiological pH and a nonionic linear or branched polymer side chain. Target p
A graft copolymer negatively charged in an aqueous medium of H. 2. The graft copolymer according to claim 1, wherein the polymer main chain comprises, as a constitutional unit, a polymer negatively charged in an aqueous medium having a physiological pH. 3. The polymer according to claim 1, wherein the polymer main chain comprises, as constituent units, a polymer which is negatively charged and a nonionic polymer in an aqueous medium having a physiological pH. Graft copolymer. 4. The graft copolymer according to claim 1, wherein the polymer main chain contains polyvinyl. 5. The graft copolymer according to claim 1, wherein the polymer main chain is a copolymer of methyl vinyl ether and maleic acid. 6. The graft copolymer according to claim 1, wherein the polymer side chain has a nonionic polymer as a constituent unit. 7. The graft copolymer according to claim 1, wherein the polymer side chain contains a polyether. 8. The graft copolymer according to claim 1, wherein the side chain of the polymer is polyethylene glycol or monomethoxy polyethylene glycol. 9. The graft copolymer according to claim 1, wherein the side chain of the polymer is monomethoxypolyethylene glycol. 10. A core material, which is a substance positively charged in an aqueous medium at physiological pH, through an electrostatic bond.
A fine particle molecular assembly formed by coating with the shell material which is the graft copolymer according to any one of the above. 11. The fine particle molecular assembly according to claim 10, wherein the core substance is a polymer. 12. The fine particle molecular assembly according to claim 10, wherein the core substance is a liposome. 13. The method according to claim 13, wherein the core material is di (C _10-18 alkanoyl).
Phosphatidylcholine, stearylamine, 3-β-
[N- (N ', N'-dimethylaminoethane) carbamoyl] liposome comprising cholesterol and / or N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride. 13. The fine particle molecule aggregate according to claim 10 or 12, characterized in that: 14. The core substance is dipalmitoylphosphatidylcholine, stearylamine, 3-β- [N- (N ′,
N'-dimethylaminoethane) carbamoyl] cholesterol and / or N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride is a liposome.
14. The fine particle molecule aggregate according to 0, 12, or 13. 15. The method according to claim 15, wherein the core substance is dipalmitoylphosphatidylcholine and / or 3-β- [N- (N ′, N′-
15. The fine particle molecular assembly according to claim 10, which is a liposome composed of [dimethylaminoethane) carbamoyl] cholesterol. 16. The liposome according to claim 10, wherein the core substance is dipalmitoylphosphatidylcholine and / or N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride. 15. The aggregate of fine particle molecules according to claim 12, 13, 13 or 14. 17. The aggregate of fine particle molecules according to claim 10, wherein the core substance is a liposome to which cholesterol is added as a constituent. 18. A drug carrier in which a drug is included in the aggregate of fine particle molecules according to any one of claims 10 to 17. 19. The drug carrier according to claim 18, wherein the drug is a fat-soluble compound. 20. The drug carrier according to claim 18, wherein the drug is a compound that is negatively charged in an aqueous medium at physiological pH. 21. The drug carrier according to claim 18, wherein the drug is a compound that is positively charged in an aqueous medium at physiological pH.