JPH11269094A - Sustained release composition, its production and use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject composition containing a physiologically active substance in a high content and capable of inhibiting its excessive initial release to realize a stable release rate over a long period by allowing the physiologically active substance and a hydroxynaphthoic acid to coexist. SOLUTION: This sustained release preparation comprises (A) a physiologically active substance or its salt (preferably a physiologically active peptide), (B) a hydroxynaphthoic acid or its salt (preferably 3-hydroxy-2-naphthoic acid) and (C) a biodegradable polymer or its salt (preferably an α-hydroxycarboxylic acid polymer). The objective composition is preferably obtained by mixing and dispersing the component A in an organic solvent solution (for example, a mixture of dichloromethane with ethanol) containing the component B and the compound C and subsequently removing the organic solvent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a sustained-release preparation of a physiologically active substance, a method for producing the same, and a use as a medicine or the like.

[0002]

2. Description of the Related Art JP-A-7-97334 discloses a sustained-release preparation comprising a physiologically active peptide or a salt thereof and a biodegradable polymer having a free carboxyl group at a terminal, and a method for producing the same. I have. GB220993
No. 7, GB2234169, GB2234896,
GB2257909 and EP626170A2
Japanese Patent Application Publication No. JP-A-2003-133125 discloses a composition based on a biodegradable polymer containing a water-insoluble salt such as a pamoate of a peptide or protein prepared separately, or a method for producing the same. WO95 / 15767 discloses cetrorelix (LH
-RH antagonist) and a method for producing the same, and at the same time, even when the pamoate is encapsulated in a biodegradable polymer, the release of the peptide is limited to pamoate alone. It is described that this is the same as the case of.

[0003]

SUMMARY OF THE INVENTION The present invention provides a novel composition containing a high content of a physiologically active substance, and capable of realizing a stable release rate over a long period by suppressing its initial excessive release.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a bioactive substance and a hydroxynaphthoic acid coexist when forming a composition. When the substance is incorporated into the composition at a high content and both are encapsulated in the biodegradable polymer, it is formed from hydroxynaphthoic acid and a physiologically active substance prepared under conditions free of the biodegradable polymer. The bioactive substance is released at a rate different from the release rate of the bioactive substance from the composition, and the release rate can be controlled by the properties of the biodegradable polymer and the amount of added hydroxynaphthoic acid, even at a high content. It is possible to reliably suppress the initial excessive release and achieve a very long-term sustained release, and as a result of further research, the present invention has been completed.

That is, the present invention provides (1) a sustained-release composition comprising a physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof, and (2) a bioactive substance. (3) the sustained release composition according to (1), wherein the substance is a physiologically active peptide; (3) the sustained release composition according to (2), wherein the physiologically active substance is an LH-RH derivative; Naphthoic acid is 3-hydroxy-2
-The sustained-release composition according to item (1), which is naphthoic acid;
(5) The sustained-release composition according to (1), wherein the biodegradable polymer is an α-hydroxycarboxylic acid polymer, (6)
(5) wherein the α-hydroxycarboxylic acid polymer is a lactic acid-glycolic acid polymer, (7) the composition mol% of lactic acid and glycolic acid is from 100/0 to 40/6.
(6) the sustained release composition according to the above (6), wherein the composition mol% of lactic acid and glycolic acid is 100/0;
Item (6), wherein the polymer has a weight average molecular weight of about 3,000 to about 100,000, and (10) the weight average molecular weight is About 20,
(9) the sustained release composition according to (9), wherein the LH-RH derivative has the formula 5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro -Z wherein Y is DLeu, DAla, DTrp, DSer (tBu), D2Nal or
DHis indicates (ImBzl), Z represents an NH-C ₂ H ₅ or Gly-NH _2. (3) The sustained release composition according to (3), wherein the amount of the carboxyl group at the terminal of the polymer is 50 to 90 micromol per unit mass (gram) of the polymer. (13) The sustained release composition according to (6), (13) the sustained release composition according to (3), wherein the molar ratio of hydroxynaphthoic acid or a salt thereof to an LH-RH derivative or a salt thereof is 3: 4 to 4: 3. Release composition, (14) in a sustained release composition, LH-RH
(13) The sustained-release composition according to (13), wherein the derivative or its salt is contained in an amount of 14% (w / w) to 24% (w / w), (15) the physiologically active substance or its salt is slightly soluble or (16) The sustained release composition according to (1), which is water-soluble, (16) the sustained release composition according to (1), which is for injection, (17) a physiologically active substance or a salt thereof, and biodegradation (18) The method for producing a sustained-release composition according to (1), wherein the solvent is removed from a mixed solution of the water-soluble polymer or a salt thereof and hydroxynaphthoic acid or a salt thereof. A method comprising mixing and dispersing a physiologically active substance or a salt thereof in an organic solvent solution containing the salt and hydroxynaphthoic acid or a salt thereof, and then removing the organic solvent.
(19) The method for producing a sustained-release composition according to the above (19), wherein the physiologically active substance or a salt thereof is an aqueous solution containing the physiologically active substance or a salt thereof. (20) The production method according to (17), wherein the salt of the physiologically active substance is a salt with a free base or an acid, (21) the (1)
(22) A medicament comprising the sustained release composition according to (3), (3) a medicament comprising the sustained release composition according to (3),
Prostatic hypertrophy, endometriosis, uterine fibroids, uterine fibroids, prevention of precocious puberty, dysmenorrhea or breast cancer, therapeutic or contraceptive, (23) hydroxynaphthoate of bioactive substance and biodegradation (24) A method for suppressing the initial excessive release of a physiologically active substance from a sustained release composition, which comprises using hydroxynaphthoic acid or a salt thereof. , (2
5) A method for improving the efficiency of encapsulating a bioactive substance in a sustained-release composition, characterized by using hydroxynaphthoic acid or a salt thereof, (26) hydroxynaphthoic acid salt of a bioactive peptide, (27) water solubility Alternatively, the present invention provides a sustained-release composition comprising the slightly water-soluble hydroxynaphthoic acid salt of the physiologically active peptide according to (26) and (28) the hydroxynaphthoic acid salt of the physiologically active peptide.

Further, in the present invention, the compounding amount of (29) hydroxynaphthoic acid or a salt thereof is about 1 to about 7 mol, preferably about 1 to about 2 mol, per 1 mol of the physiologically active peptide or a salt thereof. The sustained-release composition according to item (28),
(30) A W / O emulsion is prepared in which a liquid containing a physiologically active substance or a salt thereof is used as an internal aqueous phase, and a solution containing a biodegradable polymer and hydroxynaphthoic acid or a salt thereof is used as an oil phase. (1)
7) The method for producing a sustained-release composition according to the above item, (31) a solution containing a physiologically active substance or a salt thereof and a biodegradable polymer or a salt thereof, wherein a liquid containing hydroxynaphthoic acid or a salt thereof is used as an internal aqueous phase. No. (17), wherein a W / O type emulsion is prepared in which the oil phase is an oil phase, and then the solvent is removed.
(28) The method for producing a sustained-release composition according to (28), wherein (32) the physiologically active peptide or a salt thereof and hydroxynaphthoic acid or a salt thereof are mixed and dissolved, and then the solvent is removed. (30) the method for producing a sustained-release composition according to (30), wherein the method of removing the solvent is an in-water drying method;
The present invention also provides a method for producing the sustained-release composition according to any one of Items to (32).

[0007] The physiologically active substance used in the present invention is not particularly limited as long as it is pharmacologically useful, and may be a non-peptide compound or a peptide compound. Examples of the non-peptide compound include an agonist, an antagonist, a compound having an enzyme inhibitory action, and the like. Further, as the peptide compound, for example, a physiologically active peptide is preferable, and a molecular weight of about 300 to about 40,000, preferably about 4
Preferred are bioactive peptides of from about 00 to about 30,000, more preferably from about 500 to about 20,000. Examples of such bioactive peptides include luteinizing hormone-releasing hormone (LH-RH), insulin, somatostatin, Growth hormone, growth hormone releasing hormone (G
H-RH), prolactin, erythropoietin, adrenocortical hormone, melanocyte stimulating hormone, thyroid hormone releasing hormone, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, vasopressin, oxytocin, calcitonin, gastrin, secretin, pancreozymine , Cholecystokinin, angiotensin, human placental lactogen, human chorionic gonadotropin, enkephalin, endorphin, kyotorphin, tuftsin, thymopoietin, thymosin, thymothymulin, thymic humoral factor, blood thymic factor, tumor necrosis factor, colony inducing factor, motilin , Deinorphine, bombesin, neurotensin, caerulein, bradykinin, atrial natriuretic factor, nerve growth factor, cell growth factor, neurotrophic factor, Peptides and the like, and derivatives thereof having Ndoserin antagonism, further including derivatives of these fragments or fragments. The physiologically active substance used in the present invention may be itself or a pharmacologically acceptable salt. Such salts include, when the physiologically active substance has a basic group such as an amino group, an inorganic acid (also referred to as an inorganic free acid).
(Eg, carbonic acid, bicarbonate, hydrochloric acid, sulfuric acid, nitric acid, boric acid, etc.),
Salts with organic acids (also referred to as organic free acids) (eg, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, and the like) are included. When the physiologically active substance has an acidic group such as a carboxyl group, an inorganic base (also referred to as an inorganic free base)
(Eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium) and organic bases (also referred to as organic free bases) (eg, organic amines such as triethylamine, basic amino acids such as arginine) And the like). Further, the physiologically active peptide may form a metal complex compound (eg, a copper complex, a zinc complex, etc.).

[0008] Preferred examples of the physiologically active peptide are LH-RH derivatives, which are hormone-dependent diseases,
In particular, sex hormone-dependent cancers (eg, prostate cancer, uterine cancer, breast cancer, pituitary tumor, etc.), prostatic hypertrophy, endometriosis, uterine fibroids, precocious puberty, dysmenorrhea, amenorrhea, premenstrual period LH-RH derivatives or salts thereof effective for sex hormone-dependent diseases such as the syndrome and multilocular ovary syndrome and contraception (or infertility when the rebound effect after drug withdrawal is used). . Furthermore, an LH-RH derivative or a salt thereof that is effective for benign or malignant tumors that are sex hormone-independent but LH-RH sensitive is also included. Specific examples of the LH-RH derivative or a salt thereof include:
For example, treatment with GnRH analog:
Contravasis and Perspective (Treatm
ent with GnRH analogs: Controversies and perspecti
ves) [Parthenon Publishing Group Co., Ltd.
(The Parthenon Publishing Group Ltd.) Published 1996
Year], JP-A-3-503165, JP-A-3-10
Nos. 1695, 7-97334 and 8-2594
No. 60, for example.

Examples of the LH-RH derivative include LH-RH agonists and LH-RH antagonists. Examples of the LH-RH antagonist include those represented by the general formula [I] X-D2Nal-D4ClPhe-D3Pal-Ser-AB- Leu-C-Pro-DAlaNH _{2 wherein} X is N (4H ₂ -furoyl) Gly or NAc, A is NMeTy
r, Tyr, Aph (Atz), a residue selected from NMeAph (Atz),
B is DLys (Nic), DCit, DLys (AzaglyNic), DLys (AzaglyF
ur), DhArg (Et ₂ ), a residue selected from DAph (Atz) and DhCi, and C represents Lys (Nisp), Arg or hArg (Et ₂ ), respectively.) Is used. Examples of the LH-RH agonist include, for example, a compound represented by the general formula [II] 5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z wherein Y is DLeu, DAla, DTrp, DSer (tBu), a residue selected from D2Nal and DHis (ImBzl), Z is NH-C ₂ H ₅ or
Gly-NH ₂ respectively] or a physiologically active peptide thereof or a salt thereof. In particular, Y is DLeu,
Peptides wherein Z is NH—C ₂ H ₅ (ie, 5-oxo-Pro-His-Trp
-Ser-Tyr-DLeu-Leu- Arg-Pro-NH-C peptide represented by ₂ H ₅₎ are preferred. These peptides can be produced by the method described in the literature or the gazette or a method analogous thereto.

The meanings of the abbreviations used in the present specification are as follows. Abbreviation Name N (4H ₂ -furoyl) Gly: N-tetrahydrofuroylglycine residue NAc: N-acetyl group D2Nal: D-3- (2-naphthyl) alanine residue D4ClPhe: D-3- (4-chloro) Phenylalanine residue D3Pal: D-3- (3-pyridyl) alanine residue NMeTyr: N-methyltyrosine residue Aph (Atz): N- [5 '-(3'-amino-1'H-1', 2 ', 4'-Triazolyl)] phenylalanine residue NMeAph (Atz): N-methyl- [5'-(3'-amino-1'H-1 ', 2', 4'-triazolyl)] phenylalanine Residue DLys (Nic): D- (eN-nicotinoyl) lysine residue Dcit: D-citrulline residue DLys (AzaglyNic): D- (azaglycylnicotinoyl) lysine residue DLys (AzaglyFur): D- (aza) Glycylfuranyl) lysine residue DhArg (Et ₂ ): D- (N, N′-diethyl) homoarginine residue DAph (Atz): DN- [5 ′-(3′-amino-1′H-1 ′) , 2 ', 4'-triazolyl)] phenylalanine residue DhCi: D-homocitrulline residue Lys (Nisp): (eN-isopropyl) Emissions residue _{hArg (Et 2): (N} , N'- diethyl) relates other amino homoarginine residue, when displaying by abbreviations, IUPAC-IU
B Commission of Biochemical Nomenclature
(European Journal of Biochemistry, Vol. 138, 9
To page 37 (1984)) or a common abbreviation in the relevant field, and when an amino acid may have an optical isomer, the L-form is indicated unless otherwise specified.

The hydroxynaphthoic acid used in the present invention is one in which one hydroxyl group and one carboxyl group are bonded to different carbons of naphthalene. Accordingly, there are a total of 14 isomers in which the position of the hydroxyl group differs from the position of the carboxyl group at the 1-position and the 2-position of the naphthalene ring. And any of the isomers may be used,
Also, a mixture of these arbitrary ratios may be used. As described later, those having a large acid dissociation constant are preferable, or those having a small pKa (pKa = -log ₁₀ Ka, Ka represents an acid dissociation constant) are preferable. And a slightly water-soluble one is preferred. Further, those soluble in alcohols (eg, ethanol, methanol, etc.) are preferable. "Soluble in alcohols" means, for example, 10
g / L or more. The pKa of the above hydroxynaphthoic acid isomer is 3-hydroxy-2
Naphthoic acid value (pKa = 2.708, Chemical Handbook)
Basics II, The Chemical Society of Japan, issued September 25, 1969)
Although only known, a useful finding can be obtained by comparing the pKas of the three isomers of hydroxybenzoic acid. That is, while the pKa of m-hydroxybenzoic acid and p-hydroxybenzoic acid is 4 or more, the pKa of o-hydroxybenzoic acid (salicylic acid) (= 2.75)
4) is extremely small. Therefore, among the above 14 isomers, 3-hydroxy-2-naphthoic acid, 1-hydroxy-2-naphthoic acid and 2-hydroxy-, in which a carboxyl group and a hydroxyl group are bonded to adjacent carbon atoms of a naphthalene ring, 1-Naphthoic acid is preferred. Further, 3-hydroxy-2-naphthoic acid in which a hydroxyl group is bonded to the 3-position carbon and a carboxyl group is bonded to the 2-position carbon of naphthalene is preferable. Hydroxynaphthoic acid may be a salt. Examples of the salt include inorganic bases (eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium) and organic bases (eg, organic amines such as triethylamine, and basic amino acids such as arginine). And the like, or salts and complex salts with transition metals (eg, zinc, iron, copper, etc.).

Hereinafter, a method for preparing the hydroxynaphthoic acid salt of the physiologically active substance of the present invention will be exemplified. (1) A solution of hydroxynaphthoic acid in a water-containing organic solvent is adsorbed through a weakly basic ion exchange column and saturated. Next, after removing excess hydroxynaphthoic acid through a water-containing organic solvent, ion exchange may be performed through a solution of a physiologically active substance or a salt thereof in a water-containing organic solvent, and the solvent may be removed from the obtained effluent. As the organic solvent in the aqueous organic solvent, alcohols (eg, methanol,
Ethanol, etc.), acetonitrile, tetrahydrofuran, dimethylformamide and the like are used. As a method for removing the solvent for precipitating the salt, a method known per se or a method analogous thereto is used. For example, there is a method of evaporating the solvent while adjusting the degree of vacuum using a rotary evaporator or the like. (2) The exchange ions of the strongly basic ion exchange column are exchanged for hydroxide ions in advance, and the basic groups are converted to the hydroxyl form by passing a physiologically active substance or a salt thereof in a water-containing organic solvent solution. . The recovered effluent may be dissolved by adding an equivalent or less of hydroxynaphthoic acid, then concentrated, and the precipitated salt may be washed with water, if necessary, and dried.

The hydroxynaphthoic acid salt of a physiologically active substance is slightly water-soluble, although it depends on the physiologically active substance to be used. It can be used for release preparations,
Further, a sustained-release composition can be produced. Examples of the biodegradable polymer used in the present invention include α-hydroxymonocarboxylic acids (eg, glycolic acid, lactic acid, etc.), α-hydroxydicarboxylic acids (eg, malic acid), and α-hydroxytricarboxylic acids (eg, , Citric acid) and the like, a polymer having a free carboxyl group, a copolymer or a mixture thereof, which is synthesized from one or more kinds of α-hydroxycarboxylic acids such as poly (α-cyanoacrylate); a polyamino acid ( Examples include poly (γ-benzyl-L-glutamic acid); maleic anhydride copolymers (eg, styrene-maleic acid copolymer); and the like.

The bonding modes of the monomers are random,
Any of a block and a graft may be used. In addition, the above α-
When hydroxymonocarboxylic acids, α-hydroxydicarboxylic acids, and α-hydroxytricarboxylic acids have an optically active center in the molecule, any of D-, L-, and DL-forms may be used. Among these, a lactic acid-glycolic acid polymer (hereinafter, also referred to as poly (lactide-co-glycolide), poly (lactic-co-glycolic acid) or a lactic acid-glycolic acid copolymer, unless otherwise specified, Lactic acid and glycolic acid homopolymers (polymers) and copolymers (copolymers) are collectively referred to as lactic acid homopolymers such as lactic acid polymers, polylactic acids, and polylactides, and glycolic acid homopolymers are referred to as glycolic acid polymers and polylactic acids. Glycolic acid, polyglycolide, etc.), poly (α-cyanoacrylate) and the like. More preferably, it is a lactic acid-glycolic acid polymer, more preferably, a lactic acid-glycolic acid polymer having a free carboxyl group at a terminal. The biodegradable polymer may be a salt. Examples of the salt include inorganic bases (eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium) and organic bases (eg, organic amines such as triethylamine, and basic amino acids such as arginine). etc)
Or transition metal (eg, zinc, iron, copper, etc.)
And complex salts thereof. When a lactic acid-glycolic acid polymer is used as the biodegradable polymer, the composition ratio (mol%) is preferably about 100/0 to about 40/60, and more preferably about 100/0 to about 50/50.
Also, a lactic acid homopolymer having a composition ratio of 100/0 is preferably used.

The lactic acid which is one of the minimum repeating units of the “lactic acid-glycolic acid polymer” has an optical isomer ratio of D-form / L-form (mol / mol%) of about 75/25 to about 25. / 7
A range of 5 is preferred. As the D-form / L-form (mol / mol%), those having a range of about 60/40 to about 30/70 are widely used. The “lactic acid-glycolic acid polymer”
Usually has a weight average molecular weight of about 3,000 to about 100,
000, preferably about 3,000 to about 60,000, more preferably about 3,000 to about 50,000, particularly preferably about 20,000 to about 50,000. Further, the degree of dispersion (weight average molecular weight / number average molecular weight) is usually preferably about 1.2 to about 4.0, more preferably about 1.5 to 3.5. The amount of free carboxyl groups of the “lactic acid-glycolic acid polymer” is usually about 20 to about 1000 μmol per unit mass (gram) of the polymer.
(Micromolar), more preferably from about 40 to about 10
00 μmol (micromoles) is particularly preferred. The weight average molecular weight, number average molecular weight and dispersity in the present specification mean that the weight average molecular weight is 1,110,000, 707,0.
00, 455, 645, 354,000, 189,00
0, 156, 055, 98, 900, 66, 437, 3
7,200,17,100,9,830,5,870,
The molecular weight in terms of polystyrene measured by gel permeation chromatography (GPC) using 15 types of 2,500, 1,303, and 504 monodisperse polystyrene as a reference substance and the calculated degree of dispersion are referred to. Measurement is fast G
PC device (manufactured by Tosoh, HLC-8120GPC, detection method depends on differential refractive index), GPC column KF804L ×
2 (manufactured by Showa Denko) and chloroform as the mobile phase. The flow rate is 1 ml / min.

In the present specification, the amount of free carboxyl groups means a value obtained by a labeling method (hereinafter, referred to as "amount of carboxyl groups by the labeling method"). Specifically, in the case of polylactic acid, Wmg of polylactic acid is mixed with 5N hydrochloric acid / acetonitrile (v / v = 4/96) mixture 2
and 2 ml of a 0.01 M o-nitrophenylhydrazine hydrochloride (ONPH) solution (5N hydrochloric acid / acetonitrile / ethanol = 1.02 / 35/15).
After adding 2 ml of a 15 M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride solution (pyridine / ethanol = 4 v / 96 v), the mixture is reacted at 40 ° C. for 30 minutes, and then the solvent is distilled off. After washing the residue with water (4 times),
It is dissolved in 2 ml of acetonitrile, 1 ml of a 0.5 mol / l ethanolic potassium hydroxide solution is added, and the mixture is reacted at 60 ° C. for 30 minutes. The reaction solution is diluted with a 1.5N aqueous sodium hydroxide solution to Yml, and the absorbance A (/ cm) at 544 nm is measured for the 1.5N aqueous sodium hydroxide solution. On the other hand, using an aqueous solution of DL-lactic acid as a reference substance,
The amount Cmol / L of the free carboxyl group was determined by alkali titration, and when the absorbance at 544 nm when DL-lactic acid hydrazide was determined by the ONPH labeling method as B (/ cm), the weight per unit mass (gram) of the polymer was used. The molar amount of free carboxyl groups can be determined by the following equation. [COOH] (mol / g) = (AYC) / (WB)

The "amount of carboxyl group" is determined by dissolving the biodegradable polymer in a mixed solvent of toluene, acetone and methanol, and titrating the solution with phenolphthalein as an indicator using an alcoholic potassium hydroxide solution. (Hereinafter, the value obtained by this method is referred to as “the amount of carboxyl groups by the alkali titration method”), but the end point of the titration is unclear as a result of competing hydrolysis reaction of the polyester main chain during the titration. Therefore, it is desirable to determine the amount by the above labeling method. The rate of degradation / elimination of biodegradable polymers varies greatly depending on the copolymer composition, molecular weight, or amount of free carboxyl groups, but in the case of lactic acid-glycolic acid polymers, the degradation / elimination generally decreases as the glycolic acid fraction decreases. , The release period can be extended by lowering the glycolic acid fraction or increasing the molecular weight and decreasing the amount of free carboxyl groups. However, since the amount of free carboxyl groups affects the rate of incorporation of the physiologically active substance into the preparation, it must be at least a certain value. Therefore, in order to obtain a biodegradable polymer for a long-term (for example, 6 months or more) type sustained-release preparation, in the case of a lactic acid-glycolic acid polymer, the above-mentioned weight average molecular weight is about 20,000 to about 20,000. 50,000
And the amount of free carboxyl groups is about 30 to about 95 μmo
l / g, preferably about 40 to about 95 μmol / g, more preferably about 50 to about 90 μmol / g (eg, D-lactic acid, L-lactic acid, DL-lactic acid, etc.
-Lactic acid or the like is preferred).

The "lactic acid-glycolic acid polymer" is, for example, a catalyst-free dehydration polycondensation of lactic acid and glycolic acid (JP-A-61-28521) or a catalyst derived from lactide and a cyclic diester compound such as glycolide. Ring-opening polymerization (Encyclopedic Handbook of Biomaterials and Bioengi
neering Part A: Materials, Volume 2, Marcel Dekke
r, Inc. 1995). The polymer obtained by the above-described known ring-opening polymerization method does not necessarily have a free carboxyl group at the terminal of the obtained polymer,
For example, a polymer having a certain amount of carboxyl group per unit mass can be modified by subjecting it to a hydrolysis reaction described in EP-A-0 839 525,
This can also be used.

The above-mentioned "lactic acid-glycolic acid polymer having a free carboxyl group at the terminal" can be produced without problems by a known production method (for example, non-catalytic dehydration polycondensation method, see JP-A-61-28521). Alternatively, it can also be produced by the following method. (1) First, a hydroxymonocarboxylic acid derivative having a protected carboxyl group (eg, tert-butyl D-lactate, L-
In the presence of a hydroxydicarboxylic acid derivative (eg, benzyl lactate) or a protected hydroxydicarboxylic acid derivative (eg, dibenzyl tartronate, di-tert-butyl 2-hydroxyethylmalonate), a cyclic ester compound is subjected to a polymerization reaction using a polymerization catalyst. Attach. The above “carboxyl group-protected hydroxymonocarboxylic acid derivative” or “carboxyl group-protected hydroxydicarboxylic acid derivative” means, for example, that a carboxyl group (—COOH) is amidated (—CONH ₂ ) or an ester ( Examples thereof include a hydroxycarboxylic acid derivative in which a carboxyl group (—COOH) is converted into an ester (—COOR), and the like.

Here, R in the ester is, for example, methyl, ethyl, n-propyl, isopropyl, n
-Butyl, C _1-6 alkyl groups such as tert-butyl, for example, cyclopentyl, C _3-8 cycloalkyl groups such as cyclohexyl, for example, phenyl, C _6-12 aryl groups such as α-naphthyl, for example, benzyl, A phenyl-C _1-2 alkyl group such as phenethyl or an α-naphthyl-C _1-2 alkyl group such as α-naphthylmethyl;
_7-14 aralkyl groups and the like. Above all, tert-
A butyl group and a benzyl group are preferred. The “cyclic ester compound” refers to, for example, a cyclic compound having at least one ester bond in a ring. Specific examples include a cyclic monoester compound (lactones) and a cyclic diester compound (lactides). Examples of the “cyclic monoester compound” include 4-membered lactones (β-propiolactone, β-butyrolactone, β-isovalerolactone, β-caprolactone, β-isocaprolactone, β-methyl-β-valerolactone) Such),
5-membered lactone (such as γ-butyrolactone, γ-valerolactone), 6-membered lactone (such as δ-valerolactone), 7-membered lactone (such as ε-caprolactone), p-
Dioxanone, 1,5-dioxepan-2-one and the like.

The "cyclic diester compound" includes, for example, a compound represented by the formula

Embedded image (Wherein R ¹ and R ² are the same or different,
A hydrogen atom or a compound represented by a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl), among which R ¹ is a hydrogen atom Lactide in which R ² is a methyl group and R ¹ and R ² are each a hydrogen atom is preferred. Specifically, for example, glycolide, L-lactide, D-lactide,
DL-lactide, meso-lactide, 3-methyl-1,4-dioxane-2,5-dione (including optically active forms) and the like. Examples of the “polymerization catalyst” include organotin-based catalysts (eg, tin octylate, di-n-butyltin dilaurate, tetraphenyltin, etc.), aluminum-based catalysts (eg, triethylaluminum, etc.), zinc-based catalysts (eg, , Diethyl zinc, etc.). From the viewpoint of ease of removal after the reaction, aluminum-based catalysts and zinc-based catalysts are preferable,
Furthermore, a zinc-based catalyst is preferable from the viewpoint of safety when the catalyst remains. As a solvent for the polymerization catalyst, benzene, hexane, toluene and the like are used, and among them, hexane, toluene and the like are preferable.

The "polymerization method" is a bulk polymerization method in which the reactants are melted or a solution polymerization method in which the reactants are dissolved in an appropriate solvent (eg, benzene, toluene, xylene, decalin, dimethylformamide, etc.). May be used. As the solvent, toluene, xylene and the like are preferable. Although the polymerization temperature is not particularly limited, in the case of bulk polymerization, the temperature is at least 100 ° C. to 300 ° C. at which the reaction product is brought into a molten state at the start of the reaction, and in the case of solution polymerization, it is usually room temperature to 150 ° C. If the reaction temperature exceeds the boiling point of the reaction solution, the reaction may be carried out by refluxing with a condenser or in a pressure vessel. The polymerization time is appropriately determined in consideration of the polymerization temperature, other reaction conditions, physical properties of the target polymer, and the like, and is, for example, 10 minutes to 72 hours. After the reaction, if necessary, the reaction mixture is dissolved in an appropriate solvent (eg, acetone, dichloromethane, chloroform, etc.), and the polymerization is terminated with an acid (eg, hydrochloric acid, acetic anhydride, trifluoroacetic acid, etc.). Solvents that do not dissolve the target compound by a conventional method (eg, alcohol,
The polymer having a carboxyl group protected at the ω-terminal may be isolated by mixing or the like in water, ether, isopropyl ether, or the like. In the polymerization method of the present invention, a carboxyl group-protected hydroxycarboxylic acid derivative (eg, D-tert-butyl lactate, L-benzyl lactate, or the like) or a carboxyl group is substituted for a so-called protic chain transfer agent such as methanol. A protected hydroxydicarboxylic acid derivative (eg, dibenzyl tartronate, ditert-butyl 2-hydroxyethylmalonate, etc.) is used.

The hydroxycarboxylic acid derivative having a protected carboxyl group (eg, tert-butyl D-lactate, L-benzyl lactate, etc.) or the hydroxydicarboxylic acid derivative having a protected carboxyl group (eg, dibenzyl tartronate, 2-tert-hydroxyethylmalonic acid ditert
-Butyl etc.) as the protic chain transfer agent, the molecular weight can be controlled by the charged composition,
Carboxyl groups can be liberated at the ω-terminal of the biodegradable polymer obtained by subjecting it to a deprotection reaction after polymerization.

(2) Next, by subjecting the polymer having a carboxyl group protected at the ω end obtained by the polymerization reaction (1) to a deprotection reaction, a free carboxyl group can be added at the desired ω end. A biodegradable polymer having the same can be obtained. The protecting group can be removed by a method known per se. As such a method, any method can be used as long as the protecting group can be removed without affecting the ester bond of poly (hydroxycarboxylic acid). And acid decomposition. Examples of the reduction method include catalytic reduction using a catalyst (eg, palladium carbon, palladium black, platinum oxide, etc.), reduction with sodium in liquid ammonium, reduction with dithiothreitol, and the like.
For example, when catalytically reducing a polymer having a carboxyl group protected by a benzyl group at the ω end, specifically, palladium carbon is added to a solution of the polymer in ethyl acetate, dichloromethane, chloroform, etc., while vigorously stirring. Deprotection can be achieved by bubbling hydrogen at room temperature for about 20 minutes to about 4 hours. As the acid decomposition method, for example, an inorganic acid (eg, hydrogen fluoride, hydrogen bromide, hydrogen chloride, etc.) or an organic acid (eg, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, etc.) or a mixture thereof is used. Acid decomposition and the like. If necessary, a cation scavenger (eg, anisole, phenol, thioanisole, etc.) may be appropriately added during acid decomposition. For example, when acid-decomposing a polymer having a carboxyl group protected by a tert-butyl group at the ω end, specifically, the polymer is dichloromethane, xylene,
Deprotection can be achieved by adding an appropriate amount of trifluoroacetic acid to one dissolved in toluene or the like, or by dissolving the polymer in trifluoroacetic acid and stirring at room temperature for about 1 hour. Preferably, the acid decomposition method may be performed immediately after the polymerization reaction, in which case it can also serve as a polymerization termination reaction. Further, if necessary, by subjecting the polymer obtained by the above deprotection reaction to an acid hydrolysis reaction, the weight average molecular weight, number average molecular weight or terminal carboxyl group content of the polymer can be adjusted according to the purpose. it can. Specifically, for example, it can be carried out by the method described in EP-A-0 839 525 or a method analogous thereto.

The biodegradable polymer obtained as described above can be used as a base for producing a sustained-release preparation. Furthermore, a polymer having a free carboxyl group that is not specified at the terminal can be obtained by a known production method (for example,
WO94 / 15587). Further, a lactic acid-glycolic acid polymer whose terminal is converted into a free carboxyl group by a chemical treatment after ring-opening polymerization is, for example, Boehringer Ingelheim K.
A commercially available product from G) may be used. The biodegradable polymer may be a salt (the salt of the biodegradable polymer includes, for example, the above-mentioned salts, etc.). A biodegradable polymer dissolved in an organic solvent and an inorganic base (eg, an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium) or an organic base (eg, an organic amine such as triethylamine, arginine) And (b) isolating a salted polymer after performing an ion exchange reaction by mixing an aqueous solution containing ions of basic amino acids such as Is dissolved in an organic solvent to dissolve the weak acid salt of the base listed in (a) above (for example, acetate or glycolate), (C) dissolving the biodegradable polymer having a carboxyl group in an organic solvent into a weak acid salt of a transition metal (eg, zinc, iron, copper, etc.) (eg, acetate, glycol) Acid salt) or isolating a polymer that has become a salt after mixing the oxide. As a biodegradable polymer for a long-term (for example, 6 months or more) type sustained-release preparation, a "lactic acid-glycolic acid polymer having a free carboxyl group at a terminal" produced by the above method is suitable. .

The weight ratio of the physiologically active substance in the composition of the present invention varies depending on the kind of the physiologically active substance, the desired pharmacological effect and the duration of the effect, but the physiologically active substance or its salt and hydroxynaphthoic acid or its salt And a biodegradable polymer or a salt thereof in the case of a sustained release composition, for example, in the case of a bioactive peptide or a salt thereof, about 0.001 to about 50 weight of the sum of the three. %, Preferably about 0.02 to about 40% by weight, more preferably about 0.1 to 30% by weight, most preferably about 14 to about 40% by weight.
In the case of a non-peptidic physiologically active substance or a salt thereof, it is about 0.01 to 80% by weight, preferably about 0.1 to 50% by weight. The weight ratio is the same even when the composition contains a hydroxynaphthoic acid salt of a physiologically active substance. In the case of a sustained-release composition containing a salt of a physiologically active peptide (tentatively referred to as (A)) and hydroxynaphthoic acid (tentatively referred to as (B)), the salt of (A) and (B) The weight ratio of (A) to the sum is usually about 5 to about 90% by weight, preferably about 10 to about 85% by weight, more preferably about 15 to about 80% by weight, and particularly preferably about 30 to about 80% by weight. % By weight. In the case of a sustained-release composition containing a physiologically active substance or a salt thereof and hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof, the compounding amount of the hydroxynaphthoic acid or a salt thereof is preferably Hydroxynaphthoic acid or a salt thereof is used in an amount of about 1/2 to about 2 moles, about 3/4 to about 4/3, per mole of a physiologically active substance or a salt thereof.
Mole, particularly preferably about 4/5 to about 6/5 mole.

[0027] The composition of the present invention is designed by using a bioactive substance,
The sustained-release composition containing hydroxynaphthoic acid and the biodegradable polymer will be described below, taking as an example the case where the physiologically active substance is basic. in this case,
In the composition, a physiologically active substance as a base and hydroxynaphthoic acid as an acid coexist, and when they are incorporated in the composition as a free form or a salt, a certain point in time during the production of the composition In each case, a dissociation equilibrium is established in a water-containing state or in the presence of a small amount of water. The salt formed by the slightly water-soluble hydroxynaphthoic acid with the physiologically active substance is considered to be slightly water-soluble depending on the properties of the physiologically active substance, so that the dissociation equilibrium tends toward such a slightly water-soluble salt formation. . In order to produce a composition containing a high content of a basic physiologically active substance, it is desirable to protonate most of the physiologically active substance to form the above slightly water-soluble salt in view of the above dissociation equilibrium. For this purpose, it is desirable to mix hydroxynaphthoic acid or a salt thereof in an amount equivalent to at least the physiologically active substance or its salt. Next, the sustained release mechanism of the physiologically active substance contained in the composition will be described below.
Most of the physiologically active substance is protonated in the above-mentioned composition, and exists in a state accompanied by a counter ion. The counter ion is mainly hydroxynaphthoic acid (preferably hydroxynaphthoic acid). After the composition is administered to a living body, the degradation of the biodegradable polymer starts to form its oligomers and monomers over time, but when the polymer is a lactic acid-glycolic acid polymer, the oligomers formed (Lactic acid-glycolic acid oligomer) and the monomer (lactic acid or glycolic acid) always have one carboxyl group, and these can also be counter ions of the physiologically active substance. The release of the physiologically active substance does not involve the transfer of electric charge, that is, is performed as a salt with a counter ion. As the movable counter ion species, hydroxynaphthoic acid, lactic acid-glycolic acid oligomer (transferable And the monomers (lactic acid or glycolic acid). When a plurality of acids coexist, a salt of a strong acid is generally preferentially generated, depending on the composition ratio. The pKa of hydroxynaphthoic acid is, for example, 3
-Hydroxy-2-naphthoic acid is 2.708 (Chemical Handbook Basic Edition II, Chemical Society of Japan, September 25, 1969)
Date). On the other hand, that of the carboxyl group of the lactic acid-glycolic acid oligomer is not known, but based on the pKa of lactic acid or glycolic acid (= 3.86 or 3.83),
It can be calculated according to the principle that “the change in free energy due to the introduction of a substituent can be approximated by an addition rule”. The contribution of the substituent to the dissociation constant is required and can be used (Table 4.1 in "pKa Prediction for Organic Acid an
dBases ", DDPerrin, B. Dempsey and EPSerjeant, 1
981). ΔpKa (OH) = − 0.90 ΔpKa (ester bond) = − 1.7 for the hydroxyl group and the ester bond, respectively, so that the pKa of the carboxyl group of the lactic acid-glycolic acid oligomer is the ester closest to the dissociation group. Taking into account the binding contribution, pKa = pKa (lactic or glycolic acid)
-ΔpKa (OH) + ΔpKa (ester bond) = 3.06
Or 3.03 is required. Therefore, hydroxynaphthoic acid is composed of lactic acid (pKa = 3.86) and glycolic acid (pKa
Ka = 3.83), and furthermore, since it is a stronger acid than the lactic acid-glycolic acid oligomer, it is considered that a salt of hydroxynaphthoic acid and a physiologically active substance is preferentially formed in the above composition. It is believed that the properties of the salt predominantly determine the sustained release properties of the bioactive substance from the composition. Examples of the physiologically active substance include the aforementioned physiologically active substances. Here, the fact that the salt formed by hydroxynaphthoic acid with the physiologically active substance is slightly water-soluble and not water-insoluble has a favorable effect on the sustained release mechanism. That is, as clarified in the above discussion of the acid dissociation constant, as a salt of a mobile physiologically active substance, a salt of hydroxynaphthoic acid, which is a stronger acid than the above lactic acid-glycolic acid oligomer and monomer, predominates in the early stage of release. As a result, the solubility of the salt and the distribution to the body tissue are determinants of the release rate of the physiologically active substance. Therefore, the initial release pattern of the drug can be adjusted by the amount of hydroxynaphthoic acid. afterwards,
With the decrease of hydroxynaphthoic acid and the increase of oligomers and monomers generated by the hydrolysis of biodegradable polymers, the release mechanism of biologically active substances having oligomers and monomers as counter ions gradually becomes dominant, and hydroxynaphthoic acid is virtually eliminated. Even when the "composition" disappears, stable release of the physiologically active substance is maintained. It can also be explained that the efficiency of uptake of the physiologically active substance during the production of the sustained-release composition can be increased, and that the initial excessive release after administration of the incorporated physiologically active substance can be suppressed. The role of hydroxynaphthoic acid in a sustained-release composition containing a hydroxynaphthoic acid salt of a physiologically active peptide can also be explained by the above mechanism.

The term "water-insoluble" as used herein means that when the substance is stirred in distilled water at a temperature of 40 ° C. or lower for 4 hours, the mass of the substance dissolved in 1 L of the solution is 25 mg.
Refers to the following cases. The term "slightly water-soluble" as used herein refers to a case where the mass is more than 25 mg and 5 g or less. When the substance is a salt of a physiologically active substance, the above definition is applied based on the mass of the physiologically active substance dissolved in the above operation. The form of the sustained-release composition in the present specification is not particularly limited, but is preferably in the form of fine particles, and particularly in the form of microspheres (in the case of a sustained-release composition containing a biodegradable polymer, it is also referred to as microcapsules). preferable. In addition, the microsphere in this specification refers to injectable spherical fine particles that can be dispersed in a solution. The confirmation of the form can be performed by, for example, observation with a scanning electron microscope.

[0029]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A sustained-release composition containing a physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof according to the present invention, for example, a method for producing microcapsules I do. (I) In-water drying method (i) O / W method In this method, first, an organic solvent solution of hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof is prepared. The organic solvent used for producing the sustained-release preparation of the present invention preferably has a boiling point of 120 ° C. or lower. Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, dichloroethane, trichloroethane, carbon tetrachloride, etc.), ethers (eg, ethyl ether, isopropyl ether, etc.), fatty acid esters (eg, ethyl acetate, Butyl acetate, etc.), aromatic hydrocarbons (eg, benzene, toluene, xylene, etc.),
Alcohols (eg, ethanol, methanol, etc.),
Acetonitrile and the like are used. Of these, dichloromethane is preferred as the organic solvent for the biodegradable polymer or a salt thereof. Alcohols are preferred as the organic solvent for hydroxynaphthoic acid or a salt thereof. They may be separately dissolved and then mixed together, or these may be used by dissolving the two in an organic solvent mixed at an appropriate ratio. Of these, a mixture of a halogenated hydrocarbon and an alcohol is preferable, and a mixture of dichloromethane and ethanol is particularly preferable. When ethanol is used as a mixed organic solvent with dichloromethane, the content of ethanol in the mixed organic solvent of dichloromethane and ethanol is generally about 0.01 to about 50% (v / v), and is more preferably. It is selected from about 0.05 to about 40% (v / v), particularly preferably about 0.1 to about 30% (v / v). The concentration of the biodegradable polymer in the organic solvent solution varies depending on the molecular weight of the biodegradable polymer and the type of the organic solvent.For example, when dichloromethane is used as the organic solvent, the concentration is generally about 0.5 to 0.5. It is selected from about 70% by weight, more preferably from about 1 to about 60% by weight, particularly preferably from about 2 to about 50% by weight. The concentration of hydroxynaphthoic acid or a salt thereof in an organic solvent is generally about 0.01 to about 1 when a mixture of dichloromethane and ethanol is used as the organic solvent.
0% by weight, more preferably from about 0.1 to about 5% by weight, particularly preferably from about 0.5 to about 3% by weight. A physiologically active substance or a salt thereof is added to the thus obtained solution of hydroxynaphthoic acid or a salt thereof and a biodegradable polymer in an organic solvent, and dissolved or dispersed. Next, an organic solvent solution containing the obtained physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof and a composition comprising a biodegradable polymer or a salt thereof is added to an aqueous phase, and O (oil phase) / W (Aqueous phase) After forming the emulsion, the solvent in the oil phase is volatilized or diffused in the aqueous phase to prepare microcapsules. The volume of the aqueous phase at this time is generally about 1 time to about 10,000 times the volume of the oil phase.
Times, more preferably from about 5 times to about 50,000 times, particularly preferably from about 10 times to about 2,000 times. An emulsifier may be added to the above external water phase. The emulsifier may be any as long as it can form a stable O / W emulsion. Specifically, for example, an anionic surfactant (sodium oleate, sodium stearate, sodium lauryl sulfate, etc.), a nonionic surfactant (polyoxyethylene sorbitan fatty acid ester [Tween 80, Tween (Tween) ) 60, Atlas Powder Co., Ltd.), polyoxyethylene castor oil derivative (HCO
-60, HCO-50, Nikko Chemicals]), polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like. One of these or a combination of some of them may be used. The concentration at the time of use is preferably in the range of about 0.01 to 10% by weight, more preferably in the range of about 0.05 to about 5% by weight. An osmotic pressure regulator may be added to the above external aqueous phase. The osmotic pressure adjusting agent may be any agent that exhibits an osmotic pressure when used as an aqueous solution. Examples of the osmotic pressure adjusting agent include polyhydric alcohols, monohydric alcohols, monosaccharides, disaccharides, oligosaccharides, amino acids, and derivatives thereof. Examples of the above-mentioned polyhydric alcohols include trihydric alcohols such as glycerin, pentahydric alcohols such as arabitol, xylitol and adonitol, and hexahydric alcohols such as mannitol, sorbitol and dulcitol. Among them, hexahydric alcohols are preferred, and mannitol is particularly preferred. Examples of the monohydric alcohols include, for example, methanol, ethanol,
Examples thereof include isopropyl alcohol, and among them, ethanol is preferable. Examples of the above monosaccharides include pentoses such as arabinose, xylose, ribose, and 2-deoxyribose, and hexoses such as glucose, fructose, galactose, manose, sorbose, rhamnose, and fucose. Carbohydrates are preferred. As the oligosaccharide, for example, trisaccharides such as maltotriose and raffinose sugar, tetrasaccharides such as stachyose and the like are used, and among them, trisaccharides are preferable. As the derivatives of the above monosaccharides, disaccharides and oligosaccharides, for example, glucosamine, galactosamine, glucuronic acid, galacturonic acid and the like are used. The above amino acids include L-
Any body can be used, for example,
Glycine, leucine, arginine and the like can be mentioned. Of these, L-arginine is preferred. These osmotic pressure adjusting agents may be used alone or as a mixture. These osmotic pressure regulators are used at a concentration such that the osmotic pressure of the external aqueous phase is about 1/50 to about 5 times, preferably about 1/25 to about 3 times the osmotic pressure of physiological saline. As a method for removing the organic solvent, a method known per se or a method analogous thereto is used. For example, a method of evaporating the organic solvent at normal pressure or gradually reducing the pressure while stirring with a propeller type stirrer or a magnetic stirrer or an ultrasonic generator, or adjusting the degree of vacuum using a rotary evaporator or the like. Examples thereof include a method of evaporating the solvent, and a method of gradually removing the organic solvent using a dialysis membrane. The microcapsules thus obtained are separated by centrifugation or filtration, and then the free bioactive substance or a salt thereof attached to the surface of the microcapsule, hydroxynaphthoic acid or a salt thereof, a drug holding substance, The emulsifier and the like are repeatedly washed several times with distilled water, dispersed again in distilled water or the like, and freeze-dried. During the manufacturing process, an aggregation inhibitor may be added to prevent aggregation of the particles. Examples of the anticoagulant include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (eg, corn starch and the like);
Amino acids such as glycine and proteins such as fibrin and collagen are used. Among them, mannitol is preferred. After freeze-drying, if necessary, the microcapsules may be heated under reduced pressure under conditions that do not cause fusion of the microcapsules to remove water and the organic solvent in the microcapsules. Preferably, the polymer is heated at a temperature slightly higher than the midpoint glass transition temperature of the biodegradable polymer determined by a differential scanning calorimeter under the condition of a temperature rising rate of 10 to 20 ° C. per minute. More preferably, heating is performed within a temperature range of about 30 ° C. higher than the midpoint glass transition temperature of the biodegradable polymer. In particular, lactic acid is a biodegradable polymer.
When a glycolic acid polymer is used, it is preferably at least its midpoint glass transition temperature and at least 10
Heating is carried out in a temperature range higher than the intermediate point glass transition temperature, more preferably 5 ° C. higher than the intermediate point glass transition temperature. The heating time varies depending on the amount of the microcapsules, but generally, after the microcapsules themselves reach a predetermined temperature, about 12 hours to about 168 hours, preferably about 24 hours to about 120 hours, particularly preferably About 4
8 hours to about 96 hours. The heating method is not particularly limited as long as the method is capable of uniformly heating the microcapsules. As the heating and drying method, for example, a method of heating and drying in a constant temperature bath, a fluidized bath, a moving tank or a kiln, a method of heating and drying with a microwave, and the like are used. Of these, a method of heating and drying in a thermostat is preferred.

(Ii) W / O / W method (1) First, a biodegradable polymer or a salt thereof in an organic solvent is prepared. The concentration of the organic solvent and the biodegradable polymer or a salt thereof in the organic solvent solution is as defined in the above (I).
The same as described in the item (i). When a mixed organic solvent is used, the ratio between the two is the same as described in the above section (I) (i). A physiologically active substance or a salt thereof is added to an organic solvent solution of the biodegradable polymer or a salt thereof thus obtained, and dissolved or dispersed. Subsequently, a solution of hydroxynaphthoic acid or a salt thereof is added to an organic solvent solution (oil phase) containing the obtained physiologically active substance or a salt thereof and a composition comprising a biodegradable polymer or a salt thereof (the solvent is water, Aqueous solutions of alcohols (eg, methanol, ethanol, etc.), pyridine aqueous solutions, dimethylacetamide aqueous solutions, etc.)]. This mixture is emulsified by a known method such as a homogenizer or ultrasonic wave,
Form a W / O emulsion. Next, the obtained W / O emulsion composed of a physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof is added to an aqueous phase, and W (inner aqueous phase) /
After forming an O (oil phase) / W (outer water phase) emulsion, the solvent in the oil phase is volatilized to prepare microcapsules. The volume of the external water phase at this time is generally about 1 to the volume of the oil phase.
Times to about 10,000 times, more preferably about 5 times to about 5,
000 times, particularly preferably from about 10 times to about 2,000 times. The emulsifiers and osmotic pressure regulators which may be added to the above external aqueous phase, and the subsequent preparation method are described in the above (I) (i)
The same as described in the section.

(Iii) W / O / W method (2) First, an organic solvent solution of hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof is prepared, and the obtained organic solvent solution is prepared. Called the oil phase. The preparation method is the same as described in the above (I) (i). Alternatively, hydroxynaphthoic acid or a salt thereof and a biodegradable polymer are separately prepared as organic solvent solutions,
Thereafter, they may be mixed. The concentration of the biodegradable polymer in the organic solvent solution depends on the molecular weight of the biodegradable polymer,
Depending on the type of organic solvent, for example, when dichloromethane is used as the organic solvent, generally about 0.5 is used.
To about 70% by weight, more preferably about 1 to about 60% by weight
And particularly preferably from about 2 to about 50% by weight.
Next, a solution or dispersion of a physiologically active substance or a salt thereof (the solvent is water, a mixture of water and alcohols (eg, methanol, ethanol, etc.), etc.) is prepared. The concentration of the physiologically active substance or a salt thereof is generally 0.001.
mg / ml to 10 g / ml, more preferably 0.1 mg
/ Ml to 5 g / ml, more preferably 10 mg / ml to
3 g / ml. Known dissolution aids and stabilizers may be used. For dissolving or dispersing the physiologically active substance and additives, heating, shaking, stirring and the like may be performed to such an extent that the activity is not lost, and the resulting aqueous solution is referred to as an internal aqueous phase. The inner water phase and the oil phase obtained as described above are emulsified by a known method such as a homogenizer or ultrasonic wave, and W /
An O emulsion is formed. The volume of the oil phase to be mixed is about 1 to about 1000 times, preferably about 2 to 100 times, more preferably about 3 to 10 times the volume of the internal aqueous phase. The viscosity range of the W / O emulsion obtained is generally about 1
At 5-20 ° C., about 10-10,000 cp, preferably about 100-5,000 cp. More preferably, it is about 500 to 2,000 cp. Next, the obtained W / O emulsion composed of a physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof is added to an aqueous phase, and W (inner aqueous phase) /
After forming an O (oil phase) / W (outer water phase) emulsion, the solvent in the oil phase is volatilized or diffused into the outer water phase,
Prepare microcapsules. In this case, the volume of the external water phase is generally about 1 to about 10,000 times, preferably about 5 to about 50,000 times, particularly preferably about 10 to about 2,000 times the oil phase volume. Selected from double. The emulsifiers and osmotic pressure regulators which may be added to the above external aqueous phase, and the subsequent preparation method are the same as those described in the above section (I) (i).

(II) Phase Separation Method In the case of producing microcapsules by this method, the physiologically active substance or its salt, hydroxynaphthoic acid or its salt and the biodegradable substance described in the above (I) in-water drying method are used. A microcapsule is precipitated and solidified by gradually adding a coacervation agent to an organic solvent solution containing a composition comprising a polymer or a salt thereof, with stirring. The coacervation agent has an oil phase volume of about 0.01 to 1,000
Fold, preferably about 0.05 to 500 times, particularly preferably about 0.1 to 200 times. The coacervation agent is a compound of a bioactive compound or a salt thereof, such as a polymer, a mineral oil or a vegetable oil, which is miscible with an organic solvent, such as hydroxynaphthoic acid or a biodegradable polymer or a salt thereof. There is no particular limitation as long as it does not dissolve the body. Specifically, for example, silicone oil, sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane, n-heptane and the like are used. These may be used as a mixture of two or more. After fractionating the microcapsules thus obtained, it is repeatedly washed with heptane or the like, and a physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof and a composition other than a composition comprising a biodegradable polymer or a salt thereof are used. The coacervation agent and the like are removed, and dried under reduced pressure. Alternatively, washing is performed in the same manner as described in the underwater drying method (I) (i), followed by freeze-drying and further drying by heating.

(III) Spray drying method In the case of producing microcapsules by this method, the physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof and the biodegradable substance described in the above (I) in-water drying method are used. An organic solvent solution containing a polymer or a salt thereof is sprayed into a drying chamber of a spray drier using a nozzle, and the organic solvent in the atomized droplets is volatilized within a very short time. Prepare capsules. Examples of the nozzle include a two-fluid nozzle type, a pressure nozzle type, and a rotating disk type. Thereafter, if necessary, washing may be performed in the same manner as described in the underwater drying method (I), followed by freeze-drying and further heating and drying. Organic solvent containing a physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof, and a biodegradable polymer or a salt thereof described in the in-water drying method of the microcapsule production method (I) as a dosage form other than the microcapsules described above. The solution may be evaporated to dryness by evaporating the organic solvent and water while adjusting the degree of vacuum using, for example, a rotary evaporator or the like, and then pulverized with a jet mill or the like to form fine powder (also referred to as microparticles). Further, the pulverized fine powder is converted into a microcapsule production method (I)
After washing in the same manner as described in the underwater drying method, freeze-drying and further heating and drying may be performed. The microcapsules or fine powder obtained here can achieve drug release corresponding to the decomposition rate of the biodegradable polymer or lactic acid-glycolic acid polymer used. Next, a method for producing a sustained-release composition containing the hydroxynaphthoic acid salt of the physiologically active substance of the present invention will be exemplified. In the present production method, a physiologically active peptide is preferably used as the physiologically active substance.

(IV) Two-step method: A physiologically active substance or a salt thereof is added to a solution of hydroxynaphthoic acid or a salt thereof in an organic solvent so as to have a weight ratio as defined in the definition of the amount of the physiologically active substance. Make an organic solvent solution containing the hydroxynaphthoate salt of the substance. The organic solvent is the same as described in the above (I) (i). When a mixed organic solvent is used, the ratio between the two is the same as described in the above section (I) (i). A method for removing an organic solvent for precipitating a composition containing a hydroxynaphthoic acid salt of a physiologically active substance,
A method known per se or a method analogous thereto is used. For example, a method of evaporating an organic solvent while adjusting the degree of vacuum using a rotary evaporator or the like can be used. An organic solvent solution of the composition containing the hydroxynaphthoic acid salt of the physiologically active substance thus obtained is prepared again to prepare a sustained-release composition (microspheres or fine particles). As the organic solvent,
For example, halogenated hydrocarbons (eg, dichloromethane, chloroform, dichloroethane, trichloroethane, carbon tetrachloride, etc.), ethers (eg, ethyl ether, isopropyl ether, etc.), fatty acid esters (eg, ethyl acetate, butyl acetate, etc.), aromatics Group hydrocarbons (eg, benzene,
Toluene, xylene, etc.) are used. These may be mixed and used at an appropriate ratio. Of these, halogenated hydrocarbons are preferred, and dichloromethane is particularly preferred. Next, an organic solvent solution containing the obtained composition containing the hydroxynaphthoate of a physiologically active substance is added to the aqueous phase to form an O (oil phase) / W (aqueous phase) emulsion, and then the oil phase is formed. The solvent therein is evaporated to prepare microspheres. The volume of the water phase at this time is generally about 1 to about 10,000 times, preferably about 5 to about 5,000 times, particularly preferably about 10 to about 2, times the volume of the oil phase. 000
Selected from double. The emulsifier and osmotic pressure adjusting agent which may be added to the above external aqueous phase, and the subsequent preparation method are described in the above (I)
The same as described in the item (i). As a method for removing the organic solvent, a method known per se or a method analogous thereto is used. For example, a method of evaporating the organic solvent at normal pressure or gradually reducing pressure while stirring with a propeller-type stirrer or a magnetic stirrer, evaporating the organic solvent while adjusting the degree of vacuum using a rotary evaporator or the like And the like. The microspheres obtained in this way are separated by centrifugation or filtration, and then the free bioactive substance, hydroxynaphthoic acid, emulsifier, etc. attached to the surface of the microspheres are repeatedly washed several times with distilled water. Then, it is again dispersed in distilled water and freeze-dried. During the manufacturing process, an aggregation inhibitor may be added to prevent aggregation of the particles. Examples of the anticoagulant include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (eg, corn starch and the like);
Examples include amino acids such as glycine and proteins such as fibrin and collagen. Of these, mannitol is preferred. Further, after freeze-drying, if necessary, the microspheres may be heated under reduced pressure within a condition in which the microspheres do not fuse with each other to further remove water and the organic solvent in the microspheres. The heating time varies depending on the amount of the microspheres and the like, but generally, after the microspheres themselves reach a predetermined temperature, about 12 hours to about 168 hours, preferably about 24 hours to about 120 hours, particularly preferably From about 48 hours to about 96 hours. The heating method is not particularly limited as long as the method is capable of uniformly heating the microspheres. As the heating and drying method, for example, a method of heating and drying in a constant temperature bath, a fluidized bath, a moving tank or a kiln, a method of heating and drying with a microwave, and the like are used.
Of these, a method of heating and drying in a thermostat is preferred. The obtained microspheres have a relatively uniform spherical shape, have low resistance during injection administration, and are less likely to cause needle clogging. Further, since a thin injection needle can be used, pain of the patient at the time of injection is reduced.

(V) One-step method: A physiologically active substance or a salt thereof is added to a solution of hydroxynaphthoic acid or a salt thereof in an organic solvent so as to have a weight ratio as defined in the definition of the amount of the physiologically active substance. An organic solvent solution containing the hydroxynaphthoate of the substance is made to produce a sustained release formulation (microspheres or microparticles). The organic solvent is the same as described in the above (I) (i). When a mixed organic solvent is used, the ratio between the two is the same as described in the above section (I) (i). Next, an organic solvent solution containing a hydroxynaphthoic acid salt of a physiologically active substance is added to the aqueous phase, and O (oil phase) / W
(Aqueous phase) After forming the emulsion, the solvent in the oil phase is evaporated to prepare microspheres. The volume of the aqueous phase at this time is generally about 1 time to about 10,000 times the volume of the oil phase.
Times, more preferably from about 5 times to about 5,000 times, particularly preferably from about 10 times to about 2,000 times. The emulsifiers and osmotic pressure regulators that may be added to the above external aqueous phase, and the subsequent preparation method are the same as those described in the above section (IV). The sustained-release composition of the present invention may be in any form such as microspheres, microcapsules, and fine powders (microparticles). In the case where the composition comprises a bioactive substance and hydroxynaphthoic acid, microspheres may be used. However, microcapsules are preferred when they are composed of a bioactive substance, hydroxynaphthoic acid and a biodegradable polymer. The sustained-release composition of the present invention may be formulated as it is or as a raw material into various dosage forms, intramuscularly, subcutaneously,
Solid preparations such as injections or implants for organs, transmucosal preparations for the nasal cavity, rectum, uterus, etc., oral preparations (eg, capsules (eg, hard capsules, soft capsules, etc.), granules, powders, etc.) , Syrups, emulsions, liquids such as suspensions, etc.). For example, in order to make the sustained-release composition of the present invention an injection, it is necessary to use a dispersant (for example, a surfactant such as Tween 80 or HCO-60, sodium hyaluronate, carboxymethylcellulose, sodium alginate, etc.). Polysaccharides, etc.), preservatives (eg, methylparaben, propylparaben, etc.), tonicity agents (eg, sodium chloride, mannitol, sorbitol, glucose, proline, etc.) or the like, as an aqueous suspension, or sesame oil, corn It can be dispersed with vegetable oils such as oil to give a sustained-release injection which can be actually used as an oily suspension.

When the sustained release composition of the present invention is used as a suspension injection, the particle size may be within a range that satisfies the degree of dispersion and needle penetration. 0.1-300 μm, preferably about 0.5-150 μm
, More preferably in the range of about 1 to 100 μm. In order to make the sustained-release composition of the present invention into a sterile preparation, a method of sterilizing all production steps, a method of sterilizing with gamma rays,
Examples include a method of adding a preservative, but the method is not particularly limited. Since the sustained-release composition of the present invention has low toxicity, it can be used as a drug safe for mammals (eg, human, cow, pig, dog, cat, mouse, rat, rabbit, etc.). . The dose of the sustained-release composition of the present invention varies depending on the type and content of the bioactive substance as the main drug, dosage form, duration of bioactive substance release, target disease, target animal, etc. Any effective amount may be used. As the dose per dose of the physiologically active substance as the main drug, for example, when the sustained release formulation is a 6-month formulation, preferably
The range of about 0.01 mg to 10 mg / kg body weight per adult, more preferably about 0.05 mg to 5 mg / kg.
It can be appropriately selected from a range of weight. The dose of the sustained-release composition per dose is preferably selected from the range of about 0.05 mg to 50 mg / kg body weight, more preferably the range of about 0.1 mg to 30 mg / kg body weight per adult. Can be. The frequency of administration is once every several weeks, once a month, or once every several months (eg, once every three months, four months, six months, etc.)
It can be appropriately selected depending on the dosage form, duration of release of the physiologically active substance, target disease, target animal, and the like. The sustained-release composition of the present invention can be used as a prophylactic / therapeutic agent for various diseases depending on the type of the physiologically active substance contained therein. For example, the physiologically active substance is an LH-RH derivative. In some cases, hormone-dependent diseases, especially sex hormone-dependent cancers (eg, prostate, uterine, breast, pituitary, etc.),
Benign prostatic hyperplasia, endometriosis, uterine fibroids, precocious puberty,
When using preventive or therapeutic agents for sexual hormone-dependent diseases such as dysmenorrhea, amenorrhea, premenstrual syndrome, and multilocular ovarian syndrome, and contraception (or the rebound effect after withdrawal) It can be used as an agent for preventing or treating infertility). In addition, sex hormone-independent
Prevention of H-RH sensitive benign or malignant tumors
It can also be used as a therapeutic.

[0037]

The present invention will be described more specifically with reference to the following examples, experimental examples and comparative examples, but these are not intended to limit the present invention. Example 1 N- (S) -Tetrahydrofur-2-oyl-Gly-D2Nal-D4ClPhe-D3Pal-
Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂
Acetate (hereinafter abbreviated as peptide A) (TAP)
3429.6 m and 685.2 mg of 3-hydroxy-2-naphthoic acid (manufactured by Wako Pure Chemical Industries) were dissolved in 15 ml of ethanol. (Structural formula of peptide A)

Embedded image The solution was gradually reduced in pressure using a rotary evaporator, and the organic solvent was evaporated. This residue was redissolved in 5.5 ml of dichloromethane and adjusted to 18 ° C. in advance.
It was poured into 400 ml of an aqueous solution of 0.1% (w / w) polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical) and stirred at 8,000 rpm using a turbine type homomixer to obtain an O / W emulsion. The O / W emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane, solidify the oil phase, and then
m sieve using a sieve with a mesh opening and a centrifuge (05PR-
Microspheres were sedimented and collected at 2,000 rpm for 5 minutes using Hitachi, Ltd.). This was dispersed again in distilled water, followed by further centrifugation to wash free drugs and the like, and to collect microspheres. The collected microspheres were re-dispersed by adding a small amount of distilled water and freeze-dried to obtain a powder. The microspheres had a mass recovery of 65%, and the peptide A content and the 3-hydroxy-2-naphthoic acid / peptide A molar ratio in the microspheres were 75.4% and 1.94, respectively.

Example 2 Peptide A acetate 1785.1 mg and 3-hydroxy-2
1370.4 mg of naphthoic acid was dissolved in 15 ml of ethanol. The solution was gradually reduced in pressure using a rotary evaporator, and the organic solvent was evaporated. This residue was re-dissolved in 10 ml of dichloromethane, and 0.1% previously adjusted to 18 ° C.
(w / w) Inject into 1000 ml of polyvinyl alcohol aqueous solution,
Stir at 8,000 rpm using a turbine type homomixer and
A W emulsion was obtained. Subsequent operations were performed in the same manner as in Example 1 to obtain microspheres. The mass recovery of the microspheres was 58%, and the peptide A content and the 3-hydroxy-2-naphthoic acid / peptide A molar ratio in the microspheres were 54.3% and 6.15, respectively.

Example 3 1800 mg of the acetate salt of peptide A, 173 mg of 3-hydroxy-2-naphthoic acid and a lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/50 (mol%), weight average molecular weight 10,10
0, number average molecular weight 5,670, carboxyl group content by alkali titration 268.8 μmol / g, manufactured by Wako Pure Chemical Industries, Ltd.) 2 g was dissolved in a mixed organic solvent of dichloromethane 6 ml and ethanol 0.2 ml and adjusted to 18 ° C. in advance. Contains 5% mannitol
It was poured into 900 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol, and stirred at 7,000 rpm using a turbine type homomixer to obtain an O / W emulsion. This O / W emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane and ethanol or diffuse it into the aqueous phase to solidify the oil phase, and then sieved using a 75 μm mesh sieve, and centrifuged. The microcapsules were sedimented and collected at 2,000 rpm for 5 minutes using. This was dispersed again in distilled water, and further centrifuged to wash free drugs and the like, and microcapsules were collected. 250m of collected microcapsules
After redispersion by adding g of mannitol and a small amount of distilled water, the mixture was freeze-dried to obtain a powder. The mass recovery of the microcapsules obtained by excluding the added mannitol in the calculation was 76%, the peptide A content in the microcapsules and the 3-hydroxy-2-naphthoic acid / peptide A molar ratio were 34.7% and 14.7%, respectively. .19. The encapsulation efficiency obtained by dividing the realized content by the charged content is 84.
6%.

Example 4 1900 mg of acetate of peptide A, 182 mg of 3-hydroxy-2-naphthoic acid and 1.9 g of lactic acid-glycolic acid copolymer (same as in Example 3) were mixed with 6 ml of dichloromethane and 0.2 ml of ethanol to prepare a mixed organic solvent. And 5% mannitol and 0.05% L-arginine containing 0.1% (w /
w) The mixture was poured into 900 ml of an aqueous polyvinyl alcohol solution, and stirred at 7,000 rpm using a turbine-type homomixer to obtain an O / W emulsion. Subsequent operations were performed in the same manner as described in Example 3 to obtain microcapsules. The mass recovery of the microcapsules obtained by excluding the added mannitol by calculation was 85%, the peptide A content in the microcapsules and the 3-hydroxy-2-naphthoic acid / peptide A molar ratio were 38.6% and 0, respectively. Was 0.83. The encapsulation efficiency obtained by dividing the realized content by the charged content is 8
It was 8.9%.

Example 5 The lactic acid-glycolic acid copolymer described in Example 4 was
Glycolic acid = 75/25 (mol%), weight average molecular weight 10,70
0, the number average molecular weight was 6,100, and the amount of carboxyl groups by alkali titration was changed to a lactic acid-glycolic acid copolymer having 265.3 μmol / g, and the amount of dichloromethane was changed to 6.5 ml in the same manner as described in Example 4. Microcapsules were obtained. The mass recovery of the microcapsules obtained by excluding the added mannitol by calculation was 87%, the peptide A content in the microcapsules and the 3-hydroxy-2-naphthoic acid / peptide A molar ratio were 38.3% and 0, respectively. .
92. The encapsulation efficiency calculated by dividing the realized content by the charged content was 88.3%.

Example 6 Peptide A acetate (1800 mg) and lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/50 (mol%), weight average molecular weight 12,700, number average molecular weight 7,090, carboxyl content by alkali titration) 209.2 μmol / g, manufactured by Wako Pure Chemical Industries, Ltd.) in a solution of 1.8 g in 7.2 ml of dichloromethane was added with 196 m 3 of sodium 3-hydroxy-2-naphthoate.
g was dissolved in 2.3 ml of water, and the mixture was emulsified with a homogenizer to prepare a W / O emulsion. This emulsion was adjusted to 18 ° C in advance and contained 5% mannitol 0.1% (w /
w) Pour into 800 ml of aqueous polyvinyl alcohol solution, stir at 7,000 rpm using a turbine type homomixer, and
A W emulsion was obtained. This W / O / W emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane or ethanol or diffuse it into the aqueous phase to solidify the oil phase, then sieved using a 75 μm mesh sieve, and centrifuged. The microcapsules were sedimented and collected at 2,000 rpm for 5 minutes using a separator. This was dispersed again in distilled water, and further centrifuged to wash free drugs and the like, and microcapsules were collected. 250m of collected microcapsules
After redispersion by adding g of mannitol and a small amount of distilled water, the mixture was freeze-dried to obtain a powder. The mass recovery of the microcapsules obtained by excluding the added mannitol in the calculation was 79%, the peptide A content in the microcapsules and the 3-hydroxy-2-naphthoic acid / peptide A molar ratio were 32.8% and 0, respectively. Was .91. The encapsulation efficiency obtained by dividing the realized content by the charged content is 81.
2%.

Experimental Example 1 Approximately 40 m of each microsphere obtained in Examples 1 and 2.
g or about 60 mg of each microcapsule obtained in Examples 3 to 5 in 0.5 ml of a dispersion medium (0.25 mg of carboxymethylcellulose, 0.5 mg of polysorbate 80, and 25 mg of mannitol in distilled water). 22G subcutaneously on the back of 8-10 week old male SD rats
It was administered with a needle. After a predetermined time from the administration, the rats were sacrificed and the microspheres or microcapsules remaining at the administration site were taken out. Peptide A in the microspheres was quantified and divided by the respective initial contents.

【table 1】 From the experimental results of Examples 1 and 2, the release of peptide A from the microspheres consisting of peptide A and 3-hydroxy-2-naphthoic acid was different depending on the difference between the two ratios. The higher the proportion of naphthoic acid, the faster the release of peptide A. In addition, from the experimental results of Examples 3, 4 and 5, in the case of the microcapsules composed of three members to which the lactic acid-glycolic acid copolymer was added, the results differed from the release of peptide A from the microsphere composed of only two members. It was also found that the release behavior can be controlled by combining lactic acid-glycolic acid copolymers having different compositions, weight average molecular weights and terminal carboxyl groups.

Example 7 5-oxo-Pro-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C ₂ H ₅
(Hereinafter, abbreviated as peptide B. Takeda Pharmaceutical) acetate
A solution obtained by dissolving 0.8 g in 0.8 ml of distilled water was combined with 3.08 g of DL-lactic acid polymer (weight average molecular weight 36,000, number average molecular weight 18,000, carboxyl group content 70.4 μmol / g by labeling assay) and 3-hydroxy- 0.12 g of 2-naphthoic acid was mixed with a solution of 5 ml of dichloromethane and 0.3 ml of ethanol in a mixed organic solvent and emulsified with a homogenizer to form a W / O emulsion. Next, this W / O emulsion was poured into 800 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) that had been adjusted to 15 ° C. in advance, and was then mixed with a turbine-type homomixer. The mixture was stirred at 7,000 rpm to obtain a W / O / W emulsion. The W / O / W emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane and ethanol or diffuse into the external aqueous phase to solidify the oil phase, and then sieved using a 75 μm mesh sieve.
Then, use a centrifuge (05PR-22, Hitachi, Ltd.) for 2,0
The microcapsules were sedimented and collected at 00 rpm for 5 minutes. This was dispersed again in distilled water, and further centrifuged to wash free drugs and the like, and microcapsules were collected. The collected microcapsules were redispersed by adding a small amount of distilled water and freeze-dried to obtain a powder. The mass recovery of the microcapsules was 46%, the content of peptide B in the microcapsules was 21.3%, and the content of 3-hydroxy-2-naphthoic acid was 2.96%. The encapsulation efficiency obtained by dividing these realized contents by the charged contents was 106.6% for peptide B and 106.6% for 3-hydroxy-2-naphthoic acid.
98.6%.

Example 8 A solution prepared by dissolving 1.2 g of the acetate salt of peptide B in 1.2 ml of distilled water was treated with a DL-lactic acid polymer (weight average molecular weight: 25,200, number average molecular weight: 12,800, carboxyl group content by a labeling assay).
62.5 μmol / g) 4.62 g and 0.18 g of 3-hydroxy-2-naphthoic acid were mixed with 7.5 ml of dichloromethane and 0.45 g of ethanol.
The mixture was mixed with a solution dissolved in ml of a mixed organic solvent and emulsified with a homogenizer to form a W / O emulsion. The W / O emulsion was then adjusted to 15 ° C in advance.
It was poured into 1200 ml of 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) and stirred at 7,000 rpm using a turbine type homomixer to obtain a W / O / W emulsion. The W / O / W emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane and ethanol or diffuse into the external aqueous phase to solidify the oil phase, and then sieved using a 75 μm mesh sieve. Then centrifuge (05PR-2
The microcapsules were sedimented and collected at 2,000 rpm for 5 minutes using Hitachi, Ltd.). This was dispersed again in distilled water, and further centrifuged to wash free drugs and the like, and microcapsules were collected. The collected microcapsules are redispersed in a small amount of distilled water and mannitol 0.3 g
Was added and dissolved, and then freeze-dried to obtain a powder. The mass recovery of the microcapsules obtained by excluding the added mannitol by calculation was 55.2%, the content of peptide B in the microcapsules was 21.3%, and the content of 3-hydroxy-2-naphthoic acid was 2.96%. The encapsulation efficiency obtained by dividing these realized contents by the charged contents is the peptide B
Was 99.7% and 3-hydroxy-2-naphthoic acid was 102.2%.

Example 9 The DL-lactic acid polymer described in Example 8 was changed to a DL-lactic acid polymer (weight average molecular weight: 28,800, number average molecular weight: 14,500, carboxyl group amount by labeling assay: 78.1 μmol / g). Except for the above, a microcapsule powder was obtained in the same manner as described in Example 8. The mass recovery of the microcapsules obtained by excluding the added mannitol by calculation was 50.2%, the content of peptide B in the microcapsules was 20.8%, and the content of 3-hydroxy-2-naphthoic acid was 2.78%. Met.
The encapsulation efficiency determined by dividing these realized contents by the charged contents was 103.4% for peptide B and 92.7% for 3-hydroxy-2-naphthoic acid.

Comparative Example 1 A solution prepared by dissolving 1.2 g of the acetate salt of peptide B in 1.2 ml of distilled water was mixed with a solution prepared by dissolving 4.8 g of the DL-lactic acid polymer in 7.8 ml of dichloromethane as in Example 9 to obtain a homogenizer. To form a W / O emulsion. Then, the W / O emulsion was adjusted to 0.1% previously adjusted to 15 ° C.
(w / w) Polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) was poured into 1200 ml of an aqueous solution, and a W / O / W emulsion was formed at 7,000 rpm using a turbine-type homomixer. Thereafter, the same operation as in Example 8 was performed to obtain a microcapsule powder. The mass recovery of the microcapsules obtained by excluding the added mannitol by calculation was 53.6%, and the content of peptide B in the microcapsules was 12.1%. Then, the encapsulation rate of peptide B determined by dividing the realized content by the charged content is 60.6%, which is much lower than that of Example 9. Therefore, it is clear that the addition efficiency of peptide B was increased by the addition of 3-hydroxy-2-naphthoic acid.

Example 10 A solution prepared by dissolving 1.00 g of the acetate salt of peptide B in 1.00 ml of distilled water was mixed with a DL-lactic acid polymer (weight average molecular weight 49,500, number average molecular weight 17,500, carboxyl group content 45.9 by labeling assay). μmol / g) 3.85 g and 0.15 g of 3-hydroxy-2-naphthoic acid were added to 7.5 ml of dichloromethane and 0.1 ml of ethanol.
The mixture was mixed with a solution dissolved in 4 ml of a mixed organic solvent and emulsified with a homogenizer to form a W / O emulsion. Less than
0.1% (w / w) Polyvinyl alcohol aqueous solution 1000m
1. Microcapsule powder was obtained in the same manner as in Example 8, except that the amount of mannitol added was 0.257 g. The mass recovery of the microcapsules obtained by excluding the added mannitol by calculation was 53.8%, the content of peptide B in the microcapsules was 18.02%, and the content of 3-hydroxy-2-naphthoic acid was 2.70%. The encapsulation efficiency determined by dividing these realized contents by the charged contents was 90.1% for peptide B and 9% for 3-hydroxy-2-naphthoic acid.
0.1%.

Example 11 A solution prepared by dissolving 1.202 g of the acetate salt of peptide B in 1.20 ml of distilled water was treated with a DL-lactic acid polymer (weight-average molecular weight: 19,900, number-average molecular weight: 10,700, carboxyl group content by labeling assay: 104.6%). (μmol / g) 4.619 and 0.179 g of 3-hydroxy-2-naphthoic acid were mixed with a solution of a mixed organic solvent of 7.5 ml of dichloromethane and 0.45 ml of ethanol and emulsified with a homogenizer to form a W / O emulsion.
A microcapsule powder was obtained in the same manner as in Example 8, except that the amount of mannitol added was changed to 0.303 g. The mass recovery of the microcapsules obtained by excluding the added mannitol by calculation was 61.4%, the content of peptide B in the microcapsules was 15.88%, and the content of 3-hydroxy-2-naphthoic acid was 2.23%. The encapsulation efficiency determined by dividing these realized contents by the charged contents was 77.75% for peptide B and 7 for 3-hydroxy-2-naphthoic acid.
5.05%.

Example 12 A solution prepared by dissolving 1.00 g of the acetate salt of peptide B in 1.00 ml of distilled water was used as a DL-lactic acid polymer (weight average molecular weight: 25,900, number average molecular weight: 7,100, terminal carboxyl group amount: 98.2 μmol / g) 3.
A mixture of 85 g and 0.15 g of 3-hydroxy-2-naphthoic acid in a mixed organic solvent of 5.5 ml of dichloromethane and 0.35 ml of ethanol was mixed and emulsified with a homogenizer,
A W / O emulsion was formed. The subsequent operation was the same as described in Example 7 to obtain a microcapsule powder. The mass recovery of the microcapsules was 48.8%, the content of peptide B in the microcapsules was 21.31%, and 3-hydroxy-2-
The naphthoic acid content was 2.96%. The encapsulation efficiency determined by dividing these realized contents by the charged contents was 106.5% for peptide B and 98.7% for 3-hydroxy-2-naphthoic acid.

Comparative Example 2 A solution obtained by dissolving 1.00 g of the acetate of peptide B in 1.00 ml of distilled water was mixed with a solution obtained by dissolving 4.00 g of the same DL-lactic acid polymer in 5 ml of dichloromethane as in Example 12, and mixed with a homogenizer. Emulsified to form a W / O emulsion. The subsequent operation was the same as described in Example 7 to obtain a microcapsule powder. The mass recovery of the microcapsules was 48.7%, and the content of peptide B in the microcapsules was 11.41%.
Then, the encapsulation rate of peptide B determined by dividing the realized content by the charged content is 57.1%, which is much lower than that of Example 12. Therefore, it is clear that the addition efficiency of peptide B was increased by the addition of 3-hydroxy-2-naphthoic acid.

Example 13 DL-lactic acid polymer (weight average molecular weight 30,600, number average molecular weight 1
4,400, carboxyl group amount 63.0μm by labeling quantitative method
ol / g) A solution in which 89.2 g was dissolved in 115.3 g of dichloromethane and a solution in which 3.45 g of 3-hydroxy-2-naphthoic acid was dissolved in a mixed organic solvent of 38.8 g of dichloromethane and 6.27 g of ethanol were mixed and adjusted to 28.5 ° C. did. From this organic solvent solution, weigh 224 g and add 22.3 g of the acetate salt of peptide B to 20 m
l of distilled water, mixed with an aqueous solution heated to 44.9 ° C., stirred for 5 minutes to coarsely emulsify, and then homogenized using a homogenizer.
The mixture was emulsified under the conditions of 0,000 rpm for 5 minutes to form a W / O emulsion. Next, after cooling this W / O emulsion to 16.3 ° C., it is poured into 20 liters of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by Nippon Synthetic Chemical Industry) adjusted to 15 ° C. in 5 minutes. Then, the mixture was stirred at 7,000 rpm using HOMOMIC LINE FLOW (manufactured by Tokushu Kika) to obtain a W / O / W emulsion. The W / O / W emulsion was stirred at 15 ° C. for 3 hours to evaporate dichloromethane and ethanol or diffuse it into the external water phase to solidify the oil phase, and then to 75 μm
Sieved using a sieve with a mesh opening, and then centrifuged (H-600
(S, manufactured by Domestic Centrifuge)) to continuously sediment the microcapsules at 2,000 rpm and collect. The collected microcapsules were redispersed in a small amount of distilled water, sieved using a 90 μm mesh sieve, added with 9.98 g of mannitol, dissolved, and freeze-dried to obtain a powder. The mass recovery of the microcapsules determined by excluding the added mannitol by calculation was 66.5%, the content of peptide B in the microcapsules was 22.3%, and the content of 3-hydroxy-2-naphthoic acid was 2.99%. And the encapsulation rate calculated by dividing these realized contents by the charged contents is 104.5% for peptide B,
It was 102.1% in 3-hydroxy-2-naphthoic acid.

Experimental Example 2 About 45 mg of the microcapsules described in Example 8 were dispersed in 0.3 ml of a dispersion medium (0.15 mg of carboxymethylcellulose, 0.3 mg of polysorbate 80, and distilled water in which 15 mg of mannitol was dissolved) for 7 weeks. A male male SD rat was subcutaneously administered to the back of the rat with a 22G injection needle. After a predetermined time from the administration, the rats were sacrificed and the microcapsules remaining at the administration site were taken out, and the residual ratio determined by quantifying peptide B and 3-hydroxy-2-naphthoic acid therein and dividing by the respective initial contents was determined. Table 2 shows the properties of the used DL-lactic acid polymer.

[Table 2] As is evident from Table 2, the microcapsules described in Example 8 contained a high amount of the bioactive substance, but the residual ratio of the bioactive substance was 90% one day after administration.
Above and dramatically higher. Therefore, 3-hydroxy-2-naphthoic acid not only has the effect of incorporating a high amount of a physiologically active substance into a sustained-release preparation, but also has the effect of very effectively inhibiting the initial excessive release of a physiologically active substance. It is obvious. And, the microcapsules realize the release of a physiologically active substance at a constant rate for a very long time. After 12 weeks, 3-hydroxy-2-naphthoic acid has completely disappeared from the microcapsules, but the release of the physiologically active substance has been maintained at the same constant rate as before, and is effective as a sustained-release preparation. is there.

EXPERIMENTAL EXAMPLE 3 The microcapsules obtained in Examples 7, 9 to 12 and Comparative Example 1 were administered and recovered in the same manner as described in Experimental Example 2, and then the amount of peptide B was determined and the amount was determined. Table 3 shows the residual ratio obtained by dividing by the initial content and the characteristics of the DL-lactic acid polymer used.

[Table 3] As is clear from Tables 2 and 3, all of the microcapsules described in Examples 7 to 12 have a residual rate of about 90% or more in one day after administration, which is a leap compared to that of Comparative Example 1. High. Therefore, 3-hydroxy-
It is clear that 2-naphthoic acid not only has the effect of incorporating a high amount of a physiologically active substance into a sustained-release preparation, but also has the effect of very well inhibiting the initial excessive release of the physiologically active substance. Above all, from the experimental examples using the microcapsules described in Examples 7 to 9, the weight-average molecular weight of the biodegradable polymer was about 20,000 to about 50,000, and the carboxyl group content by the labeling quantitative method was about 50-90
In the case of using DL-lactic acid of μmol / g, a physiologically active substance can be released at a constant rate for a very long time.

Experimental Example 4 The microcapsules obtained in Example 7 were subcutaneously administered to rats according to the method described in Experimental Example 2, and the blood was collected to measure the concentrations of peptide B and testosterone in the serum obtained. Table 4 shows the results.

[Table 4] As is clear from Table 4, the blood concentration of the physiologically active substance was 28
It is maintained at a constant value until after a week, which means that the bioactive substance was continuously released from the microcapsules over 28 weeks. And, during that period, the testosterone concentration showing a medicinal effect is always suppressed to a normal value level or less, and even if 3-hydroxy-2-naphthoic acid is contained in the preparation, the physiologically active substance does not impair its activity. It was clarified that the microcapsules were stably present in the microcapsules over a long period of time and sustained release.

Example 14 A chloride ion was passed through a strongly basic ion exchange column (SeP-Pak Plus QMA cartridge, manufactured by Waters) through a mixed solution of 0.5N sodium hydroxide aqueous solution / methanol (v / v = 1/5). Discharged. After the effluent no longer became cloudy even when a silver nitrate solution was added under nitric acid acidity, excess alkali was discharged through a water / methanol mixture (v / v = 1/5). After confirming that the effluent was neutral, 18.8 mg of peptide B acetate was dissolved in 2 ml of a water / methanol mixture (v / v = 1/5) and passed through the pretreated column. I let it. This effluent was combined with a further 6 ml of only the mixture, which was then mixed with 3-hydroxy-2-naphthoic acid.
A solution prepared by dissolving 91 mg in 1 ml of a mixed solution of water / methanol (v / v = 1/5) was mixed, and concentrated by a rotary evaporator. When the mixture became turbid, 2 ml of water was added and the mixture was stirred, centrifuged (3000 rpm, 20 ° C., 15 minutes) to remove the supernatant, and repeatedly washed with water several times. Thus, 4.09 mg of 3-hydroxy-2-naphthoic acid salt of peptide B was obtained. 0.5 ml of water was added to the salt, and the mixture was stirred at room temperature for 4 hours.
The solution was filtered through a 2 μm filter and quantified by HPLC. The concentrations of peptide B and 3-hydroxy-2-naphthoic acid were 2.37 g / L and 0.751 g / L, respectively.
Part of the salt remains dissolved even after stirring, and the above
Is considered to be the water solubility of 3-hydroxy-2-naphthoate, and the water solubility of the acetate of peptide B is 1000 g / L.
Compared to the above, it is reduced to 1/100 or less. This indicates that the 3-hydroxy-2-naphthoate of peptide B can be used as a sustained-release preparation of peptide B.

[0057]

Industrial Applicability The sustained-release composition of the present invention contains a high content of a physiologically active substance, can suppress the initial excessive release, and can realize a stable release rate over a long period of time.

────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/00 635 A61K 31/00 635 31/19 602 31/19 602 38/22 37/24 (72) Inventor Kazichi Yamamoto Nara 7-10-116, Ayameikeminami, Nara City, Japan

Claims (28)

[Claims] A sustained-release composition comprising a physiologically active substance or a salt thereof, hydroxynaphthoic acid or a salt thereof, and a biodegradable polymer or a salt thereof. 2. The sustained-release composition according to claim 1, wherein the physiologically active substance is a physiologically active peptide. 3. The sustained-release composition according to claim 2, wherein the physiologically active substance is an LH-RH derivative. 4. The method according to claim 1, wherein the hydroxynaphthoic acid is 3-hydroxy-
The sustained-release composition according to claim 1, which is 2-naphthoic acid. 5. The sustained-release composition according to claim 1, wherein the biodegradable polymer is an α-hydroxycarboxylic acid polymer. 6. The method of claim 1, wherein the α-hydroxycarboxylic acid polymer is lactic acid-
The sustained-release composition according to claim 5, which is a glycolic acid polymer. 7. A composition wherein the molar percentage of lactic acid and glycolic acid is 100%.
The sustained-release composition according to claim 6, wherein the ratio is in the range of / 0 to 40/60. 8. The composition according to claim 1, wherein the composition mol% of lactic acid and glycolic acid is 100%.
The sustained-release composition according to claim 7, wherein the ratio is / 0. 9. A polymer having a weight average molecular weight of about 3,000 to about 3,000.
7. The sustained release composition of claim 6, wherein the composition is about 100,000. 10. A weight average molecular weight of about 20,000 to 5
10. The sustained-release composition according to claim 9, wherein the composition is 0.000. 11. An LH-RH derivative having the formula 5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z wherein Y is DLeu, DAla, DTrp, DSer (tBu) , D2Nal or
DHis indicates (ImBzl), Z represents an NH-C ₂ H ₅ or Gly-NH _2. The sustained-release composition according to claim 3, which is a peptide represented by the formula: 12. The sustained release composition according to claim 6, wherein the amount of terminal carboxyl groups of the polymer is 50 to 90 micromol per unit mass (gram) of the polymer. 13. Hydroxynaphthoic acid or a salt thereof and LH
-RH derivative or salt thereof in a molar ratio of 3: 4 to 4: 3
The sustained-release composition according to claim 3, which is 14. The sustained release composition according to claim 1, wherein the LH-RH derivative or a salt thereof is contained from 14% (w / w) to 24% (w / w).
3. The sustained release composition according to 3. 15. The sustained-release composition according to claim 1, wherein the physiologically active substance or a salt thereof is slightly water-soluble or water-soluble. 16. The sustained-release composition according to claim 1, which is for injection. 17. The sustained-release composition according to claim 1, wherein the solvent is removed from a mixture of a physiologically active substance or a salt thereof, a biodegradable polymer or a salt thereof, and hydroxynaphthoic acid or a salt thereof. Manufacturing method. 18. A method comprising mixing and dispersing a physiologically active substance or a salt thereof in an organic solvent solution containing a biodegradable polymer or a salt thereof and hydroxynaphthoic acid or a salt thereof, and removing the organic solvent. A method for producing the sustained release composition according to claim 17. 19. The method for producing a sustained-release composition according to claim 18, wherein the physiologically active substance or a salt thereof is an aqueous solution containing the physiologically active substance or a salt thereof. 20. The method according to claim 17, wherein the salt of the physiologically active substance is a salt with a free base or an acid. 21. A pharmaceutical comprising the sustained-release composition according to claim 1. 22. A prostate cancer, prostatic hyperplasia, endometriosis, uterine fibroid, comprising the sustained-release composition according to claim 3.
Prevention, treatment or contraceptive for uterine fibroma, precocious puberty, dysmenorrhea or breast cancer. 23. A sustained release composition comprising a hydroxynaphthoic acid salt of a physiologically active substance and a biodegradable polymer or a salt thereof. 24. A method for suppressing the initial excessive release of a physiologically active substance from a sustained-release composition, comprising using hydroxynaphthoic acid or a salt thereof. 25. A method for improving the efficiency of encapsulating a physiologically active substance in a sustained-release composition, comprising using hydroxynaphthoic acid or a salt thereof. 26. A hydroxynaphthoic acid salt of a physiologically active peptide. 27. A water-soluble or slightly water-soluble compound.
Item 14. The hydroxynaphthoic acid salt of the physiologically active peptide described in Item 8. 28. A sustained-release composition comprising hydroxynaphthoate of a physiologically active peptide.