CZ20002470A3 - Preparation with permanent release, process of its preparation and use - Google Patents

Abstract

A sustained release formulation that contains salt hydroxynaphthoic acid of biologically active substance a a biodegradable polymer, a process for making the same; a pharmaceutical composition comprising a sustained-release formulation release.

Description

Sustained-release composition, process for its manufacture and use

Technical field

The present invention relates to a sustained release composition of a biologically active agent and a process for its manufacture.

BACKGROUND OF THE INVENTION

Japanese Patent Publication No. 97334/1995, which has not been investigated, discloses a sustained release formulation comprising a biologically active peptide or a salt thereof and a biodegradable polymer having a free carboxyl group at one end, and a process for producing the same.

GB 2,209,937, GB 2,234,169, GB 2,234,896, GB 2,257,909 and European Patent Application 626 170 A2 disclose biodegradable polymer compositions comprising separately produced water-insoluble a salt, such as pamoate [1,1'-methylene-bis (2-hydroxy-3-naphthoate)] peptide or protein, or methods of making the same.

WO 05/15767 describes cetrorelix (pamoate) carbonate (pamoate) (LH-RH antagonist) and a process for its manufacture, and discloses that the peptide release profile of this pamoate remains the same as when used alone, although it is included in a biodegradable polymer.

Problems to be solved by the present invention:

To obtain a novel composition which contains a biologically active substance in high amounts and which is able to control its release rate.

Troubleshooting:

After intensive research aimed at solving this problem, the present inventors have found that when a biologically active agent is included in high amounts in a composition, allowing the biologically active agent and hydroxynaphthoic acid to be present simultaneously of the composition, and if both are included in the biodegradable polymer, the biologically active agent is released at rates that differ from the rates of release of the biologically active agent from other

1 '' of a biologically active agent and a hydroxynaphthoic acid composition which is prepared in the absence of a biodegradable polymer, the release rate being controllable by selecting the appropriate type of biodegradable polymer. The present inventors undertook further research based on this finding and developed the present invention.

Thus, the present invention provides:

(1) a sustained release composition comprising a biologically active agent or salt thereof, a hydroxynaphthoic acid or salt thereof, and a biodegradable polymer or salt thereof;

(2) the sustained release composition of (1) above, wherein the biologically active agent is a biologically active peptide;

(3) the sustained release composition of (2) above, wherein the biologically active peptide is an LH-RH derivative;

4) a sustained release formulation according to the above-mentioned r

paragraph 1), wherein the hydroxynaphthoic acid is 3-hydroxy _ς -2-naphthoic acid

(5) the sustained release composition of (1) above, wherein the biodegradable polymer is an α-hydroxycarboxylic acid polymer;

(6) the sustained release composition of (5) above, wherein the α-hydroxycarboxylic acid polymer is a lactic acid-glycolic acid polymer;

(7) the sustained release composition of (6) above, wherein the ratio of lactic acid to glycolic acid is 100: 09 to 40:60 mol%,

(8) the sustained release composition of (7) above, wherein the ratio of lactic acid to glycolic acid is 100: 0 mol%;

(9) the sustained release composition of (6) above, wherein the weight average molecular weight of the polymer is about 3000 to about 100,000;

(10) the sustained release composition of (9) above, wherein the weight average molecular weight of the polymer is about 20,000 to about 50,000;

(11) the sustained release composition of (3) above, wherein the LH-RH derivative is a peptide of formula

5-oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z wherein Y is DLeu, DAla, DTrp, DSer (tBu), D2Nal or DHis (ImBzl) and Z is NH- C _and H _s or Gly-NH 4,

(12) the sustained release composition of (6) above, wherein the carboxyl end group content of the polymer is 50 to 90 micromoles per unit weight (gram) of polymer;

(13) the sustained release composition of (3) above, wherein the molar ratio of the hydroxynaphthoic acid or salt thereof to the LH-RH derivative or salt thereof is from 3 to 4 to 4 to 3;

(14) the sustained release composition of (13) above, wherein the LH-RH derivative or salt thereof is present in an amount of 14% by weight. up to 24% by weight,

(15) the sustained release composition of (1) above, wherein the biologically active agent or salt thereof is very slightly water soluble or water soluble;

(16) the sustained-release preparation referred to in paragraph 1 above, which is intended for injection;

(17) A process for the manufacture of a sustained release formulation according to (1) above, comprising removing a solvent from a mixture of a biologically active agent or salt thereof, a biodegradable polymer or salt thereof, and a hydroxynaphthoic acid or salt thereof. · * · · · · ···· · ·· ·

(18) a method for producing a sustained release formulation according to (17) above, comprising mixing and dispersing the biologically active agent or salt thereof in an organic solvent solution comprising a biodegradable polymer or salt thereof and a hydroxynaphthoic acid or salt thereof; and subsequently removing the organic solvent, 19) a process for producing a sustained release composition according to paragraph 18 above, characterized in that the biologically active substance or a salt thereof is in the form of an aqueous solution,

(20) the production method of (17) above, wherein the salt of the biologically active agent is a salt with a free base or acid;

(21) a pharmaceutical preparation containing the sustained release preparation of (1) above;

(22) an agent for the prevention or treatment of prostate cancer, prostate hypertrophy, endometriosis, hysteromyoma, metrofibroma, premature puberty, dysmenorrhea or breast cancer, or a contraceptive containing the sustained release formulation of (3) above;

(23) a sustained release formulation comprising a hydroxynaphtholate of a biologically active agent and a biodegradable polymer or salt thereof;

(24) a method for suppressing the initial rapid release of a biologically active agent from a sustained release formulation, comprising the use of a hydroxynaphthoic acid or a salt thereof;

(25) a method of increasing the efficiency of inclusion of a biologically active agent in a sustained release formulation, characterized in that hydroxynaphthoic acid or a salt thereof is used;

(26) a biologically active peptide hydroxynaphthoate,

27) a biologically active peptide naphthalate according to paragraph 26 above, which is water soluble or very slightly water soluble; and

28) a sustained release composition comprising a hydroxynaphthoate of a biologically active peptide.

The present invention further provides;

(29) the sustained release composition of (28) above, wherein the content of hydroxynaphthoic acid or salt thereof is about 1 to about 7 moles, preferably about 1 to about 2 moles, per mole of biologically active peptide or salt thereof;

(30) a method for producing a sustained release formulation according to (17) above, comprising producing a water / oil emulsion with a solution containing the biologically active agent or salt thereof as an internal aqueous phase and a solution containing the biodegradable a polymer and a hydroxynaphthoic acid or a salt thereof, as an oil phase, followed by solvent removal,

(31) A method for producing a sustained release formulation according to (17) above, comprising producing a water / oil emulsion with a solution containing hydroxynaphthoic acid or a salt thereof as the internal aqueous phase and a solution containing a biologically active agent. or a salt thereof and a biodegradable polymer or salt thereof, as an oil phase, followed by solvent removal,

(32) a method for producing a sustained release formulation according to (22) above; comprising mixing and dissolving the biologically active peptide or salt thereof and the hydroxynaphthoic acid or salt thereof, followed by solvent removal, and

33) a process for producing a sustained release composition according to any one of the above paragraphs 30) to 32), wherein the solvent removal method is a water drying process.

Although the biologically active agent used in the present invention is not subject to limitation, if it is pharmacologically useful, it may be a non-peptide agent or a peptide agent. The non-peptide agent includes an agonist, antagonist, and agent having an enzyme inhibiting activity. For example, peptide substances include biologically active peptides, and particularly preferred are those having molecular weights of about 300 to about 40,000, preferably about 400 to about 30,000, more preferably about 500 to about 20,000. · · Θ θ θ θ θ θ »» »» »» »» »» »

Such biologically active peptides include, for example, luteinizing hormone releasing hormone (LH-RH), insulin, somatostatin, growth hormones, growth hormone releasing hormone (GH-RH), prolactin, erythropoietin, adrenocorticotropic hormone, melanocyte stimulating hormone, thyroid hormone releasing hormone , thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, vasopressin, oxytocin, calcitonin, gastrin, secretin, pancreozymin, cholecystokinin, angiotensin, human placental lactogen, human chorionic gonadotropin, thecophotin, endothorphyrin, thecophotin, endothorphin, , thymic humoral factor, blood thymic factor, tumor necrosis factor, colony stimulating factor, motilin, daynorfin, bombesin, neurotensin, kaerulein, bradykinin, atrial natriuresia enhancing factor, nerve growth factor, cell growth factor, neurotrophic factor, endothelial antagonist peptides and their derivatives, fragments of these peptides and derivatives of these fragments.

The biologically active peptide used in the present invention may be used as such or may be used as a pharmacologically acceptable salt.

These salts include salts with inorganic acids (may also be called inorganic free acids) (eg, carbonic acid, bicarbonate, hydrochloric acid, sulfuric acid, nitric acid, boric acid) and organic acids (they may also be called organic free acids) (e.g., succinic acid, acetic acid, propionic acid, trifluoroacetic acid) etc. when said biologically active peptide has a basic group such as an amino group.

When said biologically active peptide has an acidic group, such as a carboxyl group, these salts include salts with inorganic bases (also called inorganic free bases) (e.g., alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium), organic bases 9 9999 999 ··· ··· ··· · · ··· (May also be called organic free bases) (eg organic amines such as triethylamine, basic amino acids such as arginine) etc. A biologically active peptide may form a complex compound with a metal (eg complex with copper, complex with zinc).

Preferred examples of the biologically active peptide described above are LH-RH derivatives or salts thereof that are effective on sex hormone dependent diseases such as prostate cancer, prostate hypertrophy, endometriosis, hysteromyoma, premature puberty and breast cancer, and are effective for contraceptive agents. .

Examples of LH-RH derivatives or salts thereof include, for example, the peptides described in Treatment with GnRH Analogs: Controversies and Perspectives (The Parthenon Publishing Group Ltd., published 1996), Japanese Patent Publication No. 5,968,549.

503 165/1991, which has undergone a survey, Japanese Patent Specifications No. 101 695/1991, 97 334/1995 and 259 460/1996, which have not undergone a survey, and elsewhere.

The LH-RH derivatives may be LH-RH agonists or LH-RH antagonists; useful LH-RH antagonists include, for example, biologically active peptides of Formula I

_{X-D2Nal-D4ClPhe-D3Pal-Ser-AB-Leu-C-Pro-DAlaNH2} (I) [X represents N (4H ₂ -furoyl) Gly or NAc, A represents a residue selected from NMeTyr, Tyr, Aph (Atz ) and NMeAph (Atz), B is a residue selected from DLys (Nic), DCit, DLys (AzaglyNic), DLys (AzaglyFur),

DhArg (Et) _a , DAph (Atz) and DhCi and C are Lys (Nisp), Arg or hArg (Et _a )] or salts thereof.

Useful LH-RH agonists include, for example, biologically active peptides of Formula II

5-Oxo-Pro-His-Trp-Ser-Tyr-Y-Leu-Arg-Pro-Z (II) 9999

98 «« 9 *

9 9 «

9999 • · • 99 9 · [Y represents a residue selected from DLeu, DAla, DTrp, DSer (tBu), D2Nal and DHis (ImBzl), Z represents NH ₂ -C ₂ H _{s, Gly-NH2]} or salts thereof. Particularly preferred are peptides wherein Y represents DLeu and Z represents NH-C H (i.e. a peptide of formula 5-oxo-Pro-His-Trp-Ser-Tyr-DLeu-Arg-Pro-NH-C ₂ H _s).

These peptides can be produced by the methods described in the above references or patents or by methods based thereon.

The abbreviations used in this invention are defined as follows:

Abbreviation

N (4H ₂ -furoyl) Gly:

NAc:

D2Nal:

D4ClPhe:

D3Pal:

NMeTyr:

Aph (Atz):

NMeAph (Atz):

DLys (Nothing):

Dcit:

DLys (Azaglynic): DLys (AzaglyFur): DhArg (Et ₂ ): DAph (Atz): azolyl)]

Lys (Nisp)

hArg (Et _2):

Name:

N-tetrahydrofuroylglycine residue

N-acetyl group

D-3- (2-naphthyl) alanine residue

D-3- (4-chloro) phenylalanine residue

D-3- (3-pyridyl) alanine residue

N-methyltyrosine residue

N- [5 '- (3-amino-l'H ·-l', 2 ^1, 4'-triazolyl)] phenylalanine residue

N-methyl- [5 '- (3'-amino-1'H-1', 2 ', 4'-triazolyl)] phenylalanine residue D- (e-N-nicotinoyl) lysine residue D-citrulline residue

D- (azaglycylnicotinoyl) lysine residue D- (azaglycylfuranyl) lysine residue D- (N, N'-diethyl) homoarginine residue DN- [5 '- (3'-amino-1 · H-1', 2 ', 4' -triphenylalanine residue,

D-homocitruline residue (eN-isopropyl) lysine residue (N, N ¹ -diethyl) homoarginine residue

Amino acid abbreviations are based on abbreviations that

9 · »

9 0 »« 9 9 »9 9

0 · 0 ·

9 «» • 9 0 9 9 ·· are specified by the IUPAC-IUB Biochemical Nomenclature Commission [European Journal of Biochemistry 1984, 138, 9-37] or abbreviations commonly used in the relevant fields. If an amino acid can be present as an optical isomer, it is an L-configuration unless otherwise indicated.

The hydroxynaphthoic acid for the present invention consists of a naphthalene ring and one hydroxyl group and one carboxyl group, both of which are bonded to different ring carbon atoms. Thus, there are a total of 14 isomers with a hydroxyl group located at different positions relative to the carboxyl group located at positions 1 and 2 of the naphthalene ring. Any of these isomers may be used and mixtures thereof in any proportions may be used. As will be described later, it is preferable that the acid dissociation constant be great, or to a pKa (pKa = - log _io wherein Ka represents acid dissociation constant) be small. Also preferred are those isomers that are poorly water soluble.

Preferred are isomers that are soluble in alcohols (e.g., ethanol, methanol). The term soluble in alcohols as used herein means that the solubility is not less than, for example, 10 g / l of methanol.

With respect to the pKa values of the above-described hydroxynaphthoic acid isomers, the only known value is 3-hydroxy-2-naphthoic acid (pKa = 2.708, Kagaku Binran Kisohen II, Chemical Society of Japan, published September 25, 1969); useful information, however, is obtained by comparing the pKa values of the three isomers of hydroxybenzoic acid. Specifically, the pKa value of m-hydroxybenzoic acid and p-hydroxybenzoic acid are not less than 4, while the pKa value of o-hydroxybenzoic acid (salicylic acid) (= 2.754) is extremely low. Of the above 14 isomers, therefore, those isomers that contain a naphthalene ring and a carboxyl group and a hydroxyl group, both attached to adjacent ring carbon atoms, i.e. 3-hydroxy-2, 4,444, 4,9444, are preferred.

naphthoic acid, 1-hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic acid. Further preferred is 3-hydroxy-2-naphthoic acid, which consists of a naphthalene ring and a hydroxyl group attached to a carbon atom at the 3-position of the ring and one carboxyl group attached to a carbon atom at the 2-position of the ring.

Hydroxynaphthoic acid may be a salt. Salts include, for example, salts with inorganic bases (e.g., alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium), organic bases (e.g. organic amines such as triethylamine, basic amino acids such as arginine) and transition metal salts and complex salts (eg, zinc, iron, copper).

The following is an example of a process for producing a hydroxynaphthoic acid salt of a biologically active agent of the present invention.

1) Pass the hydrated solution of the hydroxynaphthoic acid organic solvent through a column of weakly basic ion exchanger to adsorb the acid and saturate the column. The excess portion of the hydroxynaphthoic acid is then removed with a hydrated organic solvent. Thereafter, the hydrated organic solvent solution of the biologically active agent or salt thereof is passed through the column for ion exchange. The solvent was removed from the eluate obtained. Useful organic solvents in the hydrated organic solvent include alcohols (e.g., methanol, ethanol), acetonitrile, tetrahydrofuran, and dimethylformamide. Removal of the solvent to precipitate the salt is achieved using a generally known method or a method based thereon. Examples of such methods include a method in which the solvent is evaporated using a rotary evaporator under vacuum, etc.

(2) Pass a solution of the hydrated organic solvent of the biologically active substance or its salt in a weakly basic ion exchanger column that has been previously exchanged with hydroxide ions to convert the basic groups into hydroxynaphthoic acid hydroxide in a molar equivalent, ·· · »type. To the recovered eluate is added no greater quantity, followed by concentration. The precipitated salt is washed with water as necessary, and dried.

Since the hydroxynaphthoic acid of the biologically active substance is very slightly soluble in water, although this also depends on the biologically active substance used, said salt of the biologically active peptide itself, exhibiting a sustained release potential, can be used for the sustained release preparation of the biologically active substance and also used for the manufacture of a sustained release formulation.

Biodegradable polymers used in the present invention include, for example, polymers and copolymers that have been synthesized from one or more species selected from α-hydroxymonocarboxylic acids (e.g., glycolic acid, lactic acid), hydroxydicarboxylic acids (e.g., malic acid), hydroxytricarboxylic acids (e.g. such as citric acid), etc. having a free carboxyl group, or mixtures thereof, poly-α-cyanoacrylic acid esters, polyamino acids (e.g. poly-g-benzyl-L-glutamic acid) and maleic anhydride copolymers (e.g. copolymers) styrene - maleic acid).

The monomer binding method may be random, block or graft. If the above α-hydroxymonocarboxylic acids, α-hydroxydicarboxylic acids and α-hydroxytricarboxylic acids have an optically active center in their molecular structures, they may have a D-, L- or DL-configuration. Of these polymers, lactic acid-glycolic acid [hereinafter also referred to as poly (lactide-coglycolide), poly (lactic acid-co-glycolic acid) or lactic acid-glycolic acid copolymer; generically refer to homopolymers and copolymers of lactic acid-glycolic acid, unless otherwise specified; lactic acid homopolymers are also referred to as lactic acid polymer, polylactic acids, polylactides, etc. and homopolymers • Φ φφφφ φφ φ

φ .Ί φφ φφφφ

Polyglycolic acid polymers such as polymers of glycolic acid, polyglycolic acids, polyglycolides, etc.] are preferred poly (α-cyanoacrylic esters), etc. More preferred are lactic acid-glycolic acid polymers. More preferably, polymers of lactic acid-glycolic acid with a free carboxyl group at one end are used.

The biodegradable polymer may be a salt. Salts include, for example, salts with inorganic bases (e.g., alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium), organic bases (e.g. organic amines such as triethylamine, basic amino acids such as arginine) and transition metal salts and complex salts (eg, zinc, iron, copper).

When a lactic acid-glycolic acid polymer is used as the biodegradable polymer, the content ratio (mole%) is preferably about 100: 0 to about 40:60, more preferably about 100: 0 to about 50:50. Lactic acid homopolymers having a content ratio of 100: 0 are also preferably used.

The ratio of the optical isomers of lactic acid, one of the minimally repeating units of said lactic acid glycolic acid polymer, is preferably between about 75:25 and about 25:75 in terms of the ratio (mole%) of the D-configuration: L-configuration. In general, polymers of lactic acid-glycolic acid with a ratio (mole%) of the D-configuration: L-configuration between about 60:40 and about 30:70 are also used.

The weight average molecular weight of said lactic acid-glycolic acid polymer is normally about 3000 to about 100,000, preferably about 3000 to about 60,000, more preferably about 3000 to about 50,000, and even more preferably about 20,000 to about 50,000.

The degree of dispersion (weight average molecular weight / number average molecular weight) is normally about 1.2 to about 4.0, more preferably about 1.5 to 3.5.

• 9 «··· • 8 ····

9

4444 4

44 • «4 5 6 7 8 9 9 9 9 9 9 9 9 4 4 4 4

44 44

The free carboxyl group content of said lactic acid-glycolic acid polymer is preferably about 20 to about 2000 gmols, more preferably about 40 to about 1000 / mole, per unit weight (gram) of polymer.

The weighted average molecular weight, number average molecular weight and degree of dispersion as defined herein are based on the molecular weights and degree of polystyrene dispersion as determined by gel displacement chromatography (GPC) with 15 polystyrenes as reference substances having a number average molecular weight of 1,110 000, 707 000,

455,645, 354,000, 189,000, 156,055, 98,900, 66,437, 37,200,

100, 9830, 5870, 2500, 1303 and 504. The measurements were performed with a high speed GPC device (manufactured by Toso, KCL-8120GPC, detection: refractive index) and a GPC column KF804Lx2 (manufactured by Showa Denko) with chloroform as the mobile phase.

The term content of free carboxyl group as used herein is defined as obtained by the labeling method (hereinafter referred to as the carboxyl group content as determined by the labeling method). Specific procedures for determining this content in polylactic acid are described below. First, W milligrams of polylactic acid was dissolved in 2 ml of 5N hydrochloric acid / acetonitrile (4:96 by volume); 2 ml of a 0.01 M o-nitrophenylhydrazine hydrochloride (ONPH) solution (5N hydrochloric acid / acetonitrile / ethanol in a ratio of 1.02: 35: 15) and 2 ml of a 0.15 M solution of 1-ethyl-3- (3- dimethylaminopropyl) -carbodimide (pyridine / ethanol in a ratio of 4:96 by volume). The reaction is continued for 30 minutes at 40 ° C. The solvent was then removed. After washing with water (four times), the residue is dissolved in 2 ml of acetonitrile, 1 ml of 0.5 M ethanolic potassium hydroxide solution is added, followed by a reaction of 30 minutes at 60 ° C. The reaction mixture was diluted to 1.5 mL with aqueous sodium hydroxide solution; determine the absorbance (/ cm) at 544 nm with a 1.5N aqueous sodium hydroxide solution as a control. Separately, the aqueous solution of DL-lactic acid is determined as a reference value by alkaline titration of the C mol / l content of its free carboxyl group99

9

9 ·

• 9 • · ··

9 ·

· 9

«· Ny. By subtracting the absorbance B (/ cm) at 544 nm of DL-lactic acid hydrazide by ONPH labeling, the molar content of free carboxyl groups per unit weight (gram) of polymer can be calculated using the equation:

[COOH] (mol / g) = (AYC) / (WB);

Although said carboxyl group content may also be obtained by dissolving the biodegradable polymer in a solvent mixture of toluene-acetone-methanol and titrating this solution to carboxyl groups with an alcoholic potassium hydroxide solution with phenolphthalein as an indicator (hereinafter referred to as carboxyl group content) (as determined by the alkaline titration method), it is desirable that the quantitative evaluation be obtained by the labeling method described above, since it is possible that the endpoint of the titration is indistinct as a result of competition of the hydrolysis reaction of the polyester backbone during titration.

The degradation / elimination ratio of the biodegradable polymer varies within wide limits depending on the composition of the copolymers, the molecular weight or the free carboxyl group content. However, the duration of drug release can be prolonged by reducing the amount of glycolic acid or by increasing the molecular weight and reducing the free carboxylic acid content, since degradation / / elimination is usually delayed as the amount of glycolic acid is reduced for lactic acid-glycolic acid polymers. However, since the free carboxyl group content affects the rate of incorporation of the biologically active agent into the formulation, it must be above a given limit. For this reason, in obtaining a biodegradable polymer in a sustained release formulation of long duration type (e.g., 6 months or longer), a lactic acid-glycolic acid polymer such as D-lactic acid, e.g. L-lactic acid, DL-lactic acid, preferably DL-lactic acid, etc.) whose weight average molecular weight and free carboxyl group content, as determined as described above, were about 20,000 to about 50,000 and about 30 to 30,000. about 95 μιηοΐύ /

9

9 9

9 9

9

9 ·

9 9 9 9 • 999 9 99 9

9 9 9

9 · 99 / gram, preferably about 40 to about 95 / mole / gram, more preferably about 50 to about 90 microns / gram.

Said lactic acid-glycoic acid polymer can be produced, for example, by a dehydration polymerization condensation process without a catalyst (Japanese Patent 28,521/1986, which has not been investigated) from lactic acid and glycolic acid, or by ring-opening polymerization of lactide and cyclic diester compound such as is a glycolide using catalysts (Encyclopedic Handbook of Biomaterials and Bioengineering Part A: Materials, Vol. 2, Marcel Dekker, Inc., 1995.).

Although the polymer obtained according to the above-described ring-opening polymerization process does not always contain a free carboxyl group at one end, it can also be used after modification to a polymer having a given amount of carboxyl groups per unit weight by undergoing the hydrolysis reaction described in European Patent Application A 0 839 525.

The above-described lactic acid-glycolic acid polymer with a free carboxylic acid at one end can be produced without any problem by a generally known method (e.g., dehydration polymerization condensation without catalyst, Japanese Patent Specification No. 28,521 / 1986, which has not been investigated) or .

1) First, in the presence of a hydroxymonocarboxylic acid derivative (e.g. tert-butyl D-lactate, benzyl L-lactate) with a carboxyl group protected thereof or a hydroxydicarboxylic acid derivative (e.g. dibenzyl tartronate (2-hydroxypropanedioate), di-tert-butyl- 2-hydroxyethyl malonate) with its carboxyl-protected group, the cyclic ester is subjected to a polymerization reaction using a polymerization catalyst.

As the above-described hydroxymonocarboxylic acid derivative with its carboxyl group protected or a hydroxydicarboxylic acid derivative with its carboxyl group protected there may be mentioned, for example, hydroxycarboxylic acid derivatives 9 9 99 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 With their carboxyl group (-COOH) amidated (-CONH4) or esterified (-COOR), preferably hydroxycarboxylic acid derivatives with their carboxylic group (-COOH) esterified (-COOR), etc.

Examples of R for the ester herein are C 1 -C 6 alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl and tert-butyl, C 3 -C 8 cycloalkyl such as cyclopentyl and cyclohexyl, aryl groups (C 6 -C 12) atoms, such as phenyl and α-naphthyl, and (C 7 -C 14) aralkyl groups such as phenyl (C 1 -C 2) alkyl, such as benzyl and phenethyl; and α-naphthyl-alkyl (1 to 2 carbon atoms) groups such as α-naphthylmethyl. Of these, tert-butyl, benzyl, etc. are preferred.

Said cyclic ester compound means a cyclic compound having at least one ester bond in its ring. In particular, these compounds include cyclic monoester compounds (lactones) or cyclic diester compounds (lactides).

Examples of said cyclic monoester compound are 4-membered cyclic lactones (B-propiolactone, B-butyrolactone, B-isovalerolactone, B-caprolactone, B-isocaprolactone, B-methyl-B-valerolactone etc.), 5-membered cyclic lactones (gamma) -butyrolactone, gamma-valerolactone, etc.), 6-membered cyclic lactones (d-valerolactone, etc.), 7-membered cyclic lactones (epsilon-caprolactone, etc.), p-dioxanone and 1,5-dioxepan-2-one.

An example of said cyclic diester compound is a compound of formula

O ιι

** ** «• • * * *« * * * * * * * * * * * * * * * * * * * * * Wherein R ¹ and R ² , whether identical or not, represent a hydrogen atom or a C 1 -C 6 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl or tert-butyl), preferably lactides, wherein R ¹ is hydrogen and R ² is methyl, or both R ¹ and R ² are hydrogen, etc.

Specifically, these compounds include glycolides, L-lactides, D-lactides, DL-lactides, meso-lactides and 3-methyl-1,4-dioxane-2,5-dione (including optically active configurations).

Examples of said polymerization catalyst are organotin compounds (e.g., tin octylate, di-butyltin-dilaurylate, tetrafenyltin), aluminum catalysts (e.g., triethylaluminum), and zinc catalysts (e.g., diethylzinc).

In terms of ease of removal after the reaction, aluminum catalysts and zinc catalysts are preferred; zinc catalysts are preferred for retention safety.

Useful solvents for polymerization catalysts include benzene, hexane and toluene, preferably hexane, toluene, and the like.

The polymerization process refers to a bulk polymerization process that is carried out with the reaction product in the molten state, or a solution polymerization process that is carried out with the reaction product dissolved in an appropriate solvent (e.g. benzene, toluene, xylene, decalin, dimethylformamide). Although the polymerization temperature is not limited, it is not lower than the temperature at which the reaction product is in the molten state at the start of the reaction, normally fcfcfcfcfcfcfcfcfcfcfcfcfcfc fcfcfc fc fcfc ·· · fcfc · · · fcfc · fc fcfcfcfcfc · ♦ · ♦ fcfc

100 to 300 ° C for block polymerization, and normally room temperature to 150 ° C for solution polymerization; if the reaction temperature exceeds the boiling point of the reaction solution, the reaction is carried out under reflux or in a pressure-resistant reactor. A suitable polymerization time, determined by taking into account the polymerization temperature, other reaction conditions, the physical properties of the desired polymer, etc., is, for example, 10 minutes to 72 hours. After complete reaction, the polymerization is terminated with an acid (e.g. hydrochloric acid, acetic anhydride, trifluoroacetic acid) with the reaction mixture dissolved in an appropriate solvent (e.g. acetone, dichloromethane, chloroform), if necessary, followed by stirring in the solvent, which does not dissolve the desired product (e.g., alcohol, water, ether, isopropyl ether) or otherwise precipitates. This is followed by isolation of a polymer having an omega-protected carboxyl group.

The polymerization process of the present invention uses carboxylic acid protected hydroxycarboxylic acid derivatives (e.g., tert-butyl-D-lactate, benzyl L-lactate) or hydroxydicarboxylic acid derivatives (e.g., dibenzyl tartronate, di-tert-butyl-L-2). (carboxy-protected hydroxyethyl malonate) instead of conventional proton chain transfer agents such as methanol.

Use of a protected carboxylic acid-protected hydroxycarboxylic acid derivative (e.g., tert-butyl D-lactate, benzyl L-lactate) or a hydroxydicarboxylic acid derivative (e.g., dibenzyl tartronate, di-tert-butyl-L-2-hydroxyethylmalonate) protected With carboxyl as proton chain transfer agents, 1) molecular weight control can be achieved by the composition of the starting materials, and 2) releasing the carboxyl group at the omega-end of the biodegradable polymer obtained by the deprotection reaction after polymerization.

2) Second, by subjecting the polymer obtained by the polymerization reaction described in paragraph 1 above and having a «» • · • *

9999 9

99

9 · 999 · 9 9 9 9 9

9 9 9 9 9 9

9 9 9 9 9

At the omega-terminated protected carboxyl group, the deprotection reaction can yield the desired biodegradable polymer having a free carboxyl group.

Said protecting group may be removed by generally known methods. These methods include all methods which allow deprotection without affecting the ester bond of the poly (hydroxycarboxylic acid), a specific example being reduction and acid decomposition.

Such reduction methods include, for example, catalytic reduction using catalysts (e.g., palladium on carbon, palladium black, platinum oxide), reduction using sodium in liquid ammonia, and reduction with dithiothreitol. When a polymer having a carboxyl group at the omega-terminus is subjected to catalytic reduction, deprotection can be achieved, for example, by adding palladium on carbon to a solution of the polymer in ethyl acetate, dichloromethane, chloroform, or the like, and supplying hydrogen at room temperature for about 20 minutes to about 4 hours with vigorous shaking.

Such acid decomposition methods include, for example, decomposition with inorganic acids (e.g., hydrofluoric acid, hydrobromic acid, hydrochloric acid), organic acids (e.g., trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid), or mixtures thereof. If necessary, a cation scavenger (e.g. anisole, phenol, thioanisole) may also be added as suitable. If the polymer at the omega-terminus has a carboxyl group protected by a tert-butyl group, it will undergo acid decomposition. Deprotection may be achieved, for example, by adding a suitable amount of trifluoroacetic acid to a solution of the polymer in dichloromethane, xylene, toluene or the like, or by dissolving the polymer in trifluoroacetic acid and stirring for about one hour at room temperature.

Preferably, said acid decomposition is carried out immediately after

9 9 9

> 9,999

9 · ·

9 • 9 polymerization reaction. In this case, it can serve as a termination of the polymerization reaction.

In addition, by subjecting the polymer obtained by the deprotection reaction described above to an acid hydrolytic reaction as necessary, the weighted average molecular weight, number average molecular weight, or terminal carboxyl group content of said polymer can be controlled accordingly. In particular, this can be achieved, for example, by the method described in or based on European patent application A 0 839 525.

The biodegradable polymer obtained as described above can be used as a basis for the manufacture of a sustained release formulation.

In addition, a polymer having a given free carboxyl group at one end can be produced by known production methods (e.g., see WO94 / 15587).

The lactic acid-glycolic acid polymer with one end providing the free carboxyl group by chemical treatment after ring-opening polymerization can also be, for example, a commercial product of Boehringer Ingelheim KG.

The biodegradable polymer may be a salt (salts of the biodegradable polymers include, for example, the above salts). Useful methods for their preparation include, for example, a) a method according to which a solution of the above-described biodegradable polymer having a carboxyl group in an organic solvent and an aqueous solution containing inorganic base ions (e.g. alkali metals such as sodium and potassium, alkali metals soils such as calcium and magnesium) or organic bases (e.g., organic amines such as triethylamine, basic amino acids such as arginine) are mixed together to form an ion exchange reaction to isolate the polymer, now in the form of a salt, b ) a process in which a salt of a weak acid (e.g., acetate, glycolate) of the base referred to under a) is dissolved in a solution from above titi titi ti ti ti ti ti ti ti ti

The described biodegradable polymer having a carboxyl group in an organic solvent, whereupon the polymer is isolated, now in the form of a salt, and c) a process in which a weak acid salt (e.g. acetate, glycolate) or a transition metal oxide (e.g., zinc, iron, copper) is stirred in a solution of the above described biodegradable polymer having a carboxyl group in an organic solvent, whereupon the polymer is isolated, now in the form of a salt.

As a biodegradable polymer for a sustained release type of long-acting type (e.g., 6 months or longer), a lactic acid-glycolic acid having a free carboxylic acid group at one end, which is produced as described above, is preferred.

The weight ratio of the biologically active agent in the composition of the present invention varies according to the type of biologically active agent, the desired pharmacological effect, the duration of action, and other factors. In the case of a sustained release composition comprising three components (a biologically active agent or a salt thereof, a hydroxynaphthoic acid or a salt thereof, and a biodegradable polymer or a salt thereof), the amount by weight of the biologically active peptide or salt thereof is about 0.001 to about 50% by weight. preferably about 0.02 to about 40 wt.%, more preferably about 0.1 to 30 wt. % and most preferably about 14 to 24 wt. based on the sum of the three components. In the case of the non-peptide biplogically active agent or salt thereof, this amount is about 0.01 to 80% by weight, preferably about 0.1 to 50% by weight. If a hydroxynaphthoic acid salt of a biologically active substance is included in the composition, similar weight ratios are applicable. In the case of a sustained release composition comprising a salt of a biologically active peptide (referred to as A) with a hydroxynaphthoic acid (referred to as B), the amount (w / w) of Compound A) is normally about 5 to about 90% by weight, preferably about 10 to about 85 wt.%, more preferably about 15 to about 80 wt. % and even more preferably about 30 to about 80 wt. based on the sum of salt A) with B).

4 44 ** 4 4 4 4 4 4 4 4

In the case of a sustained release composition comprising three components (a biologically active agent or a salt thereof, a hydroxynaphthoic acid or salt thereof, and a biodegradable polymer or a salt thereof), the amount of the hydroxynaphthoic acid or salt thereof in the composition is preferably about 1/2 to about 2 moles, more preferably about 3/4 to about 4/3 moles, and even more preferably about 4/5 to about 6/5 moles, per mole of biologically active agent or salt thereof.

The design of the composition of the present invention is further described herein for a sustained release composition comprising three components: a basic biologically active system, a hydroxynaphthoic acid, and a biodegradable polymer. In this case, the biologically active agent, such as base, and hydroxynaphthoic acid, such as acid, are present in the composition simultaneously; whether in the form of free configurations or in the form of salts, a dissociation equilibrium exists for each component in the hydrated state or in the presence of a trace amount of water. Since a salt formed by any hydroxynaphthoic acid that is very sparingly soluble in water with a biologically active agent is expected to be very sparingly soluble in water, although solubility also depends on the properties of said biologically active agent, its dissociation equilibrium is shifted towards formation salts very slightly soluble in water.

In the manufacture of a composition having high levels of basic biologically active agent, it is desirable that most of the biologically active agent be protonated. This results in the salt being very slightly soluble in water, as mentioned above, which can be judged from the dissociation equilibrium described above. For this purpose, it is desirable that the hydroxynaphthoic acid or salt thereof be prepared in an amount at least nearly equivalent to the amount of biologically active agent or salt thereof.

Further, the mechanism of release of the biologically active agent contained in the composition is described below. In the composition described above, the biologically active agent is usually protonated present with the counterion. The main counterion is the hydroxy ················. ί ί. ^The naphthoic acid (preferably hydroxynaphthoic acid). After the composition is administered to a living body, over time, due to the degradation of the biodegradable polymer, its oligomers and monomers begin to produce. When said polymer is a lactic acid-glycolic acid polymer, the resulting oligomer (lactic acid-glycolic acid oligomer) and monomer (lactic acid or glycolic acid) each have a single carboxyl group which can also serve as a counterion for the biologically active substance. The biologically active substance is released without charge transfer or in the form of a counter-charge salt; transferable counterions include hydroxynaphthoic acids, oligomers of lactic acid glycolic acid (with such molecular weights as possible) and monomers (lactic acid or glycolic acid) as described above.

If more acids are present at the same time, it is usually preferable to produce salts of stronger acids, although the result also depends on the ratio of their amounts. For example, with respect to pKa values for hydroxynaphthoic acids, it is known that 3-hydroxy-2-naphthoic acid has a pKa value of 2.708 (Kagaku Binran Kisohen II, Chemical Society of Japan, published September 25, 1969.). On the other hand, the pKa values of the carboxyl groups of the lactic acid-glycolic acid oligomers are not known, but can be calculated based on the pKa values of lactic acid or glycolic acid (= 3.86 and 3.83, respectively), according to the theory that the introduction of the substituent can be approximately calculated according to the additive rule. The contributions of the substituents to the dissociation constants have already been determined and can be used for this purpose (Table 4.1 in pKa Prediction for Organic Acids and Bases, D. D. Perrin, B. Dempsey and E. P. Serjeant, 1981.). Since the following data is applicable to the hydroxyl group and the ester bond:

delta pKa (OH) = -0.90, delta pKa (ester bond) = -1.7, it is possible to determine the pKa values of the carboxyl group of the lactic acid-glycolic acid oligomers, taking into account the contribution of the ester bond closest to the dissociated group, as you can see

99999 ·· · «9 ·· from the following:

pKa = pKa (lactic acid or glycolic acid) = delta pKa (OH) + delta pKa (ester linkages) =

3.06 or 3.03.

Thus, since hydroxynaphthoic acids are stronger than lactic acid (pKa = 3.86), glycolic acid (pKa = 3.83), and lactic acid-glycolic acid oligomers, it is believed that the hydroxynaphthoic acid salt is preferably produced in the composition described above. with a biologically active substance? it is believed that the properties of the salt are largely determined by the nature of the sustained release of the biologically active agent from the composition. Examples of said biologically active agent are the biologically active agents described above.

The fact that the salt formed by a hydroxynaphthoic acid with a biologically active substance is very slightly soluble in water, rather than insoluble in water, serves here in favor of a sustained release mechanism. In other words, as demonstrated in the above discussion on acid dissociation constants, the initial release stage is dominated by the hydroxynaphthoic acid salt, the stronger acid than the oligomers and lactic acid-glycolic acid monomers described above; the initial drug release system can be controlled by the ratio of hydroxynaphthoic acid content, since the solubility and distribution profile of the salt in body tissue serves as determinants of the rate of release of the biologically active agent. Then, as the amount of oligomers and monomers increases, due to the reduction of the hydroxynaphthoic acid and the hydrolysis of the biodegradable polymer, the mechanism of release of the biologically active agent comprising the oligomers and monomers as counter ions gradually prevails; even if the hydroxynaphthoic acid substantially disappears from said composition, a stable release of the biologically active agent is achieved. Also explained is the increased efficacy of incorporation of the biologically active agent in the manufacture of the sustained release formulation and the possibility of suppressing the initial rapid release upon administration of the incorporated biologically active agent.

·· ·· · · · · · · · · ·

The role of hydroxynaphthoic acid in a sustained release formulation containing a hydroxynaphthoic acid salt of a biologically active peptide can also be explained by the mechanism described above.

The term water insoluble as used herein means that if the substance is stirred in distilled water for 4 hours at a temperature not exceeding 40 ° C, the weight of the substance to be dissolved in one liter of this solution is not more than 25 mg.

The term "very slightly soluble in water" as used herein means that the weight described above is not less than 25 mg and not more than 5 g. If said substance is a salt of a biologically active substance, the above definition applies to the weight of the biologically active substance, which dissolves in the process described above.

Although the sustained release formulation of the present invention is not limited in form, microparticles are preferred, microspheres are preferred (also referred to as microcapsules for sustained release formulations containing biodegradable polymers). The term microsphere, as used herein, is defined as an injectable bead that can be dispersed in solutions. Its shape can be confirmed, for example, by scanning microscope.

Embodiments of the invention;

Examples of methods of making the sustained release compositions of the present invention which comprise a biologically active agent or a salt thereof, a hydroxynaphthoic acid or a salt thereof, and a biodegradable polymer or a salt thereof, microspheres are given below.

I) Water drying method

(i) O / W method

According to this method, a solution of a hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof in an organic solvent is prepared.

Examples of said organic solvent are halogenated hydrocarbons (e.g. dichloromethane, chloroform, dichloroethane, trichloroethane, carbon tetrachloride), ethers (e.g. diethyl ether, isopropyl ether), fatty acid esters (e.g. ethyl acetate, butyl acetate), aromatic hydrocarbons (e.g. benzene, toluene, xylene), alcohols (e.g. ethanol, methanol) and acetonitrile. Of these, dichloromethane is preferred as the organic solvent for the biodegradable polymer or salt thereof. Alcohols are preferred organic solvents for the hydroxynaphthoic acid or a salt thereof. These solvents can be used in mixtures in suitable proportions. Of these solvents, mixtures of halogenated hydrocarbons and alcohols are preferred, mixtures of dichloromethane and ethanol are more preferred.

When a mixture of dichloromethane and ethanol is used as the organic solvent, the concentration ratio is normally chosen to be in the range of about 0.01 to about 50% (v / v), preferably about 0.05 to about 40% (v / v). and more preferably from about 0.1 to about 30% (v / v).

The concentration of the biodegradable polymer in the organic solvent solution varies according to the molecular weight of the biodegradable polymer and the type of organic solvent. For example, when dichloromethane is used as the organic solvent, the concentration of the biodegradable polymer is normally selected to be in the range of about 0.5 to about 70 wt%, preferably about 1 to about 60 wt%. % and more preferably from about 2 to about 50 wt.

The concentration of the hydroxynaphthoic acid or salt thereof in the organic solvent solution is normally selected from, for example, from about 0.01 to about 10% by weight, preferably from about 0.1 to about 5% by weight. % and more preferably from about 0.5 to about 3 wt.

·· · »···

The biologically active substance or a salt thereof is added to the organic solvent solution thus obtained, which contains hydroxynaphthoic acid or a salt thereof and a biodegradable polymer, and is dissolved or dispersed.

The organic solvent solution thus obtained, which contains the biologically active substance or its salt, hydroxynaphthoic acid or a salt thereof and the biodegradable polymer, is then added to the aqueous phase to form an oil (oil phase) / water (water phase) emulsion, the solvent is evaporated from the oil phase. Microspheres are obtained. In this operation, the volume of the aqueous phase is normally selected from a range of from about 1 to about 10,000 times, preferably from about 5 to about 50,000 times, and more preferably from about 10 to about 2000 times the oil volume. phase.

An emulsifying agent may be added to the external aqueous phase described above. The emulsifying agent may be any agent as long as it is capable of forming a stable oil / water emulsion. Such emulsifying agents include, for example, anionic surfactants (e.g., sodium oleate, sodium stearate, sodium lauryl sulfate), nonionic surfactants [e.g. fatty acid esters of polyoxyethylene sorbitan (Tween 80, Tween 60, Atlas Powder Company), polyoxyethylene castor oil derivatives (e.g. HCO-60, HCO-50, Nikko Chemicals)], polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin and hyaluronic acid. These emulsifying agents may be used either alone or in combination with each other. In terms of concentration, it is preferred to use in the range of about 0.01% to 10% by weight, preferably from about 0.05% to about 5% by weight.

An osmotic pressure regulator may be added to the external aqueous phase described above. Said osmotic pressure regulator may be any osmotic pressure regulator if it exhibits osmotic pressure when prepared as an aqueous solution.

99 9 ·· 99 '♦ 9 «♦ · • 9

Examples of said osmotic pressure regulator are polyhydroxy alcohols, monohydroxy alcohols, monosaccharides, disaccharides, oligosaccharides, amino acids and derivatives thereof.

Useful polyhydroxy alcohols include, for example, dihydroxy alcohols such as glycerol, pentahydroxy alcohols such as arabitol, xylitol and adonitol, and hexahydroxy alcohols such as mannitol, sorbitol and dulcitol. Of these alcohols, hexahydroxy alcohols are preferred, mannitol is more preferred.

Useful mononyloxy alcohols include, for example, methanol, ethanol, and isopropyl alcohol, with ethanol being preferred.

Useful monosaccharides include, for example, pentoses, such as arabinose, xylose, ribose and 2-deoxyribose, and hexoses, such as glucose, fructose, galactose, mannose, sorbose, rhamnose, and fucose, preferred are pentoses.

Useful oligosaccharides include, for example, trisaccharides such as maltotriose and raffinose, and tetrasaccharides such as stachyose, trisaccharides being preferred.

Useful derivatives of monosaccharides, disaccharides and oligosaccharides include, for example, glucosamine, galactosamine, glucuronic acid and galacturonic acid.

Useful amino acids include, for example, glycine, leucine, and arginine, with L-arginine being preferred.

These osmotic pressure regulators can be used alone or in combination.

These osmotic pressure regulators are normally used at concentrations such that the osmotic pressure of the external aqueous phase is about 1/50 times to about 5 times, preferably about 1/25 times to about 3 times the osmotic pressure of the physiological saline solution.

Removal of the organic solvent can be achieved by methods known per se or based on them. Such methods include, for example, a process in which the organic solvent is evaporated under normal or gradually reduced pressure with stirring using a propeller stirrer, magnetic stirrer or the like, and a process in which the organic solvent is evaporated. on a rotary evaporator which is connected to a vacuum or the like.

The microspheres thus obtained are centrifuged or filtered to separate, then washed several times with distilled water to remove free biologically active substance, hydroxynaphthoic acid, drug carrier, emulsifying agent, etc. that adhere to the surface of the microspheres, then disperse again in distilled water, etc., and freeze-dried.

An anticoagulant may be added to prevent particle aggregation during the manufacturing process. Examples of said anticoagulant are water-soluble polysaccharides such as mannitol, lactose, glucose and starches (e.g. corn starch), amino acids such as glycine, and proteins such as fibrin and collagen. Of these, mannitol is preferred.

If necessary, freeze-drying may be followed by heating under reduced pressure to remove water and organic solvent from the microspheres without causing the microspheres to adhere to each other. It is preferred that the microspheres be heated to a temperature slightly higher than the immediate glass transition point of the biodegradable polymer as determined by a differential scanning calorimeter when the temperature is increased at a rate of 10 to 20 ° C per minute. More preferably, the microspheres are heated to a temperature ranging from the immediate glass transition point of the biodegradable polymer to a temperature about 30 ° C higher than the glass transition temperature. When a lactic acid-glycolic acid polymer is used as the biodegradable polymer, it is particularly preferred that the microspheres be heated at a temperature of ♦ 44

4 4 4 9 9

4 4 4 4 9 4 9 9

9

The range from the immediate glass transition point up to a temperature of 10 ° C higher than the glass transition temperature, more preferably in the temperature range from the immediate glass transition point to a temperature of 5 ° C higher than the glass transition temperature.

While the heating time may vary depending on the amount of microspheres and other factors, it is usually from about 12 hours to about 168 hours, preferably about 24 hours to about 120 hours, and more preferably about 48 hours to about 96 hours, when the microspheres reach that temperature.

Any method of heating can be used provided that the microsphere aggregates are uniformly heated.

Useful thermal drying methods include, for example, a method according to which the heat drying is carried out in a fixed temperature chamber, in a fluidized bed chamber, in a mobile chamber or in an oven, and a method using microwave heat drying. Among these methods, a method in which the heat drying is carried out in a constant temperature chamber is preferred.

(ii) Water / oil / water method (1)

First, a solution of an organic solvent containing a biodegradable polymer or a salt thereof is prepared.

The concentrations of the organic solvent and the biodegradable polymer or salt thereof are the same as those described in (i) (i) above.

When more than two types of solvents are used, the ratios of these solvents are the same as those described in paragraph (I) (i) above. The biologically active substance or a salt thereof is added to the organic solvent solution containing the biodegradable polymer and dissolved and dispersed.

«0» 0 0

• 0 0 • * 0 · ·

J · 0 0 • 0 ·

· «00 * 0» »»

Furthermore, a solution of hydroxynaphthoic acid or a salt thereof is added to a solution of the organic solvent (oil phase) of the biologically active substance and the biodegradable polymer [an example of this solvent is water, alcohols (e.g. methanol, ethanol), pyridine solution, dimethylacetamide solution, etc.]. This mixture is emulsified in a known manner such as homogenization or sonication to form a water / oil emulsion.

The water / oil emulsion thus obtained, which contains the biologically active substance or a salt thereof, the hydroxynaphthoic acid or a salt thereof and the biodegradable polymer or a salt thereof, is then added to the aqueous phase to form a water (internal aqueous phase) / oil (oil phase) emulsion. / water (external aqueous phase). The solvent is then evaporated from the oil phase to give microspheres. For this process, the volume of the external aqueous phase is normally selected to range from about 1 to about 10,000 times, preferably about 5 to about 50,000 times, and more preferably from about 10 to about 2000 times. times the volume of the oil phase.

The emulsifier and the osmotic pressure regulator described above, which may be added to the external aqueous phase, and the following procedures are the same as those described above under (I) (i).

(ii) Water / oil / water method (2)

First, an organic solvent solution is prepared which contains hydroxynaphthoic acid and a biodegradable polymer. The organic solvent solution thus obtained is called the oil phase. The method of preparation is the same as described above under (I) (i).

Alternatively, an organic solvent solution containing a hydroxynaphthoic acid and an organic solvent solution containing a biodegradable polymer may also be prepared separately. By mixing these solutions an oil phase is prepared.

• · • φφ · φφφφ ·· 94

9 9 9

9 9 «• Φ · 4 φ · · · 4

ΦΦ 94

9994

4 9

9 9 • ΦΦΦ * · * • 99

The concentration of the biodegradable polymer in the organic solvent solution varies according to the molecular weight of the biodegradable polymer and the type of organic solvent. For example, when dichloromethane is used as the organic solvent, the concentration of the biodegradable polymer is normally selected to be in the range of about 0.5 to about 70 wt%, preferably about 1 to about 60 wt%. % and more preferably from about 2 to about 50 wt.

A solution of the biologically active agent or a salt thereof is then prepared [an example of this solvent is water, alcohols (e.g. methanol, ethanol)]. The solution thus obtained is called the internal aqueous phase. The concentration of the biologically active agent is normally 0.001 mg / ml to 10 g / ml, preferably 0.1 mg / ml to 5 g / ml, more preferably 10 mg / ml to 3 g / ml. The oil phase and the internal aqueous phase are emulsified in a known manner, such as homogenization or sonication, to form a water / oil emulsion.

For this process, the volume of the oil phase is normally selected to be in the range of from about 1 to about 1000 times, preferably from about 2 to about 100 times, and more preferably from about 3 to about 10 times. volume of the internal aqueous phase.

The viscosity of the water / oil emulsion is normally selected to be in the range of from about 0.01 to about 10 Pa.s, preferably from about 0.1 Pa to about 5 Pa.s, more preferably from about 0.5 Pa. to about 2 Pa.s.

The water / oil emulsion thus obtained, which contains the biologically active substance or a salt thereof, the hydroxynaphthoic acid or a salt thereof, and the biodegradable polymer is then added to the aqueous phase to form a water (internal aqueous phase) / oil (oil phase) / water ( external aqueous phase), after which the solvent from the oil phase is evaporated to form microspheres. For this process, the volume of the external aqueous phase is normally selected from a range of from about 1 to about 10,000 times, preferably from about 2 to about 100 times, and more preferably from about 3 to about 10 times the volume of the external phase. aqueous phase.

* · · · »*« «« * * * * * * * * * * * *

The above emulsifying agent and osmotic pressure regulator, which may be added to the external aqueous phase, are as follows. the procedures are the same as those described above under (I) (i).

II) Phase separation method

In the preparation of the microspheres of this process, a coacervating agent is added to the organic solvent solution described in paragraph (I) above, a little by little, in a water-drying process, wherein the solvent comprises a composition consisting of a biologically active substance or salt, hydroxynaphthoic acid or salt thereof. and a biodegradable polymer or a salt thereof, with stirring, to precipitate and solidify the microspheres. Said coacervating agent is added in a volume amount of from about 0.01 to about 1000 times, preferably from about 0.05 to about 500 times, and more preferably from about 0.1 to about 200 times the volume. oil phase.

Said coacervating agent may be any one, when it is a polymer, a mineral oil or vegetable oil compound that is miscible with an organic solvent and which does not dissolve the complex of a biologically active substance with a hydroxynaphthoic acid and a biocompatible polymer. Useful coacervatives specifically include, for example, silicone oil, sesame oil, soybean oil, corn oil, linseed oil, coconut oil, linseed oil, mineral oil, hexane and heptane. They can also be used in combination.

The microspheres thus obtained are isolated, then washed repeatedly with heptane, etc. to remove coacervating agent, etc. other than the biologically active agent, hydroxynaphthoic acid and biodegradable polymer compositions. This is followed by drying under reduced pressure. The microspheres are also washed in the same manner as in I) (i) above of the water drying method, then freeze-dried by heat treatment.

0 · 000 · 9999 • 99 • 99 9 9 09 9 9909 _Λ. · 9,990,990 ♦ ♦ 0,999,999

0 990 0900 • 999 9 99 · 9 · 09

III) Spray drying method

In the production of microspheres according to this process, the organic solvent solution described in (I) above of the water-drying process, which comprises a composition consisting of a biologically active substance or a salt thereof, a hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof into the drying chamber of the spray dryer so that the organic solvent flows out in a very short time as fine beads. Microspheres are obtained. Examples of said nozzle are a double fluid nozzle, a pressure nozzle and a rotary disk nozzle. The microspheres can then be freeze-dried and heat dried, as necessary, after washing in the same manner as in the above-mentioned (I) method of drying from water.

For dosage forms other than the microspheres described above, the organic solvent solution described in paragraph (I) above is a water-drying process comprising a composition consisting of a biologically active substance or a salt thereof, a hydroxynaphthoic acid or a salt thereof and a biodegradable polymer or a salt thereof. may be dried by evaporation of the organic solvent and water, the degree of vacuum being adjusted using a rotary evaporator or similar device. This is followed by grinding in a jet mill or the like so as to obtain microparticles.

The milled microparticles can then be freeze-dried and heat-dried after washing in the same manner as in the above-mentioned I) drying process from water to produce microspheres.

The microspheres or microparticles thus obtained allow drug release corresponding to the rate of degradation of the biodegradable polymer or lactic acid-glycolic acid polymer used.

• 9,999

99 <9 «9« 9 9 »9 9« 9

9999 »

• 9,999

9

9

9

9

IV) Two-step method

The biologically active agent or a salt thereof is added to a solution of the hydroxynaphthoic acid or a salt thereof in an organic solvent in a weight ratio that falls within the above-described range for biologically active agents. A solution of an organic solvent of the hydroxynaphthoic acid of the biologically active substance is obtained.

Said organic solvent is the same as those described above under (I) (i). If more than two kinds of organic solvents are used as the solvent mixture, the ratio of the mixture is the same as that described in (I) (i) above.

Removal of the organic solvent upon precipitation of the hydroxynaphthoic acid composition of the biologically active substance can be achieved by conventional methods or based on them. Such methods include, for example, a process in which the organic solvent is evaporated, wherein the degree of vacuum is adjusted using a rotary evaporator or the like.

The thus obtained hydroxynaphthoic acid biologically active agent composition can be redissolved in an organic solvent to form a sustained release composition (microspheres or microparticles).

Examples of said organic solvent are halogenated hydrocarbons (e.g. dichloromethane, chloroform, dichloroethane, trichloroethane, carbon tetrachloride), ethers (e.g. diethyl ether, isopropyl ether), fatty acid esters (e.g. ethyl acetate, butyl acetate) and aromatic hydrocarbons (e.g. benzene, toluene, xylene). These solvents can be used in mixtures in appropriate proportions. Of these solvents, halogenated hydrocarbons are preferred, dichloromethane is more preferred.

Organic solvent solution containing hydroxynaphtho36 * · 9999 ♦ · 9 • 9

9 • 9 ♦ 9> 9 9

I 9 9 9 • 9 9 9 9 9

The 9% acid of the biologically active substance is then added to the aqueous phase to form an oil (oil phase) / water (water phase) emulsion. The solvent is then evaporated from the oil phase. Microspheres are obtained. For this process sc, the volume of the aqueous phase will normally be selected from a range of about 1 to about 10,000 times, preferably about 5 to about 5,000 times, and more preferably from about 10 to about 2000 times the oil phase volume. .

The emulsifying agent, the osmotic pressure regulator and the subsequent step are the same as those described in (I) (i).

Removal of the organic solvent can be achieved by methods known per se or based on them. Such methods include, for example, a process in which the organic solvent is evaporated under normal or gradually reduced pressure with stirring using a propeller stirrer, magnetic stirrer or the like, and a process in which the organic solvent is evaporated on a rotary evaporator coupled to a controllable vacuum. or similar.

The microspheres thus obtained are centrifuged or filtered to separate, then washed several times with distilled water to remove free biologically active substance, hydroxynaphthoic acid, an emulsifying agent, etc. that adhere to the surface of the microspheres, then disperse again in distilled water, etc. and freeze-dried.

An anticoagulant may be added to prevent particle aggregation during the manufacturing process. Examples of said anticoagulant are water-soluble polysaccharides such as mannitol, lactose, glucose and starches (e.g. corn starch), amino acids such as glycine, and proteins such as fibrin and collagen. Of these, mannitol is preferred.

If necessary, freeze-drying may be followed by heating under reduced pressure to further evolve from the microspheres.

99 · t ···

9

9999 9 removed the water and the organic solvent without causing the microspheres to adhere to one another.

when the heating time may vary depending on the amount of microspheres and other factors, it usually ranges from about 12 hours to about 168 hours, preferably about 24 hours to about 120 hours, and more preferably about 48 hours to about 96 hours after the microspheres reach a given temperature.

Any heating method can be used if the microsphere aggregates are uniformly heated.

Useful methods of heat drying include, for example, a method according to which the heat drying is carried out in a fixed temperature chamber, in a fluidized bed chamber, in a mobile chamber or in an oven, and a method using microwave heat drying. Among these methods, a method in which the heat drying is carried out in a constant temperature chamber is preferred. The obtained microspheres have a relatively uniform round shape and have low resistance to injection, so needle blockage is unlikely. The possible use of thin injection needles also alleviates the patient's pain during injection.

V) One-step method

The biologically active agent or salt thereof is added to a solution of the hydroxynaphthoic acid or salt thereof in an organic solvent in an amount by weight that falls within the above-described range for the biologically active agents. A solution of an organic solvent of the hydroxynaphthoic acid of the biologically active substance is obtained. A sustained-release preparation (microspheres or microparticles) is then prepared.

Said organic solvent is the same as those described above under (I) (i). If more than two types of organic solvents are used as the solvent mixture, the ratio of the mixture shall be the same as that described in (I) (i).

• 0 0000 ·· 0000

The organic solvent solution containing the hydroxynaphthoic acid of the biologically active substance is then added to the aqueous phase to form an oil (oil phase) / water (water phase) emulsion. The solvent is then evaporated from the oil phase. Microspheres are obtained. For this process, the volume of the aqueous phase is normally selected from a range of about 1 to about 10,000 times, preferably about 5 to about 5,000 times, and more preferably from about 10 to about 2000 times the oil phase volume. .

The emulsifier and the osmotic pressure regulator described above, which may be added to the external aqueous phase, and the following procedures are the same as those described above under (IV).

The sustained release composition of the present invention can be administered as such or in the form of various dosage forms prepared by using it as a starting material, specifically as intramuscular, subcutaneous, visceral and other injectable or implantable formulations, nasal, rectal, uterine and other transdermal formulations. oral preparations [e.g. solid preparations such as capsules (eg hard capsules, soft capsules), granules and powders; liquids such as syrups, emulsions and suspensions] etc.

For example, the sustained release formulation of the present invention can be prepared as injectables by suspending in water with a dispersing agent (e.g., surfactants such as Tween 80 and HCO-60, polysaccharides such as sodium hyaluronate, carboxymethylcellulose and alginate sodium), a preservative (e.g. methylparaben, propyl paraben), a solution isotonicity adjusting agent (e.g. sodium chloride, mannitol, sorbitol, glucose, proline), etc. to obtain an aqueous suspension. Alternatively, it may be dispersed in a vegetable oil such as sesame oil or corn oil. An oily suspension is obtained. In this way, a practically useful sustained-release preparation that can be injected is obtained.

· «*» »» »» »♦

When the sustained release composition of the present invention is used in the form of a suspension that can be injected, the diameter of its particles is chosen to be within a range that satisfies the requirements for the degree of dispersion and the passage through the needle. For example, the average particle diameter is normally in the range of about 0.1 to 300 µm, preferably from about 0.5 to 150 µm, more preferably from about 1 to 100 µm.

The sustained release formulation of the present invention can be manufactured as a sterile formulation by methods in which the entire method of manufacture is aseptic, by a method that uses gamma rays to sterilize, and by a method in which a preservative is added, restrictive.

Due to its low toxicity, the sustained release formulation of the present invention can be used as a safe pharmaceutical preparation, etc. in mammals (e.g., human, bovine, swine, dogs, cats, mice, rats, rabbits).

While the dosage can vary within wide limits depending on the species, amount and dosage form of the active ingredient of the biologically active agent, the duration of release of the biologically active agent, the target disease, the species and other factors, the dosage of the sustained release formulation can be adjusted to any level. if the biologically active substance is active. The dosage of active ingredient of the biologically active agent for administration may preferably be selected as suitable from the range of about 0.01 mg to 10 mg / kg body weight, more preferably from about 0.05 mg to 5 mg / kg body weight in an adult for the formulation. with one month release.

The dosage of the sustained release composition for administration may preferably be selected as suitable in the range of about 0.05 mg to 50 mg / kg body weight, more preferably from about 0.1 mg to 30 mg / kg body weight in an adult.

♦ ti ·· »» ♦ ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti

The frequency of administration may be selected as appropriate according to the species, amount and dosage form of the active ingredient of the biologically active agent, the duration of release of the biologically active agent, the target disease, the animal species and other factors, e.g. once every several weeks, once a month or once every few months (e.g., 3 months, 4 months, 6 months).

The sustained release composition of the present invention is useful, according to the biologically active agent contained in the sustained release composition, as an agent for treating or preventing various kinds of diseases. When LH-RH derivatives are used as the biologically active agent, the sustained release composition of the present invention is useful as an agent for the treatment or prevention of hormone-dependent diseases, particularly sex hormone-dependent diseases such as sex hormone-dependent cancer (eg, cancer) prostate, hysterocarcinoma, breast cancer, hypophysoma, etc.), prostate hypertrophy, endometriosis, hysteromyoma, premature puberty, dysmenorrhoea, amenorrhoea, premenstrual syndrome, and multi-site ovarian syndrome. The sustained release composition of the present invention is also useful for a contraceptive agent. When a re-binding effect after treatment is used, the sustained release composition of the present invention is useful as a treatment agent

The sustained release composition of the present invention is further useful as an agent for the treatment or prevention of a benign or cacoetic neoplasm independent of sex hormones but sensitive to LH-RH.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is described herein in more detail by the following non-limiting examples.

94

4 4 4 9

4 4 4 4 · · ♦ 99 99 ·

4 4 9

9443 9 44

4 4 4 9

44 99

Example 1

3429.6 mg of acetate (manufactured by TAP) N- (S) -tetrahydrofur-2-oyl-Gly-D2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-SAlaNH__ (here hereafter referred to as peptide A) of the chemical formula

and 685.2 mg of 3-hydroxy-2-naphthoic acid are dissolved in 15 ml of ethanol. This solution was gradually distilled on a rotary evaporator to evaporate the organic solvent. The residue was redissolved in 5.5 mL of dichloromethane and poured into 400 mL of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by The Nippon Synthetic Chemical Industry) pretreated to 18 ° C; this solution is stirred at 8000 rpm with a turbine stirrer. An oil / water emulsion is obtained. The oil / water emulsion was stirred at room temperature for 3 hours to evaporate the dichloromethane and solidify the oil phase. Following isolation of the microspheres by centrifuge (05PR-22, Hitachi, Ltd.) at 2000 rpm. The microspheres are again dispersed in distilled water, then centrifuged and the free drug etc. rinsed. The recovered microspheres were again dispersed in a small amount of distilled water, then freeze-dried. A powder was obtained. The yield was 65%. The amount of peptide A in the microspheres was 75.4% and the molar ratio of 3-hydroxy-2-naphthoic acid / peptide A in the microspheres was 1.94.

• 9 »9

9 9 9 9 9

9 9 99 994 9 9 9 9 9 9 ·

9 9

9 • 99 · 4 · 99 ·

9 9 • 9 9

9999 8 · * * ···

Example 2

1785.1 mg of peptide A acetate and 1370.4 mg of 3-hydroxy-2-naphthoic acid are dissolved in 15 ml of ethanol. This solution was gradually distilled on a rotary evaporator to evaporate the organic solvent. The residue was redissolved in 10 ml of dichloromethane and poured into 1000 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol, the temperature of which was previously adjusted to 18 ° C. Following the same procedures as those in Example 1, microspheres were obtained. 58% by weight was obtained, the peptide content in the microspheres was 54.3% and the molar ratio of 3-hydroxy-2-naphthoic acid / peptide A in the microspheres was 6.15.

Example 3

1800 mg of peptide A acetate, 173 mg of 3-hydroxy-2-naphthoic acid and 2 grams of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/50 (mol%)), weighted average molecular weight 10,100, number average molecular weight 5670, number average molecular weight 3720 (as determined by end-group quantification, manufactured by Wako Pure Chemical Industries) were dissolved in a mixture of 6 mL of dichloromethane and 0.2 mL of ethanol. This solution was poured into 900 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol containing 5% mannitol, the temperature of which had been previously adjusted to 18 ° C, and stirred with a turbine stirrer at a speed of 7000 rpm. An oil / water emulsion is obtained. The oil / water emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane and ethanol and solidify the oil phase. This is followed by isolation of the microspheres by a centrifuge at 2000 rpm. The microspheres are again dispersed in distilled water, then centrifuged and the free drug etc. rinsed. The recovered microspheres were again dispersed in 250 mg mannitol and a small amount of distilled water. They were then freeze-dried. A powder was obtained. The yield was 76%. The inclusion of peptide A in the microspheres was 84.6%, the content of peptide A in the microspheres was 34.7% and the molar ratio of 3-hydroxy-2-naphthoic acid / peptide A in the microspheres was fc. · 9 9 9 e

· «· ♦ c c c c c c c c c c c

1.19.

Example 4

1900 mg of peptide A acetate, 182 mg of 3-hydroxy-2-naphthoic acid and 1.9 g of lactic acid-glycolic acid copolymers (as in Example 3) were dissolved in a mixture of 6 ml of dichloromethane and 0.2 ml of ethanol. This solution is poured into 900 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol containing 5% mannitol and 0.05% L-arginine, the temperature of which has been previously adjusted to 18 ° C. Following the same procedures as those in Example 3, microspheres were obtained. 85% by weight was obtained, the inclusion of peptide A into the microspheres was 88.9%, the amount of peptide A in the microspheres was 38.6% and the molar ratio of 3-hydroxy-2-naphthoic acid / peptide A in the microspheres was 0.83.

Example 5

The microspheres were obtained in the same manner as in Example 4 except that the lactic acid-glycolic acid copolymer used in Example 4 was replaced by a lactic acid-glycolic acid copolymer with a lactic acid / glycolic acid ratio of 75/25 (mol%). a weight average molecular weight of 10,700, a number average molecular weight of 6100, a number average molecular weight of 3770, as determined by end group quantification, and that the amount of dichloromethane was changed to 6.5 ml. 87% by weight was obtained, the inclusion of peptide A in the microspheres was 88.3%, the amount of peptide A was 38.3% and the molar ratio of 3-hydroxy-2-naphthoic acid / peptide A in the microspheres was 0.92.

Example 6

To a solution of 1800 mg of peptide A acetate and 1.8 g of lactic acid-glycolic acid copolymers (lactic acid / glycolic acid = 50/50 (mole%), weight average molecular weight 12,700, number average molecular weight 7090, number average molecular weight mass 4780, as determined by the quantification of 2 · 2 · 2 · 2 · · * ·· «·

Industries) prepared by Wako Pure Chemical in 7.2 ml of dichloromethane, a solution of 196 mg of 3-hydroxy-2-naphthoic acid sodium salt in water (2.3 ml) was added. This is followed by emulsification with a homogenizer. A water / oil emulsion is obtained. This emulsion is poured into 800 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol containing 5% mannitol, the temperature of which has been previously adjusted to 18 ° C, and stirred with a turbine stirrer at a speed of 7000 rpm. This gives a water / oil / water emulsion. Following the same procedures as those in Example 3, microspheres were obtained. The yield was 79%. The inclusion of peptides A into the microspheres was 81.2%, the content of peptides A in the microspheres was 32.8% and the molar ratio of 3-hydroxy-2-naphthoic acid / peptide A in the microspheres was 0.91.

Experimental Example 1

About 40 mg of the microspheres obtained in each of Examples 1 and 2 or about 60 mg of the microspheres obtained in each of Examples 3 to 5 were dispersed in 0.5 ml of a dispersant (distilled water with 0.25 mg carboxymethylcellulose, 0.5 mg Polysorbates 80). and 25 mg mannitol, all dissolved therein) and subcutaneously administered to the backs of male SD rats aged 8-10 weeks with a 22G needle. After administration, each rat was sacrificed. The microspheres remaining at the site of administration were collected and tested for peptide content

A. The results are shown in Table 1.

Table 1

1 day 1 week 2 weeks 3 weeks 4 weeks Example 1 73% 30% 11% 6% 6% Example 2 85% 37% 9% 1% example 3 70% 31% 14% 9% Example 4 77% 29% 11% 10% 6% Example 5 81% 44% 25% 17% 13 %

• · · · · · · ·

The experimental results of Examples 1 and 2 show that the release rate of peptide A from microspheres consisting of two components, i.e. peptide A and 3-hydroxy-2-naphthoic acid, varies according to their ratio; peptide A is released more rapidly as the 3-hydroxy-2-naphthoic acid content is increased. The experimental results in Examples 3, 4 and 5 also show that the microspheres, which consist of the three components, ie the above two components and the lactic acid-glycolic acid copolymer, show a different release profile of peptide A compared to the microspheres consisting of two ingredients. It has also been shown that release from microspheres can be controlled by combining various lactic acid-glycolic acid formulations and weighted molecular weight averages. The results in Example 7 and Reference Example 1 show that 3-hydroxy-2-naphthoic acid increases the content of peptide B in microspheres.

Example 7

A solution of 0.8 g of 5-oxo-Pro-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NH-C H acetate (hereinafter referred to as peptide B, manufactured by

5 my Takeda Chemical) in 0.8 ml of distilled water is mixed with a solution of 3.08 g of DL-lactic acid polymer (36,000 molecular weight average, 18,000 number average molecular weight, carboxyl group content according to the 70 quantitative evaluation method), 4 gmol / g) and 0.12 g of 3-hydroxy-2-naphthoic acid in a mixture of organic solvents consisting of 5 ml of dichloromethane and 0.3 ml of ethanol. This mixture is emulsified in a homogenizer. A water / oil emulsion is obtained. This water / oil emulsion is injected into 800 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by The Nippon Synthetic Chemical Industry), the temperature of which has been previously adjusted to 15 ° C, and stirred with a turbine stirrer. at 7000 rpm. This gives a water / oil / water emulsion. The water / oil / water emulsion was stirred at room temperature for 3 hours to flow or diffuse into the external aqueous phase dichloromethane and ethanol. The oil phase solidifies. Thereafter, the oil phase is passed through a 75 micron sieve, followed by centrifugation for 5 minutes at 2000 rpm in a centrifuge (05PR-22, Hitachi, Ltd.) to sediment the microcapsules. The microcapsules are isolated, dispersed again in distilled water, then centrifuged, followed by rinsing free drug, etc., and the microcapsules are isolated. The microcapsules are again dispersed in a small amount of distilled water added and then freeze-dried. A powder is obtained. The yield of microcapsules was 46% by weight, the content of peptides B in the microcapsules was 21.3% and the content of

3-hydroxy-2-naphthoic acid was 2.96%. The inclusion efficiency, as determined by dividing these actual contents by the corresponding loading amounts, was 106.6% for peptides B and 98.6% for 3-hydroxy-2-naphthoic acid.

Example 8

A solution of 1.2 g of peptide B acetate in 1.2 ml of distilled water was mixed with a solution of 4.62 g of DL-lactic acid polymer (molecular weight average 25,200, number average molecular weight 12,800, carboxyl group content according to the method of quantitative evaluation) by labeling 62,5 μιηοΐύ / g) and 0,18 g of 3-hydroxy-2-naphthoic acid in a mixture of organic solvents consisting of 7,5 ml of dichloromethane and 0,45 ml of ethanol. This mixture is emulsified in a homogenizer. A water / oil emulsion is obtained. This water / oil emulsion is then injected into 1200 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by The Nippon Synthetic Chemical Industry), the temperature of which has been pre-adjusted to 15 ° C, and stirred with turbine. agitator at 7000 rpm. This gives a water / oil / water emulsion. The water / oil / water emulsion was stirred at room temperature for 3 hours to flow or diffuse into the external aqueous phase dichloromethane and ethanol. The oil phase solidifies. Then the oil phase is passed through a 75 µm sieve, followed by centrifugation for 5 minutes at 2000 rpm in a centrifuge (05PR-22, Hitachi, Ltd.) to sediment the microcapsules. The microcapsules are isolated, dispersed again in distilled water, then centrifuged, followed by rinsing free drug, etc., and the microcapsules are isolated. The microcapsules are re-dispersed in a small amount of distilled water. 0.3 g of mannitol is added and dissolved. The solution is then freeze-dried. The yield of microcapsules (determined by subtracting the amount of mannitol added) was

55.2% by weight, the content of peptide B in the microcapsule was 21.3% and the content of 3-hydroxy-2-naphthoic acid was 2.96%. The inclusion efficiency, as determined by dividing these actual contents by the corresponding loading amounts, was 99.7% for peptide B and 102.2% for 3-hydroxy-2-naphthoic acid.

Example 9

The microcapsule powder was obtained in the same manner as in Example 8 except that the DL-lactic acid polymer described in Example 8 was replaced by another DL-lactic acid polymer (molecular weight average 28,800, number average molecular weight 14,500, carboxyl group content) according to a quantitative evaluation method of labeling of 78.1 gmol / g). The yield of microcapsules (determined by subtracting the amount of mannitol added) was

50.2% by weight, the content of peptide B in the microcapsule was 20.8% and the content of 3-hydroxy-2-naphthoic acid was 2.78%. The inclusion efficiency, as determined by dividing these actual contents by the corresponding loading amounts, was 103.4% for peptide B and 92.7% for 3-hydroxy-2-naphthoic acid.

Comparative Example 1

A solution of 1.2 g of peptide B acetate in 1.2 ml of distilled water was mixed with a solution of 4.8 g of the same DL-lactic acid polymer as in Example 9 in 7.8 ml of dichloromethane. This mixture was injected into 1200 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by The Nippon Synthetic Chemical Industry), the temperature of which was pre-adjusted to 15 ° C, and stirred with a turbine stirrer at 7000 rpm. minute. This gives a water / oil / water emulsion. This water / oil / water emulsion was treated in the same manner as in Example 8 to obtain microcapsule powder. The yield of microcapsules (determined by subtracting the amount of mannitol added) was 53.6% by weight, the peptide B content in the microcapsules was 12.1%. Efficacy of peptide B inclusion, as • · ··· ·

was determined by dividing this actual content by the amount of fill, it was 60.6%, the efficacy much lower than that obtained in Example 9. Thus, it is evident that the efficiency of peptide B inclusion was enhanced by the addition of .beta.-hydroxy-2-nitro-cycloolefin.

Example 10

A solution of 1.00 g of peptide B acetate in 1.00 ml of distilled water was mixed with a solution of 3.85 g of DL-lactic acid polymer (MW 49.500, number average molecular weight 17.500, carboxyl group content according to the method of quantitative evaluation). by labeling 45.9 µmol / g) and 0.15 g of 3-hydroxy-2-naphthoic acid in a mixture of organic solvents consisting of 7.5 ml of dichloromethane and 0.4 ml of ethanol. This mixture was emulsified with a homogenizer. A water / oil emulsion is obtained. This water / oil emulsion was then reacted in the same manner as in Example 8, except that the amount of 0.1% (w / w) aqueous polyvinyl alcohol solution was changed to 1000 ml and the amount of mannitol to 0.257 g. The microcapsule yield (determined by subtracting the amount of mannitol added) was 53.8 wt%, the peptide B content in the microcapsule was 18.02%, and the 3-hydroxy-2-naphthoic acid content was 2.70%. The inclusion efficiency, as determined by dividing these actual contents by the respective loading amounts, was 90.1% for peptide B and 90.1% for 3-hydroxy-2-naphthoic acid.

Example 11

A solution of 1.202 g of peptide B acetate in 1.20 ml of distilled water was mixed with a solution of 4.619 g of DL-lactic acid polymer (molecular weight average 19,900, number average molecular weight 10,700, carboxyl group content according to the 104.6 quantitative assay method). μπιοΐύ / g) and 0,179 g of 3-hydroxy-2-naphthalic acid in a mixture of organic solvents consisting of 7,5 ml of dichloromethane and 0,45 ml of ethanol. This mixture was emulsified with a homogenizer. A water / oil emulsion is obtained. This water / oil emulsion is then reacted in the same manner.

as in Example 8, except that the amount of mannitol added was 0.303 g. This gave a microcapsule powder. The microcapsule yield (determined by subtracting the amount of mannitol added) was 61.4 wt%, the peptide B content in the microcapsule was 15.88%, and the 3-hydroxy-2-naphthoic acid content was 2.23%. The inclusion efficiency, as determined by dividing these actual contents by fill amounts, was 77.75% for peptide B and 75.05% for 3-hydroxy-2-naphthoic acid.

Example 12

A solution of 1.00 g of peptide B acetate in 1.00 ml of distilled water was mixed with a solution of 3.85 g of DL-lactic acid polymer (molecular weight average 25,900, number average molecular weight 7,100, carboxyl group content according to the method of quantitative evaluation) labeling 98.2 moles / g) and 0.15 g

3-hydroxy-2-naphthoic acid in a mixture of organic solvents consisting of 5.5 ml of dichloromethane and 0.35 ml of ethanol. This mixture was emulsified with a homogenizer. A water / oil emulsion is obtained. This water / oil emulsion was then reacted in the same manner as in Example 7. A microcapsule powder was obtained. The microcapsule yield was 48.8% by weight, the peptide B content in the microcapsule was 21.31% and the 3-hydroxy-2-naphthoic acid content was 2.96%. The inclusion efficiency, as determined by dividing these actual contents by the respective loading amounts, was 106.5% for peptide B and 98.7% for 3-hydroxy-2-naphthoic acid.

Comparative Example 2

A solution of 1.00 g of peptide B acetate in 1.00 ml of distilled water was mixed with a solution of 4.00 g of the same DL-lactic acid polymer as in Example 12 in 4.0 ml of dichloromethane. This mixture was emulsified with a homogenizer. A water / oil emulsion is obtained. This water / oil emulsion was then reacted in the same manner as in Example 7. A microcapsule powder was obtained. The yield of microcapsules was 48.7% by weight, the content of peptide B in the microcapsules was 11.41%. The inclusion efficiency, as determined by dividing this actual content by the amount of fill, was 57.1%, thus much lower than that obtained in Example 12. Thus, it is evident that the peptide inclusion efficiency B was increased by the addition of 3-hydroxy-2-naphthoic acid.

Example 13

A solution of 89.2 g of DL-lactic acid polymer (molecular weight average 30,600, number average molecular weight 14,400, carboxyl group content according to the quantitative evaluation method of 63.0 μπιοΐύ / g) in 115.3 g of dichloromethane is mixed with the solution 3.45 g of 3-hydroxy-2-naphthoic acid in a mixture of organic solvents consisting of 38.8 g of dichloromethane and 6.27 g of ethanol. This mixture was adjusted to 28.5 ° C. 224 g of this organic solvent is weighed and mixed with an aqueous solution of 22.3 g of peptide B acetate in 20 ml of distilled water, previously heated to 44.9 ° C. Stirring for 5 minutes yields a crude emulsion which is then emulsified in a homogenizer at 10,000 rpm for 5 minutes. A water / oil emulsion is obtained. The water / oil emulsion is then cooled to 16.3 ° C and injected into 20 liters of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, manufactured by The Nippon Synthetic Chemical Industry) at a pre-temperature adjusted to 15 ° C, and mixed with the HOMOMIC LINE FLOW (manufactured by Tokushu Kika) at 7000 rpm. A water / oil / water emulsion is thus obtained. The water / oil / water emulsion was stirred at 15 ° C for 3 hours to flow or diffuse into the external aqueous phase dichloromethane and ethanol. The oil phase solidifies. The oil phase is then sieved through a 75 μηι sieve, followed by centrifugation at 2000 rpm in a centrifuge (H-600S, manufactured by Kokusan Enshinki). Microcapsules that continuously settle are isolated. The microcapsules are again dispersed in a small amount of distilled water, then sieved through a 90 µm sieve and freeze-dried. A powder is obtained. The microcapsule yield (determined by subtracting the amount of mannitol added) was 66.5 wt%, the peptide B content in the microcapsule was 22.3%, and the 3-hydroxy-2-naphthoic acid content was 2.99%. Inclusion effectiveness,

0 «0000

0 0 0 0 00000 00 · as determined by dividing these actual contents by the respective fill amounts, it was 104.5% for peptides B and 102.1% for

3-hydroxy-2-naphthoic acid.

Experimental Example 2

Dispersion of about 45 mg microcapsules as described in Example 8 in 0.3 ml of dispersant (distilled water containing 0.15 mg carboxymethylcellulose, 0.3 mg polysorbate 80 and 15 mg mannitol; all dissolved therein) was subcutaneously administered to the back of male SD rats at 7 weeks of age using a 22G injection needle. After given time, rats were sacrificed and microcapsules remaining at the injection site were collected and peptide B and 3-hydroxy-2-naphthoic acid were quantitated. The retention amounts that were determined by dividing the values obtained by the respective starting amounts and the profile properties of the DL-lactic acid polymer employed are shown in Table 2.

• »10 99 · ·

9999 ·· 99 • 9 9 • 9 9 • 9 9 • 9 9 9

99

Table 2

Properties of microcapsules with the DL-lactic acid polymer described in Example 8 mol. wt. 25,200 [COOH] ([mu] mol / gram polymer) 62.5

Retention amount:

peptide B 3-hydroxy-2-naphthoic acid

1 day 93.1 % 91.0 2 weeks 84.2 % 54.1 4 weeks 75.7 % 34.5 8 weeks 63.0 % 5.1 12 weeks 46.9 % 0.0 16 weeks 31.7 % 0.0 20 May weeks 24.0 % 0.0

As can be seen from Table 2, the microcapsule described in Example 8 shows a dramatically high amount of biologically active agent retention not less than 90% one day after administration, although the biologically active agent content was high. Thus, it is apparent that 3-hydroxy-2-naphthoic acid is effective not only by allowing the inclusion of the biologically active agent in sustained release formulations in high amounts, but also by the very well suppressed initial rapid release of biologically active agents. This microcapsule is capable of releasing the biologically active agent at a constant rate for a very long time. In addition, even though 3-hydroxy-2-naphthoic acid was completely removed from the capsule after 12 weeks, the same release rate of the biologically active agent was maintained, indicating the efficacy of the sustained release formulation.

* ·· «

0 «···· 9

9

0 0 0 0

9 9

9 ·· 99 • 9 * ♦ • 0 0 0

9 9 9

9 99

Experimental Example 3

After the microcapsules obtained in Examples 7, 9 to 12 and Comparative Example 1 were administered and recovered in the same manner as in Experimental Example 2, the protein B was quantitatively determined in them. The retention amount was determined by dividing the values obtained. and the properties of the profiles of the DL-lactic acid polymer used are given in Table 3.

Table 3

Properties of DL-lactic acid polymers

Ex. 7 Ex. 9 Ex. 10 Ex. 11 ex. 12 cf. Ex mol. wt. 36000 28800 49500 19900 25900 28800 [COOH] (pmoles / g polymer) 70.4 78.1 45.9 104.6 98.2 78.1

retention amount:

1 day 92.9 % 94.6 % 93.0 % 92.3 % 89.4 % 83.1 % 2 weeks 82.2 % 82.2 % 80.4 % 37.5 % 34.3 % 73.0 % 4 weeks 69.6 % 69.2 % 58.3 % 30.7 % 29.7 % 65.3 % 8 weeks 62.1 % 56.0 % 36.6 % 24.6 % 20.8 % 12 weeks 47.9 % 39.4 % 30.8 % 18.6 % 16 weeks 32.2 % 28.0 % 20 May weeks (not specified) 22.9 %

weeks 11,6% weeks 4,1%

As can be seen from Tables 2 and 3, the microcapsules described in Examples 7-12 exhibit a dramatically higher retention amount of about 90 or more% one day after administration as compared to Comparative Example 1. Thus, it is apparent that 3-hydroxy-2-naphtholates • ti titititi »9999 ti ti · ti ti ti ti ti ti

99

9 9 9

The three titanium titanium selins are effective not only in allowing the incorporation of the biologically active agent in high amounts into sustained release formulations, but also in suppressing the early rapid release of biologically active agents very well. In particular, experiments using the microcapsules described in Examples 7 to 9 demonstrate that when using DL-lactic acids having a weight average molecular weight of from about 20,000 to about 50,000 and a carboxyl group content of about 50 to 90 Mole / g, as has been quantitatively determined by the labeling method, it is possible to release the biologically active substance at a constant rate for a very long time.

Experimental example 4

After the microcapsule obtained in Example 7 was subcutaneously administered to rats according to the method described in Experimental Example 2, blood was collected and serum B and testosterone concentrations were determined. The results are shown in Table 4.

Table 4

12 weeks 16 weeks 24 weeks 26 weeks 28 weeks peptide B (ng / ml) 1.10 1.65 1.46 2.73 1.30 testosterone (ng / ml) 0.18 0.45 0.68 0.41 0.71

As can be seen from Table 4, the concentration of the biologically active agent in the blood was maintained at constant levels up to 28 weeks after administration, meaning that the biologically active agent was continuously released from the microcapsule over a period of 28 weeks. It was shown that pharmacologically active testosterone concentrations were constantly suppressed below normal levels during this time, and that the biologically active agent was in the microcapsule sta • fc fc ··· Fcfcfcfc fcfc fcfcfc fcfcfc has been vigorously present and released from this microcapsule for a long time without losing its activity, albeit 3-hydroxy -2-naphthoic acid was included.

Example 14

A 0.5 N aqueous solution of sodium hydroxide / methanol (1: 5 by volume) was passed through a strongly basic ion exchange column (SeP-Pak Plus QMA pack, manufactured by Waters) to remove chloride ions. After the eluate did not respond to the addition of a solution of silver nitrate acidified with nitric acid so that it became cloudy in white, a water / methanol mixture (1: 5 by volume) was passed through to remove excess alkali. After confirming the neutrality of the eluate, 18.8 mg of peptide B acetate was dissolved in 2 mL water / methanol (1: 5 by volume) and passed through a column that had been pretreated as above. This eluate and the other eluate resulting from the shedding of 6 ml of the mixture itself were combined. This mixture was mixed with a solution of 5.91 mg of 3-hydroxy-2-naphthoic acid in 1 mL of water / methanol (1: 5 by volume). Concentration on a rotary evaporator follows. After a white cloudiness is observed in the mixture, 2 ml of water are added, followed by stirring. After centrifugation (3000 rpm, 15 minutes, 20 ° C), the supernatant was removed, followed by several washes with water, and then the precipitate was dried under vacuum (overnight, 40 ° C). 4.09 mg of the peptide B 3-hydroxy-2-naphthoic acid salt was obtained.

To this salt was added 0.5 mL of water, followed by stirring at room temperature for 4 hours. The liquid was then filtered through a 0.2 µm filter and quantitatively evaluated by HPLC. The concentration of peptide B was 2.37 g / L and 3-hydroxy-2-naphthoic acid was 0.751 g / L. The salt remained partially undissolved, even after stirring. The above values are believed to represent the water solubility of the peptide B 3-hydroxy-2-naphthoic acid salt, no more than 1/100 less than the water solubility, not less than 1000 g / l, of the peptide B acetate. that the 3-hydroxy-2-naphthoic acid salt of peptide B can be used as a peptide B preparation with a sustained

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Effect of the invention;

The sustained release composition of the present invention contains the biologically active agent in high amounts and is capable of controlling its rate of release.

Claims (28)

PATENT CLAIMS A sustained release composition comprising a biologically active agent or a salt thereof, a hydroxynaphthoic acid or a salt thereof, and a biodegradable polymer or a salt thereof. 2. The sustained release composition of claim 1, wherein the biologically active agent is a biologically active peptide. The sustained release composition of claim 2, wherein the biologically active peptide is an LH-RH derivative. 4. The sustained release composition of claim 1, wherein the hydroxynaphthoic acid is 3-hydroxy-2-naphthoic acid. The sustained release composition of claim 1, wherein the biodegradable polymer is an α-hydroxycarboxylic acid polymer. The sustained release composition of claim 5, wherein the α-hydroxycarboxylic acid polymer is a lactic acid-glycolic acid polymer. The sustained release composition of claim 6, wherein the ratio of lactic acid to glycolic acid is 100: 0 to 40:60 mol%. The sustained release composition of claim 7, wherein the ratio of lactic acid to glycolic acid is 100: 0 mol%. The sustained release composition of claim 6. 0 «0000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 000 00 00 0 0 0 0 0 0 0 0 The weighted average molecular weight of the polymer is about 3000 to about 100,000. 10. The sustained release composition of claim 9, wherein the weight average molecular weight of the polymer is about 20,000 to about 50,000. 11. The sustained release composition of claim 3, wherein the LH-RH derivative is a peptide of 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 (ImBzl) and Z is NH- C H or Gly-NH. The sustained release composition of claim 6, wherein the carboxyl end group content of the polymer is 50 to 90 micromoles per unit weight (gram) of polymer. The sustained release composition of claim 3, wherein the molar ratio of the hydroxynaphthoic acid or salt thereof to the LH-RH derivative or salt thereof is from 3 to 4 to 4 to 3. The sustained release composition of claim 13, wherein the LH-RH derivative or salt thereof is present in an amount of 14% by weight. to. 24 wt. The sustained release composition of claim 1, wherein the biologically active agent or salt thereof is very slightly water soluble or water soluble. The sustained release composition of claim 1, wherein the composition is for injection. 9 9 · 9 9 9 9 9 9 9 9 999 9 9 99 98 99 • 9 «· 9 9 9 9 9 9 9 9 9 99 99 17. A method for producing a sustained release formulation according to claim 1, comprising removing the solvent from the mixture of the biologically active agent or salt thereof, the biodegradable polymer or salt thereof, and the hydroxynaphthoic acid or salt thereof. 18. A method of making a sustained release formulation according to claim 17, comprising mixing and dispersing the biologically active agent or salt thereof in an organic solvent solution comprising the biodegradable polymer or salt thereof and the hydroxynaphthoic acid or salt thereof, followed by removal. organic solvent. 19. The method of manufacture of the sustained release composition of claim 18, wherein the biologically active agent or salt thereof is in the form of an aqueous solution. 20. The method of manufacture of the sustained release composition of claim 17, wherein the salt of the biologically active agent is a salt with a free base or acid. 21. A pharmaceutical composition comprising the sustained release composition of claim 1. An agent for preventing or treating prostate cancer, prostate hypertrophy, endometriosis, hysteromyoma, metrofibroma, premature puberty, dysmenorrhea or breast cancer, or a contraceptive comprising the sustained release composition of claim 3. 23. A sustained release composition comprising a hydroxynaphtholate of a biologically active agent and a biodegradable polymer or salt thereof. A method of suppressing the initial rapid release of biolo94 4449 94 9499 99 »9 • 9 9 4 9 4 · 9 9 9 9 9 9 99 99 99 9 • 9 9 9 9 9 99 9 49 49 gically active agents from a sustained release formulation comprising the use of a hydroxynaphthoic acid or a salt thereof. 25. A method of enhancing the effectiveness of inclusion of a biologically active agent in a sustained release formulation, which comprises using hydroxynaphthoic acid or a salt thereof. 26. Hydroxynaphthate of a biologically active peptide. The biologically active peptide hydroxynaphthoate of claim 26, which is water soluble or very slightly water soluble. 28. A sustained release composition comprising a hydroxynaphthoate of a biologically active peptide.