RU2128055C1 - Pharmaceutical composition of the delayed agent releasing and a method of its preparing - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention relates to a composition that has a physiologically active peptide of the general formula:

where X means acyl-group; R₁, R₂ and R₄ each means aromatic cyclic group; R₃ means a residue of D-amino acid or a group of the formula:

where

means heterocyclic group; R₅ means a group of the formula

where n = 2 or 3, and

means amino-group that can be substituted optionally, aromatic cyclic group or O-glycosyl-group; R₆ means a group of the formula

where n = 2 or 3, and

means amino-group that can be substituted optionally; R₇ means a residue of D-amino acid or azaglycyl-residue; Q means hydrogen atom or lower alkyl-group, or its salt and biodegradable polymer bearing a terminal carboxyl-group. Composition is prepared by dissolving of components mixture in water-insoluble solvent followed by solvent removing. Preparation of delayed releasing ensures to obtain noncontinuous release of peptide for the long time without initial release as compared with the known similar compositions. EFFECT: improved method of preparing, enhanced effectiveness of composition proposed. 25 cl, 9 tbl, 45 ex

Description

 The present invention relates to pharmaceutical compositions for sustained release containing a physiologically active peptide, and methods for its preparation.

The prior art includes, as disclosed in EP-A-481732, a sustained release formulation containing a drug, polylactic acid and a glycolic acid-hydroxy carboxylic acid copolymer [HOCH (C _2-8 alkyl) COOH]. The disclosed method includes preparing a W / O (water-in-oil emulsion) emulsion containing an internal aqueous phase comprising an aqueous solution of a physiologically active peptide and an external oil phase comprising a solution of a biodegradable polymer in an organic solvent, adding said W / O emulsion to water or an aqueous medium and processing the resulting W / O / W emulsion into sustained release microcapsules (method of drying in water).

 Japanese Patent Laid-open No. 118512/1982 describes a microcapsule comprising a hormone-active polypeptide, a biodegradable polymer and a hydrolysis regulating substance. The method disclosed for its production is a coacervation method, which comprises adding a coacervating agent to a W / O emulsion consisting of an aqueous solution of a polypeptide as an internal aqueous phase and a halogenated organic solvent as an oil phase for the production of microcapsules.

 Japanese Patent Laid-open No. 121222/1989 describes a pharmaceutical composition comprising polylactide, polyglycolide, a lactic acid-glycolic acid copolymer or a mixture of these polymers and a water-insoluble peptide. A manufacturing method is also disclosed which involves dispersing a salt of a water-insoluble peptide in a solution of the named polylactide, polyglycoside, a lactic acid-glycolic acid copolymer or a mixture of these polymers, removing the solvent by evaporation and fusing the resulting mixture into solid particles.

 Japanese Patent Laid-open No. 1506091/1982 describes a method for producing a pharmaceutical composition comprising a polylactide and an acid-resistant polypeptide, which, for example, includes dissolving tetra-gastrin hydrochloride and polylactide in aqueous dioxane, casting the solution into a film, and evaporating the solvent.

 EP-A-0467389 provides a technology for providing a drug dispensing system for proteins and polypeptides using a polymer precipitation method or a microsphere method. However, this literature does not contain a specific description of the system including the LH-RH derivative.

 The hormone-releasing lutenizing hormone known as LH-RH (or GnRH) is secreted by the hypothalamus and binds to the pituitary receptors. LH (luteinizing hormone) and FSH (follicle-stimulating hormone), which are subsequently released, act on the gonad by synthesizing steroid hormones. The existence of both agonistic and antagonistic peptides in the form of derivatives of LH-RH is known. When a highly agonistic peptide is administered repeatedly, the number of available receptors is reduced, so that gonad-produced steroid hormones are suppressed. Therefore, it is assumed that LH-RH derivatives will have value as therapeutic agents for diseases associated with hormonal dependence, such as prostate cancer, benign prostatic hypertrophy, endometriosis, uterine fibroids, uterine fibroids, premature puberty, breast cancer, etc. or as a contraceptive.

 In particular, the problem of histamine-releasing activity was noted for LH-RH antagonists of the so-called first and second generations (The Pharmaceuticols 32, 1599-1605, 1990), but after this a number of compounds were synthesized and recently obtained LH-RH-antagonistic peptides having a negligible histamine-releasing activity (cf., for example, USP 5110904). In order for any such LH-RH counteracting peptide to exhibit its pharmacological effect, a release control system is needed so that competing inhibition of endogenous LH-RH can be sustained. In addition, since histamine-releasing activity may be low but is still present in such peptides, there is a need for a sustained-release preparation with an inhibited initial impulse immediately following administration.

 In particular, in the case of a sustained release preparation (for example, 1-3 months), it is important to provide a more reliable and consistent release of the peptide in order to achieve this desired effectiveness with a greater degree of confidence and safety.

 At the same time, there has long been a need for a method for preparing a sustained release preparation having a high peptide retention for a physiologically active peptide, in particular LH-RH reactive peptides.

в которой X обозначает ацилгруппу;
R₁, R₂ и R₄ каждый представляет ароматическую циклическую группу;
R₃ представляет D-аминокислотный остаток или группу формулы

в которой R'₃ обозначает гетероциклическую группу;
R₅ представляет группу формулы -(CH₂)_n-R'₅, в которой n = 2 или 3 и R'₅ обозначает аминогруппу, которая необязательно может быть замещенной, ароматическую циклическую группу или O-гликозилгруппу.According to the present invention, it is provided:
1) A sustained release preparation that includes a physiologically active peptide of general formula I

in which X represents an acyl group;
R ₁ , R ₂ and R ₄ each represents an aromatic cyclic group;
R ₃ represents a D-amino acid residue or a group of the formula

in which R ' ₃ denotes a heterocyclic group;
R ₅ represents a group of the formula - (CH ₂ ) _n —R ′ ₅ in which n = 2 or 3 and R ′ ₅ represents an amino group which may optionally be substituted, an aromatic cyclic group or an O-glycosyl group.

R ₆ represents a group of the formula - (CH ₂ ) _n —R ′ ₆ in which n = 2 or 3, R ′ ₆ is an amino group which may be optionally substituted;
R ₇ is a D-amino acid residue or an azaglycyl residue;
Q is hydrogen or a lower alkyl group,
or a salt thereof and a biodegradable polymer having a terminal carboxyl group.

2) The sustained release preparation according to claim 1 above, wherein X is a C _2-7 alkanoyl group, which may be optionally substituted with a 5 or 6 membered heterocyclic carboxamido group.

3) The sustained release formulation of claim 2 above, wherein X is a C _2-4 alkanoyl group, which may optionally be substituted with a tetrahydrofuryl carboxamide group.

 4) A sustained release formulation according to the above 1, wherein X is acetyl.

в которой R представляет алкилгруппу с 2-8 атомами углерода,
и (B) полимолочной кислоты.5) A sustained release preparation according to the above 1, wherein the biodegradable polymer is a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the general formula II

in which R represents an alkyl group with 2-8 carbon atoms,
and (B) polylactic acid.

 6) The sustained release formulation of claim 1 above, wherein X is acetyl and the biodegradable polymer is a mixture (A) of a glycolic acid-hydroxy carboxylic acid copolymer of the general formula II.

в которой R представляет алкил группу с 2-8 атомами углерода,
и (B) полимолочной кислоты.

in which R represents an alkyl group with 2-8 carbon atoms,
and (B) polylactic acid.

 7) The sustained release formulation of claim 5 above, wherein the copolymer has a weight average molecular weight of about 2000 to 50,000, as determined by GPC (gel permeation chromatography).

 8) The sustained release preparation according to the above p. 5, in which the copolymer has a degree of dispersion of from about 1.2 to 4.0.

 9) The sustained release formulation of claim 5 above, wherein the polylactic acid has a weight average molecular weight of about 1,500 to 30,000 GPCs.

 10) The sustained release formulation of claim 5 above, wherein the polylactic acid has a degree of dispersion of from about 1.2 to 4.0.

 11) The sustained release formulation of claim 1 above, wherein the biodegradable polymer is a copolymer of lactic acid and glycolic acid.

 12) The sustained release formulation of claim 11 above, wherein the copolymer has a weight average molecular weight of from about 5,000 to about 25,000, as determined by GPC.

 13) The sustained release formulation of claim 11 above, wherein the copolymer has a degree of dispersion of from about 1.2 to 4.0.

 14) The sustained release formulation of claim 1 above, wherein the physiologically active peptide is in the range of about 0.01 to 50% (w / w) relative to the biodegradable polymer.

 15) A sustained release preparation according to the above 1, wherein the physiologically active peptide is an LH-RH antagonist.

или его ацетат.16) a sustained release preparation according to the above p. 1, in which the physiologically active peptide is:

or its acetate.

17) The sustained release formulation of claim 1 above, wherein the physiologically active peptide is NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ or its acetate.

18) The sustained release preparation according to the above 1, wherein the physiologically active peptide is NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg (Et ₂ ) -Leu-hArg (Et ₂ ) -Pro-DAlaNH ₂ or its acetate .

в которой X представляет ацилгруппу;
R₁, R₂ и R₄ каждый представляет ароматическую циклическую группу;
R₃ представляет D-аминокислотный остаток или группу формулы

в которой R'₃ обозначает гетероциклическую группу;
R₅ представляет группу формулы -(CH₂)_n-R'₅, в которой n = 2 или 3 и R'₅ является аминогруппой, которая может быть необязательно замещенной, ароматической циклической или O-гликозил группой;
R₆ представляет группу формулы -(CH₂)_n-R'₆, в которой n = 2 или 3 и R'₆ является аминогруппой, которая может быть необязательно замещенной;
R₇ представляет D-аминокислотный остаток или азаглицил остаток; и
Q обозначает водород или низшую алкильную группу,
или его соли и биоразлагаемого полимера, имеющего концевую гидроксильную группу в растворителе, который является в основном не смешиваемым с водой, и затем удаление названного растворителя.19) A method for producing a sustained release preparation, which comprises dissolving a physiologically active peptide of general formula I

in which X represents an acyl group;
R ₁ , R ₂ and R ₄ each represents an aromatic cyclic group;
R ₃ represents a D-amino acid residue or a group of the formula

in which R ' ₃ denotes a heterocyclic group;
R ₅ represents a group of the formula - (CH ₂ ) _n —R ′ ₅ in which n = 2 or 3 and R ′ ₅ is an amino group which may be an optionally substituted, aromatic cyclic or O-glycosyl group;
R ₆ represents a group of the formula - (CH ₂ ) _n —R ′ ₆ in which n = 2 or 3 and R ′ ₆ is an amino group which may be optionally substituted;
R ₇ represents a D-amino acid residue or an azaglycyl residue; and
Q is hydrogen or a lower alkyl group,
or a salt thereof and a biodegradable polymer having a terminal hydroxyl group in a solvent that is substantially immiscible with water, and then removing said solvent.

в которой R представляет алкилгруппу с 2-8 атомами углерода,
и (B) полимолочной кислоты.20) The method according to the above p. 19, in which the biodegradable polymer is a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the General formula II

in which R represents an alkyl group with 2-8 carbon atoms,
and (B) polylactic acid.

в которой R обозначает алкилгруппу с 2-8 атомами углерода,
и (B) полимолочной кислоты.21) The method according to the above p. 19, wherein X is acetyl and the biodegradable polymer is a mixture of (A) a copolymer of glycolic acid and hydroxy carboxylic acid of the general formula II

in which R denotes an alkyl group with 2-8 carbon atoms,
and (B) polylactic acid.

 22) The method according to the above p. 19, wherein the biodegradable polymer is a copolymer of lactic acid and glycolic acid.

 23) The method according to the above p. 19, which comprises dissolving a biodegradable polymer and a substantially water insoluble physiologically active peptide in a solvent that is substantially immiscible with water, and adding the resulting solution to the aqueous medium to obtain an O / W emulsion (emulsion type "oil in water").

в которой R представляет алкилгруппу с 2-8 атомами углерода,
и (B) полимолочной кислоты и в основном водонерастворимого физиологически активного пептида или его соли в растворителе, который в основном является не смешиваемым с водой, и затем удаление названного растворителя, и
25) Способ по приведенному выше п. 24, который дополнительно включает после этого растворение биоразлагаемого полимера и в основном водонерастворимого физиологически активного пептида или его соли в растворителе, который является в основном не смешиваемым с водой, и затем добавление образовавшегося раствора к водной среде с целью получения O/W эмульсии.24) A method for producing a sustained release preparation which comprises dissolving a biodegradable polymer comprising a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the general formula II

in which R represents an alkyl group with 2-8 carbon atoms,
and (B) polylactic acid and a substantially water insoluble physiologically active peptide or salt thereof in a solvent that is substantially water immiscible, and then removing said solvent, and
25) The method according to the above p. 24, which further includes then dissolving the biodegradable polymer and the substantially water-insoluble physiologically active peptide or its salt in a solvent that is substantially immiscible with water, and then adding the resulting solution to the aqueous medium to obtaining O / W emulsions.

The designations used in this description have the following meanings:
NAcD2Nal - N-Acetyl-D-3- (2-naphthyl) alanyl
D4ClPhe - D-3- (4-Chlorophenyl) Alanyl
D3Pal - D-3- (3-Pyridyl) Alanyl
NMeTyr - N-Methyl Tyrosyl
DLys (Nic) - D- (Ypsilon-N-Nicotinoyl) Lysyl
Lys (Nisp) - (Ypsilon-N-Isopropyl) Lysyl
DLys (AzaglyNic) - D- [1-Aza- (N-nicotinoyl) glycyl] lysyl
DLys (AzaglyFur) - D- [1-Aza- (N-2-furoyl) glycyl] lysyl
For any other amino acids, the designations recommended by the IUPAC-IUB by the Committee on Biochemical Nomenclature (European Journal of Biochemistry 138, 9-37, 1984) or the designations generally accepted in the relevant technique are used. In the case of the existence of optical isomers for any compound, unless otherwise specified, we mean the L-isomer.

 In the present invention, peptide (I) exhibits LH-RH antagonistic activity and is effective in the treatment of hormone-dependent diseases such as prostate cancer, prostate hypertrophy, endometriosis, uterine fibroids, uterine fibroids, premature puberty, breast cancer, and etc. or as a contraceptive.

Referring to the formula [I], the acyl group X is preferably an acyl group of a carboxylic acid. Examples of the acyl group include C _2-7 alkanoyl, C _7-15 cycloalkanoyl (e.g. cyclohexanoyl), C _1-6 alkylcarbamoyl (e.g. ethylcarbamoyl), 5- or 6-membered heterocyclic carbonyl (e.g. piperidinocarbonyl), and a carbomoyl group, which may optionally to be substituted. The acyl group is preferably a C _2-7 alkanoyl group (e.g., acetyl, propionyl, butyryl, isobutyryl, pentanoyl, hexanoyl or heptanoyl), which may optionally be substituted, most preferably a C _2-4 alkanoyl group (e.g., acetyl, propionyl, butyryl, isobutyryl) which may optionally be substituted. Substituents are, for example, a C _1-6 alkylamino group (e.g. methylamino, ethylamino, diethylamino, propylamino), a C _1-3 alkanoylamino group (e.g. formylamino, acetylamino, propionylamino), a C _7-15 cycloalkanoyl amino group (e.g. cyclohexenoylamino), C _7-15 arylcarbonyl-amino group (e.g., benzoylamino), 5- or 6-membered heterocyclic carboxamido (e.g., tertagidrofurilkarboksamido, pipiridilkarboksamido, furilkarboksamido), hydroxyl group, karbomoilgruppa, formyl, tkarboksilgruppa, 5- or 6-membered heterocycle parameter group (e.g., pyridyl, morpholino). The substituents are preferably a 5- or 6-membered heterocyclic carboxamido group (e.g. tetrahydrofurylcarboxamido, pyridylcarboxamido, furylcarboxamido).

X is preferably a C _2-7 alkanoyl group, which may be preferably substituted with a 5- or 6-membered heterocyclic carboxamido group.

More preferably, X represents a C _2-4 alkanoyl group, which may optionally be substituted with a tetrahydrofuryl carboxamido group.

-CO HCH CO - (тетрагидрофурилкарбоксамидоацетил) и тому подобное.Specific examples of X are acetyl,

-CO HCH CO - (tetrahydrofurylcarboxamidoacetyl) and the like.

The aromatic cyclic group R ₁ , R ₂ or R ₄ may be, for example, an aromatic cyclic group with 6-12 carbon atoms. Examples of the aromatic cyclic group are phenyl, naphthyl, antryl and the like. Preferred are aromatic cyclic groups with 6-10 carbon atoms, such as phenyl and naphthyl. These aromatic cyclic groups can each have from 1 to 5, preferably from 1 to 3, suitable substituents on the ring in the corresponding positions. Such substituents include hydroxy, halogen, aminotriazolyl substituted amino, alkoxy and the like. Preferred are hydroxy, halogen and aminotriazolyl substituted amino.

 The halogens mentioned above include fluorine, chlorine, bromine and iodine.

The aminotriazolyl moiety of the named aminotriazolyl substituted amino includes, among others, 3-amino-1H-1,2,4-triazol-5-yl, 5-amino-1H-1,3,4-triazol-2-yl, 5-amino 1H-1,2,4-triazol-3-yl, 3-amino-2H-1,2,4 triazol-5-yl, 4-amino-1H-1,2,3-triazol-5-yl, 4 amino-2H-1,2,3-triazol-5-yl and the like. The alkoxy group is preferably an alkoxy group with 1-6 carbon atoms (e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, etc.). More preferably, R ₁ is naphthyl or halophenyl. More preferably, R ₂ is halophenyl. More preferably, R ₄ is hydroxyphenyl or aminotriazolylamino substituted phenyl.

The D-amino acid residue R _{3 is} preferably an α- -D-amino acid residue with 3-12 carbon atoms. Examples of amino acids are leucine, isoleucine, norleucine, valine, norvaline, 2-aminobutyric acid, phenylalanine, serine, threonine, methionine, alanine, tryptophan and aminoisobutyric acid. These amino acids may have suitable protecting groups (protecting groups commonly used in the art, such as t-butyl, t-butoxy, t-butoxycarbonyl, etc.).

The heterocyclic group R ′ ₃ includes 5- or 6-membered heterocyclic groups, each containing from 1 to 2 nitrogen or sulfur atoms as heteroatoms, which may optionally be fused to the benzene ring. In particular, thienyl, pyrrolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, pyridyl, 3-pyridyl, pyridavinyl, pyrimidinyl, pyrazinyl, 3-benzo [b] thienyl, 3-benzo [b] -3-thienyl, indolyl can be mentioned , 2-indolyl, isoindolyl, 1H-indazolyl, benzoimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, etc. Particularly preferred species of R ′ ₃ are pyridyl or 3-benzo [b] thienyl. The aromatic cyclic group R ₅ may be the same as the aromatic cyclic group R ₁ , R ₂ or R ₄ . The aromatic cyclic group may have from 1 to 5, preferably from 1 to 3, suitable substituents at the corresponding positions on the ring. The substituents may also be such as the substituents mentioned for R ₁ , R ₂ or R ₄ . A particularly preferred substituent is aminotriazolyl substituted amino.

The glycosyl group for O-glycosyl R _{5 is} preferably hexose or a derivative thereof. Hexose includes D-glucose, D-fructose, D-mannose, D-galactose, L-galactose and the like. As the named derivative, deoxy sugars (L- and D-fructose, D-quinose, L-ramnose, etc.) and amino sugars (D-glucosamine, D-galactosamine, etc.) can be mentioned. Most preferred are deoxy sugars (L- and D-fructose, D-quinose, L-rhamnose, etc.). L-ramnose is even more desirable.

A substituent on an amino group, which may optionally be substituted, R ′ ₅ includes, among others, acyl, carbamoyl, carbazoyl, which may be substituted by acyl or amidino, which may be mono- or di-substituted with alkyl.

 The aforementioned acyl and acyl for the aforementioned carbazoyl, which may be substituted with acyl, include nicotinoyl, furoyl, tenoyl and the like.

 The aforementioned alkyl moiety of mono- or dialyamidino includes linear or branched alkyl groups containing from 1 to 4 carbon atoms, including so methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl and the like. A preferred alkyl moiety is methyl or ethyl.

A substituent for an amino group, which may optionally be substituted, R ′ ₆ includes alkyl and amidino, which may optionally be mono- or di-substituted with alkyl.

The above-mentioned alkyl and mono- or dialkylamidino alkyl mentioned above include those alkyl groups mentioned for R ′ ₅ .

The D-amino acid residue R ₇ is preferably a D-amino acid residue with 3 to 9 carbon atoms such as D-alanyl, D-leucyl, D-valyl, D-isoleucyl, D-phenylalanil and the like. More preferred are D-amino acid residues with 3-6 carbon atoms, such as D-alanyl, D-valyl and the like.

The most preferred variant of R ₇ is D-alanyl.

The lower alkyl group may be an alkyl group defined for R ′ ₅ . The most preferred type of Q is methyl.

и тому подобные
Конкретными примерами R₂ являются

и тому подобные
Конкретными примерами R₃ являются

и тому подобные
Конкретными примерами R₄ являются

и тому подобные
Конкретными примерами R₅ являются

-(CH₂)₃-NH₂
-(CH₂)₂-NH -CO-NH₂
-(CH₂)₃-NH -CO-NH₂

и тому подобные.Specific examples of R ₁ are

and the like
Specific examples of R ₂ are

and the like
Specific examples of R ₃ are

and the like
Specific examples of R ₄ are

and the like
Specific examples of R ₅ are

- (CH ₂ ) ₃ -NH ₂
- (CH ₂ ) ₂ -NH -CO-NH ₂
- (CH ₂ ) ₃ -NH -CO-NH ₂

and the like.

и тому подобные
Конкретными примерами R₇ являются

и тому подобные
Когда пептид [I] содержит один или более асимметрических углеводных атомов, существует два или более стереоизомеров. Любой из таких стереоизомеров равно, как и их смесь, находятся в рамках объема данного изобретения и приложенных пунктов.Specific examples of R ₆ are

and the like
Specific examples of R ₇ are

and the like
When the peptide [I] contains one or more asymmetric carbohydrate atoms, there are two or more stereoisomers. Any of these stereoisomers, as well as their mixture, are within the scope of this invention and the attached items.

 The peptide of the general formula [I] is prepared essentially by known methods. Typical specific methods are described in US Pat. No. 5,110,904.

 The peptide [I] can be used in the form of a salt, preferably a pharmacologically acceptable salt. In the case where the peptide has basic groups such as amino, the salt includes salts with inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, and the like) or organic acids (e.g., carbonic acid, hydrogen carbonic acid, succinic acid , acetic acid, propionic acid, trifluoroacetic acid, etc.). When the peptide contains acid groups such as carboxyl, salts with inorganic bases (for example, alkali metals such as sodium, potassium, etc. and alkaline earth metals such as sodium, potassium, etc., and alkaline earth metals such as calcium, magnesium, etc.) or with organic bases (for example, organic amines such as triethylamine and basic amino acids such as arginine). The peptide [I] may be in the form of a metal complex compound (eg, copper complex, zinc complex, etc.). Preferred salts of peptide [I] are salts with organic acids (for example, carbonic acid, hydrocarbonic (hydrogen carbonic) acid, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.). Most preferred is acetate.

Specific preferred types of peptide [I] or salt are as follows:
(1) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Noc) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ or its acetate.

(2) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyNic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ or its acetate.

(3) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ or its acetate.

CONHCH₂COD2Nal-D4ClPhe-D3Pal-Ser- NMeTyr-DLys(Noc)-Leu-Lys(Nisp)-Pro-DAlaNH₂ или его ацетат.(4)

CONHCH ₂ COD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Noc) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ or its acetate.

(5) NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg (Et ₂ ) -Leu-hArg (Et ₂ ) -Pro-DAlaNH ₂ or its acetate.

 In a sustained release preparation, the ratio of peptide [I] can vary, in particular, depending on the type of peptide, the expected pharmacological effect, and the exposure time, and can range from about 0.01% to 50% (weight / weight) relative to biodegradable polymer. A preferred range is from about 0.1 to 40% (w / w) and a most preferred range is from about 1 to 30% (w / w).

 A biodegradable polymer having a terminal carboxyl group is described.

A biodegradable polymer, approximately 1 to 3 g, was dissolved in a mixture of acetone (25 ml) and methanol (5 ml) and, using phenolphthalein as an indicator, the carboxyl groups in the solution were quickly titrated with 0.05 and an alcoholic solution of hydroxy potassium with stirring in room temperature conditions (20 ^o C). Then, the number average molecular weight was calculated based on the determination of the end groups using the following equation.

End-group average number molecular weight = 20,000 • A / B
where A corresponds to the mass of biodegradable polymer (g)
B represents an amount of 0.05 n. alcohol solution of hydroxy potassium (ml) used for titration.

 The result of the above calculation gives the number average molecular weight determined by the end groups.

 As an illustration, if we take a polymer having a terminal carboxyl group synthesized, for example, from one or more α-hydroxy acids by the method of non-catalytic dehydrative polycondensation, then the number average molecular weight determined from the end groups is approximately equal to the number average molecular weight found GPC method. In contrast, in the case of a polymer substantially free of free terminal carboxyl groups obtained by synthesis from a cyclic dimer by the polymerization ring opening method and using catalysts, the number average molecular weight determined from the end groups is much higher than the number average molecular weight determined by the method GPC Based on this difference, a polymer having a terminal carboxyl group can be clearly distinguished from a polymer having no terminal carboxyl group. Thus, the term “biodegradable polymer” having a terminal carboxyl group, as used herein, means a biodegradable polymer showing a significant correspondence between the number average molecular weight determined by the GPC method and the number average molecular weight determined by the end groups.

 While the number average molecular weight determined by the end groups is an absolute value, the number average molecular weight determined by the GPC method is a relative value that varies depending on analytical and technological conditions (such as the type of mobile phase and column, standard substance, selected width layer, selected zero line, etc.). Therefore, two values cannot be numerically correlated based on general laws. However, the term “substantial correspondence” between the number average molecular weight determined by the GPC and the number average molecular weight determined at the end groups means that the number average molecular weight found at the end groups is about 0.4-2 times, more preferably about 0, 8-1.5 times, most preferably 0.8-1.5 times more than the number average molecular weight determined by the method.

 The term "much higher" in the sense that it is used by your means that the number average molecular weight determined by the end groups is approximately two or more higher than the number average molecular weight determined by the GPC.

 A preferred polymer for the purposes of the present invention is a polymer showing a substantial correspondence between the number average molecular weight determined by the GPC and the number average molecular weight determined at the end groups.

 As specific examples of a biodegradable polymer having a terminal carboxyl group, mention may be made of polymers and copolymers, as well as mixtures thereof, which are synthesized from one or more types of α-hydroxy acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid, and t etc.), hydroxycarboxylic acids (e.g. citric acid), etc. by the reaction of non-catalytic dehydrative polycondensation, poly - α - cyanoacrylic esters, polyamino acids (e.g. poly - γ - benzyl-L-glutamic acid, etc.), maleic anhydride copolymers (e.g. styrene-maleic copolymer acids, etc.) and the like.

 According to the method, the polymerization can be statistical, block or grafted. In the case where any of the aforementioned α-acids, oxydicarboxylic acids and oxytricarboxylic acids have an optically active center in the molecule, any D-, L- and DL-forms can be used. The biodegradable polymer having a terminal carboxyl group is preferably a biodegradable polymer comprising a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the general formula II.

в которой R представляет алкилгруппу с 2-3 атомами углерода,
и (B) и полимолочной кислоты, или сополимера молочной кислоты - гликолевой кислоты.

in which R represents an alkyl group with 2-3 carbon atoms,
and (B) and polylactic acid, or a copolymer of lactic acid — glycolic acid.

 With respect to the general formula [II], linear or branched alkyl groups of 2 to 3 carbon atoms represented by P include, inter alia, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl , tert-pentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl.

 Among them, linear or branched alkyls with 2-5 carbon atoms are preferred. Especially ethyl, propyl, isopropyl, butyl and isobutyl are preferred.

 Oxycarboxylic acid of general formula II includes, but is not limited to, 2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid, 2-hydroxyisocaproic acid, and 2-hydroxycaproic acid. Preferred are 2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, and 2-hydroxycaproic acid.

 Most preferably, hydroxycarboxylic acid of general formula [II] is 2-hydroxybutyric acid. Although these hydroxycarboxylic acids may be in the form of D-, L- and D, L-compounds, the D- / L-ratio (mol%) is preferably in the range of about 75/25 to 25/75. A more preferred embodiment of the invention is oxycarboxylic acid with a D- / L ratio (mol%) in the range of about 60/40 to 40/60. Most preferred is oxycarboxylic acid with a D- / L ratio (mol%) in the range of about 55/45 to 45/55.

 With respect to the copolymer of glycolic acid and the named hydroxycarboxylic acid of the general formula [II] (hereinafter referred to as the glycolic acid copolymer), the copolymerization method may be random, block or grafted. Static copolymers are preferred.

 Hydroxycarboxylic acids of the general formula [II] may be used singly or in combination.

 Preferred ratios of glycolic acid to hydroxycarboxylic acid [II] in said glycolic acid copolymer (A) are from about 10 to 75 mol% and the remaining to 100% of hydroxy carboxylic acid. More preferably, the copolymer consisted of approximately 20-75 mol% of glycolic acid and the rest of hydroxycarboxylic acid. Most preferably, the copolymer consisted of approximately 40-70% glycolic acid and the rest of the hydroxycarboxylic acid. The weight average molecular weight of said glycolic acid copolymer can vary from about 2,000 to 50,000. A preferred range is from about 3,000 to 40,000. The most preferred range is from about 8,000 to 30,000. The degree of dispersion (weight average molecular weight / number average molecular weight) is preferably in the range of about from 1.2 to 4.0. Particularly preferred are copolymers with a degree of dispersion in the range of from about 1.5 to 3.5.

 The glycolic acid copolymer (A) can be synthesized by a known method, for example, by the method described in Japanese Patent Application Laid-Open No. 28521/1986.

 The polylactic acid for use in the present invention may be an L or D compound and any mixture thereof. Preferred are mixtures with a D- / L ratio (mol%) in the range of about 75/25 to 20/80. Most preferred is the D- / L ratio (mol%) of polylactic acid from about 60/40 to 25/75. The best D / L ratio (mol%) of polylactic acid is from about 55/45 to 25/75. The weight average molecular weight of polylactic acid is preferably in the range of from 1,500 to 30,000, most preferably from 2,000 to 20,000, and even more preferably from about 3,000 to 15,000. The degree of dispersion of polylactic acid is from about 1.5 to 3.5.

 Polylactic acid can be synthesized by two known alternative methods, namely, a method involving polymerization with the opening of a ring of lactide, which is a dimer of lactic acid, and a method comprising dehydrative polycondensation of lactic acid. To obtain a relatively low molecular weight polylactic acid for use in the present invention, a method comprising direct dehydrative polycondensation of lactic acid is preferred. This method is described, for example, in Japanese Patent Laid-Open No. 28521/1986.

 In the pharmacological basis used in the present invention, a copolymer of glycolic acid (A) and polylactic acid (B) is used in a (A) / (B) ratio (by weight) of from about 10/90 to 90/10. Preferably, the mixture ratio is about 20/80 to 80/20. The most desirable mix ratio is from about 30/70 to 70/30. If the proportion of either (A) or (B) is too large, the final drug will show a drug release pattern that is slightly different from that obtained separately when using either (A) or (B), which means that the linear release pattern on the final release stage, which is achievable with a mixed base, cannot be obtained. The degree and rate of release of the glycolic acid copolymer and polylactic acid varies significantly with their molecular weights and composition, but generally speaking, since the decomposition and release rates of the glycolic acid copolymer are relatively high, the release period can be increased by increasing the molecular weight of the polylactic acid or by reducing the mixture ratio (A ) / (B). In contrast, the duration of release can be reduced by reducing the molecular weight of polylactic acid or by increasing the (A) / (B) ratio of the mixture. In addition, the duration of release can be controlled by changing the type or relative amount of hydroxycarboxylic acid of the general formula II.

 When a copolymer of lactic acid and glycolic acid is used as a biodegradable polymer, its polymerization ratio (lactic acid / glycolic acid) (mol%) is preferably from about 100/0 to 40/60. Most preferably, a ratio of about 90/10 to 50/50.

 The weight average molecular weight of said copolymer is preferably from 5000 to 25000. The most desirable range is from about 7000 to 20,000.

 The degree of dispersion (weight average molecular weight) / number average molecular weight) of said copolymer is preferably from about 1.2 to 4.0. A more desirable range is from about 1.5 to 3.5.

 The aforementioned copolymer of lactic acid and glycolic acid can be synthesized by a known method, for example, by the method described in Japanese Patent Application Laid-Open No. 28521/86. The decomposition and rate of disappearance of the copolymer of lactic acid and glycolic acid varies significantly with composition and molecular weight, but generally speaking, the smaller the proportion of glycolic acid, the lower the decomposition and rate of disappearance. Therefore, the duration of release of the drug can be increased by reducing the proportion of glycolic acid or by increasing the molecular weight.

 On the contrary, the duration of release can be reduced by increasing the proportion of glycolic acid or reducing the molecular weight. To obtain a drug with a long (e.g. 1-4 month) delayed release period, it is preferable to use a copolymer of lactic acid and glycolic acid with a polymerization ratio in the aforementioned range and a weight average molecular weight in the aforementioned range. With a copolymer of lactic acid and glycolic acid having a higher decomposition rate than is observed for the above ranges of polymerization ratio and weight average molecular weight, it is difficult to control the initial momentum. On the contrary, for a copolymer of lactic acid and glycolic acid, showing a lower decomposition rate than the rate for the indicated ranges of the polymerization ratio and weight average molecular weight, there is a tendency to form periods during which the drug is not released in an effective amount.

 In the present description, the weight average molecular weight and degree of dispersion mean the molecular weight for polystyrene, determined by gel permeation chromatography (GPC), using 9 polystyrenes with weight average molecular weights of 120,000, 52,000, 22,000, 9,200, 5050, 2950, 1050, 580 and 162 as standards and degree of dispersion calculated using the obtained molecular weight, respectively. The above determination was carried out using a GPC Column KF804 L • 2 (Showa Denko), PI Monitor L-3300 (Hitachi) and chloroform as the mobile phase.

 The sustained release preparation of the present invention is prepared by dissolving a peptide [I] and a biodegradable polymer having a terminal carboxyl group in a solvent that is substantially immiscible with water, and then removing the title solvent.

The solvent, which is mainly immiscible with water, is a solvent that is not only essentially immiscible with water and capable of dissolving the biodegradable polymer, but a solvent that makes the resulting polymer solution capable of dissolving the peptide [I]. Preferably, it is a solvent with a solubility in water of not more than 3% (w / w) at atmospheric temperature (20 ^o C). The boiling point of such a solvent is preferably not higher than 120 ^° C. Thus, the solvent includes halogenated hydrocarbons (e.g. dichloromethane, chloroform, chloroethane, trichloroethane, carbon tetrachloride and so on), alkyl ethers with 3 or more carbon atoms (e.g. isopropyl ether etc.), alkyl fatty acid (of 4 carbon atoms or more), esters (e.g., butyl acetate, etc.), aromatic hydrocarbons (e.g., benzene, toluene, xylene, etc.) and the like like that. These solvents may be used in a suitable combination of 2 or more varieties. More desirable solvents are halogenated hydrocarbons (e.g. dichloromethane, chloroform, chloroethane, trichloroethane, carbon tetrachloride, etc.). Dichloromethane is most desirable.

 Solvent removal can be performed by known techniques. For example, by a method involving evaporation of the solvent at atmospheric pressure or by gradually reducing the pressure with constant stirring using a propeller stirrer or a magnetic stirrer, or by a method including evaporation of the solvent by controlled pressure reduction in a rotary evaporator.

According to the method of the invention, for the preparation of a sustained release preparation, dissolution of the peptide [I] and the biodegradable polymer with a terminal carboxyl group means the achievement of conditions under which there is no visually observed precipitate of insoluble peptide in the resulting solution at ordinary temperature (20 ^° C). In this ternary system consisting of peptide [I], a biodegradable polymer and a solvent, the amount of peptide that can be dissolved depends on the number of terminal carboxyl groups per unit weight of the biodegradable polymer. In the case where the peptide and the terminal carboxyl group interact in a 1: 1 ratio, the same molar amount of the peptide can theoretically be dissolved as the amount of the terminal carboxyl group. Therefore, generalization is difficult due to the fact that it is necessary to combine the solvent and the molecular weight of the peptide and the biodegradable polymer. However, upon receipt of a sustained release preparation, the peptide can be dissolved in the range of about 0.1 to 100% (weight / weight), preferably about 1 to 70% (weight / weight), most preferably about 2 to 50% (weight / weight), relative to a biodegradable polymer that dissolves in a solvent.

в которой R обозначает алкилгруппу из 2-8 углеродных атомов,
и (B) полимолочной кислоты и в значительной степени водонерастворимого физиологически активного пептида или его соли в растворителе, который в основном не смешивается с водой, и затем удаление названного растворителя.The present invention further relates to a method for producing a sustained release preparation, which comprises dissolving a biodegradable polymer comprising a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the general formula

in which R denotes an alkyl group of 2-8 carbon atoms,
and (B) polylactic acid and a substantially water-insoluble physiologically active peptide or salt thereof in a solvent that is substantially immiscible with water, and then removing said solvent.

 A substantially water-insoluble physiologically active peptide is not strictly defined and includes naturally-occurring, synthetic, and semi-synthetic peptides. Physiologically active peptides containing one or more aromatic groups (for example, groups derived from benzene, naphthalene, phenanthrene, anthracene, pyridine, pyrrole, indole, etc.) in the side chains are preferred. More preferred physiologically active peptides are peptides having 2 or more aromatic groups in the side chains. Physiologically active peptides having 3 or more aromatic groups in the side chains are particularly preferred. These aromatic groups may be further substituted.

 A substantially water-insoluble physiologically active peptide for use in the present invention is preferably a peptide having a solubility of not more than 1% in water, consisting of two or more amino acids and having a molecular weight of from about 200 to 30,000. The region of a more preferred molecular weight is from about 300 to 20,000 and even more preferably from about 500 to 10,000.

 As examples of the physiologically active peptides mentioned, antagonists of the hormone-releasing hormone luteinizing hormone (LH-RH) can be mentioned (cf. U.S. Patent N 4,086,219, N 4,124,577, N 4,253,997 and N 4,317,815, etc.), insulin, somatostatin, somatostatin derivatives (compare US patent N 4.087.390, N 4.093.574, N 4.100.117, N 4.253.998, etc.), growth hormone prolactin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone ( MSH), salts and derivatives of thyroid hormone releasing hormone (cf. JP Kokai S-50-121273 and S-52-116465), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), vasopressin, derivatives of vasopressin, oxytocin, calcitonin, nastrin, secretin, pancreosimin, cholecystokinin, angiotensin, human placental lactogen, human choreonic gonadotropin (HCG), enkephalin, patent 4.2, enkefalin, enkephalin, enke. 394, EP-A N 31.567), endorphin, kiotfin, tuftein, thymopoietin, thymosin, thymostimulin, thymic humoral factor, THF (THF), serum thymic factor, STF (FTS), and its derivatives (cf. U.S. Patent No. 4,229,438), other thymic factors, tumor necrosis factor (TNF), colony stimulating factor (CSF), methyline, dynorphin, bombesin, neurotensin, cerulein, bradykinin, atrial (natruetic) factor, nervous growth factor, cell growth factor , neurotrophic factor, peptides having endothelin antagonistic activity (cf. EP-A N 436189, N 457195 and N 496452, JP Kokai H-3-94692 and 03-130299) and fragments of derivatives of these physiologically active peptides.

 Specific examples of a physiologically active peptide are physiologically active peptides and salts, which are antagonists of the hormone-releasing luteinizing hormone (LH-RH) and are useful for treating hormone dependence diseases such as prostate cancer, prostate hypertrophy, endometriosis, uterine fibroids, premature puberty, breast cancer, etc., and as contraceptives.

 The physiologically active peptide for use in the present invention may be in the form of a salt, preferably a pharmacologically acceptable salt. When the named peptide has a basic group, such as amino, the aforementioned salt can be, for example, a salt with an inorganic acid (e.g., hydrochloric acid, sulfuric acid, nitric acid, etc.) or an organic acid (e.g., carbonic acid, hydrogen carbonic acid, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.). When the peptide has an acidic group such as carboxyl, the salt can, for example, be a salt with an inorganic base (e.g. alkali metals such as calcium, magnesium, etc.) or an organic base (e.g. organic amines such as triethylamine and etc., and basic amino acids such as arginine, etc.). In addition, the peptide may be in the form of a metal complex compound (e.g., copper complex, zinc complex, etc.).

 Specific examples of a physiologically active peptide or its salt can be found in US Pat. No. 5,110,904, Journal of Medicinal Chemistry 34, 2395-2402, 1991, Recent results in Cancer Research 124, 113-136, 1992, and other literature).

 In addition, physiologically active peptides of the general formula [I] and their salts may also be mentioned among others.

 In addition, even when the physiologically active peptide is water-soluble, it can be converted to a derivative that will be soluble, or converted to an insoluble salt with a water-insoluble acid (e.g. pamoic acid, tannic acid, stearic acid, palmitic acid, etc. ) and used in the method of the invention.

 The amount of physiologically active peptide in the preparations of the present invention depends on the type of peptide, the expected pharmacological effect and the desired duration of action, etc. However, it is usually used in a ratio of about 0.001 to 50% (weight / weight), preferably about 0.01 to 40% (weight / weight), most preferably about 0.1 to 30% (weight / weight) , relatively biodegradable polymer base.

 The solvent used in the method is as described above.

 Solvent removal can be performed in the same manner as described above.

 A preferred method for producing the sustained release preparation of the present invention is a microencapsulation method using water-drying technology or phase separation technology, which are described below, or any similar method.

 The method described below can be performed with the peptide [I] or a substantially water-insoluble physiologically active peptide, which includes the peptide [I].

 Thus, the peptide [I] adds to the solution of the bioorganic polymer in the organic solvent in the final weight ratio mentioned earlier for such a peptide to prepare an organic solvent solution containing the peptide [I] and a biodegradable polymer. In this regard, the concentration of the biodegradable polymer in the organic solvent varies in accordance with the molecular weight of the biodegradable polymer and the type of organic solvent, but is usually selected from a range of from about 0.01 to 80% (weight / weight). A preferred region is from about 0.1 to 70% (w / w). An even more desirable range is from about 1 to 60% (w / w).

 Then this organic solvent solution containing the peptide [I] and biodegradable polymer (oil phase) is added to the aqueous phase to obtain an O (oil phase) / W (aqueous phase) emulsion. The solvent of the oil phase is then evaporated to obtain microcapsules. The volume of the aqueous phase for this method is usually selected from the range of from about 1 to 10,000 times the volume of the oil phase. A preferred range is from about 2 to 5,000 times, and even more preferably from about 5 to 2,000 times.

 An emulsifier may be added to the above aqueous phase. The emulsifier can usually be any substance that leads to the formation of a stable O / W emulsion. T.O. here may be mentioned anionic surfactants (sodium oleate, sodium stearate, sodium lauryl sulfate, etc.), nonionic surfactants (polyoxyethylene sorbitan fatty acid ester / Tween 80 and Tween 60, Atlas Powder polyoxyethylene- castor oil derivatives / HCO-60 and HCO-50, Nikko Chemicals, etc.), polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, lecithin, gelatin, hyaluronic acid, etc. These emulsifiers can be used independently or in combination. The concentration can be selected from the range of from about 0.001 to 20% (weight / weight). A preferred range is from about 0.01 to 10% (w / w) and an even more desirable range is from about 0.05 to 5% (w / w).

The resulting microcapsules are isolated by centrifugation or filtration and washed with several portions of distilled water to remove the free peptide, emulsifier filler from the surface, then redispersed in distilled water or the like, and lyophilized. Then, if necessary, the microcapsules are heated under reduced pressure to further remove the remaining water and organic solvent from the inside of the microcapsules. Preferably, this method is carried out by heating the microcapsules at a temperature of several (5 ^° C. or more) above the average glass transition temperature of the biodegradable polymer, determined using a differential scanning calorimeter at temperature increments of 10 to 20 ^° C./min, usually not more than 1 week or 2- 3 days, preferably no more than 24 hours, then the capsules reach the target temperature.

 When microcapsules are prepared by the phase separation method, a coacerving agent is gradually added to a solution of the physiologically active peptide and biodegradable polymer in an organic solvent with constant stirring, i.e., so that the biodegradable polymer can be separated and converted into a solid product. This coacervative agent is added in a volume of from about 0.01 to 1000 times the volume of the solution in the organic solvent of the peptide [I] and the biodegradable polymer. A preferred range of from about 0.05 to 500 times, and even more preferably from about 0.1 to 200 times.

 The coacervative agent must be a polymer compound such as mineral oil or vegetable oil, which is mixed with a solvent for a biodegradable polymer, but at the same time does not dissolve the polymer. In particular, silicone oil, sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexene, n-heptane, etc. may be mentioned. These substances can be used in combination.

 The resulting microcapsules are isolated by filtration and washed repeatedly with heptane or the like in order to remove the coacervating agent. Then the free peptide and solvent are removed by drying in water, as described previously.

 In the method of drying in water or in the coacervative method, an aggregation inhibitor can be added to prevent particle aggregation. Aggregation inhibitors include water-soluble polysaccharides, such as mannitol, lactol, glucose, starch (e.g. corn starch), etc., glycine, proteins, such as fibrin, collagen, etc., and inorganic salts, such as sodium chloride , sodium hydrogen phosphate and the like.

 In the production of microcapsules by spray drying, the aforementioned organic solvent solution of the peptide (I) and the biodegradable polymer is sprayed in the form of a mist through a nozzle and a drying chamber of the spray dryer to evaporate the organic solvent from the smallest separating liquid drop in a short time to obtain small capsules. The nozzle can be two-fluid, pneumatic, rotating disk, etc. It is advisable in the method to spray an aqueous solution of the aforementioned aggregation inhibitor from another nozzle in order to prevent intercapsular aggregation in time with the aforementioned atomization of the organic solvent solution of the peptide (I) and the biodegradable polymer.

 If necessary, the residual water and the organic solvent are removed by heating the resulting microcapsules under reduced pressure in the same manner as previously described here.

 Microcapsules can be administered as they are, or processed into a variety of pharmaceutical preparations for administration by routes other than the oral route (for example, intramuscularly, subcutaneously and by injection into the body or as implants, such as nasal, rectal or uterine transmucosal release systems, etc.) or oral administration (e.g., solid preparations, such as capsules, e.g., hard capsules, soft capsules, etc.) granules, powders, etc. and liquid preparations such as syrups, emulsions, suspensions, etc.).

 To obtain microcapsules for injection, for example, microcapsules can be formulated with a dispersant (for example, a surfactant such as Tween 80, HCO-60, etc., carboxymethyl cellulose, polysaccharide such as sodium alginate, etc.), a preservative ( for example, methylparaben, propylparaben, etc.), or substances for producing an isotonic solution (for example, sodium chloride, mannitol, sorbitol, glucose, etc.) to prepare an aqueous suspension, or they can be dispersed in vegetable oil, such as sesame oil, corn oil or similarly, to obtain an oily suspension for use as a controlled release injection.

 The particle size of the microcapsules for such injection suspensions should be in the range necessary so that they can form a dispersion, and determined by the necessary throughput equipment, and may be, for example, in the range of about 0.1 to 500.0 μm. A preferred particle size range is from about 1 to 300 microns, and even more preferably from about 2 to 200 microns.

 For the production of microcapsules in the form of a sterile product, the entire production method is subjected to sterility control, microcapsules are sterilized by gamma irradiation or a preservative is added, although these techniques are not exclusive.

 In addition to the aforementioned microcapsules, in order to obtain a sustained release preparation of the present invention, a biodegradable polymer composition containing a peptide as an active ingredient, well dispersed in a suitable manner, can be fused or pressed into a spherical, strip-like, needle-shaped, granular or film form.

 The above biodegradable polymer composition can be obtained by the method described in JP Publication S-50-17525. More specifically, the peptide drug and polymer are dissolved in a solvent, and the solvent is removed by a suitable method (e.g., spray drying, flash evaporation, etc.) to obtain a desired biodegradable polymer composition.

 The sustained release preparation of the present invention can be administered as an intramuscular, subcutaneous or organ injection, or as an implant, as a transmucosal dispensing system for the nasal cavity, rectum or uterus, or an oral preparation, for example, a solid preparation, such as a capsule (e.g. , hard or soft), granule, powder, etc., or a liquid preparation, such as syrup, emulsion, suspension, etc.

 The sustained release preparation of the present invention has low toxicity and can be used successfully for mammals (e.g., human, cow, pig, dog, cat, mouse, rat and rabbit).

 The dosage of the sustained release preparation depends on the type and percentage of the active drug peptide, the final dosage form, the duration of the release of the peptide, the purpose of the treatment (such as hormone-dependent diseases, for example, prostate cancer, prostate hypertrophy, endometriosis, uterine fibroma, premature puberty , breast cancer, etc., or for contraception) and specific animal species, but in any case it is necessary that an effective amount of the peptide deliver moose continuously. The standard dose of the active drug peptide taken as a drug delivery system for one month can be selected predominantly from the range of about 0.01 to 100 mg / kg body weight for an adult. Preferably, the range is from about 0.05 to 50 mg / kg body weight. The most desirable range is from 0.1 to 10 mg / kg body weight.

 In addition, a standard dose of a sustained release preparation in an adult can be selected from a range of from about 0.1 to 500 mg / kg body weight. A preferred range is from about 0.2 to 300 mg / kg body weight. The frequency of administration may, for example, vary depending on once every several weeks, months or once every several months, and may be selected in accordance with the type and percentage of active drug peptide, final dosage form, estimated duration of release of the peptide, disease, run, and subject to treatment of the animal.

 The following reference and working examples are provided for the purpose of a more detailed description of the invention and should not be construed as limiting the scope of the invention and the attached paragraphs. (Unless otherwise specified,% means weight%).

The following notation has the following meanings:
BOC - tert-butoxycarbonyl
FMOC - 9-fluorenylmethoxycarbonyl
Cbz - benzyloxycarbonyl
Reference example 1
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 300 g of a 90% aqueous solution of D, L-lactic acid and 100 g of a 90% aqueous solution of L-lactic acid were added and the contents were heated under reduced pressure nitrogen gas from 100 ^o C / 500 mm Hg to 150 ^o C / 30 mm Hg for 4 hours with constant removal of distillate water. Next, the reaction mixture was heated under reduced pressure at 3-5 mm Hg / 150-180 ^o C for 7 hours, after which it was cooled to obtain an amber-colored polylactic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 30 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC and the number average molecular weight found by determining the end groups of the above lactic acid were 3000; 1790 and 1297, respectively.

 These data showed that the polymer had terminal carboxyl groups.

Reference example 2
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 500 g of a 90% aqueous solution of D, L-lactic acid were added and the contents were heated under reduced pressure in a stream of nitrogen gas from 100 ^o C / 500 mm Hg to 150 ^o C / 30 mm Hg for 4 hours with continuous removal of distillate water. Next, the reaction mixture was heated under reduced pressure at 3-5 mm Hg / 150-180 ^o C for 12 hours, after which it was cooled to obtain an amber-colored polylactic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 30 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC, the number average molecular weight found by determining the end groups of the above lactic acid were 5,000; 2561 and 1830, respectively.

 These data showed that the polymer had terminal carboxyl groups.

Reference example 3
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 300 g of a 90% aqueous solution of D, L-lactic acid and 100 g of a 90% aqueous solution of L-lactic acid were added and the contents were heated under reduced pressure nitrogen gas from 100 ^o C / 500 mm Hg to 150 ^o C / 30 mm Hg for 5 hours with the constant removal of distilled water. Next, the reaction mixture was heated under reduced pressure at 5-7 mm Hg / 150-180 ^o C for 18 hours, after which it was cooled to obtain an amber-colored polylactic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 30 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC; the number average molecular weight found by determining the end groups of the above lactic acid was 7500; 3563 and 2301, respectively.

 These data showed that the polymer had terminal carboxyl groups.

Reference example 4
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 300 g of a 90% aqueous solution of D, L-lactic acid and 100 g of a 90% aqueous solution of L-lactic acid were added and the contents were heated under reduced pressure nitrogen gas from 100 ^o C / 500 mm Hg to 150 ^o C / 30 mm Hg for 5 hours with constant removal of distillate water. Next, the reaction mixture was heated under reduced pressure at 5-7 mm Hg / 150-180 ^o C for 26 hours, after which it was cooled to obtain an amber-colored polylactic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 30 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC and the number average molecular weight found by determining the end groups of the above lactic acid were 9000; 3803 and 2800 respectively.

 These data showed that the polymer had terminal carboxyl groups.

Reference example 5
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 182.5 g of glycolic acid and 166.6 D, L-2-hydroxybutyric acid were added and the contents were heated under reduced pressure in a stream of nitrogen gas from 100 ^o C / 500 mm Hg up to 150 ^o C / 30 mm Hg for 3.5 hours with constant removal of distillate water. Next, the reaction mixture was heated under reduced pressure at 5-7 Hg 150-180 ^o C for 26 hours, after which it was cooled, obtaining a copolymer of glycolic acid-2-hydroxybutyric acid of amber color.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 25 ^o C.

 The weight average molecular weight determined by the GPC of the resulting glycolic acid-2-hydroxybutyric acid copolymer was 13,000.

Reference Example 6
197.7 glycolic acid and 145.8 g of D, L-2-hydroxybutyric acid were added to a four-necked flask equipped with a nitrogen inlet tube and a refrigerator in a 1000 ml flask, and the contents were heated under reduced pressure in a stream of nitrogen gas from 100 ^° C / 500 mm Hg up to 155 ^o C / 30 mm Hg for 4 hours with the constant removal of distilled water. Next, the reaction mixture was heated under reduced pressure at 3-6 mm Hg / 150-185 ^o C for 27 hours, after which it was cooled, obtaining a copolymer of glycolic acid - 2-hydroxybutyric acid of amber color.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 25 ^o C.

 The weight average molecular weight determined by the GPC of the resulting glycolic acid-2-hydroxybutyric acid copolymer was 13,000.

Reference example 7
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 212.9 g of glycolic acid and 124.9 g of D, L-2-hydroxybutyric acid were added and the contents were heated under reduced pressure in a stream of nitrogen gas from 100 ^o C / 500 mm Hg up to 160 ^o C / 30 mm Hg for 3.5 hours with continuous removal of distillate water. Next, the reaction mixture was heated under reduced pressure at 3-6 mm Hg / 160-180 ^o C for 27 hours, after which it was cooled, obtaining a copolymer of glycolic acid-2-hydroxybutyric acid of amber color.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 25 ^o C.

 The weight average molecular weight determined by the GPC of the resulting glycolic acid copolymer, 2-hydroxybutyric acid, was 11,000.

Reference example 8
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 300 g of a 90% aqueous solution of D, L-lactic acid and 100 g of a 90% aqueous solution of L-lactic acid were added and the contents were heated under reduced pressure in a stream of nitrogen gas from 100 ^o C / 500 mm Hg up to 150 ^o C / 30 mm Hg for 4 hours with constant removal of distilled water. Next, the reaction mixture was heated under reduced pressure at 3-5 mm Hg and 150-180 ^o C for 10 hours, after which it was cooled to obtain an amber-colored polylactic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 30 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC and the number average molecular weight found by determining the end groups of the above polylactic acid were 4200; 2192 and 1572 respectively.

 These data showed that the polymer had terminal carboxyl groups.

Reference example 9
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 182.5 g of glycolic acid and 166.6 D, L-2-hydroxybutyric acid were added and the contents were heated under reduced pressure in a stream of nitrogen gas from 100 ^o C / 500 mm Hg up to 150 ^o C / 30 mm Hg for 3.5 hours with a constant removal of distilled water. Next, the reaction mixture was heated under reduced pressure at 5-7 mm Hg and 150-180 ^o C for 32 hours, after which it was cooled, obtaining a copolymer of glycolic acid-2-hydroxybutyric acid of amber color.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 25 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC and the number average molecular weight found by determining the end groups of the resulting glycolic acid-2-hydroxybutyric acid copolymer were 16300; 5620 and 2904, respectively.

 These data showed that the polymer had terminal carboxyl groups.

Reference Example 10
Synthesis of NacD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -Leu-Lys (Nisp) -Pro-DAlaNH ₂
Reference examples 10 and 11 are made in accordance with the description of US patent N 5.110.904 and US patent application N 07 / 987.921.

 In order to synthesize the title peptide, 1 g of D-Ala-NH-resin (4-methyl-benzohydrylamine resin) was added to the reactor of the peptide synthesis apparatus, and then amino acids were sequentially added according to the following synthesis procedure.

 1. The reaction of the removal of protective groups.

 To remove the protective BOC group from the alpha amino acid of the peptide, a solution containing 45% trifluoroacetic acid (hereinafter referred to as TFA later), 2.5% anisole, 2.0% dimethyl phosphite and 50.5% dichloromethane was used. After pre-washing the resin with the solution for 1 minute, the deprotection reaction was carried out for 20 minutes.

 2. Rinsing with a basic solution.

 In order to remove and neutralize trifluoroacetic acid, a dichloromethane solution containing 10% N, N'-diisopropylethylamine was used. In each deprotection reaction, the resin was washed three times in 1 minute.

3. The reaction of accession
The addition reaction was carried out using, as activators, a 3-fold molar amount of a 0.3 M diisopropylcarbodiimide / dichloromethane solution and a 3-fold molar amount of a 0.3 M solution of BOC derivative of amino acid / DMF (N, N-dimethylformamide). The activated amino acid was attached to the free alpha-amino group of the peptide on the resin. The interaction time is given below.

4. Flushing
After completion of each reaction process, the resin was washed with dichloromethane, dichloromethane / DMF and DMF each for 1 minute.

Synthesis protocol
Amino acids with protected amino groups are attached to the resin in the order, frequency and for the time specified in table A.

 After completion of the synthetic reaction, the resin was treated with a 30% solution of piperidine in DMF for 4 to 24 hours in order to remove the protective FMOC group. The resin was washed several times with dichloromethane and then reacted with carbonyldiimidazole (0.9 g) dissolved in DMF (18 ml) for 15 minutes and washed three times with dichloromethane, after which it was allowed to react overnight with 2-furancarboxylic acid hydrazide (0.53 g ) dissolved in DMF (18 ml).

The resulting peptide resin was washed three times with dichloromethane and then dried in the presence of phosphorus pentoxide overnight, after which the resin was treated with dry hydrogen fluoride at 0 ^° C. for 1 hour in the presence of anisole to separate the peptide from the resin. Excess interacting reagent was removed under reduced pressure. The resin thus obtained was washed first with diethyl ether, then stirred at room temperature with diethyl ether, then stirred at room temperature for 15 minutes in 50 ml of a mixture of water / acetonitrile / acetic acid (1: 1: 0.1) and filtered. The filtrate was lyophilized to obtain the purified peptide as a loose powder. This peptide was purified by high performance liquid chromatography (HP C) using the following conditions:
1) Column: dynamo C-18 (25 • 2.5 cm, 8 μm)
2) Solvent: the acetonitrile gradient of ascension is 20 minutes from a mixture of 89% water / 11% acetonitrile / 0.1% TFA
3) Determination at a wavelength: 260 nm (UV method)
The peptide determined by an individual peak at 25.7 minutes of retention time was collected and lyophilized to obtain the purified product NacD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -Leu-Lys (Nisp) -Pro -DAlaNH ₂ in the form of trifluoroacetate . The physical property data for the purified product are as follows:
FAB (fast atomic bombardment, same applied below) mass spectrometry: m / e 1591 (M + H) ⁺
Amino Acid Analysis: 0.98 Ala, 1.02 Pro, 1.58 Lys, 1.00 Leu, 1.12 NMeTyr, 0.52 Ser.

The above peptide trifluoroacetate was converted to acetate using a gel filtration column previously equilibrated with 1N acetic acid. Gel filtration conditions are as follows:
1) Supernatant: Sephadex G-25, column inner diameter 16 mm, nozzle layer height 40 mm)
2) Solvent: 1 n. acetic acid
3) Determination at a wavelength: 254 nm (UV method). The fraction of the first washable peak was collected and lyophilized to give the purified NacD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ product as an acetate.

Reference example 11
Synthesis of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyNic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂
The peptide named in the title was synthesized according to the same procedure as in Reference Example 10, with the exception that 2-furancarboxylic acid hydrazide was replaced with hydrazide-2-nicotinic acid (0.575 g). The resulting MPLC retention time of the purified product was 16.0 minutes. The physical properties of the purified product are as follows:
FAB mass spectrometry: m / e 1592 (M + H); amino acid analysis 1.02 Ala, 1.01 Pro, 1.61 Lys, 0.99 Leu, 1.12 NMeTyr, 0.48 Ser.

 The above peptide trifluoroacetate was converted to acetate by the method of Example 10 above.

Reference example 12
322 g of a 90% aqueous solution of D, L-lactic acid and 133 g of glycolic acid were added to a four-necked flask equipped with a nitrogen inlet tube and a refrigerator in a 1000 ml flask, and using a ring mantle heater (So-go Rikagaku Glass Co.), the contents were heated under reduced pressure. pressure in a stream of nitrogen from 100 ^o C / 500 mm Hg to 150 ^o C / 30 mm Hg for 4 hours with a constant removal of distillate water. Next, the reaction mixture was heated under reduced pressure at 3-30 mm Hg / 150-185 ^o C for 23 hours, after which it was cooled, obtaining a copolymer of lactic acid-glycolic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 30 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC and the number average molecular weight found by determining the end groups of the resulting lactic acid / glycolic acid copolymer were 10,000; 4000 and 4000 respectively.

 These data showed that the copolymer had terminal carboxyl groups.

Reference Example 13
In a 1000 ml four-necked flask equipped with a nitrogen inlet tube and a refrigerator, 347 g of a 90% aqueous solution of D, L-lactic acid and 266 g of glycolic acid were added using a mantle heater (So-go Rikagaku Galss Co.), the contents were heated at reduced pressure in a stream of nitrogen from 100 ^o C / 500 mm Hg to 150 ^o C / 30 Hg mm for 5 hours with constant removal of distillate water. Next, the reaction mixture was heated under reduced pressure at 3-30 mm Hg / 150-185 ^o C for 23 hours, after which it was cooled, obtaining a copolymer of lactic acid - glycolic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into warm water at 60 ^° C. with constant stirring. The resulting viscous polymer precipitate was collected and dried under reduced pressure at 30 ^o C.

 The weight average molecular weight and number average molecular weight determined by the GPC and number average molecular weight found by determining the end groups of the resulting lactic acid-glycolic acid copolymer were 10,000, 3,700 and 3,900, respectively. These data showed that the copolymer had end groups.

Example 14
NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ (manufactured by TAP, hereinafter referred to as physiologically active peptide A for short) acetate, 200 mg dissolved in a solution of the mixture 50 : 50 (3.8 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 5 and polylactic acid obtained in Reference Example 1 in 5.3 g (4.0 ml) of dichloromethane. The resulting solution was cooled to 17 ^° C. and poured into 1000 ml of a 0.1% (w / w) aqueous solution of polyvinyl alcohol (EG-40, Nippon Synthetic Chemical Industry Co., Ltd), previously adjusted to 10 ^° C., and the mixture was emulsified using turbine homo mixer at 7000 rpm for the preparation of an O / W emulsion. This O / W emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane. The oil phase was allowed to solidify and collected by centrifugation (05PR-22, Hitachi, Ltd) at 2000 rpm. This solid product was redispersed in distilled water and further centrifuged to rinse the free drug, etc. The collected microcapsules were redispersed in a small amount of distilled water, followed by the addition of 0.3 g of D-mannitol and dried at a temperature below freezing to obtain a powder. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 60 μm and 4.7% (weight / weight), respectively.

 As described above, preparations of the following physiologically active peptides (1) and (2) were prepared.

(1) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyNic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂
(2) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -Leu-Lys (Nisp) -Pro-DAlaNH ₂
Example 15 In a 50:50 solution of a mixture (3.8 g) of the glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 5 and polylactic acid obtained in Reference Example 2 in 6.7 g (5.0 ml) of dichloromethane 200 mg of physiologically active peptide A acetate were dissolved. This solution was cooled to 17 ^° C. and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 17 ^° C., and the mixture was treated as in Example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 65 μm in 5.0% (weight / weight), respectively.

 Example 16

In a 50:50 solution of a mixture (3.8 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 5 and polylactic acid obtained in Reference Example 3, 6 mg was dissolved in 6.7 g (5.0 ml) of dichloromethane physiologically active peptide A. acetate. This solution was cooled to 17 ^° C. and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17 ^° C., and the mixture was treated as in Example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules were from 10 to 60 μm and 4.8% (weight / weight), respectively.

 Example 17

In a 50:50 solution of a mixture (3.8 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 5 and polylactic acid obtained in Reference Example 4 in 6.7 g (5.0 ml) of dichloromethane was dissolved 200 g of physiologically active peptide A acetate. This solution was cooled to 17 ^° C and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17 ^° C and the mixture was treated as in Example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules were from 10 to 75 μm and 4.6% (weight / weight), respectively.

 Example 18

In a 50:50 solution of a mixture (3.8 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 6 and polylactic acid obtained in Reference Example 2 in 6.0 g (4.5 ml) of dichloromethane was dissolved 200 g of physiologically active peptide A acetate. This solution was cooled to 17 ^° C. and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 10 ^° C., and the mixture was treated as in Example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 60 μm and 4.9% (weight / weight), respectively.

 Example 19

In a 50:50 solution of a mixture (3.8 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 7 and polylactic acid obtained in Reference Example 2 in 6.0 g (4.5 ml) of dichloromethane was dissolved 200 g of physiologically active peptide A acetate. This solution was cooled to 17 ^° C and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17 ^° C and the mixture was treated as in Example 1 to obtain microspheres. The particle size distribution and physiologically active peptide A content in the microcapsules ranged from 10 to 65 μm and 4.9% (weight / weight), respectively.

 Example 20

In a 50:50 solution of a mixture (3.6 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 9 and polylactic acid obtained in Reference Example 8 in 7.0 g (5.3 ml) of dichloromethane was dissolved 400 g of physiologically active peptide A acetate. This solution was cooled to 17 ^° C. and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 17 ^° C., and the mixture was treated as in Example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 65 μm and 7.2% (weight / weight), respectively.

Example 21
240 mg of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys acetate (AzaglyNic) - Leu-Lys (Nisp) -Pro-DAlaNH ₂ (hereinafter referred to as physiologically active peptide B for short) obtained in Reference Example 11 was dissolved in solution 50 : 50 mixture (1.76 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 9 and polylactic acid obtained in Reference Example 8 in 3.2 g (2.4 ml) of dichloromethane. The resulting solution was cooled to 18 ^° C. and poured into 400 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 16 ^° C., and the mixture was treated as in Example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide B content for microcapsules ranged from 5 to 70 μm and 10.3% (weight / weight), respectively.

Example 21
240 mg of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys acetate (AzaglyFur) - Leu-Lys (Nisp) -Pro-DAlaNH ₂ (hereinafter referred to as physiologically active peptide C for short) obtained in Reference Example 10 was dissolved in solution 50 : 50 mixture (1.76 g) of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference Example 9 and polylactic acid obtained in Reference Example 8 in 3.2 g (2.4 ml) of dichloromethane. The resulting solution was cooled to 18 ^o C and poured into 400 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 16 ^o C and the mixture was treated as in example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide C content for microcapsules ranged from 5 to 65 μm and 10.9% (weight / weight), respectively.

Example 22
N-Tetrahydrofur-2-oyl-Gly-D2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ (manufactured by the TAP industry; hereinafter referred to for brevity as a physiologically active peptide D) acetate [FAB mass spectrometry: m / e 1647 (M ⁺ H) ⁺ ], 240 mg, dissolved in a 50:50 solution of a mixture (1.76 g) of the glycolic acid-2-hydroxybutyric acid copolymer obtained in the reference example 9, and polylactic acid obtained in Reference Example 8 in 3.2 g (2.4 ml) of dichloromethane. The resulting solution was cooled to 18 ^° C. and poured into 400 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 16 ^° C., and the mixture was treated as in Example 1 to obtain microcapsules. The particle size distribution and physiologically active peptide D content for microcapsules ranged from 5 to 70 μm and 10.5% (weight / weight), respectively.

Example 23
200 mg of physiologically active peptide A acetate was added and dissolved in a solution of a lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25 (mol.%), GPC weight average molecular weight = 5000, GPC number average molecular weight. = 2000 , number average molecular weight determined by terminal groups = 2200; manufacturer Wako Pure Chemical (Lot. 920729)) in 5.3 g (4.0 ml) of dichloromethane. The resulting solution was cooled to 17 ^o C and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol (EG-40, Nippon Synthetic Chemical Industry Co., Ltd), previously adjusted to 16 ^o C, and the mixture was emulsified using a turbine a mixer at 7000 rpm to obtain an O / W emulsion. This O / W emulsion was stirred at room temperature for 3 hours to evaporate dichloromethane. The oil phase was allowed to solidify and collected using a centrifuge (05PR-22, Hitachi) at 2000 rpm. This solid product was redispersed in distilled water and further centrifuged to wash out the free drug, etc. The collected microcapsules were redispersed in a small amount of distilled water, followed by the addition of 0.3 g of D-mannitol and dried at a temperature below freezing to obtain a powder. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 60 μm and 4.7% (weight / weight), respectively.

The above method is used to produce a sustained release preparation of the following peptides (1) and (2):
1) acetate physiologically active peptide B;
2) acetate physiologically active peptide C.

Example 24
200 mg of physiologically active peptide A acetate was added and dissolved in a solution of 3.8 g of a lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25 (mol.%), GPC weight average molecular weight = 10000, GPC number average molecule. weight = 4400, number average molecular weight determined by end groups, 4300; manufactured by Wako Pure Chemical (Lot. 880530)) in 6.7 g (5.0 ml) of dichloromethane. The resulting solution was cooled to 17 ^° C. and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 11 ^° C. The procedure of Example 11 was then repeated to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 65 μm and 4.5% (weight / weight), respectively.

Example 25
400 mg of physiologically active peptide A acetate was dissolved in a solution of the lactic acid-glycolic acid copolymer obtained in Reference Example 12, 3.6 g in 8.0 g (6.0 ml) of dichloromethane. The resulting solution was cooled to 15 ^o C and poured into 1000 ml of a 0.1% solution of polyvinyl alcohol, previously adjusted to 14 ^o C. Next, the procedure of example 11 was repeated to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 65 μm and 8.2% (weight / weight), respectively.

Example 26
400 mg of physiologically active peptide A acetate was dissolved in a solution of the lactic acid-glycolic acid copolymer obtained in Reference Example 13, 3.6 g in 8.0 g (6.0 ml) of dichloromethane. The resulting solution was cooled to 15 ^o C and poured into 1000 ml of a 0.1% solution of polyvinyl alcohol, previously adjusted to 15 ^o C. After this, the procedure of example 11 was repeated to obtain microcapsules. The particle size distribution and physiologically active peptide A content for microcapsules ranged from 5 to 65 μm and 8.4% (weight / weight), respectively.

Example 27
Leuprolerin acetate (manufacturer: Takeda Chemical Industries), 400 mg was added to a solution of the same lactic acid-glycolic acid copolymer used in Example 12, 3.6 g in 8.0 (6.0 ml) dichloromethane to prepare a clear homogeneous solution . The resulting solution was cooled to 15 ^o C and poured into 1000 ml of a 0.1% solution of polyvinyl alcohol, previously adjusted to 15 ^o C. After this, the procedure of example 11 was repeated to obtain microcapsules.

Experimental Example 28
About 30 mg of the microcapsules obtained in Example 1 were dispersed in a dispersion medium (a solution of 2.5 mg of carboxymethyl cellulose, 0.5 mg of polysorbate 80 and 25 mg of mannitol in distilled water) and the dispersion was injected subcutaneously into the back of 10-week-old male SD rats using 22C-needle (microcapsule dosage was 60 μg / kg). Sequentially after administration, rats were sacrificed, the remains of the microcapsules were removed from the injection site, and the amount of physiologically active peptide A in the microcapsules was determined. The results are shown in table. 1.

Experimental Examples 29-33
Using the microcapsules obtained in examples 2-6, the residual amounts of the physiologically active peptide A were determined as well as in experimental example 28. The results are also shown in table. 1.

 From the table. 1 it is clear that all the microcapsules according to the invention are characterized to a large extent by the constant release of the physiologically active peptide and, in addition, are characterized by a significant degree of freedom from the initial impulse.

Tab. 2 shows linear regression models, correlation coefficients, and release periods calculated as an X-segment cut off on an axis of coordinates that are determined by bioassay method (by Akira Sakuma, Tokyo University Press, June 5, 1978, p. 111)
From the table. 2 it is clear that by varying the weight average molecular weight of polylactic acid miscible with the glycolic acid-2-hydroxybutyric acid copolymer, the duration of release can be easily controlled in the range of about 3.5 weeks to 11.5 weeks.

 Tab. Figure 3 shows the linear regression models, correlation coefficients, and release periods as a segment cut off by a curve on the X-axis, which are determined from the data in Table. 1 in the same way as used in table. 2.

 From the table. 3 it is clear that by changing the molar fraction of glycolic acid in the glycolic acid-2-hydroxybutyric acid copolymer, the duration of release can be easily controlled in the range of about 3.1 weeks to 6.6 weeks.

Experimental Examples 34-36
Using the microcapsules obtained in Examples 34-36, the residual amounts of the physiologically active peptide in the microcapsules were determined as in Reference Example 1, except that the dosage of the microcapsules was approximately 30 mg / kg. The results are shown in table. 4. Tab. 5 shows linear regression models, correlation coefficients, and release periods calculated as an X-segment on the coordinate axis, which are defined in the data in Table. 4 by the same methodology as used in table. 2.

 From the table. 4 and 5 it is clear that the microcapsules according to the present invention are characterized mainly by the constant release of the physiologically active peptide and, in addition, are characterized by a significant degree of freedom from the initial impulse.

Experimental Example 37
Using the microcapsules obtained in example 37, the residual amounts of the physiologically active peptide were determined as in Experimental example 34. The results are shown in table. 6. Tab. 7 shows linear regression models, correlation coefficients, and release periods calculated as X-segments on the coordinate axis, which are determined from the data in Table. 6 according to the methodology used in table. 2.

 From the table. 6 and 7 it is clear that the microcapsules according to the present invention are characterized mainly by the constant release of physiologically active peptide and, in addition, are characterized by a significant degree of freedom from the initial impulse.

Experimental Example 38
About 30 mg of the capsules obtained in Example 38 were dispersed in 0.5 ml of a dispersion medium (prepared by dissolving carboxymethyl cellulose (2.5 mg), polysorbate 80 (0.5 mg) and mannitol (25 mg) in distilled water) and the dispersion was injected subcutaneously in the back of 10-week-old male SD rats using a 22G needle (microcapsule dosage was 60 mg / kg). Sequentially after administration, rats were sacrificed, the remains of microcapsules were removed from the injection site, and the amount of physiologically active peptide A in the microcapsules was determined. The results are shown in table. eight.

Experimental Example 39
Using the microcapsules obtained in example 39, the rest repeated the procedure of experimental example 38 and investigated the remainder of the physiologically active peptide A. The results are shown in table. eight.

Experimental Example 40
Using the microcapsules obtained in experimental example 40, the rest repeated the procedure of experimental example 38 and investigated the remainder of the physiologically active peptide A. The results are shown in table. eight.

Experimental Example 41
Using the microcapsules obtained in example 41, the rest repeated the procedure of experimental example 38 and investigated the physiologically active peptide A. The results are shown in table. eight.

 Tab. 9 shows linear regression models, correlation coefficients, and release periods, as X-segments on the coordinate axis, which are determined from the data in Table. 8 by the same methods that are used in table. 2.

 From the table. 8 and 9, it is clear that the sustained release formulation of the present invention invariably provides substantially constant release of the peptide over various time periods.

Comparison Example 42
400 mg of physiologically active peptide A acetate was added to a solution of a lactic acid / glycolic acid copolymer ((lactic acid / glycolic acid = 50/50 (mol%), GPC weight average molecules. Weight = 58000, GPC number average molecular weight = 14000, number average molecular weight determined at the end groups = 45000; manufacturer: Boehringer-Ingelheim (Lot. RG 505-05077), 3.6 g, 33.2 g (25.0 ml) of dichloromethane, but physiologically active peptide A acetate may to be not completely dissolved.

Comparison Example 43
400 mg of physiologically active peptide A acetate was added to a solution of a lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25 (mol%), GPC weight average molecules. Weight = 18000, GPC number average molecular weight = 8400, number average molecule end weight = 30000; manufacturer: Boehringer-Ingelheim (Lot. RG 752-15057), 3.6 g, 8.0 g (6.0 ml) dichloromethane, but physiologically active peptide A may not be completely dissolved. This dispersion was cooled to 17 ^o C and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, precede nogo adjusted to 15 ^o C to prepare microcapsules in the same manner as that described in Example 38. The particle size distribution and physiologically active peptide A microcapsules ranged from 10 to 90 microns, and 2.5% (w / w), respectively.

Comparison Example 44
400 mg of physiologically active peptide A acetate was added to a solution of a lactic acid / glycolic acid copolymer (lactic acid / glycolic acid = 75/25 (mol%), GPC weight average molecular weight = 58000.

GPC number average molecule. weight = 15000, number average molecule. weight determined by end groups = 53000; manufacturer: Boehringer-Ingelheim (Lot. RG 755-05019), 3.6 g, in 21.1 g (16.0 ml) of dichloromethane, but the physiologically active peptide A may not be completely dissolved. This dispersion was cooled to 17 ^° C. and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, previously adjusted to 16 ^° C. to obtain microcapsules in the same manner as in Example 38. Particle size distribution and physiologically active content peptide A for microcapsules ranged from 10 to 90 μm and 3.6% (weight / weight), respectively.

 As comparative examples 42-44 show, the peptide [I] of the present invention cannot be successfully dissolved with a lactic acid-glycolic acid copolymer having a substantially non-terminal carboxyl group.

Comparison Example 44
Leuprolerin acetate (manufacturer: Takeda Chemical Industries) 400 mg was added to a solution of the same lactic acid-glycolic acid copolymer as used in Comparative Example 43, 3.6 g, in 8.0 g (6.0 ml) of dichloromethane, but leuprolerin acetate may not be completely dissolved.

 The sustained release preparation of the present invention shows a sustained release of the drug, especially the peptide [I] over a long time, thus providing a long and stable effect. In addition, the duration of drug release can be easily controlled and excessive release immediately after administration can be inhibited. In particular, the histamine-releasing activity of the peptide [I] after the administration of a sustained release preparation is inhibited. The sustained release preparation has excellent dispersion ability. In addition, the drug is stable (for example, with respect to light, heat, humidity, staining) and is low toxic, reliable when administered.

 In accordance with the production method of the present invention, a sustained release preparation containing a pharmacologically active peptide can be easily obtained in good yield. The sustained release preparation thus obtained has a smooth surface and has excellent mobility.

Claims (25)

A sustained release pharmaceutical composition that includes a physiologically active peptide and a biodegradable polymer, characterized in that it contains a peptide of general formula I as a physiologically active peptide

in which X represents an acyl group;
R ₁ , R ₂ and R ₄ each represents an aromatic cyclic group;
R ₃ represents a D-amino acid residue or a group of formula (II)

wherein

heterocyclic group;
R ₅ represents a group of the formula

in which n = 2 or 3, and

an amino group, which optionally may be substituted, an aromatic cyclic group or O-glycosyl;
R ₆ represents a group of the formula

in which n = 2 or 3, and

an amino group which may be optionally substituted;
R ₇ represents a D-amino acid or azaglycyl residue;
Q is hydrogen or lower alkyl,
or a salt thereof, and as a biodegradable polymer, a polymer having a terminal carboxyl group, where the average molecular weight of this polymer, determined by the end groups, is from 0.4 to 2 times greater than the average molecular weight determined by gel permeation chromatography. 2. The composition according to claim 1, characterized in that it contains a peptide of formula (I), where X is a C ₂ -C ₇ alkanoyl optionally substituted with a 5- or 6-membered heterocyclic carboxamido group. 3. The composition according to claim 2, characterized in that X is a C _2-4 alkanoyl optionally substituted with a tetrahydrofuryl carboxamido group.  4. The composition according to claim 1, characterized in that it contains a peptide of formula I, where X is acetyl. 5. The composition according to claim 1, characterized in that as a biodegradable polymer contains a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the General formula II

in which R is alkyl containing from 2 to 8 carbon atoms;
and (B) polylactic acid. 6. The composition according to claim 1, characterized in that it contains a peptide of formula I, where X is acetyl, and as a biodegradable polymer is a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the general formula II

in which R is alkyl containing from 2 to 8 carbon atoms,
and (B) polylactic acid.  7. The composition according to claim 5, characterized in that the copolymer has a weight average molecular weight of 2000 to 50,000, determined by gel permeation chromatography (GPC).  8. The composition according to p. 5, characterized in that the copolymer has a degree of dispersion of 1.2 to 4.0.  9. The composition according to claim 5, characterized in that polylactic acid has a weight average molecular weight of 1500-30000, determined by GPC.  10. The composition according to claim 5, characterized in that the polylactic acid has a degree of dispersion of 1.2 to 4.0.  11. The composition according to claim 1, characterized in that as a biodegradable polymer contains a copolymer of lactic acid and glycolic acid.  12. The composition according to claim 11, characterized in that the copolymer has a weight average molecular weight of 5000 - 25000, determined by GPC.  13. The composition according to claim 11, characterized in that the copolymer has a degree of dispersion of 1.2 to 4.0.  14. The composition according to p. 1, characterized in that it contains a physiologically active peptide in the range of 0.01 to 50% (weight / weight) relative to the content of biodegradable polymer.  15. The composition according to claim 1, characterized in that the physiologically active peptide is an LH-RH antagonist. 16. The composition according to claim 1, characterized in that as a physiological active peptide contains a peptide representing

or its acetate. 17. The composition according to claim 1, characterized in that as a physiologically active peptide contains a peptide representing NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ , or its acetate. 18. The composition according to claim 1, characterized in that as a physiologically active peptide contains a peptide representing NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg (Et ₂ ) -Leu-hArg (Et ₂ ) -Pro-DAlaNH ₂ or its acetate. 19. A method of obtaining a pharmaceutical composition for sustained release, characterized in that the physiologically active peptide of the general formula is dissolved

in which X represents an acyl group;
R ₁ , R ₂ and R ₄ each represents an aromatic cyclic group;
R ₃ represents a D-amino acid residue or a group of the formula

wherein

denotes a heterocyclic group;
R ₅ represents a group of the formula

in which n = 2 or 3 and

is an amino group, which may be an optionally substituted aromatic cyclic group or an O-glycosyl group;
R ₆ represents a group of the formula

in which n = 2 or 3 and

is an amino group which may be optionally substituted;
R ₇ represents a D-amino acid residue or an azaglycyl residue;
Q is hydrogen or a lower alkyl group,
or its salts and a biodegradable polymer having a terminal carboxyl group, where the average molecular weight determined from the terminal groups is from 0.4 to 2 times greater than the average molecular weight determined by gel permeation chromatography in a water-immiscible solvent, followed by removal solvent. 20. The method according to claim 19, characterized in that the biodegradable polymer is a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the General formula II

in which R is alkyl containing 2 to 8 carbon atoms,
and (B) polylactic acid. 21. The method according to claim 19, characterized in that they dissolve the peptide of formula I, where X is acetyl, and a biodegradable polymer, which is a mixture of (A) a copolymer of glycolic acid and hydroxycarboxylic acid of the formula II

in which R is alkyl containing 2 to 8 carbon atoms,
and (B) polylactic acid.  22. The method according to claim 19, characterized in that the biodegradable polymer is a copolymer of lactic and glycolic acids.  23. The method according to claim 19, characterized in that a physiologically active peptide insoluble in water is used, and before removing the solvent, an oil-in-water emulsion is obtained by adding the resulting solution to the aqueous medium. 24. A method of obtaining a pharmaceutical composition for sustained release, characterized in that they dissolve a biodegradable polymer comprising a mixture of (A) a copolymer of glycolic acid and a hydrocarbonic acid of the general formula II

where R represents alkyl containing 2 to 8 carbon atoms,
and (B) polylactic acid when the average molecular weight of these polymers, determined by the end groups, is from 0.4 to 2 times greater than the average molecular weight determined by gel chromatography and a water-insoluble physiologically active peptide of the general formula I

in which X represents an acyl group;
R ₁ , R ₂ and R ₄ each represents an aromatic cyclic group;
R ₃ represents a D-amino acid residue or a group of formula (2)

wherein

heterocyclic group;
R ₅ represents a group of the formula

in which n = 2 or 3 and

an amino group, which optionally may be substituted, an aromatic cyclic group or O-glycosyl;
R ₆ represents a group of the formula

in which n = 2 or 3, and

an amino group which may be optionally substituted;
R ₇ represents a D-amino acid or azaglycyl residue;
Q is hydrogen or lower alkyl;
or its salts in a solvent not miscible with water, and the solvent is removed.  25. The method according to paragraph 24, wherein the resulting solution is added to the aqueous medium before removing the solvent to obtain an oil-in-water emulsion. A priority
12/07/93. according to claim 1 - 3, 5, 7 - 14, 16, 18 - 20, 22, 24 and 25;
12/07/92 according to claim 4, 6, 17, 21 and 23
02/05/93 according to clause 15.

Images