LT3265B - Supstained-release preparation and method for its preparing - Google Patents

Abstract

The formula of the incessantly disengaging preparation, which consists of an active physiological peptide:<IMAGE>where X is acyl group; R1, R2 and R3 is an aromatic cyclic group;R3 is D - the residual of amino acid, its formula is:<IMAGE>where R3' is a heterocyclic group; R5 is a group and its formula is (CH2)n-R5', where n is 2 or 3, and R5' is amino group, which does not necessarily have to be changed, aromatic cyclic group or O - glycosyl group; R6 is a group and its formula is (CH2)n-R6', where n is 2 or 3, and R6' is an amino group, which does not necessarily have to be changed;R7 is D - the residual of amino acid or azaglycyl; and Q is hydrogen or the lower alkyl group or its salt and biodegrading polymer, which has the terminal carboxylic group.The incessantly disengaging preparation has a long-lasting, constantly disengaging peptide without the initial strong dissociation.

Description

i

The present invention relates to a sustained release formulation comprising a physiologically active peptide and to a process for the preparation thereof.

The earlier article as described in EP-A-481 732, presents a continuous - loosened preparation consisting of drugs, polyactic acid and the glycolic acid-hydroxycarboxylic acid (HOCH (C _2-8 alkyl) COOH) copolymer. Disclosed is a process comprising preparing a V / A emulsion, the aqueous phase of which is an aqueous solution of a physiologically active peptide, and the outer oily phase is a solution of a biodegradable polymer in an organic solvent by adding said V / A emulsion to water or aqueous medium and converting emulsion by continuous release microcapsules (water drying method).

EP-A-52510 discloses a microcapsule containing a hormonal active polypeptide, a biodegradable polymer and a polymer hydrolysis controlling agent. Disclosed is a process for its preparation which comprises the fusion process of adding a fusing agent to a V / A emulsion consisting of an aqueous solution of the polypeptide as an internal aqueous phase and a halogen organic solvent as an oily phase which forms microcapsules.

GB-A-2209937 discloses a pharmaceutical composition comprising a polylactide, a polyglycolide, a lactic acid-glycolic acid copolymer or a mixture thereof, and a water-insoluble peptide. Also disclosed is a process for preparing it, which comprises dispersion of a salt of a water-insoluble peptide in said polylactide, polyglycolide, lactic acid-glycolic acid copolymer solution, or a mixture of these polymers, by removing the solvent by evaporation and forming the resulting mixture into solids.

EP-A-58481 describes a process for preparing a pharmaceutical composition comprising a polylactide and an acid-resistant polypeptide, which comprises, for example, dissolving a solution of tetragastrin hydrochloride and polylactide in aqueous dioxane by melting the film and evaporating the solvent.

EP-A-0467389 discloses a technique for polymeric precipitation or microsphere technology for distributing drugs to proteins, polypeptides. However, this literature does not disclose a specific system containing the LH-AH derivative.

Luteinising hormone - a releasing hormone known as LH-AH (or GnRH), which is secreted from the adrenal cortex and bound to pituitary receptors. LH (luteinising hormone) and FSH (follicle stimulating hormone), which are released immediately, affect the glands that synthesize steroid hormones. Peptides with both positive and antagonistic activity are known to exist as LH-AH derivatives. When peptides with strong positive action are reused, the number of receptors is significantly reduced and steroid hormone production is inhibited. Thus, LH-AH derivatives are used as therapeutic agents or as contraceptives for hormone-dependent diseases such as prostate cancer, non-malignant prostatomegaly, endometriosis, hysteroma, metrofibroma, premature sexual maturation, mammary gland cancer and the like. Particularly problematic was the histamine-releasing activity of LH35 AH antagonists, the so-called first and second generations (The Pharmaceuticals Monthly 32, 1599-1605, 1990), but the number of compounds synthesized during that time and LH-AH antagonistic peptides lacking appreciable histamine releasing activity. has recently been increased (eg US P 511094). To emphasize the pharmacological effect of any LH-AH antagonistic peptides, a controlled release system is required to overcome endogenous LH-AH competing inhibition. Because histamine-releasing activity may be low but exists in such peptides, a sustained-release preparation is required, with the initial rapid, immediate disintegration of the product being inhibited.

In the case of a sustained release formulation (e.g., 1-3 months), it is particularly important to ensure a positive and steady release of the peptide to achieve the desired effect.

At the same time, there is a need for a sensitive long-term sustained release formulation with a high peptide release rate of the physiologically active peptide, especially LH-AH antagonist peptides, to provide the required degree of efficacy.

The present invention relates to:

1) A sustained release formulation comprising a physiologically active peptide of the formula:

CiKCHaJs

CFF ₂ Si ₂ CH ₂ CH ₂ -S -0H CH ₂ CH ₂ -E ₄ -S ₅ Cfl ₂ CH ₂ -B _e X-NH-Cfl- <OGW- <B - {E ^ ₃ -RAD; a -CO-K-Cfl-CO-if3-CH-CO-Na-Ca-CO-NH-Cfl-CO-15-CH-CO-B7-iffi ₂ (I)

W øDJ! (0) wherein X is an acyl group;

R _x , R ₂ and R _{4 are} each an aromatic group;

R ₃ is a D-amino acid residue or a group of the formula:

-NH-CH-CO (D) wherein R ₃ 'is a heterocyclic group;

R ₅ is a group of the formula - (CH ₂ ) _n -R ₅ 'where n is 2 or 3 and R ₅ ' is an amino group which may be optionally substituted, an aromatic cyclic group or an O-glycosyl group;

R ₆ is a group of the formula - (CH ₂ ) _n -R ₆ 'where n is 2 or 3 and R 1 is an amino group which does not need to be substituted;

R ₇ is 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 carboxyl terminus,

2) continuously released by a preparation according to claim 1, wherein X is C ₂ _ ₇ alkanoyl group which may optionally be replaced by - or 6-membered heterocyclic carboxamido group,

3) continuously released by a preparation according to claim 2, wherein X is C ₂ _ ₄ alkanoyl group which may optionally be substituted tetrahidrofurilkarboksamido group

4) A sustained release formulation according to claim 1, wherein X is acetyl,

The sustained release formulation according to claim 1, wherein the biodegradable polymer is a copolymer of (A) glycolic acid and hydroxycarboxylic acid having the formula:

{II} wherein R is an alkyl group of 2 to 8 carbon atoms and (B) a mixture of polylactic acid,

6) The sustained release formulation of claim 1, wherein X is acetyl and the biodegradable polymer is a mixture of (A) a copolymer of glycolic acid and a hydroxycarboxylic acid of formula (II) and (B) a polylactic acid,

7) The sustained release formulation according to claim 5, wherein the copolymer has an average molecular weight of between 2,000 and 50,000 as determined by GPC,

8) A sustained release formulation according to claim 5, wherein the copolymer has a dispersion value of 1.2 to 4.0,

9) A sustained release formulation according to claim 5, wherein the polylactic acid has an average molecular weight of between 1,500 and 30,000 as determined by GPC,

10) A sustained release formulation according to claim 5, wherein the dispersion of polylactic acid has a value of 1.2 to 4.0, &

11) A sustained release formulation according to claim 1, wherein the biodegradable polymer is a copolymer of lactic acid and glycolic acid,

12) A sustained release formulation according to claim 11, wherein the copolymer has an average molecular weight of 5,000 to 25,000 as determined by GPC,

13) A sustained release formulation according to claim 11, wherein the copolymer has a dispersion value of 1.2 to 4.0,

14) The sustained release formulation of claim 1, wherein the ratio of physiologically active peptide to biodegradable polymer is 0.01 to 50% (w / w),

15) The sustained release formulation of claim 1, wherein the physiologically active peptide is an LH-AH antagonist,

16) The sustained release formulation of claim 1, wherein the physiologically active peptide is (?) -CONHCH ₂ COD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ or an acetate thereof,

The sustained release formulation according to claim 1, wherein the physiologically active peptide is NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (Nisp) -Pro-DAlaNH ₂ or acetate thereof,

18) The sustained release formulation according to claim 1, wherein the physiologically active peptide is NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg-DhArg (Et ₂ ) -LeuhArg (Et ₂ ) -Pro-DAlaNH ₂ or its acetate,

(19) A process for preparing a sustained release formulation comprising dissolving a physiologically active peptide of formula (I), or a salt thereof, and a biodegradable polymer having a terminal carboxyl group in a solvent which does not miscible with water, and removing said solvent,

The process of claim 19, wherein the biodegradable polymer is a mixture of (A) a copolymer of glycolic acid and a hydrocarboxylic acid of formula (II) and (B) a polylactic acid,

21) The process of claim 19, wherein X is acetyl and the biodegradable polymer is a copolymer of (A) glycolic acid and hydrocarboxylic acid, a compound of formula (II) and (B) polylactic acid,

22) The method of claim 19, wherein the biodegradable polymer is a copolymer of lactic acid and glycolic acid,

23) A process according to claim 19 consisting of dissolving a biodegradable polymer and a physiologically active peptide in a solvent which does not miscibly mix with water and adding the resulting solution to an aqueous medium to form an A / V emulsion,

(24) A process for preparing a sustained release formulation comprising a biodegradable polymer consisting of a copolymer of glycolic acid (A) and hydroxycarboxylic acid of the formula:

wherein R is an alkyl group having from 2 to 8 carbon atoms and dissolving a mixture of (B) a polylactic acid and a water-insoluble physiologically active peptide or salt thereof in a solvent which is miscible with water and removing said solvent,

The process of claim 24, which, after dissolving the biodegradable polymer and the water-insoluble peptide or salt thereof in a solvent immiscible with water, further comprises forming an A / V emulsion by adding the resulting solution to an aqueous medium.

Meanings of abbreviations used:

NAcD2Nal:

D4ClPhe:

D3Pal:

NMeTyr:

DLys (Nic):

Lys (Nisp):

DLys (AzaglyNic) DLys (AzaglyFur)

N-Acetyl-D-3- (2-naphthyl) alanyl

D-3- (4-Chlorophenyl) alanyl

D-3- (3-Pyridyl) alanyl

N-Methyltyrosyl

D- (Ipsilon-N-nicotinoyl) lysyl (Ipsilon-N-isopropyl) lysyl D- {1-Aza- (N-nicotinoyl) glycyl} lysyl D- {1-Aza- (Ν-2-furoyl) glycyl} lysyl

All other amino acid abbreviations are used according to the IUPACIUB Commission for Biochemical Nomenclature (European Journal of Biochemistry 138.9-37, 1984) or abbreviated as Normal. In the case of optical isomers of the compounds, the L-isomer is used unless otherwise stated.

The peptide (I) of the present invention exhibits LH-AH antagonistic activity and is effectively used in the treatment of hormone-dependent disorders such as prostate cancer, prostatomegaly, endometriosis, hysteromyoma, metrofiLT 3265 B bromine, early sexual maturation, mammary gland cancer. as a contraceptive.

According to formula (I), the acyl group X is suitable when obtained from a carboxylic acid. Acyl groups include C _2-7 alkanoyl, C _{7th 15} cycloalkenoyl (e.g. cyclohexenoyl), C ₁ . ₆ alkylcarbamoyl (e.g. ethylocarbamoyl), 5- or 6-membered heterocyclic carbonyl (e.g. piperidinocarbonyl) and a carbamoyl group which does not necessarily need to be substituted. A suitable acyl group is C ₂ . _An alkanoyl group of ₇ (e.g., acetyl, propionyl, butyryl, isobutyryl, pentanoyl, hexanoyl or heptanoyl) which is not necessarily substituted is more preferably C ₂ . ₄ alkanoyl group (e.g., acetyl, propionyl, butyryl, isibutyryl) which is not necessarily substituted. It includes, for example. C _x _ ₆ alkylamino (eg., Methylamino, ethylamino, diethylamino, propylamino), C _x _ ₃ alkanoyl group (eg., Formylamino, acetiamino, propionylamino), C ₇ _ ₁₅ cikloalkenoilo amino group ( such as cyclohexenoylamino), C ₇ . ₁₅ arylcarbonylamino group (eg., Benzoylamino), 5- or 6-membered heterocyclic carboxamido group (eg., Tetrahidrofurilkarboksamido, piridilkarboksamido, furilkarboksamido) a hydroxyl group, a carbamoyl group, formyl group, carboxyl group, 5- or β-membered aromatic heterocyclic group ( e.g. pyridyl, morpholine). Suitable substituents include a 5- or β-membered heterocyclic carboxamide group (e.g., tetrahydrofurylcarboxamide, pyridylcarboxamide, furylcarboxamide).

X is more preferably C ₂ . ₇ alkanoyl group which is not necessarily substituted by a 5- or 6-membered heterocyclic carboxamide group.

X more appropriate C ₂ . ₄ alkanoyl group which does not necessarily have to be replaced by tetrahydrofurylcarboxamide group.

Specific examples of X are acetyl.

—OONHCH ₂ CD- (tetrahydrofurylcarboxamidoacetyl) and the like.

The aromatic cyclic group R _x , R ₂ or R ₄ may be, for example, an aromatic cyclic group of 6 to 12 carbon atoms. Examples of aromatic cyclic groups are phenyl, naphthyl, anthryl and the like. Suitable aromatic cyclic groups of 6 to 10 carbon atoms such as phenyl and naphthyl. These aromatic cyclic groups may each have from 1 to 5, preferably from 1 to 3, suitable substituents on the corresponding ring sites. Such substituents include hydroxyl, halogen, aminotriazolyl substituted amine, alkoxy and the like. Preferably, hydroxy, halogen and aminotriazolyl substituted amine.

The halogen mentioned above is fluorine, chlorine, bromine and iodine.

The aminotriazole side of said aminotriazolyl-substituted amino activates, among other 3-amino-1H-1,2,4-triazol-5-yl, 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.

A suitable alkoxy group is 1 to 6 carbon atoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, etc.).

More preferred R _x is naphthyl or halophenyl. More preferred R ₂ is halophenyl. More preferably R ₄ is hydroxyphenyl or aminotriazolylamino substituted phenyl.

D-amino acid residue R _{3 is a} suitable α-D-amino acid residue of 3-12 carbon atoms. Examples of amino acids u

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 (the protecting groups mentioned in the article are t-butyl, t-butoxy, t-butoxycarbonyl, etc.).

The heterocyclic group R ₃ is a 5- or 6-membered heterocyclic group containing 1 to 2 nitrogen or sulfur atoms as heteroatoms which do not necessarily have to be fused to a benzene ring. As examples may be mentioned thienyl, pyrrolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, pyridyl, 3-pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 3-benzo {b} thienyl, 3-benzo {b} -3-thienyl, indolyl, 2- indolyl, isoindolyl, ΙΗ-indazolyl, benzoimidazolyl, benzothiazolyl, quinolyl, isoquinolyl and 1.1. Particularly suitable is R ₃ '- pyridyl or 3-benzo {b} thienyl.

Aromatic cyclic group R ₅ may be the same as the aromatic cyclic group R _x, R ₂ or R _4th The aromatic cyclic group may have from 1 to 5, preferably from 1 to 3, suitable substituents at the respective ring positions. The substituents may be the same as the substituents R _x , R ₂ or R ₄ mentioned above. A particularly suitable substitution is aminotriazolyl, a substituted amine.

A suitable glycosyl group for O-Glycosyl R ₅ is hexose or a derivative thereof. The hexose may be D-glucose, Dfructose, D-mannose, D-galactose, L-galactose and 1.1. Deoxy sugars (L- and D-fucose, D-quinovose, L-rhamnose, etc.) and amino sugars (D-glucosamine, D-galactosamine, etc.) may also be mentioned. Deoxy sugars (L- and D-fucose, DLT 3265 B quinovose, L-rhamnose, etc.) are more suitable. The most suitable of these is Lramnose.

In the amino group, the substituent which does not necessarily have to be substituted, R'5, among others, may be acyl, carbamoyl, which may be substituted with acyl or amidine, and the latter may be substituted once or twice with alkyl.

The aforementioned acyl and the carbazoyl acyl mentioned above are nicotinoyl, furoyl, tenoyl and the like.

The alkyl side of the above mono- or di-alkylamino, alkyl is a straight or branched chain alkyl group of 1 to 4 carbon atoms, i.e., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl and tert-butyl and the like. A preferred alkyl side is methyl or ethyl.

The amine substituent which does not need to be substituted, R'6, is alkyl and amidine which may be substituted once or twice with alkyl.

The above alkyl and alkyl of the aforementioned mono- or dialkylamidine are alkyl groups as defined for R _s '.

D-amino acid residue R _{7 is a} suitable D-amino acid residue containing from 3 to 9 carbon atoms, namely, D 30 alanyl, D-leucine, D-valyl, D-isoleucyl, Dphenylalanyl and 1.1. More preferred amino acid residues having 3 to 6 carbon atoms, namely, D-alanyl, D-valyl and 1.1. The most suitable of these is Dalanil.

The lower alkyl group Q may be an alkyl group represented by R _s '. A preferred group Q is methyl.

Examples of R _x are:

Characteristic

etc.

Examples of R ₂ :

etc.

Examples of R ₃ :

Buding

-NB-CH-CO (D)

Examples of R ₄ :

-KB-Cfl-CO- et al.

(D)

etc.

Typical examples of R ₅ are:

- (CH ₂ ) 3 -NH-CO-NH-NH-CO

- CCH 2) ₃ -nh-co-nh-w-co- / - (CH ₂ ) ₃ -NB ₂ ,

- (CH ₂ ) ₂ -NE-CO-NS ₂ , - (CH ₂ ) ₃ -NH-CO-NH ₂ nc ₂ b ₅

- (CH ₂ ) ₃ -NE-C-NB - C ₂ H ₅ , etc.

Typical examples of R ₆ are:

- (CB ₂ ) ₃ -Nfi-CB ': b ₃ ή ₃ nh (CB ₂ ) ₂ -NB-C-NB ₂ , nc ₂ h ₅

- (CH ₂ ) s-NB-C-NH-C ₂ H ₅ and others.

Typical examples of R ₇ are:

If peptide (I) has one or more asymmetric carbon atoms, two or more stereoisomers are formed. Both stereoisomers and mixtures thereof are also an object of the present invention.

A peptide of formula (I) is obtained by known methods. A typical method of preparation is described in U.S. Pat. 5110904.

Peptide (I) may be used in the form of a salt, more preferably a pharmaceutically acceptable salt. In the case of peptide basic groups such as amino, inorganic acids (e.g. hydrochloric acid, sulfuric acid, nitric acid, etc.) or organic acids (e.g. carbonic acid, hydrocarbonic acid, succinic acid, acetic acid) are incorporated into the salts. , propionic acid, trifluoroacetic acid, etc.). If the peptide has acidic groups such as carboxyl, then the salts have inorganic bases (e.g., alkali metals such as sodium, potassium, etc., and alkaline earth metals such as calcium, magnesium, etc.) and organic bases ( such as organic amines such as triethylamine such as arginine). more complex peptide and basic amino acids,

The peptide (I) may be in the form of a metal (e.g. copper, zinc, etc.).

with organic hydrocarbon with salts are acids (e.g. carbonic acid, acid, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.). Most suitable is acetate.

Particularly suitable peptides (I) or salts:

1) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -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

4) OCONHCH ₂ COD2Nal-D4ClPhe-D3Pal-Ser-NMeTyrDLys (Nic) -Leu-Lys- (Nisp) -Pro-DAlaNH ₂ or its acetate

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

The ratio of peptide (I) in the sustained release formulation depends, among other factors, on the type of peptide, the expected pharmacological effect and its duration, and is about 0.01-50% (w / w) of the amount of biodegradable polymer. Suitable limits are about 0.1-40% (w / w), and most suitable are 1-30% (w / w).

A polymer having a terminal carboxylic group is described herein.

The biodegradable polymer, about 1-3 g, was dissolved in a mixture of acetone (25 mL), methanol (5 mL) using phenolphthalein as indicator, and the carboxyl groups of the solution were rapidly titrated with 0.05 N potassium hydroxide solution under constant stirring at room temperature (20 ° C). The number of molecular weight averages, as determined by the terminal group, is calculated from the following equation:

Number of molecular weights as determined by the terminal group = 20000xA / B where A is the mass (g) of biodegradable polymer,

B is the titrated volume (ml) of 0,05 N potassium hydroxide solution

The above calculation is the number of molecular weight averages as determined by the terminal group.

This can be illustrated by taking, as an example, a polymer having a terminal carboxyl group obtained from non-catalytic dehydration polycondensation of one or more α-hydroxy acids, the number average molecular weights of the terminal group being approximately equal to the number of molecular weight averages determined by GPC. Conversely, when the polymer does not have enough free carboxyl terminus formed from the cyclic dimer during open-ring polymerization and using catalysts, the number of molecular weight averages determined by the terminal groups is significantly higher than the number of molecular weight averages determined by GPC. According to this difference, a polymer having a carboxyl-terminus may be clearly distinguished from a polymer having no carboxyl-terminus. Thus, as used herein, the term biodegradable polymer having a terminal carboxyl group refers to a biodegradable polymer having a number average molecular weight determined by GPC that fully corresponds to the number average molecular weight determined by the end group determination.

If the number of molecular weight averages determined by the terminal group is absolute, then the number of molecular weight averages determined by GPC is a relative value that depends on analytical and performance conditions (such as mobile phase type and column, substance choice, slice width, baseline etc.). In addition, the two numerical values cannot be correlated in general. Thus, using the term, the number of molecular weight averages determined by GPC fully corresponds to the number of molecular weight averages as determined by the end group, meaning that the latter number of molecular weight averages is 0.4-2 times, more appropriate 0.5-2 times, most appropriate 0.8 -1.5 times the number of molecular weight averages determined by GPC. The term used above to mean that the number of molecular weight averages by the terminal group is significantly higher means that it is two or more times greater than the GPC obtained.

The present invention provides a polymer having an average number of molecular weights as determined by GPC, which is fully consistent with the average number of molecular weights as determined by the terminal group.

Representative examples of a biodegradable polymer having a terminal carboxyl group include polymers and copolymers, as well as mixtures thereof, which are synthesized from one or more types of hydroxy acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid, and the like) in a non-catalytic dehydration polycondensation reaction. etc.), from hydroxydicarboxylic acids (e.g. malic acid, etc.), from hydroxytricarboxylic acids (e.g. citric acid), etc., poly-a-cyanoacrylic esters, polyamino acids (e.g. poly-y-benzyl-). Lglutamic acid, etc.), copolymers of malic acid anhydride (e.g. styrene-maleic acid copolymer), etc.

Polymerization can be random, blocked and graft-polymerization. When any of the aforementioned α-hydroxy acids, hydroxydicarboxylic acids and hydroxytricarboxylic acids have an active optical center in the molecule, any form of D-, L- and DL- can be used.

A biodegradable polymer having a carboxyl terminus suitable as such comprising a copolymer of (A) glycolic acid and hydroxycarboxylic acid having the formula:

R

I (ii)

HOCHCOOH wherein R is an alkyl group of 2 to 8 carbon atoms and a mixture of (B) polylactic acid or a copolymer of lactic acid glycolic acid.

According to formula (II), a straight or branched chain alkyl group of 2 to 8 carbon atoms which is R is ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl. , 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl and 2-ethylbutyl. Of these, straight or branched chain alkyls of 2 to 5 carbon atoms are suitable. Ethyl, propyl, butyl and isobutyl are more suitable. Most preferred R is ethyl.

The hydroxycarboxylic acid of formula (II) is 2-hydroxybutyric acid, 2-hydroxy valeric acid,

2-hydroxy-3-methylbutyr? acid, 2-hydroxycaproic acid, 2-hydroxyisocaproic acid and 2-hydroxycapric acid. More preferred are 2-hydroxybutyric acid, 2-hydroxyvivaleric acid, 2-hydroxy-3-methyl-butyric acid, and 2-hydroxycaproic acid. Most preferred is hydroxycarboxylic acid of formula (II) which is 2-hydroxybutyric acid. Since these hydroxycarboxylic acids can be both D- and L- and D, L-compounds, a D- / L- (mol%) ratio within the 75/2525/75 range is suitable. Hydroxycarboxylic acid having a D- / L- (mol%) ratio of 60/40-40/60 is more suitable. Most preferred is hydroxycarboxylic acid with a D / L- (mol%) ratio of 55 / 45-45 / 55.

According to the copolymer of glycolic acid and said hydroxycarboxylic acid of formula (II) (hereinafter referred to as the glycolic acid copolymer), the type of copolymerization may be random, blocked or graft-copolymerization. Random copolymers are more suitable.

The hydroxycarboxylic acids of formula (II) may be used alone or in combination.

Glycolic acid and hydroxycarboxylic acid (II) in said glycolic acid copolymer (A) have a suitable ratio of about 10-75 mol% of glycolic acid to hydroxycarboxylic acid balance. A copolymer having a ratio of 20-75 mol% is more suitable. The preferred copolymer containing this ratio is 40-70 mol%. The said glycolic acid copolymer has an average molecular weight in the range of 2,000-50,000. A good average is between 3,000 and 40,000. More appropriate in the range of 8,000-30000. Valid dispersion value (average molecular weight / number average molecular weight) within the range 1.2-4.0. Particularly suitable copolymers having a dispersion value of 1.5-3.5.

The glycolic acid copolymer (A) can be synthesized according to known technology, e.g. 28521/1986.

The polylactic acid used in the present invention can be L- and D-compounds or any mixture thereof. Suitable are those with a D / L ratio (mol%) within the 75/2520/80 range. A more preferred D / L- (mol%) ratio of polylactic acid is in the range of 60/40-25/75. A preferred ratio of D-L- (mol%) of polylactic acid is 55 / 45-25 / 75. Suitable mean molecular weights of polylactic acid are in the range of 1,500-30,000, more preferably in the range of 2,000-20,000, and most appropriate 3,000LT 3265 B

15,000. The dispersion value for polylactic acid is in the range 1.2-4.0, more preferably 1.5-3.5.

Polylactic acid can be synthesized by two alternative methods, namely, open ring polymerization of lactide, which is a dimer of lactic acid, and polycondensation of lactic acid by dehydration. The present invention employs a suitable method for the production of relatively low molecular weight polylactic acid by direct dehydration of polycondensation of lactic acid. This method is described in Japanese Patent Application 28521/1986.

In the present invention, the ratio (A) / (B) (by weight) used in the pharmaceutical glycolic acid copolymer (A) and polylactic acid (B) is in the range of 10 / 90-90 / 10. Suitable mixture ratio within the range 20/80-80/20. Preferred ratios are within the 30 / 70-70 / 30 range. If the ratio of either (A) or (B) is too high, the release formulation in the resulting formulation will not be very different from the one obtained when (A) or (B) is used alone, so that the sample can be obtained using a suitable ( A) and (B). The rate of degradation or elimination of the glycolic acid copolymer and the polylactic acid is significantly dependent on their molecular weights and composition, but generally, at relatively high rates of degradation and elimination of the glycolic acid copolymer, the release period can be extended by increasing the molecular weight of the polylactic acid. A) / (B). Conversely, the release time may be reduced by reducing the molecular weight of the polylactic acid or by increasing the mixture (A) / (B) ratio. Further, the release time can be adjusted by changing the species or relative amount of hydroxycarboxylic acid of 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 suitable in the range 100/0 to 40/60. A ratio of 90 / 10-50 / 50 is more appropriate.

The said copolymer has an average molecular weight in the range of 5,000-25,000. Better ranges from 7,000 to 20,000 are more appropriate.

The degree of dispersion (average molecular weight / number average molecular weight) of said copolymer is in the range of 1.2 to 4.0. Better 1.5 to

3.5.

The abovementioned copolymer of lactic acid and glycolic acid can be synthesized according to known technology, for example according to the method disclosed in Japanese Patent Application 28521/1986.

The degradation and extinction rates of the lactic acid and glycolic acid copolymer are highly dependent on the composition and molecular weight, and generally have a lower degradation and extinction rate at a lower glycolic acid fraction. Thus, the drug release time may be extended by reducing the glycolic acid fraction or by increasing the molecular weight. Conversely, the release time may be reduced by increasing the glycolic acid fraction or by reducing the molecular weight. For prolonged release (e.g. up to 1-4 months), a copolymer of lactic acid and glycolic acid having a polymerization rate within the above ranges and a suitable average molecular weight within the ranges mentioned above is suitable. If the cleavage rate of the lactic acid and glycolic acid copolymer is higher than the above mentioned polymerization ratio and molecular weight average, it is difficult to control the initial cleavage. Conversely, if the degradation rate of the lactic acid-glycolic acid copolymer is lower than the above-mentioned polymerization ratio and average molecular weight limits, there will be periods during which the effective amount of drug is not released.

In this arrangement, the molecular weight average and degree of dispersion represent the molecular weight under polystyrene conditions as determined by gel permeation chromatography (GPC) using 9 polystyrenes having average molecular weights of 120,000, 52,000, 22,000, 9,200, 5,050, 2950, 1,050, 580, and 162. and the value of the dispersion is calculated using the same molecular weight, respectively. This was determined using a GPC KF804Lx2 column (Showa Denko), RI Monitor L-3300 (Hitachi) and chloroform as the mobile phase.

The sustained release formulation of the present invention is obtained by dissolving the peptide (I) and the biodegradable polymer having the terminal carboxyl group in a solvent immiscible with water and then removing said solvent.

A solvent which is immiscible with water is a solvent which is not only miscible with water and capable of dissolving a biodegradable polymer, but also one which renders the resulting polymer solution capable of dissolving the peptide (I). A solvent having a water solubility of not more than 3% (w / w) at atmospheric temperature (20 ° C) is suitable. The boiling point of such a solvent is preferably not higher than 120 ° C. Thus, such solvents include halogenated hydrocarbons (e.g., dichloromethane, chloroform, chloroethane, trichloroethane, carbon tetrachloride, etc.), alkyl ethers of 3 or more carbon atoms (e.g., isopropyl ether, etc.), an alkyl ester of a fatty acid (e.g. of 4 or more carbon atoms) (eg butyl acetate), aromatic hydrocarbons (eg benzene, toluene, xylene, etc.); and 1.1. These solvents can be combined from 2 or more species. More preferred solvents are halogenated hydrocarbons (e.g., dichloromethane, chloroform, chloroethane, trichloroethane, carbon tetrachloride, etc.). Dichloromethane is preferred.

Removal of the solvent may be accomplished by methods known in the art. For example, evaporation of the solvent at atmospheric pressure or gradual reduction of pressure, constant stirring with a propeller stirrer or magnetic stirrer, or evaporation of the solvent in a rotary evaporator under vacuum control.

According to the present invention, the process for preparing a sustained release formulation by dissolving a biodegradable polymer having peptide (I) having a carboxyl terminal end, is such that the resulting solution does not visually detect visible residues of the insoluble peptide at normal temperature (20 ° C). The amount of peptide dissolved in this ternary system consisting of peptide (I), biodegradable polymer, and solvent depends on the number of terminal carboxyl groups per unit weight of biodegradable polymer. In the case of a 1: 1 ratio of peptide to terminal carboxyl group, in theory, the same molar amount of peptide may be dissolved as there are terminal carboxyl groups. However, generalization is difficult due to the combination of solvent, molecular weight of the peptide and biodegradable polymer. However, for continuous release formulation, peptide dissolution may be in the range of 0.1-100% (w / w) suitable for

70% (w / w), most preferably 2-50% (w.w) based on the biodegradable polymer dissolved in the solvent.

The present invention further relates to a process for preparing a sustained release formulation by dissolving a biodegradable polymer consisting of a copolymer of (A) glycolic acid and hydroxycarboxylic acid of formula

R ho £ hcooh wherein R is an alkyl group of a mixture of 2 to 8 carbon atoms and (B) a polylactic acid and a water-insoluble physiologically active peptide or salt thereof in a solvent miscible with water and then removing said solvent.

The physiologically active water-insoluble peptide is not limited, it can be a naturally occurring peptide, it can be a synthetic or a semi-synthetic peptide. Suitable physiologically active peptides having one or more aromatic groups in the side chains (e.g., benzene, naphthalene, phenanthrene, anthracene, pyridine, pyrrole, indole, etc.). Physiologically active peptides having 2 or more aromatic groups in the side chains are more suitable. Physiologically active peptides having 3 or more aromatic groups in the side chains are particularly suitable. These aromatic groups may be further substituted.

The water-insoluble physiologically active peptide used in the present invention is more preferably one having a water solubility of up to 1% and consisting of two or more amino acids and a molecular weight of 200-30000. Molecular weight ranges of 300-20000 are more suitable, 50010000 are more suitable.

Examples of physiologically active peptide include luteinising hormone-releasing hormone (LHAH) antagonists (U.S. Patents 4,086,219, 4,124,577, 4,253,997 and 4,317,815, etc.), insulin, somatostatin, somatostatin derivatives (U.S. Pat. No. 4,093,574, No. 4,100,117, No. 4,253,998, etc.), growth hormone, prolactin, adrenocorticotropic hormone (ΑΚΤΗ), melanocyte-stimulating hormone (MSH), salts and derivatives of thyroid hormone releasing hormone (see JP Kokai S-50-121273 and S-52-116465), thyroid stimulating hormone (TSH), luteinising hormone (LH), toliculin stimulating hormone (FSH), vasopressin, vasopressin derivatives, oxytocin, calcitonin, gastrin, secretin, pancreosimine, cholecystokinin, human angiotensin, , human chorionic gonadotropin (HCG), enkephalins, enkephalin derivatives (U.S. Patent No. 4,277,394, EP-A No. 31,567), endorphin, chiotrifin as, tuftsin, thymopoietin, thymosin, thymostimulin, thymus humoral factor (ULHF), (FTS) and its derivatives (U.S. Pat. 4,229,439), other thymus gland factors, tumor necrosis factor (ANF), colony stimulating factor (CSF), motilin, dynorphin, bombesin, neurotensin, cerulein, bradykinin, atrial natruretic factor, nerve growth factor, cell growth factor, neurotropic factor having endothelial antagonistic activity (EP-A Nos. 436189, 457195 and 496452, JP Kokai H-3-94692 and 03-130299) and fragments or derivatives of these physiologically active peptides.

Specific examples of physiologically active peptides may include physiologically active peptides and salts that are antagonists of luteinizing hormone releasing hormone (LH-AH) and are used in hormone-related diseases such as prostate cancer, prostatic hypertrophy, endometriosis, uterine subgenus, early sex. breast cancer, etc., for treatment and as a contraceptive.

The physiologically active peptide used in the present invention may be in the form of a salt, more preferably a pharmaceutically acceptable salt. When said peptide contains a basic group such as amino, the above salt may be, for example, a salt with an inorganic acid (e.g., hydrochloric acid, sulfuric acid, nitric acid, etc.) or an organic species (e.g., carbon) acid, hydrocarbonic acid, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.). When the peptide has an acidic group such as carboxyl, the salt may be present, for example, with an inorganic base (e.g., alkaline metals such as sodium, potassium, as well as alkaline earth metals such as calcium, magnesium, etc.). or with an organic base (e.g., organic amines such as triethylamine as well as basic amino acids such as arginine, etc.). The peptide may be a complex compound with a metal (e.g., a complex with copper, zinc, etc.).

Specific examples of physiologically active peptide or salt thereof are disclosed in U.S. Pat. 5110904, Journal of Medical Chemistry 34,2395-2402, 1991, Recent Results in Cancer Research 124, 113-136, 1992 et al. in the literature.

Physiologically active peptides and their salts of formula (I) may be mentioned below.

However, even when the physiologically active peptide is water soluble, it can be converted into an insoluble derivative or converted into an insoluble salt containing a water insoluble acid (e.g., pamoic acid, tannic acid, stearic acid, palmitic acid, etc.) and used in the invention. .

The amount of physiologically active peptide used in the formulation of the present invention will depend on the type of peptide, the expected pharmacological effect, the duration of the desired effect, and so on. However, the following proportions are generally used: 0.001-50% (w / w), more preferably 0.01-40% (w / w), most preferably 0.1-30% (w / w) compared to a biodegradable polymer.

The solvent used in this method is the same as described above.

The solvent may be removed as described above.

The process for preparing the sustained release formulation of the present invention is by microencapsulation using a water drying technique or a phase separation technique described below, or an analogous technique.

The method described below can be applied to peptide (I) or to a water insoluble physiologically active peptide containing peptide (I).

Thus, peptide (I) is added to the solution of the biodegradable polymer in an organic solvent at a final weight ratio as above to prepare an organic solvent solution containing the peptide (I) and the biodegradable polymer.

As a result, the concentration of the biodegradable polymer in the organic solvent varies depending on the molecular weight of the biodegradable polymer and the type of organic solvent, but is usually selected in the range of 0.01-80% (w / w). Suitable limits are 0.1-70% (w / w). Most suitable limits are 1-60% (w / w).

Thus, this organic solvent solution containing the peptide (I) and the biodegradable polymer (oily phase) is added to the aqueous phase and an A (oily phase) / V (aqueous phase) emulsion is prepared. The oil phase solvent is then evaporated to prepare the microcapsules. In this method, the volume of the aqueous phase is usually used in the range of 1 to 1000 times the oil phase volume. Suitable limits are 2-5000 times and most appropriate limits are 5-2000 times.

The emulsifier may be added to the aforementioned aqueous phase. The emulsifier can generally be any material that helps to form a stable A / V emulsion. Thus, anionic surfactants (sodium oleate, sodium stearate, sodium lauryl sulfate, etc.), non-anionic surfactants (polyoxyethylene sorbitol fatty acid esters (Tween 80 and Tween 60, Atlas powder), polyoxyethylene castor oil may be mentioned here. derivatives (HCO-60 and HCO-50, Nikko chemicals), polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid, etc. These emulsifiers can be used singly and in combination. ), 0.0110% (w / w), 0.05-5% (w / w) being most suitable.

The resulting microcapsules are reconstituted by centrifugation or filtration and washed with several portions of distilled water to remove free peptide, solvent, and emulsifier from the surface, then redispersed in distilled water or the like. and by lyophilization. The microcapsules are then heated, if necessary, under reduced pressure to remove any residual water and organic solvent from the microcapsules. A more preferred method is heating the biodegradable polymer microcapsules to a temperature of 5 ° C or more above the average melting point of the glass as determined by an increase in the temperature of the scanning calorimeter from 10 to 20 ° C / min. a differential, usually no more than 1 week or 2 to 3 days, more preferably 24 hours after the microcapsules have reached a set temperature.

In the manufacture of microcapsules according to the phase separation technique, the crosslinking agent is gradually added to a solution of said peptide (I) and a biodegradable polymer in an organic solvent, with constant agitation such that the biodegradable polymer can separate and solidify. This crosslinking agent is added in a volume of 0.01 to 1000 times the volume of solution of the peptide (I) and the biodegradable polymer in the organic solvent. Suitable limits are 0.05-500 times, and even more suitable limits are 0.1-200 times.

The crosslinking agent should be a polymeric compound, a type of mineral oil or vegetable oil that does not mix with the solvent of the biodegradable polymer and does not dissolve the polymer yet. Typical examples include silicone, juniper oil, soybean oil, cotton oil, coconut oil, sunflower oil, mineral oil, n-hexane, n-heptane and the like. These materials can be used in combinations.

The resulting microcapsules are reconstituted by filtration and rinsing with heptane or the like to remove the fusing agent. The free peptide and solvent are then removed by the same method as described for water drying techniques.

According to the drying water technique or the adhesion technique, an adhesion inhibitor is added to protect the particle adhesion. The adhesion inhibitors may be water-soluble polysaccharides such as mannitol, lactose, glucose, starch (e.g. corn starch), etc., glycine such as fibrin, collagen, etc., inorganic salts such as sodium chloride, sodium. hydrophosphate and 1.1.

In microcapsule preparation by spray drying, said solution of the peptide and the biodegradable polymer in an organic solvent is injected through a nebulizer into a spray dryer chamber, evaporating from the droplets of finely divided liquid over a short period of time to form thin microcapsules. Sprayer types can be two-fluid, pressurized, rotary and 1.1. In this process, it is more advantageous to spray an aqueous solution of said adhesion inhibitor from another nebulizer to prevent internal adhesion of the capsules, adjusting the time to spraying a solution of said peptide (I) and a biodegradable polymer in an organic solvent.

If necessary, the residual water and the organic solvent are removed by heating the resulting microcapsules under reduced pressure as described above.

The microcapsules may be used as such or in the form of various pharmaceutical preparations for non-oral administration (eg intramuscular, subcutaneous, intravenous injection or implantation, nasal, posterior, uterine, etc.) or orally. (e.g., solid preparations such as capsules) (solid capsules, soft capsules, etc.), granules, powders, etc., liquid preparations such as syrups, emulsions, suspensions, and the like. in the form of.

In preparing the microcapsules for injection, they may be formulated, for example, with a dispersing agent (e.g., a surfactant such as Tween 80, HCO-60, etc., carboxymethylcellulose, a polysaccharide such as sodium alginate, etc.), preservatives ( (e.g., methylparaben, propylparaben, etc.) or an isotonic agent (e.g., sodium chloride, mannitol, sorbitol, glucose, etc.) in aqueous solution, or they may be dispersed in a vegetable oil such as juniper oil, corn oil, or etc., and using the oil suspension for a controlled release injection.

The microcapsule particle size of such injection suspensions must meet the dispersion and needle penetration requirements and may be in the range of 0.1 to 500 µm. A suitable particle size is in the range of 1-300 μιη, most preferably in the range of 2-200 μτη.

To make a microcapsule a sterile product, it has the entire sterility control process, γ-ray sterilization or the addition of a preservative agent, although these are not exceptional procedures.

In addition to the above-mentioned microcapsules, the biodegradable polymer compound containing the active peptide may be well dispersed, conveniently dissolved and converted into a spherical, stick, needle, bead, film to form a sustained release formulation of the present invention. The above biodegradable polymer compound can be obtained by the method described in JP Publication S-50-17525. For specific activity, the peptide drug and polymer are dissolved in a solvent and then removed by appropriate means (e.g., spray drying, evaporation, etc.) to provide the desired biodegradable polymer compound.

The sustained release formulation of the present invention may be administered intramuscularly, subcutaneously, or by injection into the internal organs, or by implantation through the internal mucosal system, nasal cavity, posterior or uterine mucosal system, or the oral preparation (such as a solid preparation such as as a capsule (e.g. hard or soft), granule powder, etc. or a liquid preparation such as a syrup, emulsion, suspension, etc.}.

The sustained release formulation of the present invention is of low toxicity and can be used in mammals (e.g., humans, cattle, pigs, dogs, cats, rats and rabbits).

The dosage of a sustained release formulation depends on the type and composition of the active drug peptide, the final dosage form, the duration of peptide release, the subject being treated (hormone-related diseases such as prostate cancer, prostatomegaly, endometriosis, metrofibroma, premature sexual maturation, mammary gland cancer or as a contraceptive), depending on the type of subject being treated, but in any case an effective amount of the peptide is required. A unit dose of the active drug peptide, for example, for a 1 month delivery system, is a more useful dose when the range is 0.01 to 100 mg / kg for an adult. Appropriate dose 0.05LT 3265 B mg / kg body weight. The most suitable range is 0.1-10 mg / kg body weight.

The dosage unit for continuous release in an adult can be selected from 0.1 to 500 mg / kg body weight. A suitable dose is 0.2-300 mg / kg body weight. Frequency of administration may be, for example, once every few weeks, monthly or once every several months, depending on the type and composition of the active drug peptide, the final dosage form, the duration of peptide release, the disease to be treated and the subject being treated.

The following references and working examples are intended to further illustrate the invention in more detail and should also be understood as defining the object of the invention. (Unless otherwise stated,% means% by weight).

The abbreviations used in the present invention have the following meanings:

BOC: tert-butoxycarbonyl

FMOC: 9-fluorenylmethoxycarbonyl

Cbz: Benzyloxycarbonyl

References Example 1

To a 1000 mL four-necked flask with a nitrogen tube and a condenser was added 300 g of 90% aqueous D, L-lactic acid solution and 100 g of 90% aqueous L-lactic acid solution, all under reduced pressure in a stream of nitrogen gas. heated at 100 ° C / 500 mmHg to 150 ° C / 30 mmHg for 4 hours with constant removal of distilled water. The reaction mixture was further heated under reduced pressure to 3-5 mmHg / 150LT 3265 B

180 ° C for 7 hours to form an amber-colored polylactic acid.

This polymer was dissolved in 1000 ml of dichloromethane and the solution was poured into hot water at 60 ° C with constant stirring. The resulting paste polymer particles were collected and dried under vacuum at 30 ° C.

The mean molecular weight and number of molecular weight averages of the polylactic acid obtained above as determined by the GPC and the number of molecular weight averages determined by the terminal group were 3,000; 1,750; and 1,297 respectively.

These data indicate that the polymer has carboxyl end groups.

References Example 2

500 g 90% aqueous D, L-lactic acid solution were added to a 1000 ml 4-necked flask with nitrogen and the condenser heated to 100 ° C / 500 mmHg to 150 ° C under nitrogen pressure. ° C / 30 mmHg for 4 hours with constant removal of distilled water. The reaction mixture was then further heated under reduced pressure at 3-5 mmHg / 150-180 ° C for 12 hours, then cooled to give amber polylactic acid.

This polymer is dissolved in 1000 ml of dichloromethane and the solution is poured into hot water at 60 ° C with constant stirring. The resulting paste polymer particles are collected and dried under vacuum at 30 ° C.

The average molecular weight and number of molecular weight averages of the above polylactic acid as determined by the GPC and the number of molecular weight averages determined by the terminal group were 5,000, respectively; 2,561; and 1,830.

These data indicate that the polymer has carboxyl end groups.

References Example 3

300 g 90% aqueous D, L-lactic acid solution and 100 g 90% 90% aqueous D, L-lactic acid solution were added to a 1000 ml 4-necked nitrogen flask and condenser, all this under reduced pressure. , is heated at 100 ° C / 500 mmHg to 150 ° C / 30 mmHg for 5 hours in a stream of nitrogen gas, continuously removing distilled water. The reaction mixture was heated under reduced pressure for 18 hours. 5-7 mmHg / 15Q-180 ° C, then cooled to give amber-colored polylactic acid.

This polymer is dissolved in 1000 ml of dichloroethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 30 ° C.

The mean molecular weight and number of molecular weight averages of the above polylactic acid as determined by the GPC and the number of molecular weight averages determined by the terminal group were 7,500, respectively; 3,563; and 2,301.

These data indicate that the polymer has carboxyl end groups.

References Example 4

To 1000 ml of 4-necked nitrogen inlet tube and condenser add 300 g of 90% aqueous D, L-lactic acid solution and 100 g of 90% aqueous L-lactic acid solution, all under reduced pressure in a stream of nitrogen gas for 5 hours. . heated from 100 ° C / 500mmHg to 150 ° C with constant removal of distilled water. The reaction mixture was heated under reduced pressure for 26 h. at 5-7 mmHg / 150180 ° C, then cooled to form an amber-colored polylactic acid.

This polymer is dissolved in 1000 ml of dichloromethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 30 ° C.

The mean molecular weight and number of molecular weight averages of the above polylactic acid as determined by GPC and number of molecular weight averages by terminal group were 9,000, 3, 803, respectively; and 2,800.

These data indicate that the polymer has carboxyl end groups.

References Example 5

Place 182.5 g of glycolic acid and 166.6 g of D, L-2-hydroxybutyric acid in a 1000 ml 4-necked flask with a nitrogen tube and a condenser, all at reduced pressure in a stream of nitrogen for 4 hours. heated from 100 ° C / 500 mmHg to 155 ° C / 30 mmHg with constant removal of distilled water. The reaction mixture was heated under reduced pressure for 27 hours. 3-6 mmHg / 150-185 ° C, then cooled to form an amber-colored copolymer of glycolic acid-2-hydroxybutyric acid.

This polymer is dissolved in 1000 ml of dichloromethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 25 ° C.

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

References Example 6

Add 197.7 g of glycolic acid and 145.8 g of D, L-hydroxybutyric acid, all at reduced pressure, under a stream of nitrogen gas for 4 hours to a 1000 ml 4-necked flask with a nitrogen tube and a condenser. heated from 100 ° C / 500 mmHg to 155 ° C / 30 mmHg with constant removal of distilled water. The reaction mixture was kept under reduced pressure for 27 h. heated to 3-6 mmHg / 150-185 ° C, then cooled to form an amber-colored glycolic acid-2-hydroxybutyric acid copolymer.

This copolymer is dissolved in 1000 ml of dichloromethane and the solution is poured into hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 25 ° C.

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

References Example 7

Add 212.9 g of glycolic acid and 124.9 g of D, L-2-hydroxybutyric acid, all under reduced pressure, under a stream of nitrogen for 3.5 hours to a 1000 ml 4-necked flask with a nitrogen tube and a condenser. heated from 100 ° C / 500 mmHg to 160 ° C / 30 mmHg with constant removal of distilled water. The reaction mixture was kept under reduced pressure for 27 h. heated at 3-6 mmHg / 160-180 [deg.] C., then cooled to give a yellow copolymer of glycolic acid-2-hydroxybutyric acid.

This polymer is dissolved in 1000 ml of dichloromethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 25 ° C.

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

References Example 8

Add 300 g of 90% aqueous D, L-lactic acid solution and 100 g of 90% aqueous L-lactic acid solution, all at reduced pressure, under a stream of nitrogen gas into a 1000 ml 4-necked flask with a nitrogen tube and a condenser. 4 or. heated from 100 ° C / 500 mmHg to 150 ° C / 30 mmHg with constant removal of distilled water. The reaction mixture was then under reduced pressure for 10 hours. heated at 3-5 mmHg and 150-180 ° C, then cooled to give amber-colored polylactic acid.

This polymer is dissolved in 1,000 ml of dichloromethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 30 ° C.

The molecular weight average of the above polylactic acid and the number of molecular weight averages as GPC and the number of molecular weight averages determined by the terminal group were 4,200, respectively; 2,192; and 1,572.

These data indicate that the polymer has terminal caboxyl groups.

References Example 9

Add 182.5 g of glycolic acid and 166.6 g of D, L-2-hydroxybutyric acid, all at reduced pressure, under a stream of nitrogen for 3.5 hours to a 1000 ml 4-necked nitrogen flask with a condenser. heated from 100 ° C / 500 mmHg to 100 ° C

150 ° C / 30 mmHg with constant removal of distilled water. The reaction mixture was then under reduced pressure for 32 hours. heated at 5-7 mmHg and 150-180 ° C, cooled to form an amber-colored copolymer of glycolic acid-2-hydroxybutyric acid.

The polymer is dissolved in 1,000 ml of dichloromethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 25 ° C.

The average molecular weight and number of molecular weights of the resulting glycolic acid-2-hydroxybutyric acid copolymer as determined by the GPC and the number of molecular weight averaged by the terminal group were 16,300, respectively; 5,620; and 2,904.

These data indicate that the polymer has carboxyl end groups.

References Example 10

Synthesis of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -LeuLys (Nisp) -Pro-DAlaNH ₂

Examples 10 and 11 are given in U.S. Pat. 5, 110, 904 and according to U.S. Patent Application Ser. 07/987, 921.

1 g of D-AlaNH-resin (4-methyl-benzohydrylamine resin) is added to the reactor to synthesize the peptide, followed by the gradual addition of amino acids throughout the synthesis until said peptide is formed.

1. The deprotection reaction

45% trifluoroacetic acid (hereinafter referred to as TFA), 2.5% anisole, 2.0% dimethylphosphite and 45% trifluoroacetic acid were used to remove the BOC protecting group from the peptide alpha amino group.

50.5% dichloromethane solution. The resin is rinsed with the solution for 1 min, after which the deprotection reaction is continued for 20 min.

2. Rinse with base solution

A solution of 10% N, Ndiisopropylethylamine in dichloromethane was used to remove, remove and neutralize the trifluoroacetic acid used to remove the protecting group. The resin is rinsed for 1 min. three times for each deprotection reaction.

3. Coupling reaction

The reaction is carried out using triple molar solution of 0.3 M diisopropylcarbodiimide / dichloromethane solution and triple molar amount of 0.3 M BOC amino acid derivative / DMF (N, N'-dimethylformamide) as activator. The activated amino acid in the resin is attached to the free alpha amino group of the peptide. The reaction time is shown below.

4. Flushing

After completion of each reaction, the resin was washed with dichloromethane, dichloromethane / DMF, and DMF for 1 min each.

Synthesis Protocol

The amino group protecting the amino acids is attached to the resin in the following order, frequency, time.

Queues order Amino acid Frequency Time 1 BOC-Pro 2 times 1 or. 2 BOC-Lys (N-epsilon-Cbz, isopropyl) 2 times 1 or. 3 BOC-Leu 2 times 1 or. 4 BOC-D-Lys (N-epsilon-FMOC) 2 times 1 or. 5 BOC-NMeTyr 0-2,6-diCl-Bzl) 2 times 1 or. 6th BOC-Ser (OBzl) 2 times 1 or. 7th BOC-D-3Pal 2 times 6 or. 8th BOC-D-4ClPhe 2 times 2 or. 9th BOC-D2Nal 2 times 2 or. 10th Acetic acid 2 times 2 or.

After the synthesis reaction, removal of the FMOC protecting group, resin for 4-24 hours. was treated with 30% piperidine solution in DMF. The resin is rinsed several times with dichloromethane and then exposed for 15 min. of carbonyldiimidazole (0.9 g) in DMF (18 mL) was washed three times with dichloromethane, after being reacted with 2-furancarbonate hydrazine (0.53 g) in DMF (18 mL) overnight. The resulting peptide resin is washed three times with dichloromethane and then dried overnight with phosphorus pentoxide, followed by 1 hour. dry hydrogen fluoride at 0 ° C using an anisole to separate the peptide from the resin. Excess reaction reagent is removed under vacuum. The resin thus obtained is first washed with ether and then stirred at room temperature for 15 minutes. 50 ml of water / acetonitrile / acetic acid (1: 1: 0.1) and filtered. The filtrate is lyophilized to give the crude peptide as a fluffy powder. this peptide is purified by high performance liquid chromatography (HPLC) under the following conditions.

(1) Column: Dynamax C-18 (25x2.5 cm, 8 microns) (2) Solvent: Acetonitrile before gradient over 20 min. from 89% water / 11% acetonitrile / 0.1% TFA (3) wavelength: 260 nm (UV method)

Peptide set as a single peak at 25.7 min. retention time were collected, freeze dried and the resulting product purified NAcD2NalD4ClPhe-D3Pal-SerNMeTyr-DLys (AzaglyFur) -Leu-Lys (NISP) ₂ -Pro-DALaNH as trifluoroacetate. The physical details of the purified product are as follows:

FAB (Atomic Bomb Speed) 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 pre-equilibrated with 1 N acetic acid. The gel filtration conditions are as follows:

(1) Loading: Sephadex G-25 (inside column 16 mm, trough height 40 mm) (2) Solvent: 1 N acetic acid (3) Wavelength: 254 nm (UV method)

The first eluted peaks are collected and lyophilized fraction obtained purified product NAcD2NalD4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -Leu-Lys (NISP) -Pro-DAlaNH ₂ as the acetate.

References Example 11

Synthesis of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyNic) -LeuLys (Nisp) -Pro-DAlaNH ₂ :

Said peptide was synthesized in the same manner as in Example 10 except that 2-furancarbonate hydrazide was replaced with 2-nicotine hydrazide (0.575 g). The HPLC retention time of the purified product was 16.0 min. The physical data 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 is converted to acetate in the same manner as in Example 10.

References Example 12

To a 1000 ml 4-necked nitrogen flask with a condenser was added 322 g of 90% aqueous D, L-lactic acid solution and 133 g of glycolic acid, using reduced pressure (So-go Rikagaku Glass Co.), in a stream of nitrogen gas for 4 hours. heated from 100 ° C / 500 mmHg to 150 ° C / 30 mmHg with constant removal of distilled water. Reaction mixture under reduced pressure for 23 h. heated at 3-30 mmHg / 150-185 ° C, then cooled to form a lactic acid-glycolic acid copolymer.

This polymer is dissolved in 1000 ml of dichloromethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 30 ° C.

The average molecular weight and number of molecular weights determined by GPC for the lactic acid-glycolic acid copolymer obtained and the mean number of molecular weights as determined by the terminal group were 10,000: 4,000, respectively; and 4,000. These data indicate that the polymer has terminal carboxyl groups.

References Example 13

To a 1000 ml 4-necked nitrogen flask with a condenser was added 347 g of 90% aqueous D, L-lactic acid and 266 g of glycolic acid and all this using a so-go Rikagaku Glass Co ), under reduced pressure, in a stream of nitrogen gas for 5 hours. heated from 100 ° C / 500 mmHg to 150 ° C / 30 mmHg with constant removal of distilled water. The reaction mixture was then under reduced pressure for 23 hours. heated at 3-30 mmHg / 150185 ° C, then cooled to form a copolymer of lactic acid-glycolic acid.

This polymer is dissolved in 1000 ml of dichloromethane and the solution is added to hot water at 60 ° C with constant stirring. The resulting polymer particle paste is collected and dried under vacuum at 30 ° C.

The average molecular weight and average number of molecular weights of the resulting lactic acid-glycolic acid copolymer as determined by the GPC and the number of molecular weights determined by the terminal group were 10,000, respectively; 3,700: 3,900. These data indicate that the copolymer has carboxyl end groups.

example

200 mg of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys- (Nic) Leu-Lys (Nisp) -Pro-DAlaNH ₂ acetate (manufactured by TAP; hereinafter abbreviated as physiologically active peptide A) is dissolved in the mixture (3.8 g). consisting of a 50:50 glycolic acid-2-hydroxybutyric acid copolymer obtained by reference in Example 5 and a polylactic acid obtained by reference in Example 1 in a solution of 5.3 g (4.0 ml) of dichloromethane. The resulting solution is cooled to 17 ° C and added to 1000 ml of 0.1% (w / w) aqueous polyvinyl alcohol solution (EG-40, Nippon Synthetic Chemical Industry Co., Ltd), after adjusting the temperature to 10 ° C, and the mixture is emulsified. am using a turbine homomixer at 7000 rpm to produce an A / V emulsion. This A / V emulsion was stirred at room temperature for 3 hours by evaporation of dichloromethane. The oil phase is solidified and centrifuged (05PR-22, Hitachi, Ltd) at 2000 rpm. This solid phase was redispersed in distilled water and further centrifuged, washing off the free drugs and so forth. The collected microcapsules were redispersed in a small amount of distilled water by the addition of 0.3 g of D-mannitol and freeze-dried to form a powder. The particle size distribution of the microcapsules and the amount of physiologically active peptide were 5 to 60 µm and 4.7% (w / w), respectively.

Other physiologically active peptides (1) and (2) are prepared as described above.

(1) NAcD2Nal-D4ClPhe-D3Pal-Ser-NmeTyr-DLys (AzaglyFur) Leu-Lys (Nisp) -Pro-DAlaNH ₂ .

(2) NAcDNal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur) Leu-Lys (NISP) -Pro-DAlaNH _second

example

A solution of 6.7 g (5.0 ml) of dichloromethane in a 50:50 (3.8 g) mixture of glycolic acid-2-hydroxybutyric acid copolymer obtained in Example 5 and Reference Polylactic acid obtained in Example 2 is dissolved in 200 mg physiologically. active peptide A acetate. This solution is cooled to 17 ° C and added to 1000 ml of 0.1% aqueous polyvinyl alcohol solution previously adjusted to 17 ° C and the mixture is treated as in Example 1 to obtain microcapsules. In the microcapsules, the particle size distribution and the amount of physiologically active peptide were 5 to 65 µm and 5.0% (w / w), respectively.

example

In a solution of 6.7 g (5: 0 ml) of dichloromethane in a 50:50 (3.8 g) mixture of a glycolic acid-2-hydroxybutyric acid copolymer obtained in Example 5 in reference and a polylactic acid obtained in Example 3 in reference are dissolved in 200 mg. physiologically active peptide A acetate. This solution is cooled to 17 ° C and added to 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, after adjusting its temperature to 17 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The microcapsules had a particle size of 10 to 60 µm and a physiologically active peptide A content of 4.8% (w / w).

example

6.7 (5.0 ml) in a dichloromethane solution consisting of

50:50 (3.8 g) of a mixture of glycolic acid-2-hydroxybutyric acid copolymer obtained in Example 5 in Reference and polylactic acid obtained in Example 4 in Reference are dissolved in 200 mg of physiologically active peptide A acetate. This solution is cooled to 17 ° C and added to 1000 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 17 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The microcapsules had a particle size of 10 to 75 µm and a physiologically active peptide A content of 4.6% (w / w).

example

6.0 g (4.5 ml) of a dichloromethane solution consisting of a 50:50 (3.8 g) mixture of glycolic acid-2-hydroxybutyric acid copolymer obtained according to Example 6 and the polylactic acid obtained according to example 2 are dissolved in physiologically active peptide A. acetate. This solution is cooled to 17 ° C and added to 1000 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 10 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The microcapsules had a particle size of 5 to 60 µm and a physiologically active peptide content of 4.9% (w / w).

example

6.0 (4.5 ml) in a dichloromethane solution consisting of

50:50 (3.8 g) of a mixture of glycolic acid-2-hydroxybutyric acid copolymer of Example 7 in Reference and polylactic acid of Example 2 in Reference are dissolved in 200 g of physiologically active peptide A acetate. This solution is cooled to 17 ° C and added to 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol, after having been adjusted to 17 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The particle size in the microcapsules was 10 to 65 µm and the content of physiologically active peptide A was 4.9% (w / w).

example

7.0 (5.3 ml) in a solution of dichloromethane consisting of

50:50 (3.6 g) of a mixture of glycolic acid-2-hydroxybutyric acid copolymer obtained in Example 9 in Reference and polylactic acid obtained in Example 8 in Reference are dissolved in 400 g of physiologically active peptide A acetate. This solution is cooled to 17 ° C and added to 1,000 ml of a 0.1% aqueous solution of polyvinyl alcohol, after adjusting the temperature to 17 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The particle size in the microcapsules was 5 to 65 µm, and the amount of physiologically active peptide A was 7.2% (w / w).

example

A solution of 3.2 g (2.4 ml) of dichloromethane in a 50:50 (1.76 g) copolymer of glycolic acid-2-hydroxybutyric acid obtained in Example 9 and Reference Example 8 is dissolved in 240 mg. NAcD2NalD4ClPhe-D3Pal-Ser-NmeTyr-DLys (AzaglyNic) -Leu-Lys (Nisp) Pro-DAlaNH ₂ acetate (hereinafter abbreviated as physiologically active peptide B) obtained according to Example 11 in ref. The resulting solution is cooled to 18 ° C and poured into 400 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 16 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The particle size in the microcapsules ranged from 5 to 70 μιη and the content of physiologically active peptide B was 10.3% (w / w).

example

A solution of 3.2 g (2.4 ml) of dichloromethane in a 50:50 (1.76 g) copolymer of glycolic acid-2-hydroxybutyric acid obtained in Example 9 and Reference Example 8 is dissolved in 240 mg of NAcD2NalD4ClPhe-. D3Pal-Ser-NMeTyr-DLys (AzaglyFur) -Leu-Lys (Nisp) Pro-DAlaNH ₂ acetate (hereinafter abbreviated as physiologically active peptide C) obtained according to Example 10 in the reference. The resulting solution is cooled to 18 ° C and added to 400 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 16 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The particle size in the microcapsules was from 5 to 65 gm and the content of physiologically active peptide C was 10.9% (w / w).

example

A solution of 3.2 g (2.4 ml) of dichloromethane in a 50:50 (1.76 g) copolymer of glycolic acid-2-hydroxybutyric acid obtained in Example 9 and Reference Example 8 and dissolved in 240 mg of N- Tetrahydrofur2-oil-Gly-D2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-Dlys (Nic) -LeuLys (NISP) -Pro-DAlaNH ₂ (manufactured according to the TAP, hereinafter termed the physiologically active peptide D) acetate {FAB mass spectrometry: m / e 1647 (M + H)). The resulting solution is cooled to 18 ° C and added to 400 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 16 ° C, and the mixture is subjected to the procedure of Example 1 to form microcapsules. The particle size in the microcapsules was from 5 to 70 gm, and the amount of physiologically active peptide D was 10.5% (w / w).

example

5.3 g (4.0 ml) of a dichloromethane solution consisting of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25 (mol%), average molecular weight by GPC = 5,000, number of molecular weight averages by GPC = 2,000, molecular weight average number of weights by end group = 2,200; manufacturer, Wako Pure Chemical (Lot 920729)) dissolved in 200 mg of physiologically active peptide A acetate. The resulting solution was cooled to 17 ° C and poured into 1000 ml of 0.1% aqueous polyvinyl alcohol solution (EG-40, Nippon Synthetic Chemical Industry Co., Ltd), after adjusting the temperature to 16 ° C, and the mixture was emulsified with a turbine mixer at 7000 rpm. / min to prepare an A / V emulsion. This A / V emulsion is stirred for 3 hours. at room temperature by evaporation of dichloromethane. The oil phase is solidified and centrifuged at 2000 rpm. (05Pr-22, Hitachi). The solid phase is redispersed in distilled water and further centrifuged to wash off the free drugs, etc. The collected microcapsules are redispersed in a small amount of distilled water, then 0.3 g of D-mannitol is added and dried to freeze-dry. The microcapsules had a particle size of 5 to 60 μτη with 4.7% (w / w) of physiologically active peptide A.

The sustained release formulation of peptides (1) and (2) is prepared as described above.

(1) Physiologically active peptide B acetate (2) Example of physiologically active peptide C acetate

6.7 g (5.0 ml) of a dichloromethane solution consisting of 3.8 g of lactic acid-glycolic copolymer solution (lactic acid / glycolic acid = 75/25 (mol%), average molecular weight by GPC = 10,000, number of molecular weight averages by GPC = 4,400 , mean number of molecular weights per terminal group = 4,300, manufacturer; Wako Pure Chemical (Lot 880530)) is dissolved in 200 mg of physiologically active peptide A acetate. The resulting solution is cooled to 17 ° C and poured into 1000 ml of 0.1% aqueous polyvinyl alcohol solution, after which the temperature is adjusted to 11 ° C. The process of Example 11 is then repeated to obtain the microcapsules. In the microcapsules, the particle size distribution and the content of physiologically active peptide A were between 5 and 65 μιη and 4.5% (w / w), respectively.

example

Dissolve 400 mg of physiologically active peptide A acetate in 8.0 g (6.0 ml) of a solution of 3.6 g of lactic acid-glycolic acid copolymer obtained in Reference Example 12. The resulting solution is cooled to 15 ° C and poured into 1000 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 14 ° C. The process of Example 11 is then repeated to obtain microcapsules. The particle size distribution in the microcapsules and the content of physiologically active peptide A were between 5 and 65 μπι and 8.2% (w / w), respectively.

example

Dissolve 400 g of physiologically active peptide A acetate in 8.0 g (6.0 ml) of a solution of 3.6 g of lactic acid-glycolic acid copolymer obtained according to Example 13 in the reference. The resulting solution is cooled to 15 ° C and poured into 1000 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 15 ° C. The process of Example 11 is then repeated to form microcapsules. The microcapsules had a particle size of 5 to 65 µm and the content of physiologically active peptide A was 8.4% (w / w).

example

Dissolve 400 mg of leuprolerine acetate (manufactured by Takeda Chemical) in 8.0 g (60 ml) of dichloromethane containing 3.6 g of the lactic acid-glycolic acid copolymer solution used in Example 12.

Industries) and prepare a clear homogeneous solution. The resulting solution is cooled to 15 ° C and poured into 1000 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 15 ° C. The same process as in Example 11 is then repeated to form microcapsules.

Example 1 of Experiment

About 30 mg of the microcapsules obtained in Example 1 were dispersed in a medium (in a solution of 2.5 mg carboxymethylcellulose dissolved in distilled water, 0.5 mg polysorbite 80 and 25 mg mannitol) and the dispersion solution was injected subcutaneously with a 22G needle into the back of 10 weeks old SD male rats. (dose of microcapsules 60 mg / kg). Following administration, a series of rats were sacrificed, residues of the microcapsules removed from the site of administration, and the amount of physiologically active peptide A was determined in the microcapsules. The results are shown in Table 1.

Examples of Experiment 2-6

Using the microcapsules obtained in Examples 2-6, the amount of physiologically active peptide A remaining in the microcapsules is determined in the same manner as in Experiment 1. The results are also presented in Table 1.

table

Physiologically active peptide A residue (%)

Day 1 Weekly dad 1 Weekly dad 2 Weekly dad 3 Weekly dad 4 Weekly dad 5 Weekly dad 6th Weekly dad 8th Experiment 1 example 88.0 66.5 42.3 15.2 Experiment 2 example 92.8 76.6 62.6 48.7 38.6 26.5 Experiment 3 example 96.5 90.5 77.5 64.9 59.2 46.9 38.7 20.3 Experiment 4 example 99.4 94.5 87.2 76.3 66.0 46.6 30.7 Experiment 5 example 92.9 75.0 45.7 17.5 Expert 92.3 61.3 33.5 6.4

Example 6 The data presented in the table in the microcapsule according to the present invention, at a sufficiently constant physiological level, show that all are characterized by the release of the active peptide and exhibit rapid initial degradation.

the data in the table show the linear regression models, the correlation coefficients, the release periods calculated as X-samples determined by the method described in Methods of

Bioassay (by Akira Shaunia, Tokyo University Press, June 5, 1978, p. 111).

table

Polylactic acid molecular weight average Linear regressions model Correlated cations coefficients enth Release nimo period (weekly parents) The experiment Example 1 3000 Residue (%) = 95.4- (26.9x weeks number) (R ² = 0.992) 3.5 The experiment Example 2 5000 Residue (%) = 94.4- (14.2x weeks number) (R ² = 0.975) 6.6 The experiment Example 3 7500 Remain (%) = 98.4- (10.0xweeks number) (R ² = 0.996) 9.8 The experiment Example 4 9000 Residue (%) = 102.1- (8.9x weeks number) (R ² = 0995) 11.5 In Table 2 submit data show that with the change polylactic of acid for molecular for weight, after mixing

with glycolic acid-2-hydroxybutyric acid 10 copolymer, the release time can be freely controlled over a period of 3.5-11.5 weeks.

The data presented in Table 2 show the linear regression models, correlation coefficients, and release periods as X-samples, which were determined from the data in Table 1 using the calculation methods in Table 2.

table

Glycolic acid molar fraction glycolic acid copolymer Linear regression model Correlation coefficient Release period (weeks) Example 2 of Experiment 60% Residue (%) = 94.4- (14.2x weeks number) (R ² = 0.975) 6.6 Experiment 5 example 65% Residue (%) = 95.7- (20.6x weeks number) (R ² = 0.975) 4.6 Experiment 6 example 70% Residue (%) = 96.6- (30.9x weeks number) (R ² = 0.994) 3.1

The data in the table below show that the change in the molar fraction of glycolic acid in the copolymer of glycolic acid-2-hydroxybutyric acid, when mixed with polylactic acid, can be freely controlled over a period of 3.1 to 6.6 weeks.

Examples of Experiment 7-9

Using the microcapsules obtained in Examples 7-9, the residual active peptide in the microcapsules was determined as in Experiment 1 except that the microcapsule dose was about 30 mg / kg. The results are shown in Table 4. The data in Table 5 shows the linear regression models, correlation coefficients, and release periods calculated as X -strings determined from the data in Table 4, as shown in Table 2.

table

Residue of physiologically active peptide (%)

Physiological giai active 1 day 1 week 2 weeks 3 weeks 4 weeks The experiment A 99.3 74.5 51.4 33.2 24.1 Example 7 The experiment B 87.4 75.0 52.3 32.8 25.1 Example 8 The experiment C 89.4 73.6 54.9 37.7 23.4

example table

Physiological giai active peptide Linear regressions model Correlation coefficient Release nimo period The experiment Example 7 A Residue (%) = 97.8- {20.lxsav. number) (R ² = 0.975) 4.9 The experiment Example 8 B Residue (%) = 93.5- (18.6xav. number) (R ² = 0.971) 5.0

Physiological giai active peptide Linear regressions model Correlation coefficient Release nimo period The experiment Example 9 C Residue (%) = 94.4- (18.5xav. number) (R ² = 0.987) 4.9

and Table 5 show that the microcapsules of the present invention are characterized by a relatively constant release of physiologically active peptide and an initial rapid degradation.

Example 10 of Experiment

Using the microcapsules obtained in Example 10, the residues of the physiologically active peptide were determined in the same manner as in Example 7 of the experiment. The results are shown in Table 6. Table 7 shows the linear regression models, correlation coefficients, and release periods calculated as X-samples, which were determined from the data in Table 6 according to the method shown in Table 2.

table

Physiologically active peptide D (%) residue Day 1 Weeks 1 Weeks 2 Weeks 3 The week 4 The experiment 93.5 + 0.5 69.9 + 3.6 37.3 + 1.6 17.0 + 1.4 7.9 + 0.5 Example 10

table

Linear regression model Correlations coefficient Release periods (weeks) The experiment Example 10 Residue (%) = 95.0— (24.lxweeks number) (R ² = 0.969) 3.9

and Table 7 show that the microcapsules of the present invention are characterized by a sufficiently constant release of the physiologically active peptide, irrespective of the initial rapid degradation.

Example 11 of Experiment

About 30 microcapsules obtained in Example 11 were dispersed in 0.5 ml dispersion medium {prepared by dissolving carboxymethylcellulase (2.5 mg) in distilled water}, polysorbite 80 (0.5 mg) and mannitol (25 mg), and the dispersion solution was injected subcutaneously into a 22G needle. 10 weeks old SD rat male hindquarters (microcapsule dose 60 mg / kg). After administration, rats were serially sacrificed, residues of the microcapsules were removed from the site of administration, and residues of active peptide A were determined in the microcapsules. The results are shown in Table 8.

Example 12 of Experiment

Using the microcapsules obtained in Example 12, the procedure of Experiment 11 was otherwise repeated and the residue of physiologically active peptide A was determined. The results are shown in Table 8.

Example 13 of Experiment

Using the microcapsules obtained in Example 13, otherwise repeating the procedure of Experiment 11, and determining the physiologically active peptide A residue, the results are shown in Table 8.

Example 14 of Experiment

Using the microcapsules obtained in Example 14, the procedure of Example 11 of Experiment 11 was otherwise repeated and the residue of physiologically active peptide A was determined. The results are shown in Table 8.

Table 8

Physiologically active peptide A residue (%)

Day Sa- whore Weekly parents Weekly parents Weekly parents Weekly Weekly parents parents 1 1 2 3 4 6th 8th Example 11 of Experiment 82.8 21.8 - - - - - The experiment Example 12 96.7 91.7 79.5 69.2 59.2 - 22.8 The experiment Example 13 100.0 84.3 43.9 31.9 - - - The experiment Example 14 96.3 67.5 38.0 23.5 - - -

(-: not determined) The data in the table show linear regression models, correlation coefficients, and release periods as X -strings determined from the data in Table 8 following the procedure in Table 2.

table

Linear regression model Correlation coefficient Periods of liberation (weeks) The experiment Residue (%) = 97.1- (R ² = 0994) 1.3 Example 11 (75.7xweeks) The experiment Residue (%) = 92.2- (R ² = 0.998) 10.3 Example 12 (9.7x week number) The experiment Remaining (%) = 102.4- (R ² = 0.982) 4.1 Example 13 (24.8x weeks) The experiment Residue (%) = 97.7- (R ² = 0.989) 3.7 Example 14 (26.5x weeks) 8 and 9. in the tables data shows that

the sustained release formulation of the present invention provides for a consistent, sustained release of the peptide at various times.

Comparison Example 1

400 mg of physiologically active peptide A acetate is added to a solution of lactic acid-glycolic acid copolymer {lactic acid / glycolic acid = 50/50 (mol%), mean molecular weight by GPC = 58,000, number of molecular weight average by GPC = 14,000, molecular weight number of averages according to end group setting = 45,000; manufacturer: BoehringerIngelheim (Lot.RG505-05077)}, 3.6 g of which are dissolved in 33.2 g (25.0 ml) of dichloroethane, but the physiologically active peptide A acetate cannot be well dissolved.

Comparison Example 2

400 mg of physiologically active peptide A acetate is added to a solution of lactic acid-glycolic acid copolymer {lactic acid / glycolic acid = 75/25 (mol%), average molecular weight by GPC = 18,000, number of molecular weight by GPC = 8,400, molecular weight average number of weights according to end group determination = 30,000: Manufacturer: BoehringerIngelheim (Lot RG752-15057)} with 3.0 g dissolved in 8.0 g (6.0 ml) dichloroethane but physiologically active peptide A cannot be dissolved well. This solution is cooled to 17 ° C and added to 1,000 ml of a 0.1% aqueous solution of polyvinyl alcohol, after adjusting its temperature to 15 ° C, to obtain the microcapsules as described in Example 11. The particle sizes in the microcapsules ranged from 10 to 90 μιη and the content of physiologically active peptide was 2.5% (w / w).

Comparison Example 3

400 mg of physiologically active peptide A acetate is added to a solution of lactic acid-glycolic acid copolymer {lactic acid / glycolic acid = 75/25 (mol%), mean molecular weight based on GPC = 58,000, number of molecular weight average based on GPC = 15,000, molecular weight mean number by end group determination = 53,000: Manufacturer: BoehringerIngelheim (Lot RG755-05019)} with 3.6 g dissolved in 21.2 g (16.0 ml) dichloromethane but physiologically active peptide A cannot be dissolved well. This solution is cooled to 17 ° C and added to 1,000 ml of 0.1% aqueous polyvinyl alcohol solution, after adjusting its temperature to 16 ° C, to obtain the microcapsules in the manner described in Example 11. The particle size of the microcapsules ranged from 10 to 90 µm and the content of physiologically active peptide A was 3.6% (w / w).

Comparison Example 4

400 mg of leuprolerine acetate (manufactured by Takeda Chemical Industries) are added to the same solution of the lactic acid-glycolic acid copolymer used in Comparison Example 2, containing 3.6 g of 8.0 g (6.0 ml) of dichloromethane, but the leuprolerine acetate cannot be well dissolved.

The sustained release formulation of the present invention provides sustained release of drugs, particularly peptide (I), over a relatively long period of time, thereby providing a sustained and stable effect. Thus, the duration of drug release can be easily controlled and the excess released immediately after administration can be suppressed. Typically, histamine-releasing activity in peptide (I) is inhibited shortly after continuous administration of the preparation. The sustained release formulation is highly dispersible. In addition, the preparation is stable (eg light, heat, humidity, color), non-toxic and therefore non-hazardous in use.

In accordance with the method of the present invention, a sustained release formulation containing a physiologically active peptide can be readily obtained in bulk. The resulting sustained release formulation has a smooth and highly mobile surface.

DEFINITION OF INVENTION

Claims (10)

DEFINITION OF INVENTION 1. Continuous release formulation containing a physiologically active peptide of the formula CH (CH 1), CHrRi Clli-8j CHj-OH CHi-ίϊ * CHrRj CHj CHr8 < Q where X is an acyl group, R _1z R ₂ , and R ₄ are an aromatic cyclic group, R ₃ is a damminic acid residue or a group of the formula CH2-R3 I -NH - CH «CO (D) where R ₃ 'is a heterocyclic group, R ₅ is a group of the formula - (CH ₂ ) _n -R ₅ 'wherein n is 2 or 3 and R ₅ ' is an amino group which may be optionally substituted, an aromatic cyclic group or an O-glycosyl group, R ₆ is a group of the formula - (CH ₂ ) _n -R ₆ 'where n is 2 or 3 and R ₆ ' is an amino group which may not be optionally substituted, R ₇ is 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 carboxyl terminal group. Second continuous loosened preparation according to claim 1, characterized in that X is C ₂ _ ₇ alkanoyl group which may optionally be substituted 5arba 6- membered heterocyclic carboxamido group. 3. A sustained release formulation according to claim 1, wherein the biodegradable polymer is a copolymer of (A) glycolic acid and hydroxycarboxylic acid of formula R HOCHCOOH wherein R is an alkyl group of 2 to 8 carbon atoms and a mixture of (B) polylactic acid. 4. A sustained release formulation according to claim 1, wherein X is acetyl and the biodegradable polymer is a copolymer of (A) glycolic acid and hydroxycarboxylic acid of formula R I HOCHCOOH wherein R is an alkyl group of 2 to 8 carbon atoms and a mixture of (B) polylactic acid. 5.Continuously claiming that the glycolic acid liberated polymer is biodegradable is a copolymer. A preparation according to claim 1 wherein the lactic acid and 6. The sustained release formulation according to claim 1, wherein the ratio of physiologically active peptide to biodegradable polymer is from 0.01 to 50% by weight. A sustained release claim, the distinctive physiologically active peptide is a formulation according to claim 1, wherein the LH-AH antagonist. 8.Nepertraukiamai loosened preparation according to claim 1, wherein the physiologically active peptide is <~ ^ - CONHCH ₂ COD2NalD4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic) -Leu-Lys (NISP) -ProDAlaNH ₂ or its acetate. 9. A process for preparing a sustained release formulation comprising the step of providing a physiologically active peptide of formula CH (CH 3), CHrRi Cllj-Rj CH, -OH CH _r R <CHrRj CH, ^ • ^ 'CH-CO-NH-Cfi ^ CO-Ri-HH-CH-CO-N-Cyl-CO-NH-CH-CO-NH -Ca-CO-.VH-CH-CO-N-CH-CO-RTNH,! (D) wherein X is an acyl group, R _x , R ₂ and R ₄ are aromatic, R ₃ is a D-amino acid residue or a group of formula CH2 - Rq '• NH · CH · COT (U), wherein R _3' is a heterocyclic group R ₅ is a group of the formula - (CH ₂ ) _n -R ₅ 'where n is 2 or 3 and R ₅ ' is an amino group which may be optionally substituted, an aromatic cyclic group or an O-glycosyl group, R ₆ is a group of the formula - (CH ₂ ) _n -R ₆ ^/ where n is 2 or 3 and R ₆ 'is an amino group which may not be optionally substituted, R ₇ is a D - amino acid residue or an azaglycyl residue, and Q is hydrogen or a lower alkyl group or a salt thereof and dissolves the biodegradable polymer in a solvent which is miscible with water and removes said solvent. 10. A process for preparing a sustained release formulation comprising biodegrading a polymer containing a copolymer of (A) glycolic acid and a hydroxycarboxylic acid of the formula R I HOCHCOOH wherein R is a mixture of an alkyl group of 2 to 8 carbon, and (B) a polylactic acid, and the water-insoluble physiologically active peptide or salt thereof is dissolved in a solvent which is not miscible 5 with water, then removing said solvent.