U.S. patent application number 09/386232 was filed with the patent office on 2003-02-27 for sustained-release preparation.
Invention is credited to IGARI, YASUTAKA, KAMEI, SHIGERU, OGAWA, YASUAKI.
Application Number | 20030039698 09/386232 |
Document ID | / |
Family ID | 27282444 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030039698 |
Kind Code |
A1 |
KAMEI, SHIGERU ; et
al. |
February 27, 2003 |
SUSTAINED-RELEASE PREPARATION
Abstract
A sustained-release preparation which comprises a
physiologically active peptide of general formula 1 wherein X
represents an acyl group; R.sub.1, R.sub.2 and R.sub.4 each
represents an aromatic cyclic group; R.sub.3 represents a D-amino
acid residue or a group of the formula 2 wherein R.sub.3' is a
heterocyclic group; R.sub.5 represents a group of the formula
--(CH.sub.2).sub.n--R.sub.5' wherein n is 2 or 3, and R.sub.5' is
an amino group which may optionally be substituted, an aromatic
cyclic group or an O-glycosyl group; R.sub.6 represents a group of
the formula --(CH.sub.2).sub.n--R.sub.6' wherein n is 2 or 3, and
R.sub.6' is an amino group which may optionally be substituted;
R.sub.7 represents a D-amino acid residue or an azaglycyl residue;
and Q represents hydrogen or a lower alkyl group, or a salt thereof
and a biodegradable polymer having a terminal carboxyl group. The
sustained-release preparation shows a constant release of the
peptide over a long time and is substantially free from an initial
burst.
Inventors: |
KAMEI, SHIGERU; (TAKARAZUKA,
JP) ; IGARI, YASUTAKA; (KOBE, JP) ; OGAWA,
YASUAKI; (OHYAMAZAKI-CHO, JP) |
Correspondence
Address: |
FOLEY & LARDNER
3000 K STREET N W
SUITE 500
WASHINGTON
DC
200075109
|
Family ID: |
27282444 |
Appl. No.: |
09/386232 |
Filed: |
August 31, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09386232 |
Aug 31, 1999 |
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08892315 |
Jul 14, 1997 |
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5972891 |
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Current U.S.
Class: |
424/489 ;
514/10.3; 514/10.4 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 38/09 20130101; A61K 38/00 20130101; C07K 7/23 20130101; A61K
9/1647 20130101; A61P 5/24 20180101 |
Class at
Publication: |
424/489 ;
514/2 |
International
Class: |
A01N 037/18; A61K
038/00; A61K 009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 7, 1992 |
JP |
327070/1992 |
Feb 5, 1993 |
JP |
018978/1993 |
Jun 16, 1993 |
JP |
145134/1993 |
Claims
What is claimed is:
1. A sustained-release preparation which comprises a
physiologically active peptide of the general formula 13wherein X
represents an acyl group; R.sub.1, R.sub.2 and R.sub.4 each
represents an aromatic cyclic group; R.sub.3 represents a D-amino
acid residue or a group of the formula 14wherein R.sub.3' is a
heterocyclic group; R.sub.5 represents a group of the formula
--(CH.sub.2).sub.n--R5' wherein n is 2 or 3 and R.sub.5' is an
amino group which may optionally be substituted, an aromatic cyclic
group or an O-glycosyl group; R.sub.6 represents a group of the
formula --(CH.sub.2).sub.n--R.sub.6' wherein n is 2 or 3 and
R.sub.6' is an amino group which may optionally be substituted;
R.sub.7 represents a D-amino acid residue or an azaglycyl residue;
and Q represents 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, wherein
X is a C.sub.2-7 alkanoyl group which may optionally be substituted
by a 5- or 6-membered heterocyclic carboxamido group.
3. The sustained-release preparation according to claim 2, wherein
X is a C.sub.2-4 alkanoyl group which may optionally be substituted
by a tetrahydrofurylcarboxamide group.
4. The sustained-release preparation according to claim 1, wherein
X is acetyl.
5. The sustained-release preparation according to claim 1, wherein
the biodegradable polymer is a mixture of (A) a copolymer of
glycolic acid and a hydroxycarboxylic acid of the general formula
15wherein R represents an alkyl group of 2 to 8 carbon atoms and
(B) a polylactic acid.
6. The sustained-release preparation according to claim 1, wherein
X is acetyl, and the biodegradable polymer is a mixture of (A) a
copolymer of glvcolic acid and a hydroxycarboxylic acid of the
general formula 16wherein R represents an alkyl group of 2 to 8
carbon atoms and (B) a polylactic acid.
7. The sustained-release preparation according to claim 5, wherein
the copolymer has a weight average molecular weight of about 2,000
to 50,000, as determined by GPC.
8. The sustained-release preparation according to claim 5, wherein
the copolymer has a dispersion value of about 1.2 to 4.0.
9. The sustained-release preparation according to claim 5, wherein
the polylactic acid has a weight average molecular weight of about
1,500 to 30,000 as determined by GPC.
10. The sustained-release preparation according to claim 5, wherein
the polylactic acid has a dispersion value of about 1.2 to 4.0.
11. The sustained-release preparation according to claim 1, wherein
the biodegradable polymer is a copolymer of lactic acid and
glycolic acid.
12. The sustained-release preparation according to claim 11,
wherein the copolymer has a weight average molecular weight of
about 5,000 to 25,000, as determined by GPC.
13. The sustained-release preparation according to claim 11,
wherein the copolymer has a dispersion value of about 1.2 to
4.0.
14. The sustained-release preparation according to claim 1, wherein
the proportion of the physiologically active peptide ranges from
about 0.01 to 50% (w/w) based on the biodegradable polymer.
15. The sustained-release preparation according to claim 1, wherein
the physiologically active peptide is a LH-RH antagonist.
16. The sustained-release preparation according to claim 1, wherein
the physiologically active peptide is 17or its acetate.
17. The sustained-release preparation according to claim 1, wherein
the physiologically active peptide is
NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic)-Leu-Lys
(Nisp)-Pro-DAlaNH.sub.2 or its acetate.
18. The sustained-release preparation according to claim 1, wherein
the physiologically active peptide is
NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg (Et.sub.2)-Leu-hArg
(Et.sub.2)-Pro-DAlaNH.sub.2 or its acetate.
19. A method of producing a sustained-release preparation which
comprises dissolving a physiologically active peptide of the
general formula 18wherein X represents an acyl group; R.sub.1,
R.sub.2 and R.sub.4 each represents an aromatic cyclic group;
R.sub.3 represents a D-amino acid residue or a group of the formula
19wherein R.sub.3' is a heterocyclic group; R.sub.5 represents a
group of the formula --(CH.sub.2).sub.n--R.su- b.5' wherein n is 2
or 3, and R.sub.5' is an amino group which may optionally be
substituted, an aromatic cyclic group or an O-glycosyl group;
R.sub.6 represents a group of the formula --(CH.sub.2).sub.n--R.su-
b.6' wherein n is 2 or 3, and R.sub.6' is an amino group which may
optionally be substituted; R.sub.7 represents a D-amino acid
residue or an azaglycyl residue; and Q represents hydrogen or a
lower alkyl group or a salt thereof and a biodegradable polymer
having a terminal carboxyl group in a solvent which is
substantially immiscible with water and then removing said
solvent.
20. The method according to claim 19, wherein the biodegradable
polymer is a mixture of (A) a copolymer of glycolic acid and a
hydroxycarboxylic acid of the general formula 20wherein R
represents an alkyl group of 2 to 8 carbon atoms and (B) a
polylactic acid.
21. The method according to claim 19, wherein X is acetyl, and the
biodegradable polymer is a mixture of (A) a copolymer of glycolic
acid and a hydroxycarboxylic acid of the general formula 21wherein
R represents an alkyl group of 2 to 8 carbon atoms and (B) a
polylactic acid.
22. The method according to claim 19, wherein the biodegradable
polymer is a copolymer of lactic acid and glycolic acid.
23. A method according to claim 19, which comprises dissolving the
biodegradable polymer and the physiologically active peptide in a
solvent which is substantially immiscible with water and adding the
resulting solution to an aqueous medium to provide an O/W
emulsion.
24. A method of producing a sustained-release preparation which
comprises dissolving a biodegradable polymer comprising a mixture
of (A) a copolymer of glycolic acid and a hydroxycarboxylic acid of
the general formula 22wherein R represents an alkyl group of 2 to 8
carbon atoms and (B) a polylactic acid and a substantially
water-insoluble physiologically active peptide or a salt thereof in
a solvent which is substantially immiscible with water and then
removing said solvent.
25. A method according to claim 24, which further comprises after
dissolving the biodegradable polymer and the substantially
water-insoluble peptide or salt thereof in the solvent adding the
resulting solution to an aqueous medium to provide an O/W emulsion.
Description
[0001] The present invention relates to a sustained-release
preparation containing a physiologically active peptide and to a
method of producing the same.
BACKGROUND OF THE INVENTION
[0002] The prior art includes, as disclosed in EP-A-481,732, a
sustained-release preparation comprising a drug, a polylactic acid
and a glycolic acid-hydroxycarboxylic acid [HOCH(C.sub.2-8
alkyl)COOH] copolymer. The disclosed process comprises preparing a
W/O emulsion consisting of an internal water 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 (drying-in-water method).
[0003] EP-A-52510 describes a microcapsule comprising a hormonally
active polypeptide, a biodegradable polymer and a polymer
hydrolysis control agent. The disclosed, process for its production
is a coacervation process which comprises adding a coacervation
agent to a W/O emulsion consisting of an aqueous solution of the
polypeptide as the internal water phase and a halogenated organic
solvent as the oil phase to provide microcapsules.
[0004] GB-A-2209937 describes a pharmaceutical composition
comprising a polylactide, a polyglycolide, a lactic-acid-glycolic
acid copolymer or a mixture of these polymers and a water-insoluble
peptide. Also disclosed is a production process which comprises
dispersing a salt of the water-insoluble peptide in a solution of
said polylactide, polyglycolide, a lactic acid-glycolic acid
copolymer or a mixture of these polymers, removing the solvent by
evaporation and molding the resulting mixture into solid
particles.
[0005] EP-A-58481 describes a process for producing a
pharmaceutical composition comprising a polylactide and an
acid-stable polypeptide which, for instance, comprises dissolving
tetragastrin hydrochloride and a polylactide in aqueous dioxane,
casting the solution into a film and evaporating the solvent.
[0006] EP-A-0467389 teaches a technology for providing a drug
delivery system for proteins and polypeptides by the polymer
precipitation technique or the microsphere technique. However, this
literature contains no specific disclosure about a system
containing an LH--RH derivative.
[0007] The luteinizing hormone-releasing hormone, known as LH-RH
(or GnRH), is secreted from the hypothalamus and binds to receptors
on the pituitary gland. The LH (luteinizing hormone) and FSH
(folicle stimulating hormone), which are released thereon, act on
the gonad to synthesize steroid hormones. As derivatives of LH-RH,
the existence of both agonistic and antagonistic peptides is known.
When a highly agonistic peptide is repeatedly administered, the
available receptors are reduced in number so that the formation of
gonad-derived steroidal hormones is suppressed. Therefore, LH--RH
derivatives are expected to be of value as therapeutic agents for
hormone-dependent diseases such as prostate cancer, benign
prostatomegaly, endometriosis, hysteromyoma, metrofibroma,
precocious puberty mammary cancer, etc. or as contraceptives.
Particularly, the problem of histamine-releasing activity was
pointed out for LH-RH antagonists of the so-called first and second
generations (The Pharmaceuticals Monthly 32, 1599-1605, 1990) but a
number of compounds have since been synthesized and recently
LH-RH-antagonizing peptides having no appreciable
histamine-releasing activity have been developed (cf. U.S. Pat. No.
5,110,904, for instance). In order for any such LH-RH antagonizing
peptide to mani-fest its pharmacological effect, there is a need
for a controlled release system so that the competitive inhibition
of endogenous LH-RH may be persistent. Moreover, because of
histamine-releasing activity which may be low but is not
non-existent in such peptides, a demand exists for a
sustained-release preparation with an inhibited initial burst
immediately following administration.
[0008] Particularly, in the case of a sustained-release (e.g. 1-3
months) preparation, it is important to insure a more positive and
constant release of the peptide in order that the desired efficacy
may be attained with greater certainty and safety.
[0009] At the same time, there is a long-felt need for a method of
producing a sustained-release preparation having a high peptide
trap rate for a physiologically active peptide, particularly
LH-RH-antagonizing peptides.
SUMMARY OF THE INVENTION
[0010] According to the present invention, there is provided:
[0011] 1) A sustained-release preparation which comprises a
physiologically active peptide of the general formula 3
[0012] wherein X represents an acyl group;
[0013] R.sub.1, R.sub.2 and R.sub.4 each represents an aromatic
cyclic group;
[0014] R.sub.3 represents a D-amino acid residue or a group of the
formula 4
[0015] wherein R.sub.3' is a heterocyclic group;
[0016] R.sub.5 represents a group of the formula
--(CH.sub.2).sub.n--R.sub- .5' wherein n is 2 or 3, and R.sub.5' is
an amino group which may optionally be substituted, an aromatic
cyclic group or an O-glycosyl group;
[0017] R.sub.6 represents a group of the formula
--(CH.sub.2).sub.n--R.sub- .6' wherein n is 2 or 3, and R.sub.6' is
an amino group which may optionally be substituted;
[0018] R.sub.7 represents a D-amino acid residue or an azaglycyl
residue; and
[0019] Q represents hydrogen or a lower alkyl group or a salt
thereof and a biodegradable polymer having a terminal carboxyl
group,
[0020] 2) The sustained-release preparation according to the above
paragraph 1, wherein X is a C.sub.2-7 alkanoyl group which may
optionally be substituted by a 5- or 6-membered heterocyclic
carboxamido group,
[0021] 3) The sustained-release preparation according to the above
paragraph 2, wherein X is a C.sub.2-4 alkanoyl group which may
optionally be substituted by a tetrahydrofurylcarboxamide
group,
[0022] 4) The sustained-release preparation according to the above
paragraph 1, wherein X is acetyl,
[0023] 5) The sustained-release preparation according to the above
paragraph 1, wherein the biodegradable polymer is a mixture of (A)
a copolymer of glycolic acid and a hydroxycarboxylic acid of the
general formula 5
[0024] wherein R represents an alkyl group of 2 to 8 carbon atoms
and (B) a polylactic acid,
[0025] 6) The sustained-release preparation according to the above
paragraph 1, wherein X is acetyl, and the biodegradable polymer is
a mixture of (A) a copolymer of glycolic acid and a
hydroxycarboxylic acid of the general formula [II] and (B) a
polylactic acid,
[0026] 7) The sustained-release preparation according to the above
paragraph 5, wherein the copolymer has a weight average molecular
weight of about 2,000 to 50,000, as determined by GPC,
[0027] 8) The sustained-release preparation according to the above
paragraph 5, wherein the copolymer has a dispersion value of about
1.2 to 4.0,
[0028] 9) The sustained-release preparation according to the above
paragraph 5, wherein the polylactic acid has a weight average
molecular weight of about 1,500 to 30,000 as determined by GPC,
[0029] 10) The sustained-release preparation according to the above
paragraph 5, wherein the polylactic acid has a dispersion value of
about 1.2 to 4.0,
[0030] 11) The sustained-release preparation according to the above
paragraph 1, wherein the biodegradable polymer is a copolymer of
lactic acid and glycolic acid,
[0031] 12) The sustained-release preparation according to the above
paragraph 11, wherein the copolymer has a weight average molecular
weight of about 5,000 to 25,000, as determined by GPC,
[0032] 13) The sustained-release preparation according to the above
paragraph 11, wherein the copolymer has a dispersion value of about
1.2 to 4.0,
[0033] 14) The sustained-release preparation according to the above
paragraph 1, wherein the proportion of the physiologically active
peptide ranges from about 0.01 to 50% (w/w) based on the
biodegradable polymer,
[0034] 15) The sustained-release preparation according to the above
paragraph 1, wherein the physiologically active peptide is a LH-RH
antagonist,
[0035] 16) The sustained-release preparation according to the above
paragraph 1, wherein the physiologically active peptide is 6
[0036] or its acetate,
[0037] 17) The sustained-release preparation according to the above
paragraph 1, wherein the physiologically active peptide is
NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic)-Leu-Lys
(Nisp)-Pro-DAlaNH.sub.2 or its acetate,
[0038] 18) The sustained-release preparation according to the above
paragraph 1, wherein the physiologically active peptide is
NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg(Et.sub.2)-Leu-hArg
(Et.sub.2)-Pro-DAlaNH.sub.2 or its acetate,
[0039] 19) A method of producing a sustained-release preparation
which comprises dissolving a physiologically active peptide of the
general formula [I] or a salt thereof and a biodegradable polymer
having a terminal carboxyl group in a solvent which is
substantially immiscible with water and then removing said
solvent,
[0040] 20) The method according to the above paragraph 19, wherein
the biodegradable polymer is a mixture of (A) a copolymer of
glycolic acid and a hydroxycarboxylic acid of the general formula
[II] and (B) a polylactic acid,
[0041] 21) The method according to the above paragraph 19, wherein
X is acetyl, and the biodegradable polymer is a mixture of (A) a
copolymer of glycolic acid and a hydroxycarboxylic acid of the
general formula [II] and (B) a polylactic acid,
[0042] 22) The method according to the above paragraph 19, wherein
the biodegradable polymer is a copolymer of lactic acid and
glycolic acid,
[0043] 23) A method according to the above paragraph 19, which
comprises dissolving the biodegradable polymer and the
physiologically active peptide in a solvent which is substantially
immiscible with water and adding the resulting solution to an
aqueous medium to provide an O/W emulsion,
[0044] 24) A method of producing a sustained-release preparation
which comprises dissolving a biodegradable polymer comprising a
mixture of (A) a copolymer of glycolic acid and a hydroxycarboxylic
acid of the general formula 7
[0045] wherein R represents an alkyl group of 2 to 8 carbon atoms
and (B) a polylactic acid and a substantially water-insoluble
physiologically active peptide or a salt thereof in a solvent which
is substantially immiscible with water and then removing said
solvent, and
[0046] 25) A method according to the above paragraph 24, which
further comprises after dissolving the biodegradable polymer and
the substantially water-insoluble peptide or salt thereof in the
solvent adding the resulting solution to an aqueous medium to
provide an O/W emulsion.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The abbreviations used in this specification have the
following meanings.
[0048] NAcD2Nal: N-Acetyl-D-3-(2-naphtyl)alanyl
[0049] D4ClPhe: D-3-(4-Chlorophenyl)alanyl
[0050] D3Pal: D-3-(3-Pyridyl)alanyl
[0051] NMeTyr: N-Methylthyrosyl
[0052] DLys(Nic): D-(Ipsilon-N-nicotinoyl)lysyl
[0053] Lys(Nisp): (Ipsilon-N-isopropyl)lysyl
[0054] DLys(AzaglyNic): D-[1-Aza-(N-nicotinoyl)glycyl]lysyl
[0055] DLys(AzaglyFur): D-[1-Aza-(N-2-furoyl)glycyl]lysyl
[0056] Where any other amino acids are expressed by abbreviations,
the abbreviations recommended by IUPAC-IUB Commission on
Biochemical Nomenclature (European Journal of Biochemistry 138,
9-37, 1984) or the abbreviations in common usage in the art are
used. Where optical isomers exist for any compound, the L-isomer is
meant unless otherwise indicated.
[0057] In the present invention, the peptide [I] shows LH--RH
antagonistic activity and is effective for the treatment of
hormone-dependent diseases such as prostatic cancer,
prostatomegaly, endometriosis, hysteromyoma, metrofibroma,
precocious puberty, mammary cancer, etc. or for contraception.
[0058] Referring to general formula [I], the acyl group X is
preferably an acyl group derived from carboxylic acid. Examples of
the acyl group include a C.sub.2-7 alkanoyl, C.sub.7-15
cycloalkenoyl (e.g., cyclohexenoyl), C.sub.1-6 alkylcarbamoyl
(e.g., ethyl carbamoyl), 5- or 6-membered heterocyclic carbonyl
(e.g. piperidinocarbonyl) and carbamoyl group which may optionally
be substituted. The acyl group is preferably a C.sub.2-7 alkanoyl
group (e.g., acetyl, propionyl, butyryl, isobutyryl, pentanoyl,
hexanoyl or heptanoyl) which may optionally be substituted, more
preferably C.sub.2-4 alkanoyl group (e.g., acetyl, propionyl,
butyryl, isobutyryl) which may optionally be substituted. The
substituents are for example C.sub.1-6 alkylamino group (e.g.,
methylamino, ethylamino, diethylamino, propylamino), C.sub.1-3
alkanoyl amino group (e.g., formylamino, acetylamino,
propionylamino), C.sub.7-15 cycloalkenoyl amino group (e.g.,
cyclohexenoylamino), C.sub.7-15 arylcarbonyl-amino group (e.g.,
benzoylamino), 5- or 6-membered heterocyclic carboxamido group
(e.g., tetrahydrofurylcarboxamido, pyridylcarboxamido,
furylcarboxamido), hydroxyl group, carbamoyl group, formyl group,
carboxyl group, 5- or 6-membered heterocyclic group (e.g., pyridyl,
morpholino). The substituents are preferably 5- or 6-membered
heterocyclic carboxamido group (e.g., tetrahydrofurylcarboxamido,
pyridylcarboxamido, furylcarboxamido).
[0059] X is preferably a C.sub.2-7 alkanoyl group which may
optionally be substituted by a 5- or 6-membered heterocyclic
carboxamido group.
[0060] X is more preferably a C.sub.2-4 alkanoyl group which may
optionally be substituted by a tetrahydrofuryl carboxamido
group.
[0061] Specific examples of X are acetyl, 8
[0062] (tetrahydrofurylcarboxamidoacetyl) and so on.
[0063] The aromatic cyclic group R.sub.1, R.sub.2 or R.sub.4 may
for example be an aromatic cyclic group of 6 to 12 carbon atoms.
Examples of the aromatic cyclic group are phenyl, naphthyl, anthryl
and so on. Preferred are aromatic cyclic groups of 6 to 10 carbon
atoms, such as phenyl and naphthyl. These aromatic cyclic groups
may each have 1 to 5, preferably 1 to 3, suitable substituents in
appropriate positions on the ring. Such substituents include
hydroxyl, halogen, aminotriazolyl-substituted amino, alkoxy and so
on. Preferred are hydroxy, halogen and aminotriazolyl-substituted
amino.
[0064] The halogens mentioned above include fluorine, chlorine,
bromine and iodine.
[0065] The aminotriazolyl moiety of said 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 so on.
[0066] The alkoxy group is preferably an alkoxy group of 1 to 6
carbon atoms (e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy,
isobutoxy, etc.).
[0067] More preferably, R.sub.1 is naphthyl or halophenyl. More
preferably, R.sub.2 is halophenyl. More preferably, R.sub.4 is
hydroxyphenyl or aminotriazolylamino-substituted phenyl.
[0068] The D-amino acid residue R.sub.3 is preferably an
.alpha.-D-amino acid residue of 3 to 12 carbon atoms. Examples of
the amino acid are leucine, isoleucine, norleucine, valine,
norvaline, 2-aminobutyric acid, phenylalanine, serine, threonine,
methionine, alanine, tryptophan and aminoisobutyric acid. These
amino acids may have suitable protective groups (the protective
groups used conventionally in the art, such as t-butyl, t-butoxy,
t-butoxycarbonyl, etc.).
[0069] The heterocyclic group R.sub.3' includes 5- or 6-membered
heterocyclic groups each containing 1 to 2 nitrogen or sulfur atoms
as hetero-atoms, which may optionally be fused to a benzene ring.
Specifically, thienyl, pyrrolyl, thiazolyl, isothiazolyl,
imidzolyl, pyrazolyl, pyridyl, 3-pyridyl, pyridazinyl, pyrimidinyl,
pyrazinyl, 3-benzo[b]thienyl, 3-benzo[b]-3-thienyl, indolyl,
2-indolyl, isoindolyl, 1H-inda-zolyl, benzoimidazolyl,
benzothiazolyl, quinolyl, iso-quinolyl, etc. may be mentioned. The
particularly preferred species of R.sub.3' is pyridyl or
3-benzo[b]thienyl.
[0070] The aromatic cyclic group R.sub.5 may be the same as the
aromatic cyclic group R.sub.1, R.sub.2 or R.sub.4. The aromatic
cyclic group may have 1 to 5, preferably 1 to 3, suitable
substituents in appropriate positions on the ring. The substituents
may also be the same as the substituents mentioned for R.sub.1,
R.sub.2 or R.sub.4. The particularly preferred substituent is
aminotriazolyl-substituted amino.
[0071] The glycosyl group for O-glycosyl R.sub.5 is preferably a
hexose or a derivative thereof. The hexose includes D-glucose,
D-fructose, D-mannose, D-galactose, L-galactose and so on. As said
derivative, deoxy sugars (L- and D-fucose, D-quinovose, L-rhamnose,
etc.) and amino sugars (D-glucosamine, D-galactosamine, etc.) can
be mentioned. More preferred are deoxy sugars (L- and D-fucose,
D-quinovose, L-rhamnose, etc.). Still more preferred is
L-rhamnose.
[0072] The substituent on the amino group which may optionally be
substituted, R.sub.5', includes, among others, acyl, carbamoyl,
carbazoyl which may be substituted by acyl or amidino which may be
mono- or di-substituted by alkyl.
[0073] The above-mentioned acyl and the acyl for the
above-mentioned carbazoyl which may be substituted by acyl include
nicotinoyl, furoyl, thenoyl and so on.
[0074] The alkyl moiety of the mono- or di-alkylamidino mentioned
above includes straight-chain or branched alkyl groups of 1 to 4
carbon atoms, thus including methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, sec-butyl and tert-butyl and so on. The preferred
alkyl moiety is methyl or ethyl.
[0075] The substituent for the amino group which may optionally be
substituted, R.sub.6', includes alkyl and amidino which may be
mono- or di-substituted by alkyl.
[0076] The above-mentioned alkyl and the alkyl of the mono- or
dialkylamidino mentioned above include those alkyl groups mentioned
for R.sub.5'.
[0077] The D-amino acid residue R.sub.7 is preferably a D-amino
acid residue of 3 to 9 carbon atoms, such as D-alanyl, D-leucyl,
D-valyl, D-isoleucyl, D-phenylalanyl and so on. More preferred are
D-amino acid residues of 3 to 6 carbon atoms, such as D-alanyl,
D-valyl and so on. The more preferred species of R.sub.7 is
D-alanyl.
[0078] The lower alkyl group Q may be the alkyl group defined for
R.sub.5'. The most preferred species of Q is methyl.
[0079] Specific examples of R.sub.1 are 9
[0080] When the peptide [I] has one or more asymmetric carbon
atom(s), there are two or more stereoisomers. Any of such
steroisomers as well as a mixture thereof is within the scope of
the present invention.
[0081] The peptide of general formula [I] is produced by the per se
known processes. Typical specific processes are described in U.S.
Pat. No. 5,110,904.
[0082] The peptide [I] can be used in the form of a salt,
preferably a pharmacologically acceptable salt. Where the peptide
has basic groups such as amino, the salt includes salts with
inorganic acids (e.g. hydrochloric acid, sulfuric acid, nitric
acid, etc.) or organic acids (e.g. carbonic acid, hydrogen carbonic
acid, succinic acid, acetic acid, propionic acid, trifluoroacetic
acid, etc.). Where the peptide has acidic groups such as carboxyl,
salts with inorganic bases (e.g. alkali metals such as sodium,
potassium, etc. and alkaline earth metals such as calcium,
magnesium, etc.) or organic bases (e.g. 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 (e.g. copper
complex, zinc complex, etc.). The preferred salts of peptide [I]
are salts with organic acids (e.g. carbonic acid, hydrogen carbonic
acid, succinic acid, acetic acid, propionic acid, trifluoroacetic
acid, etc.). The most preferred is the acetate.
[0083] Particularly preferred species of peptide [I] or salt are as
follows. (1) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-
Lys(Nisp)-Pro-DAlaNH.sub.2 or its acetate (2)
NAcD2Nal-D4ClPhe-D3Pal-Ser-- NMeTyr-DLys(AzaglyNic)-
Leu-Lys(Nisp)-Pro-DAlaNH.sub.2 or its acetate (3)
NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyFur)-
Leu-Lys(Nisp)-Pro-DAlaNH.sub.2 or its acetate (4) 10
[0084] or its acetate (5)
NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg(Et.sub.2)-L- eu-
hArg(Et.sub.2)-Pro-DAlaNH.sub.2 or its acetate
[0085] In the sustained-release preparation, the proportion of the
peptide [I] may vary with the type of peptide, the expected
pharmacological effect and duration of effect, among other factors,
and may range from about 0.01 to about 50% (w/w) based on the
biodegradable polymer. The preferred range is about 0.1 toabout 40%
(w/w) and a more preferred range is about 1 to about 30% (w/w).
[0086] The biodegradable polymer having a terminal carboxyl group
is now described.
[0087] A biodegradable polymer, about 1 to 3 g, was dis- solved in
a mixture of acetone (25 ml) and methanol (5 ml) and using
phenolphthalein as the indicator, the carboxyl groups in the
solution were quickly titrated with 0.05N alcoholic potassium
hydroxide solution under stirring at room temperature
(20.degree.C.). The number average molecular weight by end-group
determination was then calculated by means of the following
equation.
[0088] Number average molecular weight by end-group
[0089] determination =20000 .times.A/B
[0090] where A is the mass of biodegradable polymer (g) B is the
amount of O.05N alcoholic potassium hydroxide solution (ml) added
to react the titration end-point.
[0091] The result of the above calculation is referred to as the
number average molecular weight by end-group determination.
[0092] By way of illustration, taking a polymer having a terminal
carboxyl group as synthesized from one or more .alpha.-hydroxy
acids by the non-catalytic dehydrative poly-condensation process as
an example, the number average molecular weight by end-group
determination is approximately equal to the number average
molecular weight found by GPC. In contrast, in the case of a
polymer substantially not containing free terminal carboxyl groups
as synthesized from a cyclic dimer by the ring-opening
polymerization process and using catalysts, the number average
molecular weight by end-group determination is by far greater than
the number average molecular weight by GPC determination. By this
difference, a polymer having a terminal carboxyl group can be
clearly discriminated from a polymer having no terminal carboxyl
group. Thus, the term biodegradable polymer having a terminal
carboxyl group is used herein to mean a biodegradable polymer
showing a substantial agreement between the number average
molecular weight by GPC determination and the number average
molecular weight by end-group determination.
[0093] Whereas the number average molecular weight by end-group
determination is an absolute value, the number average molecular
weight by GPC determination is a relative value which varies
according to analytical and procedural conditions (such as types of
mobile phase and column, reference substance, selected slice width,
selected baseline, etc.). Therefore, the two values cannot be
numerically correlated by generalization. However, the term
`substantial agreement` between the number average molecular weight
by GPC determination and the number average molecular weight by
end-group determination means that the number average molecular
weight found by end-group determination is about 0.4 to 2 times,
more preferably about 0.5 to 2 times, most preferably about 0.8 to
1.5 times, the number average molecular weight by GPC
determination. The term `by far greater` as used above means that
the number average molecular weight by end-group determination is
about twice or greater than the number average molecular weight by
GPC determination.
[0094] The preferred polymer for the purpose of the present
invention is a polymer showing a substantial agreement between the
number average molecular weight by GPC determination and the number
average molecular weight by end-group determination.
[0095] As specific examples of the biodegradable polymer having a
terminal carboxyl group can be mentioned polymers and copolymers,
as well as mixtures thereof, which are synthesized from one or more
species of .alpha.-hydroxy acids (e.g. glycolic acid, lactic acid,
hydroxybutyric acid, etc.), hydroxydicarboxylic acids (e.g. malic
acid etc.), hydroxytricarboxylic acids (e.g. citric acid etc.),
etc. by the non-catalytic dehydrative polycondensation reaction,
poly-.alpha.-cyanoacrylic esters, polyamino acids (e.g.
poly-.gamma.-benzyl-L-glutamic acid etc.), maleic anhydride
copolymers (e.g. styrene-maleic acid copolymer etc.) and so on.
[0096] The mode of polymerization may be random, block or graft.
Where any of the above-mentioned .alpha.-hydroxy acids,
hydroxydicarboxylic acids and hydroxytricarboxylic acids has an
optical activity center within the molecule, any of the D-, L- and
DL-forms c an be employed.
[0097] The biodegradable polymer having a terminal carboxyl group
is preferably a biodegradable polymer comprising a mixture of (A) a
copolymer of glycolic acid and a hydroxycarboxylic acid of the
general formula 11
[0098] wherein R represents an alkyl group of 2 to 8 carbon atoms
and (B) a polylactic acid, or a lactic acid-glycolic acid
copolymer.
[0099] Referring to the general formula [II], the straight-chain or
branched alkyl group of 2 to 8 carbon atoms, as represented by R,
includes, 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. Preferred,
among them, are straight-chain or branched alkyls of 2 to 5 carbon
atoms. Specifically, ethyl, propyl, isopropyl, butyl and isobutyl
are preferred. R is most preferably ethyl.
[0100] The hydroxycarboxylic acid of the general formula [II]
includes, inter alia, 2-hydroxybutyric acid, 2-hydroxyvaleric acid,
2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid,
2-hydroxyisocaproic acid and 2-hydroxycapric acid. Preferred are
2-hydroxybutyric acid, 2-hydroxyvaleric acid,
2-hydroxy-3-methylbutyric acid and 2-hydroxycaproic acid. The
hydroxycarboxylic acid of the general formula [II] is most
preferably 2-hydroxybutyric acid. While these hydroxycarboxylic
acids may be any of the D-, L- and D,L-compounds, the D-/L-ratio
(mol %) is preferably in the range of about 75/25 through about
25/75. The more preferred embodiment is a hydroxycarboxylic acid
with a D-/L-ratio (mol %) within the range of about 60/40 through
about 40/60. The most preferred is a hydroxycarboxylic acid with a
D-/L-ratio (mol %) within the range of about 55/45 through about
45/55.
[0101] Referring to the copolymer of glycolic acid and said
hydroxycarboxylic acid of the general formula [II] (hereinafter
referred to as glycolic acid copolymer), the mode of
copolymerization may be random, block or graft. Preferred are
random copolymers.
[0102] The hydroxycarboxylic acids of the general formula [II] can
be used alone or in combination.
[0103] The preferred proportions of glycolic acid and
hydroxycarboxylic acid [II] in said glycolic acid copolymer (A) are
about 10 to about 75 mole % of glycolic acid and the balance of
hydroxycarboxylic acid. More desirably, the copolymer consists of
about 20 to about 75 mole % of glycolic acid and the balance of
hydroxycarboxylic acid. Most desirably, the copolymer consists of
about 40 to about 70 mole % of glycolic acid and the balance of
hydroxycarboxylic acid. The weight average molecular weight of said
glycolic acid copolymer may range from about 2,000 to about 50,000.
The preferred range is about 3,000 to about 40,000. The more
preferred range is about 8,000 to about 30000. The dispersion value
(weight average molecular weight/number average molecular weight)
is preferably in the range of about 1.2 to about 4.0. Particularly
preferred are copolymers with dispersion values in the range of
about 1.5 to about 3.5.
[0104] The glycolic acid copolymer (A) can be synthesized by the
known technology, for example by the process described in Japanese
laid-open patent application 28521/1986 specification.
[0105] Polylactic acid for use in the present invention may be
whichever of L- and D-compounds and any mixture thereof. Preferred
is a species with a D-/L-ratio (mole %) in the range of about 75/25
through about 20/80. The more preferred D-/L-ratio (mole %) of
polylactic acid is about 60/40 through about 25/75. The most
advantageous D/L-ratio (mole %) of polylactic acid is about 55/45
through about 25/75. The weight average molecular weight of
polylactic acid is preferably in the range of about 1,500 to about
30,000, more preferably about 2,000 to about 20,000 and still more
preferably about 3,000 to about 15,000. The dispersion value of
polylactic acid is preferably about 1.2 to about 4.0 and more
desirably about 1.5 to about 3.5.
[0106] Polylactic acid can be synthesized by two known alternative
processes, namely a process involving a ring-opening polymerization
of lactide which is a dimer of lactic acid and a process involving
a dehydrative polycondensation of lactic acid. For the production
of a polylactic acid of comparatively low molecular weight for use
in the present invention, the process involving a direct
dehydrative polycondensation of lactic acid is preferred. This
process is described in, for example, Japanese laid-open patent
application 28521/1986.
[0107] In the pharmaceutical base for use in the present invention,
the glycolic acid copolymer (A) and polylactic acid (B) are used in
an (A)/(B) ratio (by weight) of about 10/90 through about 90/10.
The preferred blend ratio is about 20/80 through about 80/20. The
most desirable ratio is about 30/70 through about 70/30. If the
proportion of either (A) or (B) is too large, the final preparation
will show a drug release pattern not much different from the
pattern obtained when (A) or (B) alone is used, that is to say the
linear release pattern in a late stage of release which is
obtainable with the mixed base cannot be obtained. The degradation
and elimination rates of glycolic acid copolymer and polylactic
acid vary considerably with their molecular weights and composition
but generally speaking, since the decomposition and elimination
rates of glycolic acid copolymer are relatively higher, the period
of release can be prolonged by increasing the molecular weight of
polylactic acid or reducing the blend ratio (A)/(B). Conversely,
the duration of release may be shortened by reducing the molecular
weight of polylactic acid or increasing the (A)/(B) blend ratio.
Furthermore, the duration of release can be adjusted by changing
the species or relative amount of hydroxycarboxylic acid of general
formula [II].
[0108] When a copolymer of lactic acid and glycolic acid is used as
the biodegradable polymer, its polymerization ratio (lactic
acid/glycolic acid) (mole %) is preferably about 100/0 to about
40/60. The more preferred ratio is about 90/10 to about 50/50.
[0109] The weight average molecular weight of said copolymer is
preferably about 5,000 to about 25,000. The more preferred range is
about 7,000 to about 20,000.
[0110] The degree of dispersion (weight average molecular
weight/number average molecular weight) of said copolymer is
preferably about 1.2 to about 4.0. The more preferred range is
about 1.5 to about 3.5.
[0111] The above-mentioned copolymer of lactic acid and glycolic
acid can be synthesized by the known technology, for example by the
process described in Japanese laid-open patent application
28521/1986.
[0112] The decomposition and disappearance rate of a copolymer of
lactic acid and glycolic acid varies greatly with the composition
and molecular weight but generally speaking, the smaller the
glycolic acid fraction, the lower is the decomposition and
disappearance rate. Therefore, the duration of drug release can be
prolonged by reducing the glycolic acid fraction or increasing the
molecular weight. Conversely, the duration of release can be
diminished by increasing the glycolic acid fraction or reducing the
molecular weight. To provide a long-term (e.g. 1.about.4 months)
sustained-release preparation, it is preferable to use a copolymer
of lactic acid and glycolic acid with a polymerization ratio within
the above-mentioned range and a weight average molecular weight
within the above-mentioned range. With a copolymer of lactic acid
and glycolic acid having a higher decomposition rate than that
within the above ranges for polymerization ratio and weight average
molecular weight, it is difficult to control the initial burst. On
the contrary, with a copolymer of lactic acid and glycolic acid
showing a lower decomposition rate than that within said ranges for
polymerization ratio and weight average molecular weight, periods
in which the drug will not be released in an effective amount tend
to occur.
[0113] In this specification, the weight average molecular weight
and the degree of dispersion mean the molecular weight in terms of
polystyrene as determined by gel permeation chromatography (GPC)
using 9 polystyrenes with the weight average molecular weights of
120,000, 52,000, 22,000, 9,200, 5,050, 2950, 1,050, 580 and 162 as
references and the dispersion value calculated using the same
molecular weight, respectively. The above determination was carried
out using GPC Column KF804 L.times.2 (Showa Denko), RI Monitor
L-3300 (Hitachi) and, as the mobile phase, chloroform.
[0114] The sustained-release preparation of the present invention
is produced by dissolving the peptide [I] and a biodegradable
polymer having a terminal carboxyl group in a solvent which is
substantially immiscible with water and then removing said
solvent.
[0115] The solvent which is substantially immiscible with water is
a solvent which is not only substantially immiscible with water and
capable of dissolving the biodegradable polymer but one which
renders the resultant 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.degree.
C.). The boiling point of such solvent is preferably not higher
than 120.degree. C. The solvent, thus, includes halogenated
hydrocarbons (e.g. dichloromethane, chloroform, chloroethane,
trichloroethane, carbon tetrachloride, etc.), alkyl ethers of 3 or
more carbon atoms (e.g. isopropyl ether etc.), fatty acid alkyl (of
4 or more carbon atoms) esters (e.g. butyl acetate etc.), aromatic
hydrocarbons (e.g. benzene, toluene, xylene, etc.) and so on. These
solvents can be used in a suitable combination of 2 or more
species. The more preferred solvents are halogenated hydrocarbons
(e.g. dichloromethane, chloroform, chloroethane, trichloroethane,
carbon tetrachloride, etc.). The most preferred is
dichloromethane.
[0116] Removal of the solvent can be effected by the per se known
procedures. For example, the method comprising evaporating the
solvent at atmospheric pressure or under gradual decompression with
constant stirring by means of a propeller mixer or a magnetic
stirrer or the method comprising evaporating the solvent under
controlled vacuum in a rotary evaporator can be employed.
[0117] Referring to the method of the invention for the production
of the sustained-release preparation, dissolving the peptide [I]
and a biodegradable polymer with a terminal carboxyl group means
achieving a condition such that the resultant solution shows no
visually observable residue of undissolved peptide at ordinary
temperature (20.degree. C.). In this ternary system consisting of
the peptide [I], biodegradable polymer and solvent, the amount of
peptide which can be dissolved depends on the number of a terminal
carboxyl groups per unit weight of the biodegradable polymer. In
case the peptide and the terminal carboxyl group interact in the
ratio of 1 to 1, the same molar amount of the peptide as that of
the terminal carboxyl group can be dissolved in theory. Therefore,
generalization is difficult according to the combination of the
solvent and the molecular weight of the peptide and the
biodegradable polymer. However, in producing sustained-release
preparations, the peptide may be dissolved in the range of about
0.1 to about 100% (w/w), preferably about 1 to about 70% (w/w),
most preferably about 2 to about 50% (w/w), with respect to the
biodegradable polymer which is dissolved in the solvent.
[0118] The present invention is further related to a method of
producing a sustained-release preparation which comprises
dissolving a biodegradable polymer comprising a mixture of (A) a
copolymer of glycolic acid and a hydroxycarboxylic acid of the
general formula 12
[0119] wherein R represents an alkyl group of 2 to 8 carbon atoms
and (B) a polylactic acid and a substantially water-insoluble
physiologically active peptide or a salt thereof in a solvent which
is substantially immiscible with water and then removing said
solvent.
[0120] The substantially water-insoluble physiologically active
peptide is not limited and includes naturally-occurring, synthetic
and semi-synthetic peptides. Preferred are physiologically active
peptides containing one or more aromatic groups (e.g. groups
derived from benzene, naphthalene, phenanthrene, anthracene,
pyridine, pyrole, indole, etc.) in side chains thereof. More
preferred physiologically active peptides are those having 2 or
more aromatic groups in side chains thereof. Particularly preferred
physiologically active peptides are those having 3 or more aromatic
groups in side chains thereof. These aromatic groups may be further
substituted.
[0121] The substantially water-insoluble physiologically active
peptide for use in the present invention is preferably a peptide
showing a solubility of not more than 1% in water, consisting of
two or more amino acids and having a molecular weight of about 200
to 30000. The molecular weight range is more preferably about 300
to 20000 and still more preferably about 500 to 10000.
[0122] As examples of said physiologically active peptide may be
mentioned luteinizing hormone releasing hormone (LH-RH) antagonists
(cf. U.S. Pat. No. 4,086,219, No. 4,124,577, No. 4,253,997 and No.
4,317,815, etc.), in-sulin, somatostatin, somatostatin derivatives
(cf. US Pat. No. 4,087,390, No. 4,093,574, No. 4,100,117, No.
4,253,998, etc.), growth hormone, prolactin, adreno-corticotropic
hormone (ACTH), melanocyte stimulating hormone (MSH), salts and
derivatives of thyroid hormone releasing hormone (cf. JP Kokai
S-50-121273 and S-52-11646), thyroid stimulating hormone (TSH),
luteinizing hormone (LH), follicle stimulating hormone (FSH),
vaso-pressin, vasopressin derivatives, oxytocin, calcitonin,
gastrin, secretin, pancreozymin, cholecystokinin, angiotensin,
human placental lactogen, human chorionic gonadotropin (HCG),
enkepharin, enkephalin derivatives (cf. U.S. Pat. No. 4,277,394,
EP-A No. 31,567), endorphin, kyotrphin, tuftsin, thymopoietin,
thymosin, thymostimulin, thymic humoral factor (THF), facteur
thymique serique (FTS) and its derivatives (cf. U.S. Pat. No.
4,229,438), other thymic factors, tumor necrosis factor (TNF),
colony stimulating factor (CSF), motilin, dynorphin, bombesin,
neurotensin, cerulein, bradykinin, atrial natruretic factor, nerve
growth factor, cell growth factor, neurotrophic factor, peptides
having endothelin antagonistic activity (cf. EP-A No. 436189, No.
457195 and No. 496452, JP Kokai H-3-94692 and 03-130299) and
fragments or derivatives of these physiologically active
peptides.
[0123] Specific examples of the physiologically active peptide are
physiologically active peptides and salts which are antagonists of
luteinizing hormone releasing hormone (LH-RH) and useful for the
treatment of hormone-dependent diseases such as prostatic cancer,
prostatic hypertrophy, endometriosis, uterine myoma, precocious
puberty, breast cancer, etc. and for contraception.
[0124] The physiologically active peptide for use in the present
invention can be in the form of a salt, preferably a
pharmacologically acceptable salt. Where said peptide has a basic
group such as amino, the salt mentioned above may for example be
the salt formed with an inorganic acid (e.g. hydrochloric acid,
sulfuric acid, nitric acid, etc.) or an organic acid (e.g. carbonic
acid, hydrogencarbonic acid, succinic acid, acetic acid, propionic
acid, trifluoroacetic acid, etc.). Where the peptide has an acidic
group such as carboxyl, the salt may for example be the salt formed
with an inorganic base (e.g. alkali metals such as sodium,
potassium, etc. and alkaline earth metals such as calcium,
magnesium, etc.) or an organic base (e.g. organic amines such as
triethylamine etc. and basic amino acids such as arginine etc.).
The peptide may further be in the form of a metal complex compound
(e.g. copper complex, zinc complex, etc.).
[0125] Specific examples of the physiologically active peptide or
salt thereof are found in U.S. 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.
[0126] Furthermore, the physiologically active peptides of general
formula [I] and salts thereof can also be mentioned, among
others.
[0127] Moreover, even when the physiologically active peptide is
water-soluble, it can be converted to a derivative compound which
is insoluble 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 process of the invention.
[0128] The amount of said physiologically active peptide in the
preparations of the present invention depends on the species of
peptide, expected pharmacologic effect and desired duration of
effect and so on. Generally, however, it is used in a proportion of
about 0.001 to 50% (w/w), preferably about 0.01 to 40% (w/w), more
preferably about 0.1 to 30% (w/w), relative to the biodegradable
polymer base.
[0129] The solvent employed in the method is the same as described
above.
[0130] Removal of the solvent can be carried out in the same manner
as described above.
[0131] The preferred process for the production of the
sustataind-release preparation of the present invention is a
microencapsulating process utilizing the drying-in-water technique
or the phase separation technique, which is described below, or any
process analogous thereto.
[0132] The process described below may be carried out with peptide
[I] or with a substantially water-insoluble physiologically active
peptide which includes peptide [I].
[0133] Thus, the peptide [I] is added to a solution of the
biodegradable polymer in an organic solvent in the final weight
ratio mentioned hereinbefore for such peptide to prepare an organic
solvent solution containing the peptide [I] and biodegradable
polymer. In this connection, the concentration of the biodegradable
polymer in the organic solvent varies according to the molecular
weight of the biodegradable polymer and the type of organic solvent
but is generally selected from the range of about 0.01 to about 80%
(w/w). The preferred range is about 0.1 to about 70% (w/w). The
still more preferred range is about 1 to about 60% (w/w).
[0134] Then, this organic solvent solution containing the peptide
[I] and biodegradable polymer (oil phase) is added to a water phase
to prepare an O(oil phase)/W (water phase) emulsion. The solvent of
the oil phase is then evaporated off to provide microcapsules. The
volume of the water phase for this procedure is generally selected
from the range of about 1 to about 10000 times the volume of the
oil phase. The preferred range is about 2 to about 5000 times and
the still more preferred range is about 5 to about 2000 times.
[0135] An emulsifier may be added to the above water phase. The
emulsifier may generally be any substance that contributes to the
formation of a stable O/W emulsion. Thus, there can be mentioned
anionic surfactants (sodium oleate, sodium stearate, sodium lauryl
sulfate, etc.), nonionic surfactants (polyoxyethylene-sorbitan
fatty acid esters [Tween 80 and Tween 60, Atlas Powder],
polyoxyethylene-castor oil derivatives [HCO-60 and HCO-50, Nikko
Chemicals], etc.), polyvinylpyrrolidone, polyvinyl alcohol,
carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and so
on. These emulsifiers can be used independently or in combination.
The concentration may be selected from the range of about 0.001 to
about 20% (w/w). The preferred range is about 0.01 to about 10%
(w/w) and the still more preferred range is about 0.05 to about 5%
(w/w).
[0136] The resultant microcapsules are recovered by centrifugation
or filtration and washed with several portions of distilled water
to remove the free peptide, vehicle and emulsifier 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 residual water and organic
solvent from within the microcapsules. Preferably, this procedure
is carried out by heating the microcapsule at a temperature
somewhat (5.degree. C. or more) above the median glass transition
temperature of the biodegradable polymer as determined with a
differential scanning calorimeter at temperature increments of 10
to 20.degree. C./min., generally for not more than 1 week or 2 to 3
days, preferably for not more than 24 hours, after the
microcapsules have reached the target temperature.
[0137] In the production of microcapsules by the phase separation
technique, a coacervation agent is gradually added to a solution of
said peptide [I] and biodegradable polymer in an organic solvent
with constant stirring so that the biodegradable polymer may
separate out and solidify. This coacervation agent is added in a
volume of about 0.01 to about 1000 times the volume of the organic
solvent solution of peptide [I] and biodegradable polymer. The
preferred range is about 0.05 to about 500 times and the still more
preferred range is about 0.1 to about 200 times.
[0138] The coacervation agent should be a compound of polymer,
mineral oil or vegetable oil type which is miscible with the
solvent for the biodegradable polymer yet which does not dissolve
the polymer. Specifically, silicone oil, sesame oil, soybean oil,
corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil,
n-hexane, n-heptane, etc. can be mentioned. These substances can be
used in combination.
[0139] The resultant microcapsules are recovered by filtration and
washed repeatedly with heptane or the like to remove the
coacervation agent. Then, the free peptide and solvent are removed
by the same procedure as described for the drying-in-water
technique.
[0140] In the drying-in-water technique or in the coacervation
technique, an aggregation inhibitor may be added so as to prevent
aggregation of particles. The aggregation inhibitor includes
water-soluble polysaccharides such as mannitol, lactose, 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 so on.
[0141] In the production of microcapsules by the spray drying
technique, said organic solvent solution of peptide [I] and
biodegradable polymer is ejected in a mist form through a nozzle
into the drying chamber of a spray drier to evaporate the organic
solvent from the finely-divided liquid droplets in a brief time to
provide fine microcapsules. The nozzle may be a two-fluid nozzle,
pressure nozzle, rotary disk nozzle and so on. It is advantageous
to the process to spray an aqueous-solution of said aggregation
inhibitor from another nozzle for the prevention of intercapsule
aggregation in timed coordination with said spray of the organic
solvent solution of peptide [I] and biodegradable polymer.
[0142] If necessary, the residual water and organic solvent are
removed by heating the resultant microcapsules under reduced
pressure in the same manner as described hereinbefore.
[0143] The microcapsules can be administered as they are or as
processed into various pharmaceutical preparations for
administration by routes other than peroral (e.g. intramuscular,
subcutaneous and intraorgan injections or implants, nasal, rectal
or uterine transmucosal delivery systems, and so on) or for 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 and so on).
[0144] To process the microcapsules for injection, for instance,
the microcapsules can be formulated with a dispersant (e.g. a
surfactant such as Tween 80, HCO-60, etc., carboxymethylcellulose,
a polysaccharide such as sodium alginate, etc.), a preservative
(e.g. methylparaben, propylparaben, etc.), or an isotonizing agent
(e.g. sodium chloride, mannitol, sorbitol, glucose, etc.) to
prepare an aqueous suspension or they may be dispersed in a
vegetable oil such as sesame oil, corn oil or the like to provide
an oil suspension for use as a controlled release injection.
[0145] The particle size of the microcapsules for such injectable
suspensions need only be in the range satisfying the dispersibility
and needle passage requirements and may for example range from
about 0.1 to about 500 .mu.m. The preferred particle size range is
about 1 to about 300 .mu.m and the still more preferred range is
about 2 to about 200 .mu.m.
[0146] For providing the microcapsules as a sterile product, the
whole production process is subjected to sterility control, the
microcapsules are sterilized by gamma-ray irradiation or a
preservative is added, although these are not exclusive
procedures.
[0147] Aside from the above-mentioned microcapsules, a
biodegradable polymer composition containing the active ingredient
peptide well dispersed by a suitable technique can be melted and
molded into a spherical, bar-shaped, needle-shaped, pelletized or
film shape to provide a sustained-release preparation of the
present invention. The above biodegradable polymer composition can
be produced by the method described in JP Publication S-50-17525.
To be specific, the peptide drug and the polymer are dissolved in a
solvent and the solvent is then removed by a suitable method (e.g.
spray drying, flash evaporation, etc.) to provide the desired
biodegradable polymer composition.
[0148] The sustained-release preparation of the present invention
can be administered as an intramuscular, subcutaneous or intraorgan
injection or implant, a transmucosal delivery system for
application to the nasal cavity, rectum or uterus, or an oral
preparation (e.g. 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.).
[0149] The sustained-release preparation of the present, invention
has low toxicity and can be used safely in mammalian animals (e.g.
man, bovine, swine, canine, feline, murine, rat and rabbit).
[0150] The dosage of the sustained-release preparation is dependent
on the type and content of the active drug peptide, final dosage
form, the duration of release of the peptide, the object of
treatment (such as hormone-dependent diseases, e.g. prostatic
cancer, prostatomegaly, endometriosis, metrofibroma, precocious
puberty, mammary cancer, etc., or for contraception) and the
subject animal species, but in any case it is necessary that an
effective amount of peptide is successfully delivered. The unit
dosage of the active drug peptide, taking a one-month delivery
system as an example, can be selected advantageously from the range
of about 0.01 to about 100 mg/kg body weight for an adult human.
The preferred range is about 0.05 to about 50 mg/kg body weight.
The most preferred range is about 0.1 to about 10 mg/kg body
weight.
[0151] The unit dosage of the sustained-release preparation per
adult human can therefore be selected from the range of about 0.1
to about 500 mg/kg body weight. The preferred range is about 0.2 to
about 300 mg/kg body weight. The frequency of administration may
range from once in a few weeks, monthly or once in a few months,
for instance, and can be selected according to the type and content
of the active drug peptide, final dosage form, designed duration of
release of the peptide, the disease to be managed and the subject
animal.
[0152] The following reference and working examples are intended to
describe the invention in further detail and should by no means be
construed as defining the scope of the invention. (Unless otherwise
specified, % means % by weight).
[0153] Abbreviations used hereinafter have the following
definitions:
[0154] BOC: tert-butoxycarbonyl
[0155] FMOC: 9-fluorenylmethoxycarbonyl
[0156] Cbz: Benzyloxycarbonyl
REFERENCE EXAMPLE 1
[0157] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 300 g of 90% aqueous solution
of D,L-lactic acid and 100 g of 90aqueous solution of L-lactic acid
and the charge was heated under reduced pressure in a nitrogen gas
stream from 100.degree. C./500 mmHg to 150.degree. C./30 mmHg over
a period of 4 hours, with the distillate water being constantly
removed. The reaction mixture was further heated under reduced
pressure at 3-5 mmHg/150-180.degree. C. for 7 hours, after which it
was cooled to provide an amber-colored polylactic acid.
[0158] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 30.degree. C.
[0159] The weight average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the above
polylactic acid were 3,000; 1,790; and 1,297, respectively.
[0160] These data showed that the polymer had terminal carboxyl
groups.
REFERENCE EXAMPLE 2
[0161] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 500 g of 90% aqueous solution
of D,L-lactic acid and the charge was heated under reduced pressure
in a nitrogen gas stream from 100.degree. C./500 mmHg to
150.degree. C./30 mmHg for a period of 4 hours, with the distillate
water being constantly removed. The reaction mixture was further
heated under reduced pressure at 3-5 mmHg/150-180.degree. C. for 12
hours, after which it was cooled to provide an amber-colored
polylactic acid.
[0162] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 30.degree. C.
[0163] The weight average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the above
polylactic acid was 5,000; 2,561; and 1,830, respectively.
[0164] These data showed that the polymer had terminal carboxyl
groups.
REFERENCE EXAMPLE 3
[0165] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condensor was charged with 300 g of 90% aqueous solution
of D,L-lactic acid and 100 g of 90% aqueous solution of L-lactic
acid and the charge was heated under reduced pressure in a nitrogen
gas stream from 100.degree. C./500 mmHg to 150.degree. C./30 mmHg
for a period of 5 hours, with the distillate water being constantly
removed. The reaction mixture was further heated under reduced
pressure at 5-7 mmHg/150-180.degree. C. for 18 hours, after which
it was cooled to provide an amber-colored polylactic acid.
[0166] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 30.degree. C.
[0167] The weight average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the above
polylactic acid was 7,500 ; 3,563; and 2,301, respectively.
[0168] These data showed that the polymer had terminal carboxyl
groups.
REFERENCE EXAMPLE 4
[0169] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 300 g of 90% aqueous solution
of D,L-lactic acid and 100 g of 90% aqueous solution of L-lactic
acid and the charge was heated under reduced pressure in a nitrogen
gas stream from 100.degree. C./500 mmHg to 150.degree. C./30 mmHg
for a period of 5 hours, with the distillate water being constantly
removed. The reaction mixture was further heated under reduced
pressure at 5-7 mmHg/150-180.degree. C. for 26 hours, after which
it was cooled to provide an amber-colored polylactic acid.
[0170] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 30.degree. C.
[0171] The weight average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the above
polylactic acid was 9,000; 3,803; and 2,800, respectively.
[0172] These data showed that the polymer had terminal carboxyl
groups.
REFERENCE EXAMPLE 5
[0173] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 182.5 g of glycolic acid and
166.6 g of D,L-2-hydroxybutyric acid and the charge was heated
under reduced pressure in a nitrogen gas stream from 100.degree.
C./500 mmHg to 150.degree. C./30 mmHg for a period of 3.5 hours,
with the distillate water being constantly removed. The reaction
mixture was further heated under reduced pressure at 5-7
mmHg/150-180.degree. C. for 26 hours, after which it was cooled to
provide an amber-colored glycolic acid-2-hydroxybutyric acid
copolymer.
[0174] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 25.degree. C.
[0175] The weight average molecular weight, as determined by GPC,
of the resulting glycolic acid-2-hydroxybutyric acid copolymer was
13,000.
REFERENCE EXAMPLE 6
[0176] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 197.7 g of glycolic acid and
145.8 g of D,L-2-hydroxybutyric acid and the charge was heated
under reduced pressure in a nitrogen gas stream from 100.degree.
C./500 mmHg to 155.degree. C./30 mmHg for a period of 4 hours, with
the distillate water being constantly removed. The reaction mixture
was further heated under reduced pressure at 3-6
mmHg/150-185.degree. C. for 27 hours, after which it was cooled to
provide an amber-colored glycolic acid-2-hydroxybutyric acid
copolymer.
[0177] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 25.degree. C.
[0178] The weight average molecular weight, as determined by GPC,
of the resulting glycolic acid-2-hydroxybutyric acid copolymer was
13,000.
REFERENCE EXAMPLE 7
[0179] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condensor was charged with 212.9 g of glycolic acid and
124.9 g of D,L-2-hydroxybutyric acid and the charge was heated
under reduced pressure in a nitrogen gas stream from 100.degree.
C./500 mmHg to 160.degree. C./30 mmHg for a period of 3.5 hours,
with the distillate water being constantly removed. The reaction
mixture was further heated under reduced pressure at 3-6
mmHg/160-180.degree. C. for 27 hours, after which it was cooled to
provide an amber-colored glycolic acid-2-hydroxybutyric acid
copolymer.
[0180] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 25.degree. C.
[0181] The weight average molecular weight, as determined by GPC,
of the resulting glycolic acid-2-hydroxybutyric acid copolymer was
11,000.
REFERENCE EXAMPLE 8
[0182] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condensor was charged with 300 g of 90% aqueous solution
of D,L-lactic acid and 100 g of 90% aqueous solution of L-lactic
acid and the charge was heated under reduced pressure in a nitrogen
gas stream from 100.degree. C./500 mmHg to 150.degree. C./30 mmHg
for a period of 4 hour with the distillate water being constantly
removed. The reaction mixture was further heated under reduced
pressure at 3-5 mmHg and 150-180.degree. C. for 10 hours, after
which it was cooled to provide an amber-colored polylactic
acid.
[0183] This polymer was dissolved in 1,000 ml of dichloromethane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 3020 C.
[0184] The weight-average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the above
polylactic acid was 4,200;. 2,192; and 1,572, respectively.
[0185] These data showed that the polymer had terminal carboxyl
groups.
REFERENCE EXAMPLE 9
[0186] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 182.5 g of glycolic acid and
166.6 g of D,L-2-hydroxybutyric acid and the charge was heated
under reduced pressure in a nitrogen gas stream from 100.degree.
C./500 mmHg to 150.degree. C./30 mmHg for a period of 3.5-hour,
with the distillate water being constantly removed. The reaction
mixture was further heated under reduced pressure at 5-7 mmHg and
150-180.degree. C. for 32 hours, after which it was cooled to
provide an amber-colored glycolic acid-2-hydroxybutyric acid
copolymer.
[0187] The polymer was dissolved in 1,000 ml of dichloromethane and
the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in a vacuo at 25.degree. C.
[0188] The weight-average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the
resulting glycolic acid-2-hydroxybutyric acid copolymer was 16,300;
5,620; and 2,904, respectively.
[0189] These data showed that the polymer had terminal carboxyl
groups.
REFERENCE EXAMPLE 10
[0190] Synthesis of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys
(AzaglyFur)-Leu-Lys (Nisp) -Pro-DAlaNH.sub.2
[0191] Reference Examples 10 and 11 were carried out in accordance
with the description of U.S. Pat. No. 5,110,904 and U.S. patent
application No. 07/987,921.
[0192] To the reactor of a peptide synthesizer was added 1 g of
D-Ala-NH-resin (4-methyl-benzohydrylamine resin), followed by
sequential additions of amino acids per the following synthesis
procedure, to synthesize the title peptide.
[0193] 1. Deprotecting reaction
[0194] To remove the protecting BOC group from the peptide's alpha
amino acid, a solution consisting of 45% trifluoroacetic acid
(hereinafter also referred to as TFA), 2.5% anisole, 2.0% dimethyl
phosphite and 50.5% dichloromethane was used. After the resin was
pre-washed with the solution for 1 minute, a deprotecting reaction
was conducted for 20 minutes.
[0195] 2. Washing with basic solution
[0196] To remove and neutralize the trifluoroacetic acid used for
deprotection, a dichloromethane solution containing 10%
N,N'-diisopropylethylamine was used. The resin was washed for 1
minute three times for each deprotecting reaction.
[0197] 3. Coupling reaction
[0198] A coupling reaction was carried out, using as activators a
3-fold molar amount of 0.3 M
diisopropylcarbodiimide/dichloromethane solution and a 3-fold molar
amount of 0.3 M BOC amino acid derivative/DMF
(N,N'-dimethylformamide) solution. The activated amino acid was
coupled to the free alpha amino group of the peptide on the resin.
Reaction times are shown below.
[0199] 4. Washing
[0200] After completion of every reaction process, the resin was
washed with dichloromethane, dichloromethane/DMF and DMF, each for
1 minute.
[0201] Synthesis protocol
[0202] Amino-group-protected amino acids were coupled to the resin
in the order, frequency and time shown below.
1 Order Amino acid Frequency time 1 BOC-Pro 2 times 1 hour 2
BOC-Lys(N-epsilon- 2 times 1 hour Cbz, isopropyl) 3 BOC-Leu 2 times
1 hour 4 BOC-D-Lys 2 times 1 hour (N-epsilon-FMOC) 5 BOC-NMeTyr 2
times 1 hour (O-2, 6-diCl-Bzl) 6 BOC-Ser(OBzl) 2 times 1 hour 7
BOC-D-3Pal 2 times 6 hours 8 BOC-D-4ClPhe 2 times 2 hours 9
BOC-D2Nal 2 times 2 hours 10 Acetic acid 2 times 2 hours
[0203] After completion of the synthesis reaction, the resin was
treated with a 30% piperidine solution in DMF for 4 to 24 hours to
remove the protecting FMOC group. The resin was washed with
dichloromethane several times and then reacted with
carbonyldiimidazole (0.9 g) dissolved in DMF (18 ml) for 15 minutes
and washed with dichloromethane three times, after which it was
reacted overnight with 2-furoic hydrazide (0.53 g) dissolved in DMF
(18 ml). The resulting peptide-resin was washed with
dichloromethane three times and then dried in the presence of
phosphorus pentoxide overnight, after which it was treated with dry
hydrogen fluoride at 0.degree. C. for 1 hour in the presence of
anisole to cut the peptide from the resin. The excess reaction
reagent was removed under vacuum conditions. The thus-obtained
resin was first washed with ether, then stirred at room temperature
for 15 minutes in 50 ml of a water/acetonitrile/acetic acid mixture
(1:1:0.1) and filtered. The filtrate was lyophilized to yield an
unpurified peptide as a fluffy powder. This peptide was purified by
high performance liquid chromatography (HPLC) under the following
conditions.
[0204] (1) Column: Dynamax C-18 (25.times.2.5cm, 8 microns)
[0205] (2) Solvent: Acetonitrile ascending gradient over a
20-minute period from 89% water/11% acetonitrile/0.1% TFA
[0206] (3) Detection wavelength: 260 nm (UV method)
[0207] The peptide detected as a single peak at 25.7 minutes
retention time was collected and lyophilized to yield a purified
product of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys
(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAla- NH.sub.2 as a trifluoroacetate.
Physical property data on the purified product are as follows:
[0208] FAB (fast atom bombardment, the same applies below) mass
spectrometry: m/e 1591 (M+H).sup.+
[0209] Amino acid analysis: 0.98 Ala, 1.02 Pro, 1.58 Lys, 1.00 Leu,
1.12 NMeTyr, 0.52 Ser
[0210] The above trifluoroacetate of peptide was converted to an
acetate, using a gel filtration column previously equilibrated with
1 N acetic acid. Gel filtration conditions are as follows:
[0211] (1) Packing: Sephadex G-25 (column inside diameter 16 mm,
packing bed height 40 mm)
[0212] (2) Solvent: 1 N acetic acid
[0213] (3) Detection wavelength: 254 nm (UV method)
[0214] The fraction of the first eluted peak was collected and
lyophilized to yield a purified product of
NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur)-Leu-Lys
(Nisp)-Pro-DAlaNH.sub.2 as an acetate.
REFERENCE EXAMPLE 11
[0215] Synthesis of NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys
(AzaglyNic)-Leu-Lys(Nisp)-Pro-DAlaNH.sub.2
[0216] The title peptide was synthesized in the same manner as in
Reference Example 10, except that 2-furoic hydrazide was replaced
with 2-nicotinic hydrazide (0.575 g). The HPLC retention time of
the purified product thus obtained was 16.0 minutes. Physical
property data on the purified product are as follows:
[0217] FAB mass spectrometry: m/e 1592 (M+H).sup.-
[0218] Amino acid analysis: 1.02 Ala, 1.01 Pro, 1.61 Lys, 0.99 Leu,
1.12 NMeTyr, 0.48 Ser
[0219] The above trifluoroacetate of peptide was converted to an
acetate in the same manner as in Reference Example 10.
REFERENCE EXAMPLE 12
[0220] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 322 g of 90% aqueous solution
of D,L-lactic acid and 133 g of glycolic acid and using a mantle
heater (So-go Rikagaku Glass Co.), the charge was heated under
reduced pressure in a nitrogen stream from 100.degree. C./500 mmHg
to 150.degree. C./30 mmHg for a period of 4 hours the distillate
water being constantly removed. The reaction mixture was further
heated under reduced pressure at 3-30 mmHg/150-185.degree. C. for
23 hours, after which it was cooled to provide a lactic
acid-glycolic acid copolymer.
[0221] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 30.degree. C.
[0222] The weight average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the
resultant lactic acid-glycolic acid copolymer were 10,000; 4,000;
and 4,000, respectively. These data showed that the copolymer was a
polymer having terminal carboxyl groups.
REFERENCE EXAMPLE 13
[0223] A 1000 ml four-necked flask equipped with a nitrogen inlet
pipe and condenser was charged with 347 g of 90% aqueous solution
of D,L-lactic acid and 266 g of glycolic acid and using a mantle
heater (So-go Rikagaku Glass Co.), the charge was heated under
reduced pressure in a nitrogen stream from 100.degree. C./500 mmHg
to 150.degree. C./30 mmHg for a period of 5 hours, with the
distillate water being constantly removed. The reaction mixture was
further heated under reduced pressure at 3-30 mmHg/150 -185.degree.
C. for 23 hours, after which it was cooled to provide a lactic
acid-glycolic acid copolymer.
[0224] This polymer was dissolved in 1000 ml of dichloro-methane
and the solution was poured in warm water at 60.degree. C. with
constant stirring. The resulting pasty polymeric precipitates were
collected and dried in vacuo at 30.degree. C.
[0225] The weight average molecular weight and number average
molecular weight, as determined by GPC, and the number average
molecular weight, as found by end-group determination, of the
resultant lactic acid-glycolic acid copolymer were 10,000; 3,700;
and 3,900, respectively. These data showed that the copolymer was a
polymer having terminal carboxyl groups.
EXAMPLE 1
[0226] NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (Nic)-Leu-Lys
(Nisp)-Pro-DAlaNH.sub.2 (manufactured by TAP; hereinafter referred
to briefly as physiologically active peptide A) acetate, 200 mg,
was dissolved in a solution of a 50:50 mixture (3.8 g) of the
glycolic acid-2-hydroxybutyric acid copolymer obtained in Reference
Example 5 and the polylactic acid obtained in Reference Example 1
in 5.3 g (4.0 ml) of dichloromethane. The resulting solution was
cooled to 17.degree. 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.degree. C.
and the mixture was emulsified using a turbine homomixer at 7000
rpm to prepare an O/W emulsion. This O/W emulsion was stirred at
room temperature for 3 hours to evaporate the dichloromethane. The
oil phase was solidified and collected with a centrifuge (05PR-22,
Hitachi, Ltd.) at 2000 rpm. This solid was redispersed in distilled
water and further centrifuged to wash off the free drug etc. The
collected microcapsules were redispersed in a small quantity of
distilled water, followed by addition of 0.3 g of D-mannitol and
freeze-drying to provide a powder. The particle size distribution
and physiologically active peptide A content of the microcapsules
were 5 to 60 .mu.m and 4.7% (w/w), respectively.
[0227] Preparations of the following physiologically active
peptides (1) and (2) were manufactured in the same manner as
above.
[0228] (1) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys
(AzaglyNic)-Leu-Lys (Nisp)-Pro-DAlaNH.sub.2
[0229] (2) NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys
(AzaglyFur)-Leu-Lys (Nisp)-Pro-DAlaNH.sub.2
EXAMPLE 2
[0230] In a solution of a 50:50 mixture (3.8 g) of the glycolic
acid-2-hydroxybutyric acid copolymer obtained in Reference Example
5 and the polylactic acid obtained in Reference Example 2 in 6.7 g
(5.0 ml) of dichloromethane was dissolved 200 mg of physiologically
active peptide A acetate. This solution was cooled to 17.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 17.degree. C. and the mixture was
treated as in Example 1 to provide microcapsules. The particle size
distribution and physiologically active peptide A content of the
microcapsules were 5 to 65 .mu.m and 5.0% (w/w), respectively.
EXAMPLE 3
[0231] In a solution of a 50:50 mixture (3.8 g) of the glycolic
acid-2-hydroxybutyric acid copolymer obtained in Reference Example
5 and the polylactic acid obtained in Reference Example 3 in 6.7 g
(5.0 ml) of dichloromethane was dissolved 200 mg of physiologically
active peptide A acetate. This solution was cooled to 17.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 17.degree. C. and the mixture was
treated as in Example 1 to provide microcapsules. The particle size
distribution and physiologically active peptide A content of the
microcapsules were 10 to 60 .mu.m and 4.8% (w/w), respectively.
EXAMPLE 4
[0232] In a solution of a 50:50 mixture (3.8 g) of the glycolic
acid-2-hydroxybutyric acid copolymer obtained in Reference Example
5 and the polylactic acid obtained in Reference Example 4 in 6.7 g
(5.0 ml) of dichloromethane was dissolved 200 mg of physiologically
active peptide A acetate. This solution was cooled to 17.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 17.degree. C. and the mixture was
treated as in Example 1 to provide microcapsules. The particle size
distribution and physiologically active peptide A content of the
microcapsules were 10 to 75 .mu.m and 4.6% (w/w), respectively.
EXAMPLE 5
[0233] In a solution of a 50:50 mixture (3.8 g) of the glycolic
acid-2-hydroxybutyric acid copolymer obtained in Reference Example
6 and the polylactic acid obtained in Reference Example 2 in 6.0 g
(4.5 ml) of dichloromethane was dissolved 200 mg of physiologically
active peptide A acetate. This solution was cooled to 17.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 10.degree. C. and the mixture was
treated as in Example 1 to provide microcapsules. The particle size
distribution and physiologically active peptide A content of the
microcapsules were 5 to 60 .mu.m and 4.9% (w/w), respectively.
EXAMPLE 6
[0234] In a solution of a 50:50 mixture (3.8 g) of the glycolic
acid-2-hydroxybutyric acid copolymer obtained in Reference Example
7 and the polylactic acid obtained in Reference Example 2 in 6.0 g
(4.5 ml) of dichloromethane was dissolved 200 mg of physiologically
active-peptide A acetate. This solution was cooled to 17.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 17.degree. C. and the mixture was
treated as in Example 1 to provide microcapsules. The particle size
distribution and physiologically active peptide A content of the
microcapsules were 10 to 65 .mu.m and 4.9% (w/w), respectively.
EXAMPLE 7
[0235] In a solution of a 50:50 mixture (3.6 g) of the glycolic
acid.cndot.2-hydroxybutyric acid copolymer obtained in Reference
Example 9 and the polylactic acid obtained in Reference Example 8
in 7.0 g (5.3 ml) of dichloromethane was dissolved 400 mg of
physiologically active peptide A acetate. This solution was cooled
to 17.degree. C. and poured into 1,000 ml of a 0.1% aqueous
solution of polyvinyl alcohol previously adjusted to 17.degree. C.
and the mixture was treated as in Example 1, to provide
microcapsules. The particle size distribution and physiologically
active peptide A content of the microcapsules were 5 to 65 .mu.m
and 7.2% (w/w), respectively.
EXAMPLE 8
[0236] 240 mg of the acetate of
NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys
(AzaglyNic)-Leu-Lys(Nisp)-Pro-DAlaNH.sub.2 (hereinafter referred to
briefly as physiologically active peptide B) obtained in Reference
Example 11 was dissolved in a solution of a 50:50 mixture (1.76 g)
of the glycolic acid.cndot.2-hydroxybutyric acid copolymer obtained
in Reference Example 9 and the polylactic acid obtained in
Reference Example 8 in 3.2 g (2.4 ml) of dichloromethane. The
resulting solution was cooled to 18.degree. C. and poured into 400
ml of a 0.1% aqueous solution of polyvinyl alcohol previously
adjusted to 16.degree. C. and the mixture was treated as in Example
1, to provide microcapsules. The particle size distribution and
physiologically active peptide B content of the microcapsules were
5 to 70 .mu.m and 10.3% (w/w), respectively.
EXAMPLE 9
[0237] 240 mg of the acetate of
NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys (AzaglyFur)-Leu-Lys
(Nisp)-Pro-DAlaNH.sub.2 (hereinafter referred to briefly as
physiologically active peptide C) obtained in Reference Example 10
was dissolved in a solution of a 50:50 mixture (1.76 g) of the
glycolic acid.cndot.2-hydroxybutyric acid copolymer obtained in
Reference Example 9 and the polylactic acid obtained in Reference
Example 8 in 3.2 g (2.4 ml) of dichloromethane. The resulting
solution was cooled to 18.degree. C. and poured into 400 ml of a
0.1% aqueous solution of polyvinyl alcohol previously adjusted to
16.degree. C. and the mixture was treated as in Example 1, to
provide microcapsules. The particle size distribution and
physiologically active peptide C content of the microcapsules were
5 to 65 .mu.m and 10.9% (w/w), respectively.
EXAMPLE 10
[0238]
N-Tetrahydrofur-2-oyl-Gly-D2Nal-D4clPhe-D3Pal-Ser-NMeTyr-Dlys
(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH.sub.2 (Manufactured by TAP;
hereinafter referred to briefly as physiologically activepeptide D)
acetate [FAB mass spectrometry: m/e 1647 (M+H).sup.+], 240 mg, was
dissolved in a solution of a 50:50 mixture (1.76 g) of the glycolic
acid-2-hydroxybutyric acid copolymer obtained in Reference Example
9 and the polylactic acid obtained in Reference Example 8 in 3.2 g
(2.4 ml) of dichloromethane. The resulting solution was cooled to
18.degree. C. and poured into 400 ml of a 0.1% aqueous solution of
polyvinyl alcohol previously adjusted to 16.degree. C. and the
mixture was treated as in Example 1 to provide microcapsules. The
particle size distribution and physiologically active peptide D
content of the microcapsules were 5 to 70 .mu.m and 10.5% (w/w),
respectively.
EXAMPLE 11
[0239] 200 mg of physiologically active peotide A acetate was added
and dissolved in a solution of a lactic acid-glycolic acid
copolymer (lactic acid/glycolic acid=75/25 (mole %), GPC weight
average mol. wt.=5,000, GPC number average mol. wt.=2,000, number
average mol. wt. by end-group determination =2,200; manufacturer;
Wako Pure Chemical (Lot. 920729)) in 5.3 g (4.0 ml) of
dichloromethane. The resulting solution was cooled to 17.degree. 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.degree. C. and the mixture was emulsified
using a turbine mixer at 7000 rpm to prepare an O/W emulsion. This
O/W emulsion was stirred at room temperature for 3 hours to
evaporate the dichloromethane. The oil phase was solidified and
collected with a centrifuge (05PR-22, Hitachi) at 2000 rpm. This
solid was redispersed in distilled water and further centrifuged to
wash off the free drug etc. The collected microcapsules were
redispersed in a small quantity of distilled water, followed by
addition of 0.3 g of D-mannitol and freeze-drying to provide a
powder. The particle size distribution and physiologically active
peptide A content of the microcapsules were 5 to 60 .mu.m and 4.7%
(w/w), respectively.
[0240] Sustained-release preparation of the following peptides (1)
and (2) are produced in the same manner as above.
[0241] (1) Physiologically active peptide B acetate
[0242] (2) Physiologically active peptide C acetate
EXAMPLE 12
[0243] 200 mg of physiologically active peptide A acetate was added
and dissolved in a solution of 3.8 g of a lactic acid-glycolic
copolymer (lactic acid/glycolic acid=75/25 (mole %), GPC weight
average mol. wt.=10,000, GPC number average mol. wt.=4,400, number
average mol. wt. by end-group determination=4,300; manufacturer;
Wako Pure Chemical (Lot. 880530)) in 6.7 g (5.0 ml) of
dichloromethane. The resulting solution was cooled to 17.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 11.degree. C. Thereafter, the
procedure of Example 11 was repeated to provide microcapsules. The
particle size distribution and physiologically active peptide A
content of the microcapsules were 5 to 65 .mu.m and 4.5% (w/w),
respectively.
EXAMPLE 13
[0244] 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.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 14.degree. C. Thereafter, the
procedure of Example 11 was repeated to provide microcapsules. The
particle size distribution and physiologically active peptide A
content of the microcapsules were 5 to 65 .mu.m and 8.2% (w/w),
respectively.
EXAMPLE 14
[0245] 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.degree. C.
and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl
alcohol previously adjusted to 15.degree. C. Thereafter, the
procedure of Example 11 was repeated to provide microcapsules. The
particle size distribution and physiologically active peptide A
content of the microcapsules were 5 to 65 .mu.m and 8.4% (w/w) ,
respectively.
EXAMPLE 15
[0246] Leuprolerin acetate (manufacturer: Takeda Chemical
Industries), 400 mg, was added to a solution of the same lactic
acid-glycolic acid copolymer as used in Example 12, 3.6 g, in 8.0 g
(60 ml) of dichloromethane to prepare a clear homogeneous solution.
The resulting solution was cooled to 15.degree. C. and poured into
1000 ml of a 0.1% aqueous solution of polyvinyl alcohol previously
adjusted to 15.degree. C. Thereafter, the procedure of Example 11
was repeated to provide microcapsules.
EXPERIMENTAL EXAMPLE 1
[0247] About 30 mg of the microcapsules obtained in Example 1 were
dispersed in a dispersion medium (a solution of 2.5 mg of
carboxymethylcellulose, 0.5 mg of polysorbate 80 and 25 mg of
mannitol in distilled water) and the dispersion was injected
subcutaneously in the back of 10-week-old male SD rats using a 22G
needle (the dosage of microcapsules was 60 mg/kg). Serially after
administration, the rats were sacrificed, the remnants of
microcapsules were taken out from the administration site and the
amount of the physiologically active peptide A in the microcapsules
was determined. The results are shown in Table 1.
EXPERIMENTAL EXAMPLES 2-6
[0248] Using the microcapsules obtained in Examples 2 to 6, the
residual amounts of the physiologically active peptide A in the
microcapsules were determined as in Experimental Example 1. The
results are also shown in Table 1.
2 TABLE 1 Residue of physiologically active peptide A (%) Day Week
Week Week Week Week Week Week 1 1 2 3 4 5 6 8 Experimental 88.0
66.5 42.3 15.2 Example 1 Experimental 92.8 76.6 62.6 48.7 38.6 26.5
Example 2 Experimental 96.5 90.5 77.5 64.9 59.2 46.9 38.7 20.3
Example 3 Experimental 99.4 94.5 87.2 76.3 66.0 -- 46.6 30.7
Example 4 Experimental 92.9 75.0 45.7 -- 17.5 Example 5
Experimental 92.3 61.3 33.5 6.4 Example 6
[0249] It is apparent from Table 1 that all the micro-capsules
according to the present invention are characterized by
substantially constant release of physiologically active peptide
and are further characterized by being substantially free from an
initial burst.
[0250] Table 2 shows the linear regression models, correlation
coefficients, and release periods calculated as X-intercept which
were determined by the procedures described in Methods of Bioassay
(authored by Akira Sakuma, Tokyo University Press, Jun. 5, 1978, p.
111).
3 TABLE 2 Weight average molecular weight of Linear Release
polylactic regression Correlation period acid model coefficient
(weeks) Experimental 3000 Residue (%) = (R.sup.2 = 3.5 Example 1
95.4 - (26.9 .times. no. 0.992) of weeks) Experimental 5000 Residue
(%) = (R.sup.2 = 6.6 Example 2 94.4 - (14.2 .times. no. 0.975) of
weeks) Experimental 7500 Residue (%) = (R.sup.2 = 9.8 Example 3
98.4 - (10.0 .times. no. 0.996) of weeks) Experimental 9000 Residue
(%) = (R.sup.2 = 11.5 Example 4 102.1 - (8.9 .times. no. 0.995) of
weeks)
[0251] It is apparent from Table 2 that by varying the weight
average molecular weight of polylactic acid to be blended with
glycolic acid-2-hydroxybutyric copolymer, the duration of release
can be freely controlled within the range of about 3.5 weeks to
about 11.5 weeks.
[0252] Table 3 shows the linear regression models, correlation
coefficients and release periods as X-intercept which were
determined from the data in Table 1 by the same procedures as used
in Table 2.
4TABLE 3 Mole fraction of glycolic acid in Linear Release glycolic
acid regression Correlation period copolymer model coefficient
(weeks) Experimental 60% Residue (%) = (R.sup.2 = 6.6 Example 2
94.4 - (14.2 .times. no. 0.975) of weeks) Experimental 65% Residue
(%) = (R.sup.2 = 4.6 Example 5 95.7 - (20.6 .times. no. 0.976) of
weeks) Experimental 70% Residue (%) = (R.sup.2 = 3.1 Example 6 96.6
- (30.9 .times.no. 0.994) of weeks)
[0253] It is apparent from Table 3 that by varying the mole
fraction of glycolic acid in the glycolic acid-2-hydroxybutyric
acid copolymer to be blended with polylactic acid, the duration of
release can be freely controlled within the range of about 3.1
weeks to about 6.6 weeks.
EXPERIMENTAL EXAMPLES 7 - 9
[0254] Using the microcapsules obtained in Examples 7 to 9, the
residual amounts of the physiologically active peptide in the
microcapsules were determined as in Experimental Example 1, except
that the microcapsule dose was about 30 mg/kg. The results are
shown in Table 4. Table 5 shows the linear regression models,
correlation coefficients and release periods calculated as
X-intercepts, which were determined from the data in Table 4 by the
same procedure as used in Table 2.
5TABLE 4 Residue of Physiologically active peptide (%) Physiologi-
1 1 2 3 4 cally active Day Week Weeks Weeks Weeks Experimental A
99.3 74.5 51.4 33.2 24.1 Example 7 Experimental B 87.4 75.0 52.3
32.8 25.1 Example 8 Experimental C 89.4 73.6 54.9 37.7 23.4 Example
9
[0255]
6 TABLE 5 Physiologically Linear Release active regression
Correlation period Peptide model Coefficient (weeks) Experimental A
Residue (%) = (R.sup.2 = 4.9 Example 7 97.8 - (20.1 .times. 0.975)
no. of weeks) Experimental B Residue (%) = (R.sup.2 = 5.0 Example 8
93.5 - (18.6 .times. 0.971) no. of weeks) Experimental C Residue
(%) = (R.sup.2 = 4.9 Example 9 94.4 - (18.5 .times. 0.987) no. of
weeks)
[0256] It is apparent from Tables 4 and 5 that the microcapsules
according to the present invention are characterized by
substantially constant release of physiologically active peptide
and are further characterized by being substantially free from an
initial burst.
EXPERIMENTAL EXAMPLE 10
[0257] Using the microcapsules obtained in Example 10, the residual
amounts of the physiologically active peptide in the microcapsules
were determined as in Experimental Example 7. The results are shown
in Table 6. Table 7 shows the linear regression models, correlation
coefficients and release periods calculated as X-intercepts, which
were determined from the data in Table 6 by the same procedure as
used in Table 2.
7 TABLE 6 Residue of physiologically active peptide D (%) Day Week
Week Week Week 1 1 2 3 4 Experimental 93.5 .+-. 0.5 69.9 .+-. 3.6
37.3 .+-. 1.6 17.0 .+-. 1.4 7.9 .+-. 0.5 Example 10
[0258]
8 TABLE 7 Linear Release regression Correlation periods model
coefficient (weeks) Experimental Residue (%) = (R.sup.2 = 3.9
Example 10 95.0 - (24.1 .times. no. of 0.969) weeks)
[0259] It is apparent from Tables 6 and 7 that the microcapsules
according to the present invention are characterized by
substantially constant release of physiologically active peptide
and are further characterized by being substantially free from an
initial burst.
EXPERIMENTAL EXAMPLE 11
[0260] About 30 mg of the microcapsules obtained in Example 11 were
dispersed in 0.5 ml of a dispersion medium (prepared by dissolving
carboxymethylcellulose (2.5 mg), polysorbate 80 (0.5 mg) and
mannitol (25 mg) in distilled water) and, the dispersion was
injected subcutaneously at the back of 10-week-old male SD rats
using a 22G needle (the dosage as microcapsules 60 mg/kg). Serially
after administration, the rats were sacrificed, the remains of
microcapsules were taken out from the administration site and the
amount of the physiologically active peptide A in the microcapsules
was determined. The results are shown in Table 8.
EXPERIMENTAL EXAMPLE 12
[0261] Using the microcapsules obtained in Example 12, the
procedure of Experimental Example 11 was otherwise repeated and the
residue of physiologically active peptide A was assayed. The
results are shown in Table 8.
EXPERIMENTAL EXAMPLE 13
[0262] Using the microcapsules obtained in Example 13, the
procedure of Experimental Example 11 was otherwise repeated and the
residue of physiologically active peptide A was assayed. The
results are shown in Table 8.
EXPERIMENTAL EXAMPLE 14
[0263] Using the microcapsules obtained in Example 14, the
procedure of Experimental Example 11 was otherwise repeated and the
residue of physiologically active peptide A was assayed. The
results are shown in Table Table 8
9 TABLE 8 Residue of physiologically active peptide A (%) Day Week
Week Week Week Week Week 1 1 2 3 4 6 8 Experimental 82.8 21.8 -- --
-- -- -- Example 11 Experimental 96.7 91.7 79.5 69.2 59.2 -- 22.8
Example 12 Experimental 100.0 84.3 43.9 31.9 -- -- -- Example 13
Experimental 96.3 67.5 38.0 23.5 -- -- -- Example 14 (--: not
determined)
[0264] Table 9 shows the linear regression models, correlation
coefficients, and release periods as X-intercept which were
determined from the data in Table 8 by the same procedures as used
in Table 2.
10 TABLE 9 Linear Release regression Correlation periods model
coefficient (weeks) Experimental Residue (%) = (R.sup.2 = 1.3
Example 11 97.1 - (75.7 .times. no. of weeks) 0.994) Experimental
Residue (%) = (R.sup.2 = 10.3 Example 12 92.2 - (9.7 .times. no. of
weeks) 0.998) Experimental Residue (%) = Example 13 102.4 - (24.8
.times. no. of weeks) (R.sup.2 = 4.1 0.982) Experimental Residue
(%) = (R.sup.2 = 3.7 Example 14 97.7 - (25.5 .times. no. of weeks)
0.989)
[0265] It is apparent from Tables 8 and 9 that the
sustained-release preparation according to the present invention
invariably insure a substantially constant release of the peptide
over various segments of the time.
COMPARATIVE EXAMPLE 1
[0266] 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 (mole %), GPC weight average mol.
wt.=58,000, GPC number average mol. wt.=14,000, number average mol.
wt. by end-group determination=45,000; manufacturer;
Boeh-ringer-Ingelheim (Lot. RG505-05077), 3.6 g, in 33.2 g (25.0
ml) of dichloromethane but the physiologically active peptide A
acetate could not be successfully dissolved.
COMPARATIVE EXAMPLE 2
[0267] 400 mg of physiologically active peptide A acetate was added
to a solution of lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=75/25 (mole %), GPC weight average mol.
wt.=18,000, GPC number average mol. wt.=8,400, number average mol.
wt. by end-group determination=30,000; manufacturer;
Boeh-ringer-Ingelheim (Lot. RG752-15057), 3.6 g, in 8.0 g (6.0 ml)
of dichloromethane but the physiologically active peptide A could
not be successfully dissolved. This dispersion was cooled to
17.degree. C. and poured into 1,000 ml of a 0.1% aqueous solution
of polyvinyl alcohol previously adjusted to 15.degree. C. to
prepare microcapsules in the same manner as in Example 11. The
particle size distribution and physiologically active peptide A
content of the microcapsules were 10 to 90 .mu.m and 2.5% (w/w),
respectively.
COMPARATIVE EXAMPLE 3
[0268] 400 mg of physiologically active peptide A acetate, was
added to a solution of lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=75/25 (mole %), GPC weight average mol.
wt.=58,000, GPC number average mol. wt.=15,000, number average mol.
wt. by end-group determination=53,000; manufacturer;
Boehringer-Ingelheim (Lot. RG755-05019), 3.6 g, in 21.2 g (16.0 ml)
of dichloromethane but the physiologically active peptide A could
not be successfully dissolved. This dispersion was cooled to
17.degree. C. and poured into 1,000 ml of a 0.1% aqueous solution
of polyvinyl alcohol previously adjusted to 16.degree. C. to
prepare microcapsules in the same manner as in Example 11. The
particle size distribution and physiologically active peptide A
content of the microcapsules were 10 to 90 .mu.m and 3.6% (w/w),
respectively.
[0269] As shown in Comparative Examples 1 to 3, with a lactic
acid-glycolic acid copolymer having substantially no terminal
carboxyl group, the peptide [I] of the present invention could not
be successfully dissolved.
COMPARATIVE EXAMPLE 4
[0270] 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 2, 3.6
g, in 8.0 g (6.0 ml) of dichloromethane but the leuprolerin acetate
could not be successfully dissolved.
[0271] The sustained-release preparation of the present invention
shows a constant release of the drug, especially the peptide [I]
over a long time, thus being conducive to a lasting and stable
effect. Furthermore, the duration of release of the drug can be
easily controlled and excessive release immediately following
administration can be inhibited. Specifically the
histamine-releasing activity in the peptide [I] following
administration of the sustained-release preparation is inhibited.
The sustained-release preparation has excellent dispersibility.
Moreover, the preparation is stable (e.g. to light, heat, humidity,
colouring) and of low toxicity and, therefore, can be safely
administered.
[0272] In accordance with the production method of the present
invention, a sustained-release preparation containing a
physiologically active peptide can be easily obtained in good
yield. The thus obtained sustained-release preparation has a smooth
surface and is excellent in mobility.
* * * * *