U.S. patent application number 09/985925 was filed with the patent office on 2002-05-16 for sustained-release preparation.
Invention is credited to Igari, Yasutaka, Iinuma, Satoshi, Ikeda, Hitoshi, Okada, Hiroaki, Tsuda, Masao, Wakimasu, Mitsuhiro, Yamagata, Yutaka, Yamamoto, Kazumichi.
Application Number | 20020058622 09/985925 |
Document ID | / |
Family ID | 27473220 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058622 |
Kind Code |
A1 |
Igari, Yasutaka ; et
al. |
May 16, 2002 |
Sustained-release preparation
Abstract
According to a first embodiment, there is provided a
sustained-release preparation comprising a water-insoluble or
slightly water-soluble polyvalent metal salt of a water-soluble
physiologically active substance which is not an endothelin
antagonist, and a biodegradable polymer. The sustained-release
preparation of the first embodiment is highly efficient in
incorporating the water-soluble physiologically active substance
and suppresses the initial burst of the water-soluble
physiologically active substance. The sustained-release preparation
of the present invention is capable of releasing the water-soluble
physiologically active substance while retaining its bioactivity
after administration in vivo. Furthermore, the water-soluble
physiologically active substance in the sustained-release
preparation is kept stable for a long period of time, with little
loss of bioactivity. According to a second embodiment, there is
provided a sustained-release preparation comprising an
anti-endothelin substance and a biodegradable polymer. The
sustained-release preparation of the present invention sustainedly
releases an anti-endothelin substance, serving well in the
treatment of endothelin-associated diseases.
Inventors: |
Igari, Yasutaka; (Kobe,
JP) ; Yamagata, Yutaka; (Kobe, JP) ; Iinuma,
Satoshi; (Kobe, JP) ; Okada, Hiroaki; (Suita,
JP) ; Ikeda, Hitoshi; (Higashiosaka, JP) ;
Tsuda, Masao; (Kobe, JP) ; Yamamoto, Kazumichi;
(Nara, JP) ; Wakimasu, Mitsuhiro; (Osaka,
JP) |
Correspondence
Address: |
Stephen B. Maebius
FOLEY & LARDNER
Washington Harbour
3000 K Street, N.W., Suite 500
Washington
DC
20007-5109
US
|
Family ID: |
27473220 |
Appl. No.: |
09/985925 |
Filed: |
November 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09985925 |
Nov 6, 2001 |
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09426716 |
Oct 26, 1999 |
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09426716 |
Oct 26, 1999 |
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08644631 |
Apr 22, 1996 |
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6087324 |
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08644631 |
Apr 22, 1996 |
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08265124 |
Jun 24, 1994 |
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08265124 |
Jun 24, 1994 |
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PCT/JP95/01771 |
Sep 6, 1995 |
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Current U.S.
Class: |
424/85.4 ;
424/486; 424/85.1; 514/11.3; 514/13.7; 514/15.4; 514/16.1;
514/19.1; 514/5.9; 514/6.9 |
Current CPC
Class: |
A61K 9/5031 20130101;
A61K 38/212 20130101; A61K 31/00 20130101; A61K 38/193 20130101;
A61K 9/0019 20130101; A61K 38/27 20130101; A61K 38/1816 20130101;
A61K 38/28 20130101 |
Class at
Publication: |
514/12 ;
424/85.1; 424/486 |
International
Class: |
A61K 038/22; A61K
038/19; A61K 009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 1994 |
JP |
6-310291 |
Sep 9, 1994 |
JP |
6-216449 |
Jun 24, 1993 |
JP |
5-153393 |
Claims
1. A sustained-release preparation which comprises (a) a
water-insoluble or slightly water-soluble polyvalent metal salt of
a water soluble peptide type of physiologically active substance
except for an endothelin antagonist and (b) a biodegradable
polymer.
2. A preparation of claim 1, wherein the physiologically active
substance is a water soluble peptide or a derivative thereof.
3. A preparation of claim 2, wherein the peptide is a hormone,
cytokine, hematopoietic factor, growth factor, enzyme, soluble or
solubilized receptor, antibody, antigen containing peptide, blood
coagulation factor or adhesion molecule.
4. A preparation of claim 2, wherein the peptide is a hormone.
5. A preparation of claim 4, wherein the hormone is a growth
hormone.
6. A preparation of claim 4, wherein the hormone is an insulin.
7. A preparation of claim 2, wherein the peptide is a cytokine.
8. A preparation of claim 7, wherein the cytokine is an
interferon.
9. A preparation of claim 2, wherein the peptide is a growth
factor.
10. A preparation of claim 1, wherein the polyvalent metal salt is
a transition metal salt.
11. A preparation of claim 1, wherein the polyvalent metal salt is
a zinc salt.
12. A preparation of claim 1, wherein the solubility of the
polyvalent metal salt to water is about 0 to about 0.1% (w/w) at
20.degree. C.
13. A preparation of claim 1, wherein the solubility of the
polyvalent metal salt to water is about 0 to about 0.01% (w/w).
14. A preparation of claim 1, which contains about 0.1 to about 50%
(w/w) of the polyvalent metal salt.
15. A preparation of claim 1, which contains about 1 to about 30%
(w/w) of the polyvalent metal salt.
16. A preparation of claim 1, wherein the biodegradable polymer is
an aliphatic polyester.
17. A preparation of claim 16, wherein the aliphatic polyester is a
polymer of lactic acid and glycolic acid.
18. A preparation of claim 17, wherein the composition ratio of
lactic acid and glycolic acid is 100/0 to about 40/60 (mole %).
19. A preparation of claim 18, wherein the composition ratio is
about 90/10 to about 45/55 (mole %).
20. A preparation of claim 17, wherein the weight-average molecular
weight of the polymer is about 3,000 to about 20,000.
21. A preparation of claim 17, wherein the weight-average molecular
weight of the polymer is about 3,000 to about 14,000.
22. A preparation of claim 16, wherein the alihatic polyester is a
homopolymer of lactic acid.
23. A preparation of claim 22, wherein the weight-average molecular
weight of the homopolymer is about 3,000 to about 20,000.
24. A preparation of claim 22, wherein the weight-average molecular
weight of the homopolymer is about 3,000 to about 14,000.
25. A preparation of claim 1, wherein the preparation is a
microcapsule.
26. A preparation of claim 25, wherein the microcapsule is for
injection.
27. A preparation of claim 1, which is an injectable one,
28. Use of a water-insoluble or slightly water-soluble polyvalent
metal salt of a water-soluble peptide type of physiologically
active substance except for an endothelin antagonist and a
biodegradable polymer for the production of a sustained-release
preparation.
29. A method of producing a sustained-release preparation, which
comprises dispersing a water-insoluble or slightly water-soluble
polyvalent metal salt of a water-soluble peptidfe type of
physiologically active substance except for an endothelin
antagonist in an oil phase containing a biodegradable polymer to
make a solid-in-oil emulsion, adding the solid-in-oil emulsion to a
water phase to make a solid-in-oil-in-water emulsion, and then
in-water drying the soild-in-oil-in-water emulsion.
30. A sustained-release preparation which comprises an
anti-endothelin substance and a biodegradable polymer.
31. The sustained-release preparation according to claim 30,
wherein the anti-endothelin substance is an endothelin
antagonist.
32. The sustained-release preparation according to claim 31,
wherein the endothelin antagonist is a peptide.
33. The sustained-release preparation according to claim 31,
wherein the endothelin antagonist is a peptide of the general
formula: 23wherein X and Y independently represent an .alpha.-amino
acid residue; A represents a D-acidic-.alpha.-amino acid residue; B
represents a neutral-.alpha.-amino acid residue; C represents an
L-.alpha.-amino acid residue; E represents a D-.alpha.-amino acid
residue having an aromatic cyclic group, or an ester thereof, or a
salt thereof.
34. The sustained-release preparation according to claim 33,
wherein the peptide is a compound of the formula
cyclo[-D-Asp-Asp(R1')-Asp-D-Thg(2)-L- eu-D-Trp-] wherein Asp
represents aspartic acid; Asp(R1') represents aspartic acid
.beta.-4-phenylpiperazinamide; Thg(2) represents 2-thienylglycine;
Leu represents leucine; and Trp represents tryptophan.
35. The sustained-release preparation according to claim 33,
wherein A is a D-acidic-.alpha.-amino acid residue which is
esterified with an alkyl group.
36. The sustained-release preparation according to claim 33,
wherein Y is a L-acidic-.alpha.-amino acid residue.
37. The sustained-release preparation according to claim 33,
wherein Y is a L-acidic-.alpha.-amino acid residue which is
esterified with an alkyl group.
38. The sustained-release preparation according to claim 33,
wherein the peptide is a compound of the formula,
cyclo-[-D-Asp(OC.sub.2H.sub.5)-Asp(-
R1')-Asp(OC.sub.2H.sub.5)-D-Thg(2)-Leu-D-Trp-] wherein Asp
represents aspartic acid; Asp(R1') represents aspartic acid
.beta.-4-phenylpiperazin- amide; Thg(2) represents
2-thienylglycine; Leu represents leucine; and Trp represents
tryptophan.
39. The sustained-release preparation according to claim 33,
wherein the salt is a polyvalent metal salt.
40. The sustained-release preparation according to claim 39,
wherein the polyvalent metal salt is a zinc salt.
41. The sustained-release preparation according to claim 30,
wherein the biodegradable polymer is an aliphatic polyester.
42. The sustained-release preparation according to claim 41,
wherein the aliphatic polyester is a copolymer of glycolic acid and
lactic acid.
43. The sustained-release preparation according to claim 42,
wherein the copolymer has a weight-average molecular weight of
about 2,000 to 50,000, as determined by Gel Permeation
Chromatography.
44. The sustained-release preparation according to claim 42,
wherein the copolymer has a dispersity of about 0.2 to 4.0.
45. The sustained-release preparation according to claim 30, which
further comprises an organic basic substance.
46. The sustained-release preparation according to claim 30, which
further comprises a water-soluble polyvalent metal salt.
47. A method for treatment of diseases caused by endothelin
comprising administering to a patient in need thereof an effective
amount of the sustained-release preparation according to claim
30.
48. The method according to claim 47, wherein the diseases are
chronic diseases.
49. The method according to claim 48, wherein the chronic diseases
are chronic complications in diabetes mellitus.
50. The method according to claim 49, wherein the chronic
complications are diabetic nephropathy.
51. An injectable preparation which comprises the sustained-release
preparation as claimed in claim 30.
52. A peptide of the general formula: 24wherein X and Y
independently represent an .alpha.-amino acid residue; A'
represents a D-acidic-.alpha.-amino acid residue which is
esterified with an alkyl group; B represents a
neutral-.alpha.-amino acid residue; C represents an L-.alpha.-amino
acid residue; E represents a D-.alpha.-amino acid residue having an
aromatic cyclic group, or a salt thereof.
53. The peptide according to claim 52, wherein X is an
L-isomer.
54. The peptide according to claim 52, wherein Y is an
L-isomer.
55. The peptide according to claim 52, wherein A' is D-glutamic
acid or D-aspartic acid which is esterified with an alkyl
group.
56. The peptide according to claim 52, wherein B is an
D-isomer.
57. The peptide according to claim 52, wherein B is selected from
the group consisting of D-leucine, D-alloisoleucine, D-tertiary
leucine, D-gamma methyl leucine, D-phenylglycine,
D-2-thienylglycine, D-3-thienylglycine, D-2-cyclopentylglycine,
D-phenylalanine, D-2-thienylalanine, D-valine, D-2-furylglycine and
D-3-furylglycine residues.
58. The peptide according to claim 52, wherein C is selected from
the group consisting of L-leucine, L-phenylalanine and L-tryptophan
residues.
59. The peptide according to claim 52, wherein E is selected from
the group consisting of D-tryptophan or derivatives thereof,
D-1-naphthylalanine, D-2-naphthylalanine, D-benzothienylalanine,
D-4-bisphenylalanine and D-pentamethyl phenylalanine residues.
60. The peptide according to claim 52, wherein Y is an
.alpha.-amino acid residue having a carboxyl group which is
esterified with an alkyl group.
61. A peptide of the formula: cyclo-[-D-Asp(OC.sub.2H.sub.5)-Asp
(R1')-Asp(OC.sub.2H.sub.5)-D-Thg(2)-Leu-D-Trp-], wherein Asp
represents aspartic acid; Asp (R1') represents aspartic acid
.beta.-4-phenylpiperazi- namide; Thg(2) represents
2-thienylglycine; Leu represents leucine; and Trp represents
tryptophan, or a salt thereof.
62. A zinc salt of a peptide represented by the general formula:
25wherein X and Y independently represent an .alpha.-amino acid
residue; A represents a D-acidic-.alpha.-amino acid residue; B
represents a neutral-.alpha.-amino acid residue; C represents an
L-.alpha.-amino acid residue; and E represents a D-.alpha.-amino
acid residue having an aromatic cyclic group.
Description
[0001] The following disclosure through page 31 relates to a first
embodiment of the present invention.
TECHNICAL FIELD OF THE FIRST EMBODIMENT
[0002] The first embodiment of the present invention relates to a
sustained-release preparation which comprises a water-insoluble or
a slightly water-soluble polyvalent metal salt of a water-soluble
peptide type of physiologically active substance which is not an
endothelin antagonist, and a biodegradable polymer.
BACKGROUND ART OF THE FIRST EMBODIMENT
[0003] Physiologically active substances, particularly peptides or
derivatives thereof, are known to exhibit various pharmacologic
actions in vivo. Some have been produced in large amounts, for
pharmaceutical application, by chemical synthesis, or as a result
of advances in gene engineering and cell engineering technologies,
using organisms such as Escherichia coli, yeasts, animal cells and
hamsters. However, these peptides must be administered frequently,
since they generally have a short biological half-life, and so pose
a significant physical burden of injection on patents. To solve
this problem, various attempts have been made to develop
sustained-release preparations.
[0004] The first problem to solve in developing a sustained-release
preparation of a water-soluble physiologically active substance,
particularly a water-soluble peptide (hereinafter also referred to
as "peptide") is to control peptide solubility, i.e., to regulate
the peptide release rate.
[0005] Japanese Publication of the Translation of International
Patent Application No. 500286/1991 discloses an insoluble
zinc-protamine-.alpha.-interferon complex.
[0006] Japanese Patent Unexamined Publication No. 2930/1988
discloses a system comprising a polylactide in which a
macromolecular polypeptide is dispersed.
[0007] Japanese Patent Unexamined Publication Nos. 221855/1993 and
172208/1994 disclose a technology by which a water-soluble peptide
is converted to a water-insoluble peptide salt, which is then
suspended in an organic medium containing a biodegradable polymer
to efficiently incorporate the water-soluble peptide in fine
grains. The water-insoluble peptide used in these patent
publications is an organic acid salt formed at the base portion of
the water-soluble peptide molecule, and is exemplified by pamoate,
tannic acid, stearic acid or palmitate.
[0008] Although there have been various attempts to produce
sustained-release preparations of water-soluble physiologically
active substances, as stated above, no satisfactory
sustained-release preparations have been obtained; there is
therefore need for the development of a sustained-release
preparation that is highly efficient in incorporating water-soluble
physiologically active substance, suppresses initial water-soluble
physiologically active substance burst, offers a constant
water-soluble physiologically active substance release rate, and
keeps the bioactivity of water-soluble physiologically active
substance.
DISCLOSURE OF THE FIRST EMBODIMENT
[0009] Through extensive investigation to solve the above problems,
the present inventors found that a sustained-release preparation,
having dramatically increased efficiency of water-soluble peptide
type of physiologically active substance except for an endothelin
antagonist incorporation in a biodegradable polymer and showing
little drug burst just after administration to the living body, can
be obtained by producing a water-insoluble or a slightly
water-soluble polyvalent metal salt of a water-soluble peptide type
of physiologically active substance except for an endothelin
antagonist (hereinafter also referred to as "complex"), which salt
is formed from a combination of a water-soluble peptide type of
physiologically active substance except for an endothelin
antagonist having an acidic group, or a water-soluble salt thereof
(hereinafter also referred to as "physiologically active
substance"), with a water-soluble polyvalent metal salt, and
dispersing or dissolving it in a biodegradable polymer. After
further investigations based on this finding, the inventors
developed the present invention.
[0010] Accordingly, the present invention relates to:
[0011] (1) a sustained-release preparation which comprises
[0012] (a) a water-insoluble or slightly water-soluble polyvalent
metal salt of a water-soluble peptide type of physiologically
active substance except for an endothelin antagonist and
[0013] (b) a biodegradable polymer,
[0014] (2) a preparation of term 1 above, wherein the
physiologically active substance is a water-soluble peptide or a
derivative thereof,
[0015] (3) a preparation of term 2 above, wherein the peptide is a
hormone, cytokine, hematopoietic factor, growth factor, enzyme,
soluble or solubilized receptor, antibody, antigen containing
peptide, blood coagulation factor or adhesion molecule,
[0016] (4) a preparation of term 2 above, wherein the peptide is a
hormone,
[0017] (5) a preparation of term 4 above, wherein the hormone is a
growth hormone
[0018] (6) a preparation of term 3 above, wherein the hormone is an
insulin,
[0019] (7) a preparation of term 2 above, wherein the peptide is a
cytokine,
[0020] (8) a preparation of term 7 above, wherein the cytokine is
an interferon,
[0021] (9) a preparation of term 2 above, wherein the peptide is a
growth factor,
[0022] (10) a preparation of term 1 above, wherein the polyvalent
metal salt is a transition metal salt,
[0023] (11) a preparation of term 1 above, wherein the polyvalent
metal salt is a zinc salt,
[0024] (12) a preparation of term 1 above, wherein the solubility
of the polyvalent metal salt to water is about 0 to about 0.1%
(w/w) at 20.degree. C.,
[0025] (13) a preparation of term 1 above, wherein the solubility
of the polyvalent metal salt to water is about 0 to about 0.01%
(w/w),
[0026] (14) a preparation of term 1 above, which contains about 0.1
to about 50% (w/w) of the polyvalent metal salt,
[0027] (15) a preparation of term 1 above, which contains about 1
to about 30% (w/w) of the polyvalent metal salt,
[0028] (16) a preparation of term 1 above, wherein the
biodegradable polymer is an aliphatic polyester,
[0029] (17) a preparation of term 16 above, wherein the aliphatic
polyester is a polymer of lactic acid and glycolic acid,
[0030] (18) a preparation of term 17 above, wherein the composition
ratio of lactic acid and glycolic acid is 100/0 to about 40/60
(mole %),
[0031] (19) a preparation of term 18 above, wherein the composition
ratio is about 90/10 to about 45/55 (mole %),
[0032] (20) a preparation of term 17 above, wherein the
weight-average molecular weight of the polymer is about 3,000 to
about 20,000,
[0033] (21) a preparation of term 17 above, wherein the
weight-average molecular weight of the polymer is about 3,000 to
about 14,000,
[0034] (22) a preparation of term 16 above, wherein the alihatic
polyester is a homopolymer of lactic acid,
[0035] (23) a preparation of term 22 above, wherein the
weight-average molecular weight of the homopolymer is about 3,000
to about 20,000,
[0036] (24) a preparation of term 22 above, wherein a
weight-average molecular weight of the homopolymer is about 3,000
to about 14,000,
[0037] (25) a preparation of term 1 above, wherein the preparation
is a microcapsule,
[0038] (26) a preparation of term 25 above, wherein the
microcapsule is for injection,
[0039] (27) a preparation of term 1 above, which is an injectable
one,
[0040] (28) Use of a water-insoluble or slightly water-soluble
polyvalent metal salt of a water-soluble peptide type of
physiologically active substance except for an endothelin
antagonist and a biodegradable polymer for the production of a
sustained-release preparation, and
[0041] (29) a method of producing a sustained-release preparation,
which comprises dispersing a water-insoluble or slightly
water-soluble polyvalent metal salt of a water-soluble peptide type
of physiologically active substance except for an endothelin
antagonist in an oil phase containing a biodegradable polymer to
make a solid-in-oil emulsion, adding the solid-in-oil emulsion to a
water phase to make a solid-in-oil-in-water emulsion, and then
in-water drying the soild-in-oil-in-water emulsion.
[0042] Incidentally abbreviations of amino acid, peptide or the
like used in the present invention are based on those in accordance
with IUPAC-IUB Commission on Biochemical Nomenclature or those
conventionally used in the relevant fields, and possible optical
isomers of amino acid are, unless otherwise specified,
L-isomers.
[0043] The physiologically active substance in the water-insoluble
or the slightly water-soluble polyvalent metal salt is a
physiologically active substance having an acidic group. Here, the
acidic group is exemplified by the carboxyl group and sulfo group.
The physiologically active substance is preferably a
physiologically active substance having a peptide bond or an amino
acid and acidic group. The acidic group may be derived from an
amino acid. More preferably, the physiologically active substance
is a water-soluble peptide having an acidic group or a derivative
thereof. A solubility of the physiologically active substance to
water is 1% (w/w) or more at 25.degree. C.
[0044] The physiologically active substance preferably has two or
more carboxyl groups.
[0045] The molecular weight of the physiologically active substance
is about 200 to 200,000, preferably about 200 to about 50,000, more
preferably about 500 to about 40,000.
[0046] A representative activity of a physiologically active
substance is hormone action. The physiologically active substance
may be a natural, synthetic, semi-synthetic or genetically
engineered product, or a derivative thereof. As concerns the
mechanism of action, these physiologically active substances may be
agonistic or antagonistic.
[0047] Physiologically active substances, particularly
water-soluble peptide or a derivative thereof for the present
invention include hormones, cytokines, hematopoietic factors,
growth factors, enzymes, a soluble or solubilized receptor, an
antibody or a fragment thereof, an antigen containing peptide, a
blood coagulation factor, an adhesion molecule, agonists or
antagonists capable of binding to receptors of the physiologically
active substances and so on.
[0048] Example hormones include insulin, growth hormone,
natriuretic peptide, gastrin, prolactin, adrenocorticotropic
hormone (ACTH), thyroid-stimulating hormone (TSH), luteinizing
hormone (LH), follicle-stimulating hormone (FSH), human chorionic
gonadotropin (HCG), motilin, kallikrein and so on. The hormone is
preferably insulin and growth hormone.
[0049] Example cytokines include lymphokines, monokines and so on.
Example lymphokines include interferons (alpha, beta, gamma),
interleukins (IL-2 through IL-12) and so on. Example monokines
include an interleukin 1 (IL-1), tumor necrosis factor and so on.
The cytokine is preferably a lymphokine, more preferably an
interferon (alpha, beta, gamma).
[0050] Example hematopoietic factors include erythropoietin,
granulocyte colony-stimulating factor (G-CSF), macrophage
colony-stimulating factor (M-CSF), thrombopoietin, platelet
growth-stimulating factor, megakaryocyte potentiator and so on.
[0051] Example growth factors include basic or acidic fibroblast
growth factors (FGF), members of the family thereof (e.g., FGF-9
etc.), nerve cell growth factor (NGF) or members of the family
thereof, insulin-like growth factors (e.g., IGF-1, IGF-2), bone
morphogenetic protein (BMP) or members of the family thereof and so
on.
[0052] Example enzymes include superoxide dismutase (SOD), tissue
plasminogen activator (TPA) and so on.
[0053] Example soluble receptors include soluble IL-6 receptor,
insulin-like growth factor binding protein (IGFBP), soluble TNF
receptor, soluble EGF receptor, soluble IL-1 receptor and so
on.
[0054] Example solubilized receptors include a known receptors such
as IL-1 receptor, IL-6 receptor, TNF receptor or Fas ligand etc.,
which is solubilized by a method of gene engineering.
[0055] Example antibodies include a human monoclonal antibody, a
human-mouse chimeric monoclonal antibody in which the variable
region of an antibody derived from mouse is bound to the constant
region of an antibody derived from human, or a fragment thereof and
so on. Example type of antibody include IgM, IgG, IgE and so on.
Example antigenes, which is recognized by the above described
antibody, include platelet, virus and so on.
[0056] Example blood coagulation factors include factor VIII and so
on.
[0057] Example adhesion molecules include fibronectin, ICAM-1 and
so on.
[0058] Furthermore, example physiologically active substances
include endothelin, Arg-Gly-Asp-Ser (RGDS), pituitary adenylate
cyclase activating polypeptide (PACAP) and so on.
[0059] The physiologically active substance is converted to a
water-insoluble or a slightly water-soluble polyvalent metal salt
thereof by bringing it into contact with a water-soluble polyvalent
metal.
[0060] The polyvalent metal in the water-soluble polyvalent metal
salt is exemplified by divalent, trivalent or tetravalent metal
etc. such as alkaline earth metals (e.g., calcium, magnesium etc.),
transition metals [e.g., iron (II, III), copper (II), zinc (II)
etc.), the group III.sub.b metals [e.g., aluminum (II, III) etc.],
the group IV.sub.b metals [e.g., tin (II, IV) etc.]and so on. The
polyvalent metal is preferably alkaline earth metals or transition
metals, more preferably calcium or zinc, still more preferably
zinc.
[0061] Water-soluble polyvalent metal salts include salts of
polyvalent metals and acids, e.g., salts of polyvalent metals and
inorganic acids, and salts of polyvalent metals and organic
acids.
[0062] The salt of a polyvalent metal and an acid is preferably a
salt whose water solubility at normal temperature (20.degree. C.)
is not lower than about 20 mg/ml, more preferably not lower than
about 100 mg/ml, and still more preferably not lower than about 200
mg/ml.
[0063] Inorganic acids to form salts with polyvalent metals include
hydrochloric acid, sulfuric acid, nitric acid, thiocyanic acid and
so on.
[0064] Organic acids to form salts with polyvalent metals include
aliphatic carboxylic acids and aromatic acids. The aliphatic
carboxylic acid is preferably an aliphatic carboxylic acid having 2
to 9 carbon atoms. Aliphatic carboxylic acids include aliphatic
monocarboxylic acids, aliphatic dicarboxylic acids, aliphatic
tricarboxylic acids and so on. These aliphatic carboxylic acids may
be saturated or unsaturated one.
[0065] Example aliphatic monocarboxylic acids include saturated
aliphatic monocarboxylic acids having 2 to 9 carbon atoms (e.g.,
acetic acid, propionic acid, butyric acid, valeric acid, caproic
acid, enanthic acid, caprylic acid, pelargonic acid, caprynic acid
etc.) and unsaturated aliphatic monocarboxylic acids having 2 to 9
carbon atoms (e.g., acrylic acid, propiolic acid, methacrylic acid,
crotonic acid, isocrotonic acid etc.).
[0066] Example aliphatic dicarboxylic acids include saturated
aliphatic dicarboxylic acids having 2 to 9 carbon atoms (e.g.,
malonic acid, succinic acid, glutaric acid, adipic acid, pimelic
acid etc.) and unsaturated aliphatic dicarboxylic acids having 2 to
9 carbon atoms (e.g., maleic acid, fumaric acid, citraconic acid,
mesaconic acid etc.).
[0067] Example aliphatic tricarboxylic acids include saturated
aliphatic tricarboxylic acids having 2 to 9 carbon atoms (e.g.,
tricarballylic acid, 1,2,3-butanetricarboxylic acid etc.).
[0068] The above-mentioned aliphatic carboxylic acids may have 1 or
2 hydroxyl groups. Such aliphatic carboxylic acids include glycolic
acid, lactic acid, glyceric acid, tartronic acid, malic acid,
tartaric acid, citric acid and so on.
[0069] The aliphatic carboxylic acid is preferably an aliphatic
monocarboxylic acid, more preferably an aliphatic monocarboxylic
acid having 2 to 9 carbon atoms, and still more preferably a
saturated aliphatic monocarboxylic acid having 2 or 3 carbon atoms.
Examples of particularly preferable aliphatic carboxylic acids
include acetic acid and so on.
[0070] Example aromatic acids include benzoic acid, salicylic acid
and so on, with preference given to benzoic acid.
[0071] Examples of salts of polyvalent metals and inorganic acids,
i.e., inorganic acid polyvalent metal salts, include halides (e.g.,
zinc chloride, calcium chloride), sulfates, nitrates, thiocyanates
and so on.
[0072] Examples of salts of polyvalent metals and aliphatic
carboxylic acids, i.e., aliphatic carboxylic acid polyvalent metal
salts, include calcium acetate, zinc acetate, calcium propionate,
zinc glycolate, calcium lactate, zinc lactate, zinc tartrate and so
on. Preferable aliphatic carboxylic acid polyvalent metal salts
include calcium acetate and zinc acetate. Greater preference is
given to zinc acetate and so on.
[0073] Examples of salts of polyvalent metals and aromatic acids,
i.e., aromatic acid polyvalent metal salts, include benzoates,
salicylates and so on. Greater preference is given to zinc
benzoate.
[0074] A water-insoluble or a slightly water-soluble polyvalent
metal salt of a physiologically active substance is produced by
mixing in a solvent the water-soluble physiologically active
substance and a water-soluble polyvalent metal salt. The mixing
procedure is preferably conducted in water.
[0075] The mixing ratio (mole ratio) of the physiologically active
substance and water-soluble polyvalent metal salt in water is, for
example 1:1 to 1:1000, preferably 1:1 to 1:100, more preferably 1:1
to 1:50, still more preferably 1:1 to 1:10. The concentrations of
both components in water may be optional, as long as they exceed
the solubility of the resulting complex, within their respective
solubility ranges.
[0076] The pH of the aqueous solution resulting from the above
mixing must be such that the bioactivity of the physiologically
active substance is not affected, and that the solubilities of the
physiologically active substance and water-soluble polyvalent metal
salt are not lowered in excess. Although the mixing procedure is
normally conducted in distilled water, it may be conducted in water
adjusted to weakly acidic, neutral, or weakly alkaline pH as
necessary.
[0077] "Being water insoluble or slightly water-soluble" as
mentioned herein is not irreversible but reversible, meaning that
water solubility is very low. Water solubility is about 0 to about
0.1% (w/w), preferably about 0 to about 0.01% (w/w) at ordinary
temperature (20.degree. C.).
[0078] The thus-obtained water insoluble or slightly water-soluble
polyvalent metal salt of a water-soluble physiologically active
substance is used after being vacuum dried or freeze dried as
necessary.
[0079] In the sustained-release preparation of the present
invention, the content of the water-insoluble or slightly
water-soluble polyvalent metal salt of the physiologically active
substance is normally about 0.1 to about 50% (w/w), preferably
about 1 to about 30% (w/w).
[0080] The biodegradable polymer is exemplified by high-molecular
polymers slightly soluble or insoluble in water, such as aliphatic
polyesters (e.g., homopolymers, copolymers or mixtures thereof
synthesized from one or more a -hydroxycarboxylic acids such as
glycolic acid, lactic acid, hydroxybutyric acid etc.),
hydroxydicarboxylic acids such as malic acid etc.,
hydroxytricarboxylic acids such as citric acid etc. and others,
poly-.alpha.-cyanoacrylic acid esters, polyamino acids such as
poly-.gamma.-benzyl-L-glutamic acid and so on. These may be used in
mixture at appropriate ratios. The type of polymerization may be
random, block or graft.
[0081] The biodegradable polymer is preferably an aliphatic
polyester (e.g., a homopolymer, copolymer or mixture thereof
synthesized from one or more .alpha.-hydroxycarboxylic acids such
as glycolic acid, lactic acid, hydroxybutyric acid etc.,
hydroxydicarboxylic acids such as malic acid etc.,
hydroxytricarboxylic acids such as citric acid etc. and others.
[0082] Of the above-mentioned aliphatic polyesters, homopolymers or
copolymers synthesized from one or more .alpha.-hydroxycarboxylic
acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid etc.)
are preferred from the viewpoint of reliable biodegradability and
biocompatibility. More preferably, the aliphatic polyester is a
copolymer synthesized from one or more .alpha.-hydroxycarboxylic
acids (e.g., glycolic acid, lactic acid, hydroxybutyric acid etc.).
Also, these copolymers may be used in mixture.
[0083] The biodegradable polymer for the present invention is
produced by a commonly known method.
[0084] Although the above-described .alpha.-hydroxycarboxylic acid
may be of the D-, L- or D,L-configuration, it is preferable that
the ratio of the D-/L-configuration (mole %) fall within the range
from about 75/25 to about 25/75. The ratio of the
D-/L-configuration (mole %) is more preferably about 60/40 to about
30/70.
[0085] Example copolymers of the above-described
.alpha.-hydroxycarboxylic acid include copolymers of glycolic acid
with another .alpha.-hydroxy acid, which is preferably lactic acid
or 2-hydroxybutyric acid.
[0086] The .alpha.-hydroxycarboxylic acid copolymer is preferably a
lactic acid-glycolic acid copolymer or a 2-hydroxybutyric
acid-glycolic acid copolymer.
[0087] More preferably, the .alpha.-hydroxycarboxylic acid
copolymer is a lactic acid-glycolic acid copolymer.
[0088] With respect to the lactic acid-glycolic acid copolymer, it
is preferable that the content ratio (lactic acid/glycolic acid)
(mole %) be about 100/0 to about 40/60. The content ratio is more
preferably about 90/10 to about 45/55, and more preferably about
80/20 to about 45/55. The weight-average molecular weight of the
lactic acid-glycolic acid copolymer is about 3,000 to about 20,000,
preferably about 3,000 to about 14,000 more preferably about 3,000
to about 12,000.
[0089] Also, the degree of dispersion of the lactic acid-glycolic
acid copolymer (weight-average molecular weight/number-average
molecular weight) is preferably about 1.2 to about 4.0, more
preferably about 1.5 to about 3.5.
[0090] The lactic acid-glycolic acid copolymer can be synthesized
by a known process, such as the method described in Japanese Patent
Unexamined Publication No. 28521/1986. It is preferable that the
copolymer be synthesized by catalyst-free dehydration
polymerization condensation.
[0091] With respect to the 2-hydroxybutyric acid-glycolic acid
copolymer, it is preferable that glycolic acid account for about 10
to about 75 mole % and 2-hydroxybutyric acid for the remaining
portion. More preferably, glycolic acid accounts for about 20 to
about 75 mole %, and still more preferably about 30 to about 70
mole %. The weight-average molecular weight of the 2-hydroxybutyric
acid-glycolic acid copolymer is preferably about 2,000 to about
20,000. The degree of dispersion of the 2-hydroxybutyric
acid-glycolic acid copolymer (weight-average molecular
weight/number-average molecular weight) is preferably about 1.2 to
4.0, more preferably about 1.5 to 3.5. A 2-hydroxybutyric
acid-glycolic acid copolymer can be synthesized by a known process,
such as that described in Japanese Patent Unexamined Publication
No. 28521/1986. It is preferable that the copolymer be synthesized
by catalyst-free dehydration polymerization condensation.
[0092] Preferable example homopolymers of the above-described
.alpha.-hydroxycarboxylic acid include homopolymer of lactic acid.
The weight-average molecular weight of the homopolymer of lactic
acid is about 3,000 to about 20,000, preferably about 3,000 to
about 14,000. A homopolymer of lactic acid can be synthesized by a
known process, such as that described in Japanese Patent Unexamined
Publication No. 28521/1986. It is preferable that the homopolymer
be synthesized by catalyst-free dehydration polymerization
condensation.
[0093] The above-described 2-hydroxybutyric acid-glycolic acid
copolymer may be used in a mixture with polylactic acid. Although
the polylactic acid may be of the D- or L-configuration or a
mixture thereof, it is preferable that the ratio of the
D-/L-configuration (mole %) fall within the range from about 75/25
to about 20/80. The ratio of the D-/L-configuration (mole %) is
more preferably about 60/40 to about 25/75, and still more
preferably about 55/45 to about 25/75. The weight-average molecular
weight of polylactic acid is preferably about 1,500 to about
20,000, more preferably about 1,500 to 10,000. Also, the degree of
dispersion of the polylactic acid is preferably about 1.2 to 4.0,
more preferably about 1.5 to 3.5.
[0094] For producing polylactic acid, two methods are known:
ring-opening polymerization of lactide, a dimer of lactic acid, and
dehydration polymerization condensation of lactic acid. For
obtaining a polylactic acid of relatively low molecular weight for
the present invention, direct dehydration polymerization
condensation of lactic acid is preferred. This method is, for
example, described in Japanese Patent Unexamined Publication No.
28521/1986.
[0095] When a 2-hydroxybutyric acid-glycolic acid copolymer and
polylactic acid are used in mixture, their mixing ratio is about
10/90 to about 90/10 (% by weight). The mixing ratio is preferably
about 20/80 to 80/20, and more preferably about 30/70 to 70/30.
[0096] In the present specification, weight-average molecular
weight is defined as the molecular weight obtained by gel
permeation chromatography (GPC) with 9 polystyrenes as reference
substances with respective weight-average molecular weights of
120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and 162.
Number-average molecular weight based on GPC measurement is also
calculated. The degree of dispersion is calculated from the
weight-average molecular weight and the number-average molecular
weight. Measurements were taken using a GPC column KF804L.times.2
(produced by Showa Denko) and an RI monitor L-3300 (produced by
Hitachi, Ltd.) with chloroform as the mobile phase.
[0097] The above-described copolymer, synthesized by catalyst-free
dehydration polymerization condensation, usually has a terminal
carboxyl group.
[0098] In the present invention, the biodegradable polymer
preferably has a terminal carboxyl group.
[0099] A biodegradable polymer having a terminal carboxyl group is
a polymer in which the number-average molecular weight by GPC
determination and that by terminal group determination almost
agree.
[0100] By terminal group quantitation, number-average molecular
weight is calculated as follows:
[0101] About 1 to 3 g of the biodegradable polymer is dissolved in
a mixed solvent of acetone (25 ml) and methanol (5 ml), and the
solution is quickly titrated with a 0.05-N alcoholic solution of
potassium hydroxide while being stirred at room temperature with
phenolphthalein as an indicator to determine the terminal carboxyl
group content; the number-average molecular weight based on
terminal group quantitation is calculated using the following
equation:
Number-average molecular weight based on terminal group
quantitation=20,000 A/B
[0102] A: Weight mass (g) of the biodegradable polymer
[0103] B: Amount (ml) of the 0.05 N alcoholic solution of potassium
hydroxide added until the titration end point is reached
[0104] For example, in the case of a polymer having a terminal
carboxyl group, and synthesized from one or more .alpha.-hydroxy
acids by catalyst-free dehydration polymerization condensation, the
number-average molecular weight based on GPC measurement and the
number-average molecular weight based on terminal group
quantitation almost agree. On the other hand, in the case of a
polymer having essentially no terminal carboxyl group, and
synthesized from a cyclic dimer by ring-opening polymerization
using a catalyst, the number-average molecular weight based on
terminal group quantitation is significantly higher than the
number-average molecular weight based on GPC determination. This
difference makes it possible to clearly differentiate a polymer
having a terminal carboxyl group from a polymer having no terminal
carboxyl group.
[0105] While the number-average molecular weight based on terminal
group quantitation is an absolute value, the number-average
molecular weight based on GPC determination is a relative value
that varies depending on various analytical conditions (e.g., kind
of mobile phase, kind of column, reference substance, slice width
chosen, baseline chosen etc.); it is therefore difficult to have an
absolute numerical representation of the latter. However, the fact
that the number-average molecular weight based on GPC determination
and that based on terminal group quantitation almost agree means
that the number-average molecular weight based on terminal group
quantitation falls within the range from about 0.5 to about 2
times, preferably from about 0.8 to about 1.5 times, the
number-average molecular weight based on GPC determination. Also,
the fact that the number-average molecular weight based on terminal
group quantitation is significantly higher than that based on GPC
determination means that the number-average molecular weight based
on terminal group quantitation is about 2 times or more the
number-average molecular weight based on GPC determination.
[0106] The sustained-release preparation of the present invention
is produced by dispersing in a biodegradable polymer a
water-insoluble or a slightly water-soluble polyvalent metal salt
of a physiologically active substance obtained by mixing the
physiologically active substance and a water-soluble polyvalent
metal salt. Methods of producing a sustained-release preparation
include the in-water drying method, phase separation method, spray
drying method, and modifications thereof.
[0107] Methods of producing a sustained-release preparation, e.g.,
microcapsules, are described below.
[0108] (i) In-water Drying Method (o/w Method)
[0109] In this method, a solution of a biodegradable polymer in an
organic solvent is first prepared. The organic solvent used to
produce the sustained-release preparation of the present invention
preferably has a boiling point not higher than 120.degree. C. Such
organic solvents include halogenated hydrocarbons (e.g.,
dichloromethane, chloroform, carbon tetrachloride etc.), alcohols
(e.g., ethanol, methanol), acetonitrile and so on. These may be
used in mixture at appropriate ratios. For example, when a
dichloromethane and alcohols are used in mixture, their mixing
ratio (v/v) is about 1000/1 to about 1/1, preferably about 100/1 to
about 1/1, still more preferably about 10/1 to about 2/1. The
organic solvent is preferably dichloromethane and acetonitrile, and
still more preferably dichloromethane. The concentration of the
biodegradable polymer in the organic solvent solution is normally
about 0.01 to about 80% (w/w), preferably about 0.1 to about 70%
(w/w), and more preferably about 1 to about 60% (w/w), depending on
the molecular weight of the biodegradable polymer, kind of organic
solvent and so on.
[0110] To the organic solvent solution of the biodegradable polymer
thus obtained, a water-insoluble or a slightly water-soluble
polyvalent metal salt of a physiologically active substance is
added or dissolved, after being freeze-dried or vacuum dried as
necessary. In this operation, the amount of complex added is set so
that the complex:biodegradable polymer weight ratio is up to about
1:2, preferably about 1:3.
[0111] The organic solvent solution thus prepared is added to an
aqueous phase to form an o/w emulsion using a turbine type
mechanical stirrer or the like, followed by evaporation of the
solvent in the oil phase, to yield microcapsules. The volume of the
aqueous phase is normally chosen over the range of about 1 to about
10,000 times, preferably about 2 to about 5,000 times, and more
preferably about 5 to about 2,000 times, the volume of the oil
phase.
[0112] An emulsifier may be added to the external aqueous phase.
The emulsifier may be any one, as long as it is capable of forming
a stable o/w emulsion. Examples of such emulsifiers include anionic
surfactants, nonionic surfactants, polyoxyethylene castor oil
derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethyl
cellulose, lecithin, gelatin, hyaluronic acid and so on. These may
be used in combination as appropriate. The emulsifier concentration
in the external aqueous phase is preferably about 0.001 to about
20% (w/w), more preferably about 0.01-about 10% (w/w), and still
more preferably about 0.05-about 5% (w/w).
[0113] In the above-described o/w method, microcapsules may be
produced by a method in which the complex is dispersed in an
organic solvent solution of a biodegradable polymer, i.e., the
s/o/w method.
[0114] (ii) In-water Drying Method (w/o/w Method)
[0115] In this method, a solution of a biodegradable polymer in an
organic solvent is first prepared. The concentration of the
biodegradable polymer in the organic solvent solution is normally
about 0.01 to about 80% (w/w), preferably about 0.1 to about 70%
(w/w), and more preferably about 1 to about 60%, depending on the
molecular weight of the biodegradable polymer, kind of
organic-solvent and so on. An aqueous dispersion of the complex is
used as the internal aqueous phase. The concentration of the
complex in the aqueous dispersion is, for example, about 10 to
about 90% (w/v). The above-described aqueous dispersion of the
complex is emulsified and dispersed in the organic solvent solution
of the biodegradable polymer to form a w/o emulsion by known
methods of dispersion using a turbine type mechanical stirrer,
homogenizer and so on. This operation is conducted in such a way as
to bring the weight ratio of the internal aqueous phase and the
biodegradable polymer up to about 1:2, preferably about 1:3. The
ratio of the internal aqueous phase and the organic solvent
solution of the biodegradable polymer is 1:1,000 to 1:1 (v/v),
preferably 1:100 to 1:5 (v/v), and more preferably 1:50 to 1:5
(v/v).
[0116] The w/o emulsion thus prepared is then added to another
aqueous phase to form a w/o/w emulsion, followed by evaporation of
the solvent in the oil phase, to yield microcapsules. This
operation is conducted in accordance with term (i) above.
[0117] The sustained-release preparation of the present invention
is preferably used in the form of fine particles. This is because
sustained-release preparation does not cause undue pain to the
patient when administered via an injection needle for ordinary
subcutaneous or intramuscular injection. The mean particle diameter
of the sustained-release preparation, for example, is about 0.1 to
about 300 .mu.m, preferably about 1 to about 150 .mu.m, and more
preferably about 2 to about 100 .mu.m.
[0118] In the present specification, a sustained-release
preparation in fine particle form is also referred to as a
microcapsule.
[0119] As used herein the term "microcapsule" may be referred to as
"microsphere".
[0120] The sustained-release preparation of the present invention
can, for example, be administered as microcapsules as such, or in
the form of various dosage forms of non-oral preparations (e.g.,
intramuscular, subcutaneous or visceral injections or indwellable
preparations, nasal, rectal or uterine transmucosal preparations
etc.) or oral preparations (e.g., capsules such as hard capsules,
soft capsules etc., solid preparations such as granules and powders
etc., liquid preparations such as suspensions etc.).
[0121] In the present invention, the sustained-release preparation
is preferably used for injection. When the sustained-release
preparation is a microcapsule, for instance, it can be prepared as
an aqueous suspension by suspending microcapsules in water, along
with a dispersing agent (e.g., surfactants such as Tween 80 and
HCO-60, polysaccharides such as carboxymethyl cellulose, sodium
alginate and sodium hyaluronate etc.), a preservative (e.g., methyl
paraben, propyl paraben etc.), an isotonizing agent (e.g., sodium
chloride, mannitol, sorbitol, glucose etc.), etc., to yield a
sustained-release preparation for injection of practical use.
Alternatively, the sustained-release preparation of the present
invention is prepared as an oily suspension by dispersing
microcapsules, along with a vegetable oil such as sesame oil or
corn oil with or without a phospholipid such as lecithin, or a
medium-chain fatty acid triglyceride (e.g., MIGLYOL 812), to yield
a sustained-release preparation for injection of practical use.
[0122] When the sustained-release preparation is a microcapsule,
for instance, its mean particle size is chosen over the range from
about 0.1 to about 300 .mu.m as long as the requirements concerning
degree of dispersion and needle passage are met, when it is to be
used as an injectable suspension. Preferably, the particle size
falls within the range from about 1 to about 150 .mu.m, more
preferably about 2 to about 100 .mu.m.
[0123] The above-described microcapsule can be prepared as a
sterile preparation, without limitation by the method in which the
entire production process is sterile, the method in which gamma
rays is used as sterilant, and the method in which an antiseptic is
added.
[0124] With low toxicity, the sustained-release preparation of the
present invention can be safely used in mammals (e.g., humans,
bovines, swines, dogs, cats, mice, rats, rabbits etc.).
[0125] Indications for the sustained-release preparation of the
present invention vary according to the physiologically active
substance used. For example, the sustained-release preparation of
the present invention is effective in the treatment or prevention
of diabetes mellitus etc. when the physiologically active substance
is insulin; renal cancer, hepatitis C etc. when the physiologically
active substance is interferon alpha; anemia etc. when the
physiologically active substance is erythropoietin; developmental
failure when the physiologically active substance is growth
hormone, and neutropenia etc. after anticancer chemotherapy when
the physiologically active substance is granulocyte
colony-stimulating factor. When the physiologically active
substance is erythropoietin, the sustained-release preparation of
the present invention is also effective in promoting hematopoiesis
for autotransfusion.
[0126] Depending on the type and content of the physiologically
active substance, duration of physiologically active substance
release, target disease, subject animal and other factors, the dose
of the sustained-release preparation may be set at levels such that
the physiologically active substance exhibits its action. The dose
per administration of the physiologically active substance is
chosen as appropriate over the range from about 0.0001 to about 10
mg/kg body weight for each adult, when the preparation is a 1-week
preparation. More preferably, the dose may be chosen as appropriate
over the range about about 0.0005 to about 1 mg/kg body weight.
[0127] The dose per administration of the sustained-release
preparation is preferably chosen as appropriate over the range from
about 0.0005 to about 50 mg/kg body weight for each adult. More
preferably, the dose is chosen as appropriate over the range from
about 0.0025 to about 10 mg/kg body weight. Dosing frequency can be
chosen as appropriate, e.g., once weekly, once every two weeks or
once every four weeks, depending on type, content and dosage form
of the physiologically active substance, duration of
physiologically active substance release, subject disease, subject
animal and other factors.
[0128] Although the preparation of the present invention may be
stored at normal temperature or in a cold place, it is preferable
to store it in a cold place. Normal temperature and a cold place as
mentioned herein are as defined by the Pharmacopoeia of Japan,
specifically, 15 to 25.degree. C. for normal temperatures and under
15.degree. C. for cold places.
BEST MODE FOR CARRYING OUT THE INVENTION
[0129] The present invention is hereinafter described in more
detail by means of the following examples, which are not to be
construed as limitative.
REFERENCE EXAMPLE 1
[0130] A solution of 0.5 g of swine insulin (27.3 U/mg, DIOSYNTH,
Netherlands) in 22 ml of 100 mM sodium hydroxide aqueous solution
and a solution of 1 g of zinc acetate (dihydrate) in 10 ml of
distilled water were mixed together and kept standing at room
temperature for 1 hour. After centrifugation at about 3,000 rpm
(05PR-22, Hitachi, Ltd.), the supernatant was discarded. The
residue was again dispersed in distilled water and centrifuged.
After the supernatant was discarded, a small amount of distilled
water was added to the residue, which was then freeze-dried to
yield about 1 g of crude zinc salt of swine insulin as a dry
powder.
[0131] To determine the insulin content in the powder thus
obtained, the powder was extracted with a 50 mM EDTA solution
containing 30% acetonitrile being shaken for 3 hours, followed by
quantitation by high performance liquid chromatography (HPLC). It
was shown that 47.6 mg of swine insulin was contained per 100 mg of
dry powder.
REFERENCE EXAMPLE 2
[0132] To a mixture of 168 ml of 40% aqueous solution of potassium
hydroxide and 1,000 ml of ethyl ether, 104 g of nitrosoethylurea
was added little by little, while the mixture was stirred under ice
cooling conditions. The resulting yellow ether layer was separated
and dried by the addition of granular potassium hydroxide. The
potassium hydroxide was then removed to yield about 900 ml of a
diazoethane solution.
[0133] 130 g of a lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=50/50 (mole %) of about 5,800 in weight-average
molecular weight) was dissolved in 1,900 ml of methylene chloride,
stirred and cooled. Under ice cooling conditions, the above
diazoethane solution was added drop by drop, followed by stirring
at room temperature for 2 hours. After the mixture was kept
standing overnight, the solvent was distilled off under reduced
pressure; the residue was vacuum dried at room temperature to yield
131 g of the ethyl ester of the lactic acid-glycolic acid
copolymer.
REFERENCE EXAMPLE 3
[0134] A solution of 1 mg of human growth hormone (Biotechnology
General, USA) in 0.9 ml of distilled water and a solution of 9.98,
29.43, 49.88, 69.84, 79.81 or 99.77 .mu.g of zinc acetate
(dihydrate) in 0.1 ml of distilled water were mixed together. A
molar ratio of zinc atom to growth hormone are 1, 3, 5, 7, 8 and
10. In case of the molar ratio being 5, about 60% of the human
growth hormone was precipitated. In case of the molar ratio being 7
or more, almost 100% of the human growth hormone was
precipitated.
REFERENCE EXAMPLE 4
[0135] 1 g of leuprolide acetate (TAP-144) and 157.5 mg of gelatin
were dissolved in 1 ml of distilled water at 70 to 80.degree. C. To
the aqueous solution warming at the temperature being slightly
higher than the gelation temperature of the aqueous solution, 21 g
of solution of lactic acid-glycolic acid copolymer, which was
prepared by dissolving 7.85 g of the lactic acid-glycolic acid
copolymer [lactic aicd/glycolic acid: 75/25 (mole %), viscosity:
0.142 to 0.169 cP] in 13.15 g of dichloromethane, was added. The
mixture was emulsified with a compact homogenizer for several
minutes or more to provide a W/O emulsion. The obtained W/O
emulsion was cooled to 10 to 20.degree. C. The emulsion was poured
in 5000 ml of 0.1% (w/v) aqueous polyvinyl alcohol solution which
temperature was adjusted to 10 to 20.degree. C. and the mixture was
emulsified using a turbine homomixer to provide a W/O/W emulsion.
This W/O/W emulsion was stirred at room temperature (15 to
30.degree. C.) to evaporate the dichloromethane and, thereby,
solidify the internal W/O emulsion, after which the microcapsules
were collected by centrifugation. These microcapsules were
redispersed in distilled water and further centrifuged to wash off
the excess drug and polyvinyl alcohol. The recovered microcapsules
were suspended in a small amount of distilled water. To the
suspension, 1.5 g of D-mannitol was added and dissolved. The
obtained suspension was freeze-dried under reduced pressure to
provide powdery microcapsules.
[0136] After lyophilization, the obtained microcapsules as a powder
were further dried at 50.degree. C. under reduced pressure, viz. a
temperature 3.degree. C. higher than Tmg of the matrix component
lactic acid-glycolic acid copolymer, for 24, 48, 96 or 120 hours to
provide powdery sustained-release microcapsules.
EXAMPLE 1
[0137] To 200 ml of an aqueous solution of interferon alpha
(containing 40 billion IU), 1 ml of an aqueous solution of zinc
acetate (dihydrate) (200 mg/ml) and 1 ml of 1 N sodium hydroxide
were added; after mixing, the mixture was kept standing at
4.degree. C. overnight. After centrifugation at 3,000 rpm, the
insoluble complex was recovered and freeze-dried to yield about 200
mg of crude zinc salt of interferon alpha.
[0138] To a solution of 1.5 g of a lactic acid-glycolic acid
copolymer (lactic acid/glycolic acid ratio=50/50, molecular weight
5,800, produced by Wako Pure Chemical Industries) and 1.5 g of the
ethyl ester of lactic acid-glycolic acid copolymer obtained in
Reference Example 2 in 4 ml of dichloromethane, 200 mg of the
above-described crude zinc salt of interferon alpha was added,
followed by stirring for about 30 seconds using a homogenizer
(Polytron) to yield an s/o emulsion. This emulsion was poured in
700 ml of a 0.1% (w/w) aqueous solution of polyvinyl alcohol
(EG-40, produced by The Nippon Synthetic Chemical Industry)
previously adjusted to 18.degree. C., followed by stirring in a
turbine homomixer at 6,000 rpm to yield an s/o/w emulsion. This
emulsion was stirred at room temperature for 3 hours to volatilize
the dichloromethane and solidify the oil phase. Subsequently, after
centrifugation at about 2,000 rpm (05PR-22, Hitachi, Ltd.), the
supernatant was discarded. The resulting residue was again
dispersed in distilled water and centrifuged. After the collected
microcapsules were re-dispersed in a small amount of distilled
water in the presence of 50 mg of D-mannitol, the dispersion was
freeze-dried to yield powder microcapsules.
EXAMPLE 2
[0139] 3.6 g of a lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=75/25 (mole %), weight-average molecular weight
13,585, number-average molecular weight 4,413, produced by Wako
Pure Chemical Industries) was dissolved in 6.6 g (5 ml) of
dichloromethane. 420 mg of the crude zinc salt of swine insulin
obtained in Reference Example 1 (containing 200 mg of swine
insulin) was dispersed in 6.6 g (5 ml) of dichloromethane. Both
were mixed and stirred for about 10 seconds in a homogenizer
(Polytron) to yield an s/o emulsion. This emulsion was poured in
800 ml of a 0.1% (w/w) aqueous solution of polyvinyl alcohol
(EG-40, produced by The Nippon Synthetic Chemical Industry),
previously adjusted to 18.degree. C., followed by stirring in a
turbine homomixer at 6,000 rpm to yield an s/o/w emulsion. This
emulsion was then stirred at room temperature for 3 hours to
volatilize the dichloromethane and solidify the oil phase. After
centrifugation at about 2,000 rpm (05PR-22, Hitachi, Ltd.), the
supernatant was discarded. The residue was again dispersed in
distilled water and centrifuged. After the collected microcapsules
were re-dispersed in a small amount of distilled water in the
presence of 50 mg of D-mannitol, the dispersion was freeze-dried to
yield powder microcapsules (about 3 g recovered).
[0140] To determine the insulin content in the microcapsules thus
obtained, the powder was extracted by shaking with a 50 mM EDTA
solution containing 30% acetonitrile for 3 hours, followed by
quantitation by high performance liquid chromatography (HPLC). It
was shown that 6.2 mg of insulin was contained per 100 mg of
microcapsules.
EXAMPLE 3
[0141] To 8 ml of an erythropoietin injection solution (Espo.TM.
Injection 3000, produced by Sankyo) (containing 12,000 IU), 1 g of
zinc chloride was added little by little; the mixture was kept
standing at room temperature for 1 hour. After the mixture was
centrifuged at 3,000 rpm, the precipitate was again dispersed in
distilled water and centrifuged to yield a precipitate. To this
precipitate, a small amount of distilled water was added, followed
by freeze-drying, to yield 60 mg of a mixture of crude zinc salt of
erythropoietin and crude zinc salt of albumin as a powder.
[0142] To a solution of 0.5 g of a lactic acid-glycolic acid
copolymer (lactic acid/glycolic acid ratio=50/50, molecular weight
14,000, produced by Wako Pure Chemical Industries) in 1.5 ml of
dichloromethane, 60 mg of the above-described mixture of crude zinc
salt of erythropoietin and crude zinc salt of albumin was added,
followed by stirring for about 30 seconds using a homogenizer
(Polytron), to yield an s/o emulsion. This emulsion was then
treated in the same manner as in Example 1 to yield 152 mg of
powder microcapsules.
EXAMPLE 4
[0143] Human growth hormone (Genotropin.TM. 161U, produced by
Sumitomo Pharmaceuticals) was dissolved in 1 ml of distilled water.
To this solution, 100 .mu.l of an aqueous solution of zinc chloride
(10 mg/ml) was added; the mixture was kept standing at room
temperature for 1 hour. The mixture was then centrifuged; the
precipitate was again dispersed in distilled water and centrifuged
to yield a precipitate. To this precipitate, a small amount of
distilled water was added, followed by freeze-drying, to yield 5.6
mg of crude zinc salt of human growth hormone as a powder.
[0144] To a solution of 0.5 g of a lactic acid-glycolic acid
copolymer (lactic acid/glycolic acid ratio=75/25, molecular weight
9,800, produced by Wako Pure Chemical Industries) in 1.5 ml of
dichloromethane, 5.6 mg of the above-described crude zinc salt of
human growth hormone was added, followed by stirring for about 30
seconds using a homogenizer (Polytron), to yield an s/o emulsion.
This emulsion was then treated in the same manner as in Example 1
to yield 121 mg of powder microcapsules.
EXAMPLE 5
[0145] After 10 ml (containing 3.times.10.sup.8 IU) of a
granulocyte colony-stimulating factor (G-CSF) injection solution
(Filgrastin Neupogen, trade name, Amgen, USA) was neutralized with
a dilute aqueous solution of sodium hydroxide, 1 ml of an aqueous
solution of zinc chloride (10 mg/ml) was added; the mixture was
kept standing at room temperature for 1 hour. The mixture was then
centrifuged; the precipitate was again dispersed in distilled water
and centrifuged to yield a precipitate. To this precipitate, a
small amount of distilled water was added, followed by
freeze-drying, to yield 4 mg of crude zinc salt of granulocyte
colony-stimulating factor as a powder.
[0146] To a solution of 0.5 g of a lactic acid-glycolic acid
copolymer (lactic acid/glycolic acid ratio=50/50, molecular weight
8,000, produced by Wako Pure Chemical Industries) in 1.5 ml of
dichloromethane, 4 mg of the above-described crude zinc salt of
granulocyte colony stimulating factor was added, followed by
stirring for about 30 seconds using a homogenizer (Polytron), to
yield an s/o emulsion. This emulsion was then treated in the same
manner as in Example 1 to yield 110 mg of powder microcapsules.
EXAMPLE 6
[0147] After 5.21 mg (26 U/mg) of human insulin (human recombinant
insulin, purchased from Wako Pure Chemical Industries) was
dissolved in 0.63 ml of a 57 mM aqueous solution of hydrochloric
acid, 0.35 ml of a 0.05 N aqueous solution of sodium hydroxide was
added to yield a human insulin solution of nearly neutral pH. To
this human insulin solution, 0.2 ml of an aqueous solution of zinc
acetate (20 mg/ml) was added; the mixture was kept standing at
4.degree. C. overnight. The mixture was then centrifuged at about
3,000 rpm; the precipitate was again dispersed in distilled water
and centrifuged to yield a precipitate. To this precipitate, a
small amount of distilled water was added, followed by
freeze-drying, to yield 11 mg of crude zinc salt of human insulin
as a powder.
[0148] To a solution of 0.5 g of a lactic acid-glycolic acid
copolymer (lactic acid/glycolic acid ratio=50/50, molecular weight
6,000, produced by Wako Pure Chemical Industries) in 1.5 ml of
dichloromethane, 11 mg of the above-described crude zinc salt of
human insulin was added, followed by stirring for about 30 seconds
using a homogenizer (Polytron), to yield an s/o emulsion. This
emulsion was then treated in the same manner as in Example 1 to
yield 105 mg of powder microcapsules.
COMPARATIVE EXAMPLE
[0149] To a solution of 0.9 g of lactic acid-glycolic acid
copolymer [lactic acid/glycolic acid ratio=50/50 (mole %),
weight-average molecular weight 6,000, produced by Wako Pure
Chemical Industries] in 1.5 ml of dichloromethane, 100 mg of a
substantially zinc free human insulin [zinc content being under
0.0001% (w/w)] was added, followed by stirring for about 10 seconds
using a homogenizer (Polytron), to yield an s/o emulsion. This
emulsion was then treated in the same manner as in Example 1 to
yield powder microcapsules (470 mg).
[0150] To determine the insulin content in the microcapsules thus
obtained, the powder was extracted by shaking with a 50 mM EDTA
solution containing acetonitrile for 3 hours, followed by
quantitation by high performance liquid chromatography (HPLC). It
was shown that 8.7 mg of insulin was contained per 100 mg of
microcapsules.
EXPERIMENTAL EXAMPLE 1
[0151] 323 mg of powder microcapsules as obtained in Example 2 was
dispersed in a 1 ml of dispersant for injection (5 mg of
carboxymethyl cellulose, 1 mg of polysorbate 80 and 50 mg of
mannitol dissolved per ml distilled water). The resulting
dispersion was subcutaneously administered to the backs of
6-week-old male SD rats (insulin administered at about 20 mg per
rat). After administration, blood was collected via the tail at
constant intervals and assayed for serum swine insulin
concentration using an enzyme immunoassay (EIA) kit (produced by
Sanko Junyaku). Active swine insulin was detected in serum for 1
week or more after administration.
EXPERIMENTAL EXAMPLE 2
[0152] 70 mg of powder microcapsules as obtained in Example 4 was
dispersed in a 0.5 ml of dispersant for injection (5 g of
carboxymethyl cellulose, 2 g of polysorbate 80 and 50 g of mannitol
dissolved per liter distilled water). The resulting dispersion was
subcutaneously administered to the backs of 6-week-old male SD rats
(growth hormone administered at about 3 mg per rat). After
administration, blood was collected via the tail at constant
intervals and assayed for serum growth hormone concentration by
radio immunoassay. Active growth hormone was detected in serum for
1 week or more after administration.
COMPARATIVE EXPERIMENTAL EXAMPLE
[0153] 154.7 mg of powder microcapsule as obtained in Comparative
Example was dispersed in a 1.75 ml of dispersant for injection (5
mg of carboxymethyl cellulose, 1 mg of polysorbate 80 and 50 mg of
mannitol dissolved per ml distilled water). The resulting
dispersion was subcutaneously administered to the backs of
6-week-old male SD rats (insulin administered at about 44 mg per
rat). After administration, blood was collected via the tail at
constant intervals and assayed for serum insulin concentration by
an enzyme immunoassay (EIA). Active insulin was detected in serum
only at 1 day after administration.
INDUSTRIAL APPLICABILITY
[0154] According to the present invention, it is possible to
provide a sustained-release preparation that is highly efficient in
incorporating physiologically active substance and suppresses
initial physiologically active substance burst. The
sustained-release preparation of the present invention is capable
of releasing the physiologically active substance while retaining
its bioactivity after administration in vivo. Furthermore, the
physiologically active substance in the sustained-release
preparation is kept stable for a long period of time, with little
loss of bioactivity.
[0155] The following disclosure on pages 32-86 relates to a second
embodiment of a sustained-release preparation which comprises an
anti-endothelin substance.
FIELD OF THE SECOND EMBODIMENT
[0156] The second embodiment of the present invention relates to a
sustained-release preparation of an anti-endothelin substance, such
as an endothelin antagonist, used to treat endothelin associated
diseases, particularly chronic diseases, such as chronic
complications in diabetes mellitus.
BACKGROUND OF THE SECOND EMBODIMENT
[0157] Showing various potent physiological actions, peptides have
been applied as pharmaceuticals in numerous attempts. Their
biological half-life, however, is usually very short. Therefore,
for a sustained pharmacologic effect, peptides must be frequently
administered, resulting in severe suffering by the patient.
Endothelin, a peptide secreted by the vascular endothelium, shows
vascular smooth muscle constricting action, both potent and
sustainable. Endothelin is therefore important both physiologically
and pathologically. Also, there have been reports of the
development of peptide-based endothelin antagonists, with the
strong expectation that anti-endothelin substances such as
endothelin receptor antagonists will contribute to the treatment of
various diseases associated with endothelin. For the reasons
described above, however, the application of such peptide-based
antagonists as pharmaceuticals has been limited. Also, in
therapeutic application of conventional endothelin antagonists,
there have been attempts to prevent the onset and progress of
pathologic states by antagonizing endothelin-associated reactions
in acute diseases such as attacks and shocks of acute myocardial
infarction. Although application to the treatment of hypertension,
cardiac/cerebral circulatory diseases, renal diseases and other
diseases has been suggested, there is no specific exemplification.
Nor has there been any finding that administration of endothelin
antagonists is effective in preventing the onset and progress of
endothelin-associated pathologic states in chronic diseases such as
diabetic nephropathy.
[0158] Various sustained-release preparations are known, including
the release rate controlling system based on a polymeric matrix
containing a polypeptide dispersed in a poly(lactide-glycolide)
copolymer, described in Japanese Patent Unexamined Publication No.
2930/1988 (EP-A-251476).
[0159] Japanese Patent Examined Publication No. 40329/1992
(Japanese Patent Unexamined Publication No. 118512/1982,
EP-A-52510) discloses a composition comprising a biodegradable
poly(lactide-glycolide) copolymer which is biologically compatible
with luteinizing hormone-releasing hormone (LH-RH) or an analog
thereof, a water-soluble polypeptide, and which is capable of
sustained release of an effective amount of the polypeptide over a
period of at least 1 month.
[0160] Japanese Patent Unexamined Publication No. 124814/1990
(EP-A-350246) discloses an art in which a water-soluble drug is
effectively packed in microcapsules by adding drug retaining
substance comprising an organic basic substance such as a basic
amino acid and using a wall made of polymer, and excessive drug
release just after administration is suppressed.
[0161] There is no sustained-release preparation which comprises a
combination of an anti-endothelin substance and a biodegradable
polymer and which is capable of effective sustained release of the
anti-endothelin substance at an almost constant rate.
[0162] Against the above background there is a need for an
excellent sustained-release preparation for the treatment of
chronic diseases caused by endothelin.
SUMMARY OF THE SECOND EMBODIMENT
[0163] According to the present invention, there is provided:
[0164] (1) a sustained-release preparation which comprises an
anti-endothelin substance and a biodegradable polymer,
[0165] (2) the sustained-release preparation according to (1)
above, wherein the anti-endothelin substance is an endothelin
antagonist,
[0166] (3) the sustained-release preparation according to (2)
above, wherein the endothelin antagonist is a peptide,
[0167] (4) the sustained-release preparation according to (2)
above, wherein the endothelin antagonist is a peptide of the
general formula: 1
[0168] wherein X and Y independently represent an .alpha.-amino
acid residue; A represents a D-acidic-.alpha.-amino acid residue; B
represents a neutral-.alpha.-amino acid residue; C represents an
L-.alpha.-amino acid residue; E represents a D-.alpha.-amino acid
residue having an aromatic cyclic group, or an ester thereof, or a
salt thereof,
[0169] (5) the sustained-release preparation according to (4)
above, wherein the peptide is a compound of the formula
cyclo[-D-Asp-Asp(R1')-As- p-D-Thg(2)-Leu-D-Trp-] wherein Asp
represents aspartic acid; Asp(R1') represents aspartic acid
.beta.-4-phenylpiperazinamide; and Thg(2) represents
2-thienylglycine; Leu represents leucine; Trp represents
tryptophan,
[0170] (6) the sustained-release preparation according to (4)
above, wherein A is a D-acidic-.alpha.-amino acid residue which is
esterified with an alkyl group,
[0171] (7) the sustained-release preparation according to (4)
above, wherein Y is a L-acidic-.alpha.-amino acid residue,
[0172] (8) the sustained-release preparation according to (4)
above, wherein Y is a L-acidic-.alpha.-amino acid residue which is
esterified with an alkyl group,
[0173] (9) the sustained-release preparation according to (4)
above, wherein the peptide is a compound of the formula,
cyclo-(-D-Asp(OC.sub.2H-
.sub.5)-Asp(R1')-Asp(OC.sub.2H.sub.5)-D-Thg(2)-Leu-D-Trp-], wherein
Asp represents aspartic acid; Asp(R1') represents aspartic acid
.beta.-4-phenylpiperazinamide; Thg(2) represents 2-thienylglycine;
Leu represents leucine; and Trp represents tryptophan,
[0174] (10) the sustained-release preparation according to (4)
above, wherein the salt is a polyvalent metal salt,
[0175] (11) the sustained-release preparation according to (10)
above, wherein the polyvalent metal salt is a zinc salt,
[0176] (12) the sustained-release preparation according to (1)
above, wherein the biodegradable polymer is an aliphatic
polyester,
[0177] (13) the sustained-release preparation according to (12)
above, wherein the aliphatic polyester is a copolymer of glycolic
acid and lactic acid,
[0178] (14) the sustained-release preparation according to (13)
above, wherein the copolymer has a weight-average molecular weight
of about 2,000 to 50,000, as determined by Gel Permeation
Chromatography,
[0179] (15) the sustained-release preparation according to (13)
above, wherein the copolymer has a dispersity of about 0.2 to
4.0,
[0180] (16) the sustained-release preparation according to (1)
above, which further comprises an organic basic substance,
[0181] (17) the sustained-release preparation according to (1)
above, which further comprises a water-soluble polyvalent metal
salt,
[0182] (18) a method for treatment of diseases caused by endothelin
comprising administering to a patient in need thereof an effective
amount of the sustained-release preparation according to (1)
above,
[0183] (19) the method according to (18) above, wherein the
diseases are chronic diseases,
[0184] (20) the method according to (19) above, wherein the chronic
diseases are chronic complications in diabetes mellitus,
[0185] (21) the method according to (20) above, wherein the chronic
complications are diabetic nephropathy,
[0186] (22) an injectable preparation which comprises the
sustained-release preparation according to (1) above.
[0187] (23) a peptide of the general formula: 2
[0188] wherein X and Y independently represent an .alpha.-amino
acid residue; A' represents a D-acidic-.alpha.-amino acid residue
which is esterified with an alkyl group; B represents a
neutral-.alpha.-amino acid residue; C represents an L-.alpha.-amino
acid residue; E represents a D-.alpha.-amino acid residue having an
aromatic cyclic group, or a salt thereof,
[0189] (24) the peptide according to (23) above, wherein X is an
L-isomer,
[0190] (25) the peptide according to (23) above, wherein Y is an
L-isomer,
[0191] (26) the peptide according to (23) above, wherein A' is
D-glutamic acid or D-aspartic acid which is esterified with an
alkyl group,
[0192] (27) the peptide according to (23) above, wherein B is an
D-isomer,
[0193] (28) the peptide according to (23) above, wherein B is
selected from the group consisting of D-leucine, D-alloisoleucine,
D-tertiary leucine, D-gamma methyl leucine, D-phenylglycine,
D-2-thienylglycine, D-3-thienylglycine, D-2-cyclopentylglycine,
D-phenylalanine, D-2-thienylalanine, D-valine, D-2-furylglycine and
D-3-furylglycine residues,
[0194] (29) the peptide according to (23) above, wherein C is
selected from the group consisting of L-leucine, L-phenylalanine
and L-tryptophan residues,
[0195] (30) the peptide according to (23) above, wherein E is
selected from the group consisting of D-tryptophan or derivatives
thereof, D-1-naphthylalanine, D-2-naphthylalanine,
D-benzothienylalanine, D-4-bisphenylalanine and D-pentamethyl
phenylalanine residues,
[0196] (31) the peptide according to (23) above, wherein Y is an
.alpha.-amino acid residue having a carboxyl group which is
esterified with an alkyl group,
[0197] (32) a peptide according to the formula:
cyclo-[-D-Asp(OC.sub.2H.su-
b.5)-Asp(R1')-Asp(OC.sub.2H.sub.5)-D-Thg(2)-Leu-D-Trp-], wherein
Asp represents aspartic acid; Asp(R1') represents aspartic acid
.beta.-4-phenylpiperazinamide; Thg(2) represents 2-thienylglycine;
Leu represents leucine; and Trp represents tryptophan, or a salt
thereof, and
[0198] (33) a zinc salt of a peptide represented by the general
formula: 3
[0199] wherein X and Y independently represent an .alpha.-amino
acid residue; A represents a D-acidic-.alpha.-amino acid residue; B
represents a neutral-.alpha.-amino acid residue; C represents an
L-.alpha.-amino acid residue; E represents a D-.alpha.-amino acid
residue having an aromatic cyclic group.
[0200] As the pathologic state of diabetics is better managed as a
result of advances in medicine and pharmacology, the life span of
diabetics is increasing also. The extended period of the diabetic
condition, however, has raised the problem of chronic
complications, especially vascular disorders. Vascular disorders
are known to cause various organ disorders because the former occur
in coronary arteries, cerebral arteries and microvessels such as
those in the retina and renal glomeruli. An example of a chronic
complication in diabetes mellitus is nephropathy. Although many
factors have been suggested as being involved in the onset of
diabetic nephropathy, mesangial thickening and mesangial cell
proliferation are marked pathologic factors. It has been assumed
that such mesangial thickening eventually destroys glomeruli,
causing terminal renal failure. Endothelin, secreted from vascular
endothelial cells, is known to be released in large amounts from
damaged vessels. Based on the fact that in mesangial cells
endothelin stimulates various reactions associated with cell
proliferation, such as thymidine uptake, Na.sup.+/H.sup.+ exchange
and c-fos expression, the possibility is suggested that chronic
exposure to excess endothelin can be the initial stimulation to
cause mesangial cell proliferation, suggesting the involvement of
endothelin in diabetic nephropathy. In complications other than
nephropathy (e.g., diabetic cardiomyopathy and diabetic
retinopathy) as well, endothelin resulting from vascular disorders
may be involved in the chronic fixation of the pathologic state.
Also, since arteriosclerosis and hyperlipidemia are often seen in
diabetes mellitus, with some disorder of endothelial cells,
involvement of endothelin in these pathologic states is suspected.
There are other endothelin-associated diseases, particularly
chronic ones, whose onset and progress can be prevented by applying
the therapy of the present invention for sustained retention of an
anti-endothelin substance in the living body.
DETAILED DESCRIPTION OF THE SECOND EMBODIMENT
[0201] Where 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.
[0202] In the present invention, the anti-endothelin substance is
exemplified by antibodies against endothelin, antibodies against
endothelin receptors, high molecular substances represented by
soluble endothelin receptors, endothelin antagonists obtained by
chemical synthesis or fermentation, and substances which inhibit
endothelin production (endothelin converting enzyme
inhibitors).
[0203] The anti-endothelin substance in the present invention
inhibits the binding of endothelin to its receptors. For example,
the anti-endothelin substance inhibits the binding of endothelin-1
to a membrane fraction prepared from a homogenate of swine aortic
smooth muscle. It is reported that there are at least two subtypes
of endothelin receptors, referred to as ET-A and ET-B,
respectively. The anti-endothelin substance in the present
invention antagonizes one or both of these two receptors.
[0204] The anti-endothelin substance in the present invention
inhibits vascular or muscular contraction induced by endothelin-1
administration in spiral specimens of swine coronary artery with
the endothelial cells removed, specimens of the excised guinea pig
tracheal muscle or specimens of the excised swine cerebral basal
artery, antagonizes the increase in perfusion pressure by
endothelin in excised rat hearts, and improves mortality in mice
receiving endotoxin.
[0205] The anti-endothelin substance in the present invention may
be water soluble or oil soluble. The degree of water solubility in
the present invention is preferably octanol/water ratios of not
higher than 0.1. The degree of oil solubility in the present
invention is preferably octanol/water ratios of over 0.1. Also, the
anti-endothelin substance may be soluble in polar solvents such as
acetonitrile, dichloromethane and chloroform at not less than 10
mg/ml and not more than 100 mg/ml. It may also be almost insoluble
in acetonitrile, dichloromethane and chloroform.
[0206] In the present invention, the anti-endothelin substance is
preferably an endothelin antagonist, as exemplified by non-peptide
compounds, peptides and derivatives thereof obtained by chemical
synthesis or fermentation, peptides and derivatives thereof. Here,
the peptide may be a chain or cyclic peptide or a cyclic and chain
peptide.
[0207] Examples of non-peptide compounds include the non-peptides
described in European Patent Publication Nos. 510526 and 526708 and
WO93/08799.
[0208] (1) EPA-510526:
[0209] a compound represented by the formula 4
[0210] wherein
[0211] R.sup.1: a hydrogen atom, lower-alkyl group, lower-alkoxy
group, lower-alkylthio group, a halogen atom or
trifluoromethyl;
[0212] R.sup.2: a hydrogen atom, a halogen atom, lower-alkoxy
group, hydroxy-lower-alkoxy group, or trifluoromethyl;
[0213] R.sup.3: a hydrogen atom, hydroxy group, a halogen atom,
alkylthio group, cycloalkyl group, hydroxy-lower-alkyl group,
hydroxy-lower-alkoxy group, hydroximino-lower-alkyl lower-alkenyl
group, oxo-lower-alkyl group, trifluoromethyl, trifluoromethoxy,
lower-alkoxy group, lower-alkoxy-lower-alkoxy group,
aryl-lower-alkoxy group;
[0214] R.sup.2 and R.sup.3: together to form butadienyl;
[0215] R.sup.4: a hydrogen atom, lower-alkyl group, aryl group or
heteroaryl group;
[0216] R.sup.5: a hydrogen atom, lower-alkanoyl group, benzoyl,
hetrocyclylcarbonyl group, or tetrahydropyran-2-yl;
[0217] R.sup.6 is represented by the formula (a) or (b) 5
[0218] R.sup.7: a hydrogen atom, lower-alkoxy group or nitro, and
R.sup.8 represents a hydrogen atom, a halogen atom, lower-alkyl
group, lower-alkoxy group, lower-alkylthio group, nitro, hydroxy,
amino or trifluoromethyl;
[0219] R.sup.7 and R.sup.8: together to form butadienyl;
[0220] R.sup.9: a hydrogen atom, a halogen atom, lower-alkyl group,
lower-alkoxy group, lower-alkylthio group or trifluoromethyl;
[0221] R.sup.10: a hydrogen atom, a halogen atom, lower-alkyl
group, lower-alkoxy group or lower-alkylthio group;
[0222] X and Y: independently O, S or NH;
[0223] n: 2, 3 or 4; or a salt thereof;
[0224] (2) EPA-526708:
[0225] a compound represented by the formula 6
[0226] wherein
[0227] R.sup.1: a hydrogen atom, lower-alkoxy group,
lower-alkylthio group, a halogen atom or trifluoromethyl;
[0228] R.sup.2: a hydrogen atom, a halogen atom, lower-alkoxy
group, trifluoromethyl or --OCH.sub.2COOR.sup.a;
[0229] R.sup.3: a hydrogen atom, a halogen atom, lower-alkyl group,
lower-alkylthio group, cycloalkyl group, lower-alkoxy group or
trifluoromethyl
[0230] R.sup.2 and R.sup.3: together to form butadienyl,
methylenedioxy, ethylenedioxy or isopropylidendioxy;
[0231] R.sup.4: a hydrogen atom, lower-alkyl group, cycloalkyl
group, trifluoromethyl, lower-alkoxy group, lower-alkylthio group,
lower-alkylthio-lower-alkyl group, hydroxy-lower-alkyl group,
hydroxy-lower-alkoxy group, lower-alkoxy-lower-alkyl group,
hydroxy-lower-alkoxy-lower-alkyl group,
hydroxy-lower-alkoxy-lower-alkoxy group, lower-alkylsulfinyl group,
lower-alkylsulfonyl group, 2-methoxy-3-hydroxypropoxy,
2-hydroxy-3-phenylpropyl, amino-lower-alkyl group,
lower-alkylamino-lower-alkyl group, di-lower-alkylanmino-lower-alk-
yl group, amino, lower-alkylamino group, di-lower-alkylamino group;
arylamino group, aryl group, arylthio group, aryloxy group,
aryl-lower-alkyl group or heterocyclyl group;
[0232] R.sup.5: a hydrogen atom, lower-alkyl group, lower-alkanoyl
group, benzoyl, hetrocyclyl-carbonyl group, hetrocyclyl-methyl, or
tetrahydropyran 2-yl;
[0233] R.sup.6.about.R.sup.9: a hydrogen atom, a halogen atom,
trifluoromethyl, lower-alkyl group, lower-alkoxy group,
lower-alkylthio group, hydroxy, hydroxymethyl, cyano, carboxyl,
formyl, methyl sulfinyl, methyl sulfonyl, methyl sulfonyloxy,
lower-alkoxy carbonyloxy;
[0234] R.sup.7: together with R.sup.6 or R.sup.8 to form
butadienyl, methylenedioxy, ethylenedioxy or
isopropyliedenedioxy;
[0235] Z: --O--, --S--, ethylene, vinylene, --CO--, --OCHR.sup.10--
or --SCHR.sup.10--;
[0236] R.sup.10: a hydrogen atom or lower-alkyl group;
[0237] X and Y: independently O, S or NH;
[0238] YR.sup.5: lower-alkyl sulfinyl or
--OCH.sub.2CH(OR.sup.c)CH.sub.2R.- sup.d;
[0239] R.sup.a, R.sup.b, R.sup.c and R.sup.d: a hydrogen atom or
lower-alkyl group;
[0240] R.sup.c and R.sup.d: together to form methylene, ethylene or
isopropylidene;
[0241] n: 1, 2 or 3; or a salt thereof;
[0242] (3) WO93/08799:
[0243] a compound of formula 7
[0244] wherein:
[0245] R.sub.1 is --X(CH.sub.2).sub.nAr or
--X(CH.sub.2).sub.nR.sub.8 or 8
[0246] R.sub.2 is hydrogen, Ar or (c);
[0247] P.sub.1 is --X(CH.sub.2).sub.nR.sub.8;
[0248] P.sub.2 is --X(CH.sub.2).sub.nR.sub.8, or --XR.sub.9Y;
[0249] R.sub.3 and R.sub.5 are independently hydrogen, R.sub.11,
OH, C.sub.1-8 alkoxy, S(O).sub.qR.sub.11, N(R.sub.6).sub.2, Br, F,
I, Cl, CF.sub.3, NHCOR.sub.6, --XR.sub.9--Y or
--X(CH.sub.2).sub.nR.sub.8 wherein the methylene groups of
--X(CH.sub.2).sub.bR.sub.8 may be substituted by one or more
(CH.sub.2).sub.nAr groups;
[0250] R.sub.4 is hydrogen, R.sub.11, OH, C.sub.1-5 alkoxy,
S(O).sub.qR.sub.11, N(R.sub.6).sub.2, --X(R.sub.11), Br, F, I, Cl
or NHCOR.sub.6 wherein the C.sub.1-5 alkoxy may be substituted by
OH, methoxy or halogen;
[0251] R.sub.6 is independently hydrogen or C.sub.1-4 alkyl;
[0252] R.sub.7 is independently hydrogen, C.sub.1-6 alkyl or
(CH.sub.2).sub.nAr;
[0253] R.sub.8 is hydrogen, R.sub.11, CO.sub.2H, PO.sub.3H.sub.2,
P(O)(OH)R.sub.7 or tetrazole;
[0254] R.sub.9 is C.sub.1-10 alkyl, C.sub.2-10 alkenyl or phenyl
all of which may be substituted by one or more OH,
N(R.sub.6).sub.2, COOH, halogen or XC.sub.1-5 alkyl;
[0255] R.sub.10 is R.sub.3 or R.sub.4;
[0256] R.sub.11 is C.sub.1-8 alkyl, C.sub.2-8 alkenyl, C.sub.2-8
alkynyl all of which may be substituted by one or more OH,
CH.sub.2OH, N(R.sub.6).sub.2 or halogen;
[0257] X is (CH.sub.2).sub.n, O, NR.sub.6 or S(O).sub.q;
[0258] Y is CH.sub.3 or --CH.sub.2X(CH.sub.2).sub.nAr;
[0259] Ar is 9
[0260] naphthyl, indolyl, pyridyl or thienyl, oxazolidinyl,
oxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl,
tetrazolyl, imidazolyl, imidazolidinyl, thiazolidinyl, isoxazolyl,
oxadiazolyl, thiadiazolyl, morpholinyl, piperidinyl, piperazinyl,
pyrrolyl, or pyrimidyl; all of which may be substituted by one or
more R3 or R4 groups;
[0261] A is C.dbd.O, or [C(R.sub.6).sub.2].sub.m;
[0262] B is --CH.sub.2-- or --O--;
[0263] Z.sub.1 and Z.sub.2 are independently hydrogen, C.sub.1-8
alkyl, C.sub.2-8 alkenyl, C.sub.2-8 alkynyl, OH, C.sub.1-8 alkoxy,
S(O).sub.qC.sub.1-8 alkyl, N(R.sub.6).sub.2, Br, F, I, Cl,
NHCOR.sub.6, --X(CH.sub.2).sub.nR.sub.8, phenyl, benzyl or
C.sub.3-6 cycloalkyl wherein the C.sub.1-8 alkyl, C.sub.2-8 alkenyl
or C.sub.2-8 alkynyl may be optionally substituted by COOH, OH,
CO(CH.sub.2).sub.nCH.sub.3,
CO(CH.sub.2).sub.nCH.sub.2N(R.sub.6).sub.2, or halogen; or Z.sub.1
and Z.sub.2 together may be --O--A--O-- on contiguous carbons;
[0264] Z.sub.3 is Z.sub.1 or XR.sub.9Y;
[0265] q is zero, one or two;
[0266] n is an integer from 0 to six;
[0267] m is 1, 2 or 3;
[0268] and the dotted line indicates the optional presence of a
double bond; or a pharmaceutically acceptable salt thereof;
provided that
[0269] R.sub.2 is not hydrogen when X is S(O).sub.q;
[0270] when the optional double bond is present there is only one
R.sub.10 and there is no P.sub.1;
[0271] the compound of Formula I is not
(1RS)-1,3-diphenylindene-2-carboxy- lic acid; (cis,
cis)-(1RS,3SR)-1,3-diphenylindane-2-carboxylic acid;
(1RS)-3-[3-Methyl-1-phenyl-(1H)-ind-2-en-1-yl] propionic aicd; or
(1RS)-2[1,3-diphenyl-(1H)-ind-2-en-2-yl]ethanoic acid.
[0272] Examples of chain peptides include the peptides described in
Japanese Patent Unexamined Publication Nos. 244097/1992,
283600/1992 and WO93/10144.
[0273] (1) Japanese Patent Unexamined Publication No.
244097/1992:
[0274] a peptide of the formula: 10
[0275] in which
[0276] R.sup.1 is hydrogen or acyl,
[0277] R.sup.2 is lower alkyl, optionally substituted
ar(lower)alkyl, cyclo(lower)alkyl(lower)alkyl or optionally
substituted heterocyclic(lower)alkyl,
[0278] R.sup.3 is optionally substituted heterocyclic(lower)alkyl
or optionally substituted ar(lower)alkyl,
[0279] R.sup.4 is hydrogen or optionally substituted lower
alkyl,
[0280] R.sup.5 is carboxy, protected carboxy, carboxy(lower)alkyl
or protected carboxy(lower)alkyl,
[0281] R.sup.6 is hydrogen or optionally substituted lower
alkyl,
[0282] R.sup.7 is hydrogen or lower alkyl, and
[0283] A is --O--, --NH--, lower alkylimino or lower alkylene,
[0284] provided that when R.sup.2 is (S)-isobutyl, R.sup.3 is
N-(dichlorobenzyloxycarbonyl)indol-3-ylmethyl, R.sup.4 is methyl,
R.sup.5 is methoxycarbonyl, R.sup.6 is hydrogen, R.sup.7 is
hydrogen and A is --NH--, then the partial formula: 11
[0285] has the absolute configuration of 12
[0286] or a pharmaceutically acceptable salt thereof.
[0287] (2) Japanese Patent Unexamined Publication No.
283600/1992:
[0288] a peptide derivative represented by the formula:
R1-HX.sub.1DX.sub.2IX.sub.3
[0289] wherein X.sub.1 represents leucine, arginine or glutamine
residue, X.sub.2 represents isoleucine or valine residue, X.sub.3
represents tryptophan, amidotryptophan or D-naphtylalanine residue
and R1 represents residual 15 amino acids.
[0290] (3) WO93/10144:
[0291] a compound of the formula: 13
[0292] in which
[0293] R.sup.3 is hydrogen or lower alkyl,
[0294] R.sup.4 is pyridyl(lower)alkyl; and
[0295] R.sup.1 is C.sub.3-C.sub.8 alkyleneamino,
N,N-di(lower)alkylamino, N-lower alkyl-N-arylamino, N-lower
alkyl-N-C.sub.3-C.sub.8 cycloalkylamino, or C.sub.5-C.sub.10
bycyclic alkyleneamino,
[0296] R.sup.2 is lower alkyl,
[0297] R.sup.5 is C.sub.3-C.sub.8 alkyleneamino,
N,N-di(lower)alkylamino, morpholino, thiomorpholino,
N',N'-di(lower)alkylhydrazino, morpholinoamino, lower alkylpipe
razinylamino, lower alkoxy(lower)alkylamino,
morpholino(lower)alkylamino, C.sub.3-C.sub.8
alkyleneamino(lower)-alkylamino which may be substituted by oxo, or
pyridyl(lower)alkylamino, and
[0298] A is lower alkylene; or
[0299] R.sup.1 is piperidin-1-yl, lower alkylpiperidin-1-yl,
octahydroazocin-1-yl, indolin-1-yl,
1,2,3,4-tetrahydroquinolin-1-yl, N,N-di(lower)alkyl amino, N-lower
alkyl-N-arylamino, N-lower alkyl-N-C.sub.3-C.sub.8 cycloalkylamino,
or C.sub.5-C.sub.10 bycyclic alkyleneamino,
[0300] R.sup.2 is lower alkyl,
[0301] R.sup.5 is amino or lower alkylamino, and
[0302] A is lower alkylene; or
[0303] R.sup.1 is piperidin-1-yl, octahydroazocin-1-yl,
N,N-di(lower)alkylamino, or C.sub.5-C.sub.10 bycyclic
alkyleneamino,
[0304] R.sup.2 is lower alkyl,
[0305] R.sup.5 is amino, lower alkylamino, N,N-di(lower)alkylamino,
C.sub.3-C.sub.8 alkyleneamino, or morpholino, and
[0306] A is --NH--; or
[0307] R.sup.1 hexahydro-1H-azepin-1-yl,
[0308] R.sup.2 is isobutyl,
[0309] R.sup.5 is ethylamino, and
[0310] A is methylene; or
[0311] R.sup.1 is
N-[1-(dimethylcarbamoyl)-2,2-dimethylpropyl]amino,
[0312] R.sup.2 is isobutyl,
[0313] R.sup.5 is amino, and
[0314] A is --NH--; or
[0315] R.sup.1 is N,N-di(lower)alkylamino,
1,2,3,4-tetrahydroquinolin-1-yl- , N-lower alkyl-N-arylamino, or
N-lower alkyl-N-C.sub.3-C.sub.8 cycloalkylamino,
[0316] R.sup.2 is lower alkyl,
[0317] R.sup.5 is hydroxy or CO--R.sup.5 is protected carboxy,
and
[0318] A is lower alkylene; or
[0319] R.sup.1 is C.sub.5-C.sub.10 bycyclic alkyleneamino,
[0320] R.sup.2 is lower alkyl,
[0321] R.sup.5 is hydroxy or CO-R.sup.5 is protected carboxy,
and
[0322] A is lower alkylene or --NH--; or
[0323] R.sup.1 is N-ethyl-N-(1-ethylpropyl)amino,
N-ethyl-N-isopropylamino- , N-ethyl-N-neopentylamino, or
N-(1-ethylpropyl)-N-propylamino,
[0324] R.sup.2 is isobutyl,
[0325] R.sup.5 is hydroxy or CO-R.sup.5 is protected carboxy,
and
[0326] A is --NH--; or
[0327] R.sup.1 is piperidin-1-yl,
[0328] R.sup.2 is isobutyl,
[0329] R.sup.5 is hydroxy or CO-R.sup.5 is protected carboxy,
and
[0330] A is methylene; or
[0331] R.sup.1 is hexahydro-1H-azepin-1-yl,
[0332] R.sup.2 is propyl,
[0333] R.sup.5 is hydroxy or CO-R.sup.5 is protected carboxy,
and
[0334] A is --NH--;
[0335] or a pharmaceutically acceptable salt thereof.
[0336] Examples of cyclic peptides include the peptides described
in Japanese Patent Unexamined Publication No.261198/1992.
[0337] Japanese Patent Unexamined Publication No. 261198/1992:
[0338] a cyclic pentapeptide of the formula:
cyclo (-X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-)
[0339] wherein X.sup.1.about.X.sup.5 represent amino acid residues,
respectively, and X.sup.1 is D-Phe, D-Tyr, D-Tha, D-Tza, D-Nal,
D-Bta, D-Trp, D-Trp(O), D-Trp(CHO) or
D-Trp(CH.sub.2).sub.mCOR.sup.1, wherein m is from 0 to 6, and
R.sup.1 is a hydroxyl group, a C.sub.1-C.sub.6 alkoxy group, an
amino group or a C.sub.1-C.sub.6 monoalkylamino group, provided
that when m=0, R.sup.1 is not a hydroxyl group; X.sup.2 is D-Asp,
D-Glu, or D-Cys(O.sub.3H); X.sup.3 is Pro, Hyp, Pip, Thz,
.beta.-Ala, Gly, Ala, .alpha.-Aba, Aib, Val, Nva, Leu, Ile, aIle,
Nle, Met, Met(O), Met(O.sub.2), Phe, Tza, Tha, Tyr, Trp, His, Arg,
Lys, Lys(CHO), Orn, Orn(O), Asn, Gln, Asp, Glu, Cys(O.sub.3H), Cys,
Ser or Thr wherein those .alpha.-amino acids having a hydrogen atom
on the .alpha.-amino group are optionally substituted by a
C.sub.1-C.sub.6 alkyl or C.sub.3-C.sub.7 cycloalkyl group which
optionally has a group selected from the group consisting of an
imidazolyl group, a carboxyl group, a sulfo group and a hydroxy
group; X.sup.4 is D-Ala, D-Thr, D-.alpha.-Aba, D-Val, D-Nva, D-Leu,
D-Ile, D-aIle, D-Nle, D-tert-Leu, D-Cpg, D-Chg, D-Dpg, D-Pen, Aib,
Ac.sub.3c, Ac.sub.4c, Ac.sub.5c, Ac.sub.6c, Ac.sub.7c, D-Phg,
D-Thg, D-Fug, D-Tzg or D-Itg wherein those .alpha.-amino acids
having a hydrogen atom at the .alpha.-position are optionally
substituted by a C.sub.1-C.sub.3 alkyl group; X.sup.5 is Pro, Pip,
Thz, His, Ala, .alpha.-Aba, Val, Nva, Leu, Ile, aIle, Nle, Met,
C.sub.3al, C.sub.4al, C.sub.5al or C.sub.6al wherein those
.alpha.-amino acids having hydrogen atom on the .alpha.-amino group
are optionally substituted by a C.sub.1-C.sub.6 alkyl group; or a
pharmaceutically acceptable salt thereof.
[0340] Examples of cyclic and chain peptides-containing compounds
include the peptides described in Japanese Patent Unexamined
Publication No. 288099/1992.
[0341] Japanese Patent Unexamined Publication No. 288099/1992:
[0342] a peptide represented by the formula 14
[0343] wherein Xaa.sub.1 represents Tyr, Phe or Ala, Xaa.sub.2
represents Asp or Gly, Xaa.sub.3 represents Trp or Phe.
[0344] The above-described endothelin antagonists include those
produced by microbes, such as cochinmicins, a cyclodepsipeptide
[The Journal of Antibiotics, Vol. 45, No. 11, 1709-1722
(1992)].
[0345] Examples of endothelin antagonists which antagonize both
receptors ET-A and ET-B include the cyclic peptide (I) described
hereinafter which is described in European Patent Publication No.
528312 and Japanese Patent Application No. 278722/1993.
[0346] More specifically, the anti-endothelin substance in the
present invention is preferably a peptide represented by the
general formula: 15
[0347] wherein X and Y independently represent an .alpha.-amino
acid residue; A represents a D-acidic-.alpha.-amino acid residue; B
represents a neutral-.alpha.-amino acid residue; C represents an
L-.alpha.-amino acid residue; E represents a D-.alpha.-amino acid
residue having an aromatic cyclic group.
[0348] With respect to general formula [I], the parent amino acid
for the .alpha.-amino acid residue represented by X or Y may be any
amino acid, as long as it is an .alpha.-amino acid. Such amino
acids include alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, 2-aminomalonic acid,
2-aminoadipic acid, glycine, histidine, isoleucine, leucine,
lysine, ornithine, 2,4-diaminobutyric acid, methionine,
phenylalanine, proline, 4-hydroxyproline, thioproline,
azetidine-2-carboxylic acid, pipecolic acid
(piperidine-2-carboxylic acid), indoline-2-carboxylic acid,
tetrahydroisoquinoline-3-carboxylic acid, serine, threonine,
tryptophan, 5-methyltryptophan, tyrosine, valine, alloisoleucine,
norvaline, norleucine, tertiary leucine, gamma methylleucine,
phenylglycine, 2-aminobutyric acid, cysteic acid, homocysteic acid,
1-naphthylalanine, 2-naphthylalanine, 2-thienylglycine,
3-thienylglycine, 3-benzothienylalanine, 4-biphenylalanine,
pentamethylphenylalanine, 1-aminocyclopropane-1-carboxylic acid,
1-aminocyclobutane-1-carboxylic acid,
1-aminocyclopentane-1-carboxylic acid,
1-aminocyclohexane-1-carboxylic acid and
1-aminocycloheptane-1-carb- oxylic acid. When these .alpha.-amino
acids have functional groups (e.g., hydroxyl group, thiol group,
amino group, imino group and carboxyl group), the functional groups
may be substituted for by a suitable substituent.
[0349] Hydroxyl groups which are substituted include C.sub.1-6
alkanoyloxy (e.g., formyloxy, acetoxy and propionyloxy), C.sub.4-9
alicyclic carbonyloxy (e.g., cyclopentanecarbonyloxy and
cyclohexanecarbonyloxy), C.sub.7-15 arylcarbonyloxy (e.g.,
benzoyloxy and 4-methylbenzoxloxy), C.sub.8-16 aralkylcarbonyloxy
(e.g., phenylacetoxy, 2-phenylpropionyloxy, 3-phenylpropionyloxy
and diphenylacetoxy), aromatic heterocyclic-alkylcarbonyloxy (e.g.,
indol-2-ylacetoxy and indol-3-ylacetoxy), C.sub.1-6 alkoxy (e.g.,
methoxy, ethoxy, n-propoxy and tert-butoxy), C.sub.3-8 cycloalkoxy
(e.g., cyclopentoxy and cyclohexyloxy), C.sub.6-12 aryloxy (e.g.,
phenyloxy and 4-methylphenyloxy) and C.sub.7-15 aralkyloxy (e.g.,
benzyloxy, phenethyloxy and diphenyimethoxy). .alpha.-Amino acids
in which hydroxyl group is substituted include o-acetylserine,
o-acetylthreonine, 4-acetoxyproline, o-benzoylserine,
o-benzoylthreonine, 4-benzoyloxyproline, o-phenylacetylserine,
o-phenylacetylthreonine, 4-phenylacetoxyproline, o-ethylserine,
o-ethylthreonine, 4-ethoxyproline, o-cyclohexylserine,
o-cyclohexylthreonine, 4-cyclohexyloxyproline, o-phenylserine,
o-phenylthreonine, 4-phenoxyproline, o-benzylserine,
o-benzylthreonine, 4-benzyloxyproline, o-diphenylmethylserine,
o-diphenylmethylthreonine and 4-diphenylmethoxyproline.
[0350] Thiol groups which are substituted include C.sub.1-6
alkanoylthio (e.g., formylthio, acetylthio and propionylthio),
C.sub.4-9 alicyclic carbonythio (e.g., cyclopentanecarbonylthio and
cyclohexanecarbonylthio), C.sub.7-15 arylcarbonylthio (e.g.,
benzoylthio and 4-methylbenzoylthio), C.sub.8-16
aralkylcarbonylthio (e.g., phenylacetylthio, 2-phenylpropionylthio,
3-phenylpropionyithio and diphenylacetylthio), C.sub.1-6 alkylthio
(e.g., methylthio, ethylthio, n-propylthio and tert-butylthio),
C.sub.3-8 cycloalkylthio (e.g., cyclopentylthio and
cyclohexylthio), C.sub.6-12 arylthio (e.g., phenylthio and
4-methylphenylthio) and C.sub.7-15 aralkylthio (e.g., benzylthio,
phenethylthio and diphenylmethylthio). .alpha.-Amino acids in which
thiol group is substituted include S-acetylcysteine,
S-benzoylcysteine, S-phenylacetylcysteine, S-ethylcysteine,
S-cyclohexylcysteine, S-phenylcysteine and S-benzylcysteine.
[0351] Amino groups which are substituted include C.sub.1-6
alkylamino (e.g., N-methylamino, N-ethylamino and
N-tert-butylamino), C.sub.3-8 cycloalkylamino (e.g.,
N-cyclopentylamino and N-cyclohexylamino), C.sub.6-12 arylamino
(e.g., N-phenylamino and N-{4-methyl phenyl}amino), C.sub.7-15
aralkylamino (e.g., N-benzylamino, N-phenethylamino,
N-{2-chlorobenzyl}amino, N-{3-chlorobenzyl}amino,
N-{4-chlorobenzyl}amino- , N-{2-methylbenzyil}amino,
N-{3-methylbenzyl}amino, N-{4-methylbenzyl}amino,
N-{2-methoxybenzyl}amino, N-{3-methoxybenzyl}amino and
N-{4-methoxybenzyl}amino), aromatic heterocyclic-C.sub.1-6
alkylamino (e.g., 2-furylmethylamino, 3-furylmethylamino,
2-thienylmethylamino, 3-thienylmethylamino, indol-2-ylmethylamino
and indol-3-ylmethylamino), and C.sub.1-6 aliphatic acylamido
(e.g., formamido, acetamido and propionamido), C.sub.4-9 alicyclic
acylamido (e.g., cyclopentanecarboxamido and
cyclohexanecarboxamido), C.sub.7-15 arylacylamido (e.g., benzamido
and 4-methylbenzamido), C.sub.8-16 aralkylacylamido (e.g.,
phenylacetamido, 2-phenylpropionamido, 3-phenylpropionamido,
diphenylacetamido, 1-naphthylacetamido and 2-naphthylacetamido),
aromatic heterocyclic-carboxamido (e.g., indol-2-ylcarboxamido and
indol-3-ylcarboxamido), aromatic
heterocyclic-alkylcarboxamido(e.g., indol-2-ylacetamido and
indol-3-ylacetamido), and sulfonylamido (e.g.,
benzenesulfonylamido, p-toluenesulfonylamido and
4-methoxy-2,3,6-trimethy- lbenzenesulfonylamido). Substituents in
imino or imido groups which are substituted are the same as those
in each amino or amido groups which are substituted. .alpha.-Amino
acids wherein the amino group is substituted include
N-methylglycine (sarcosine), N-ethylglycine, N-methylleucine,
N-ethylleucine, N-methylphenyl alanine, N-ethylphenylalanine,
N(.alpha.)-methyltryptophan, N(.alpha.)-ethyltryptophan,
N-cyclopentylglycine, N-cyclohexylglycine, N-phenylglycine,
N-phenylleucine, N-benzylglycine, N-benzylleucine,
N(.pi.)-benzylhistidine, N(.tau.)-benzylhistidine,
N(.pi.)-phenacyihistidine, N(.pi.)-benzyloxymethylhistidine,
N.sup.g-benzenesulfonylarginine, N.sup.g-p-toluenesulfonylarginine,
N.sup.g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine,
N(.epsilon.)-benzenesulfonyllysine,
N(.epsilon.)-p-toluenesulfonyllysine,
N(.epsilon.)-(4-methoxy-2,3,6-trimethylbenzenesulfonyl) lysine,
N.sup.in-methyltryptophan, N.sup.in-ethyltryptophan,
N.sup.in-formyltryptophan, N.sup.in-acetyltryptophan,
N(.epsilon.)-benzyllysine, N(.epsilon.)-(2-furylmethyl)lysine,
N(.epsilon.)-(2-thienylmethyl)lysine,
N(.epsilon.)-(indol-3-ylmethyl)lysi- ne,
N(.epsilon.)-phenylacetyl)lysine, N(.epsilon.)-({2-furyl}
acetyl)lysine, N(.epsilon.)-({2-thienyl}acetyl)lysine,
N(.epsilon.)-({indol-3-yl}acetyl)lysine,
N(.epsilon.)-benzoyllysine, N(.epsilon.)-(3-phenylpropionyl)lysine,
N(.delta.)-benzylornithine, N(.delta.)-(2-furylmethyl)ornithine,
N(.delta.)-(2-thienylmethyl)ornithin- e,
N(.delta.)-(indol-3-ylmethyl)ornithine,
N(.delta.)-benzoylornithine, N(.delta.)-phenylacetylornithine,
N(.delta.)-(3 -phenylpropionyl)ornithin- e,
N(.delta.)-({2-methylphenyl}acetyl))ornithine,
N(.delta.)-({3-methylphe- nyl}acetyl)ornithine,
N(.delta.)-({4-methylphenyl}acetyl) ornithine,
N(.delta.)-({2-chlorophenyl}acetyl)ornithine,
N(.delta.)-({3-chlorophenyl- }acetyl)ornithine,
N(.delta.)-({4-chlorophenyl}acetyl)ornithine,
N(.delta.)-({2-methoxyphenyl}acetyl)ornithine,
N(.delta.)-({3-methoxyphen- yl}acetyl)ornithine,
N(.delta.)-({4-methoxyphenyl}acetyl)ornithine,
N(.delta.)-(4-biphenylacetyl)ornithine,
N(.gamma.)-benzyl-2,4-diaminobuty- ric acid,
N(.gamma.)-(2-furylmethyl)-2,4-diaminobutyric acid,
N(.gamma.)-(2-thienylmethyl)-2,4-diaminobutyric acid,
N(.gamma.)-(indol-3-ylmethyl)-2,4-diaminobutyric acid,
N(.gamma.)-benzoyl-2,4-diaminobutyric acid,
N(.gamma.)-phenylacetyl-2,4-d- iaminobutyric acid,
N(.gamma.)-(3-phenylpropionyl)-2,4-diaminobutyric acid,
N(.gamma.)-(2-furylacetyl)-2,4-diaminobutyric acid,
N(.gamma.)-(2-thienylacetyl)-2,4-diaminobutyric acid and
N(.gamma.)-({indol-3-yl}acetyl)-2,4-diaminobutyric acid.
[0352] Carboxyl groups which are substituted include carbamoyl
group (--CONH.sub.2) and substituted carbamoyl group such as
N--C.sub.1-6 alkylcarbamoyl (e.g., methylcarbamoyl, ethylcarbamoyl,
n-propylcarbamoyl and tert-butylcarbamoyl), C.sub.3-8
cycloalkylcarbamoyl (e.g., cyclopentylcarbamoyl and
cyclohexylcarbamoyl), C.sub.6-12 arylcarbamoyl (e.g.,
phenylcarbamoyl and 4-methylphenylcarbamoyl), C.sub.7-15
aralkylcarbamoyl (e.g., benzylcarbamoyl, phenetyl and
1,2-diphenylethylcarbamoyl), aromatic heterocyclic-C.sub.1-6
alkylcarbamoyl (e.g., 2-{indol-2-yl}ethylcarbamoyl and
2-{indol-3-yl}ethylcarbamoyl), piperidinocarbonyl,
piperazincarbonyl, N.sup.4-C.sub.1-6 alkylpiperazincarbonyl (e.g.,
N.sup.4-methylpiperazinca- rbonyl and
N.sup.4-ethylpiperazincarbonyl), N.sup.4-C.sub.3-8
cycloalkylpiperazincarbonyl (e.g.,
N.sup.4-cyclopentylpiperazincarbonyl and
N.sup.4-cyclohexylpiperazincarbonyl), N.sup.4-5 to 7-membered
heterocyclic piperazincarbonyl (e.g.,
N.sup.4-pyridylpiperazincarbonyl, N.sup.4-furylpiperazincarbonyl
and N.sup.4-thienylpiperazincarbonyl), N.sup.4-C.sub.6-12
arylpiperazincarbonyl (e.g., N.sup.4-phenylpiperazinca- rbonyl and
N.sup.4-{4-methylphenyl}piperazincarbonyl), N.sup.4-C.sub.7-15
aralkylpiperazincarbonyl (e.g., N.sup.4-benzylpiperazincarbonyl,
N.sup.4-phenetylpiperazincarbonyl and
N.sup.4-{1,2-diphenylethyl}piperazi- ncarbonyl), N.sup.4-{aromatic
heterocyclic -C.sub.1-6 alkyl}piperazincarbonyl (e.g.,
N.sup.4-[2-{indol-2-yl}ethyl]piperazincarb- onyl and
N.sup.4-[2-{indol-3-yl}ethyl]piperazincarbonyl), N.sup.4-C.sub.1-6
aliphatic acylpiperazincarbonyl (e.g.,
N.sup.4-acetylpiperazincarbonyl and
N.sup.4-propionylpiperazincarbonyl), N.sup.4-C.sub.4-9 alicyclic
acylpiperazincarbonyl (e.g.,
N.sup.4-cyclopentanecarbonylpiperazincarbonyl and
N.sup.4-cyclohexane carbonylpiperazincarbonyl), N.sup.4-C.sub.7-15
arylacylpiperazincarbonyl (e.g., N.sup.4-benzoylpiperazincarbonyl
and N.sup.4-{4-methylbenzoyl}pipe- razincarbonyl),
N.sup.4-C.sub.8-16 aralkylacylpiperazincarbonyl (e.g.,
N.sup.4-phenylacetylpiperazincarbonyl,
N.sup.4-{2-phenylpropion}piperazin- carbonyl,
N.sup.4-{3-phenylpropionyl}piperazincarbonyl,
N.sup.4-diphenylacetylpiperazincarbonyl,
N.sup.4-{1-naphthylacetyl}pipera- zincarbonyl and
N.sup.4-{2-naphthylacetyl}piperazincarbonyl), N.sup.4-{aromatic
heterocyclic-carbonyl}piperazincarbonyl (e.g.,
N.sup.4-{indol-2-ylcarbonyl}piperazincarbonyl and
N.sup.4-{indol-3-ylcarb- onyl}piperazincarbonyl) and
N.sup.4-{aromatic heterocyclic-alkylcarbonyl}p- iperazincarbonyl
(e.g., N.sup.4-{indol-2-ylacetyl}piperazincarbonyl and
N.sup.4-{indol-3-ylacetyl}piperazincarbonyl), and C.sub.1-6
alkyloxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl and
n-propoxycarbonyl), C.sub.3-8 cycloalkyloxycarbonyl (e.g.,
cyclopentyloxycarbonyl and cyclohexyloxycarbonyl) and C.sub.7-15
aralkyloxycarbonyl (e.g., benzyloxycarbonyl, phenethyloxycarbonyl,
1-phenylethoxycarbonyl and diphenylmethoxycarbonyl). The above
substituted carbamoyl groups include amides with .alpha.-amino
acids and amides with oligopeptides (e.g., dipeptide, tripeptide
and tetrapeptide). .alpha.-amino acids wherein the carboxyl group
is substituted include N.sup.4-methylasparagine,
N.sup.4-phenylasparagine, N.sup.4-benzylasparagine,
N.sup.4-phenetylasparagine,
N.sup.4-(2-{indol-3-yl}ethyl)asparagine, N.sup.5-methylglutamine,
N.sup.5-phenylglutamine, N.sup.5-benzylglutamine,
N.sup.5-phenetylglutami- ne, N.sup.5-(2-{indol-3-yl}ethyl)
glutamine, aspartic acid .beta.-methyl ester, aspartic acid
.beta.-cyclopropyl ester, aspartic acid .beta.-benzyl ester,
aspartic acid .beta.-phenethyl ester, aspartic acid
.beta.-N.sup.4-phenylpiperazinamide, aspartic acid
.beta.-N.sup.4-(2-methylphenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(3-methylphenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(4-methylphenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(2-methoxyphenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(3-methoxyphenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(4-methoxyphenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(2-chlorophenyl) piperazinamide, aspartic acid
.beta.-N.sup.4-(3-chlorophenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(4-chlorophenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(4-nitrophenyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(4-fluorophenyl) piperazinamide, aspartic acid
.beta.-N.sup.4-(3-trifluoromethylphenyl)piperazinamide, aspartic
acid .beta.-N.sup.4-(2,3-dimethylphenyl)piperazinamide, aspartic
acid .beta.-N.sup.4-(2-pyridyl)piperazinamide, aspartic acid
.beta.-N.sup.4-(2-pyrimidyl)piperazinamide, glutamic acid
.gamma.-methyl ester, glutamic acid .gamma.-cyclopropyl ester,
glutamic acid .gamma.-benzyl ester and glutamic acid
.gamma.-phenethyl ester.
[0353] With respect to general formula [I], the parent
.alpha.-amino acid for the .alpha.-amino acid residue represented
by X or Y may be any isomer, whether D, L or DL, with preference
given to the L-isomer for both X and Y.
[0354] X preferably represents -Asp(R.sup.1)-. -Asp(R.sup.1)- is a
group of the formula: 16
[0355] wherein R.sup.1 represents a group represented by the
formula: 17
[0356] wherein X.sup.1 and X.sup.2 independently represent a
hydrogen atom, C.sub.1-6 alkyl group, C.sub.1-6 alkoxy group, a
halogen atom or a nitro group, and X.sup.1 and X.sup.2
independently may be combined together to form a ring in 18
[0357] Examples of C.sub.1-6 alkyl group represented by X.sup.1 and
X.sup.2 are methyl, ethyl, n-propyl, iso-propyl, n-butyl,
iso-butyl, tert-butyl, n-pentyl and n-hexyl, among which C.sub.1-3
alkyl group such as methyl, ethyl, n-propyl and iso-propyl is
preferred. Most preferred is methyl.
[0358] Examples of C.sub.1-6 alkoxy group represented by X.sup.1
and X.sup.2 are methoxy, ethoxy, n-propoxy, n-butoxy, n-pentyloxy
and n-hexyloxy, among which C.sub.1-3 alkoxy group such as methoxy,
ethoxy and n-propoxy is preferred. Most preferred is methoxy or
ethoxy.
[0359] Examples of halogen atom represented by X.sup.1 and X.sup.2
are fluorine, chlorine, bromine and iodine, among which chlorine is
preferred.
[0360] Examples of R.sup.1 in case X.sup.1 and X.sup.2 are combined
together to form a ring are represented by the formula; 19
[0361] Examples of ring Q are 4- to 7-membered rings which may
contain 1 to 3 hetero atom selected from O, N or S (e.g. saturated
carbon rings, aromatic carbon rings, saturated heterocyclic rings
and aromatic heterocyclic rings).
[0362] R.sup.1 is preferably represented by the formula; 20
[0363] wherein X.sup.11 represents a hydrogen atom, C.sub.1-6 alkyl
group, C.sub.1-6 alkoxy group, a halogen atom or a nitro group.
[0364] Preferred examples of R.sup.1 are 21
[0365] Above mentioned -Asp(R.sup.1)- may be any isomer, whether D,
L or DL, with preference given to L-isomer.
[0366] With respect to general formula [I], the parent amino acid
for the D-acidic-.alpha.-amino acid residue represented by A is
exemplified by amino acids having an acidic group such as the
carboxyl group, sulfo group or tetrazolyl group in the side chain
thereof, including D-glutamic acid, D-aspartic acid, D-cysteic
acid, D-homocysteic acid, D-.beta.-(5-tetrazolyl)alanine and
D-2-amino-4-(5-tetrazolyl)butyric acid, with preference given to
D-glutamic acid, D-aspartic acid and D-cysteic acid.
[0367] With respect to general formula [I], the parent amino acid
for the neutral-.alpha.-amino acid residue represented by B is
exemplified by .alpha.-amino acids such as alanine, valine,
norvaline, leucine, isoleucine, alloisoleucine, norleucine,
tert-leucine, .gamma. methylleucine, phenylglycine, phenylalanine,
1-naphthylalanine, 2-naphthylalanine, proline, 4-hydroxyproline,
azetidine-2-carboxylic acid, pipecolic acid
(piperidine-2-carboxylic acid), 2-thienylalanine, 2-thienylglycine,
3-thienylglycine, 1-aminocyclopropane-1-carboxylic acid,
1-aminocyclobutane-1-carboxylic acid,
1-aminocyclopentane-1-carboxy- lic acid,
1-aminocyclohexane-1-carboxylic acid, 1-aminocycloheptane-1-carb-
oxylic acid, 2-cyclopentylglycine and 2-cyclohexylglycine. If the
neutral-.alpha.-amino acid involves both the L- and
D-configurations, the D-configuration is preferred. Greater
preference is given to D-leucine, D-alloisoleucine, D-tert-leucine,
D-r methylleucine, D-phenylglycine, D-2-thienylalanine,
D-2-thienylglycine, D-3-thienylglycine and D-2-cyclopentylglycine.
The .alpha.-amino group of these neutral-.alpha.-amino acids may be
replaced by a C.sub.1-6 alkyl group (e.g., methyl, ethyl, n-propyl
or tert-butyl). Such .alpha.-amino acids include N-methylleucine,
N-methylalloisoleucine, N-methyl tert-leucine, N-methyl .gamma.
methylleucine and N-methylphenylglycine, preferably of the
D-configuration.
[0368] B preferably represents --NH--CHR.sup.2--CO--, wherein
R.sup.2 represents C.sub.1-6 alkyl group, C.sub.3-7 cycloalkyl
group, C.sub.3-7 cycloalkyl-C.sub.1-3 alkyl group, C.sub.1-6
alkylthio-C.sub.1-3 alkyl group, C.sub.3-7 cycloalkylthio-C.sub.1-3
alkyl group, C.sub.1-6 alkoxy-C.sub.1-3 alkyl group, C.sub.3-7
cycloalkoxy-C.sub.1-3 alkyl group, C.sub.1-6 alkylthio group,
C.sub.3-7 cycloalkylthio group, C.sub.1-6 alkoxy group or C.sub.3-7
cycloalkoxy group.
[0369] Examples of C.sub.1-6 alkyl group represented by R.sup.2 are
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, (1-methyl)
propyl, tert-butyl, n-pentyl, (2-methyl) butyl, (3-methyl) butyl,
neopentyl, n-hexyl, (2,2-dimethyl) butyl and (3,3-dimethyl) butyl,
among which C.sub.4-6 alkyl group such as n-butyl, iso-butyl,
(1-methyl) propyl, tert-butyl, n-pentyl, (2-methyl) butyl,
(3-methyl) butyl, (2-methyl) butyl, (3-methyl) butyl, neopentyl and
n-hexyl is preferred.
[0370] Examples of C.sub.3-7 cycloalkyl group represented by
R.sup.2 are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl, among which C.sub.5-7 cycloalkyl group such as
cyclopentyl, cyclohexyl and cycloheptyl is preferred.
[0371] Examples of C.sub.3-7 cycloalkyl-C.sub.1-3 alkyl group
represented by R.sup.2 are cyclopropylmethyl, cyclobutylmethyl,
cyclobutylethyl, cyclobutylpropyl, cyclopentylmethyl,
cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl,
cyclohexylethyl, cyclohexylpropyl, cycloheptylmethyl and
cycloheptylethyl, among which C.sub.3-7 cycloalkyl-methyl group
such as cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,
cyclohexylmethyl, cycloheptylmethyl is preferred.
[0372] Examples of C.sub.1-6 alkylthio-C.sub.1-3 alkyl group
represented by R.sup.2 are methylthiomethyl, methylthioethyl,
methylthiopropyl, ethylthiomethyl, ethylthioethyl,
n-propylthiopropyl, iso-propylthiomethyl, n-butylthiomethyl,
tert-butylthiomethyl, n-butylthioethyl, tert-butylthiopropyl and
(1,1-dimethyl) propylthiomethyl, among which C.sub.3-7
alkylthio-methyl group such as iso-propylthiomethyl,
n-butylthiomethyl, tert-butylthiomethyl and (1,1-dimethyl)
propylthiomethyl is preferred.
[0373] Examples of C.sub.3-7 cycloalkylthio-C.sub.1-3 alkyl group
represented by R.sup.2 are cyclopropylthiomethyl,
cyclopropylthioethyl, cyclopropylthiopropyl, cyclobutylthiomethyl,
cyclobutylthioethyl, cyclobutylthiopropyl, cyclopentylthiomethyl,
cyclopentylthioethyl, cyclohexythiomethyl and
cycloheptylthiomethyl, among which C.sub.4-7 cycloalkylthiomethyl
group such as cyclobutylthiomethyl, cyclopentylthiomethyl,
cyclohexylthiomethyl and cycloheptylthiomethyl is preferred.
[0374] Examples of C.sub.1-6 alkoxy-C.sub.1-3 alkyl group
represented by R.sup.2 are methoxymethyl, methoxyethyl,
methoxypropyl, ethoxymethyl, ethoxyethyl, n-propoxymethyl,
n-propoxyethyl, iso-propoxymethyl, iso-propoxyethyl,
n-butoxymethyl, n-butoxyethyl, tert-butoxymethyl, tert-butoxyethyl,
n-pentyloxymethyl, n-pentyloxyethyl, (1,1-dimethyl) propoxymethyl,
(1,1-dimethyl) propoxyethyl, n-hexyloxymethyl and n-hexyloxyethyl,
among which C.sub.1-6 alkoxy-methyl group such as methoxymethyl,
ethoxymethyl, n-propoxymethyl, iso-propoxymethyl, n-butoxymethyl,
tert-butoxymethyl, n-pentyloxymethyl, (1,1-dimethyl) propoxymethyl
and n-hexyloxymethyl is preferred. More preferred are
iso-propoxymethyl, tert-butoxymethyl, (1,1-dimethyl) propoxymethyl
and n-hexyloxymethyl.
[0375] Examples of C.sub.3-7 cycloalkoxy-C.sub.1-3 alkyl group
represented by R.sup.2 are cyclopropoxymethyl, cyclopropoxyethyl,
cyclobutoxymethyl, cyclobutoxyethyl, cyclopentyloxymethyl,
cyclopentyloxyethyl, cyclohexyloxymethyl and cycloheptyloxymethyl,
among which C.sub.3-7 cycloalkoxy-methyl group such as
cyclopropoxymethyl, cyclobutoxymethyl, cyclopentyloxymethyl,
cyclohexyloxymethyl and cycloheptyloxymethyl is preferred.
[0376] Examples of C.sub.1-6 alkylthio group represented by R.sup.2
are methylthio, ethylthio, n-propylthio, iso-propylthio,
n-butylthio, tert-butylthio, n-pentylthio, (1,1-dimethyl)
propylthio and n-hexylthio, among which C.sub.3-6 alkylthio group
such as n-propylthio, iso-propylthio, n-butylthio, tert-butylthio,
n-pentylthio, (1,1-dimethyl) propylthio and n-hexylthio is
preferred.
[0377] Examples of C.sub.3-7 cycloalkylthio group represented by
R.sup.2 are cyclopropylthio, cyclobutylthio, cyclopentylthio,
cyclohexylthio and cycloheptylthio, among which C.sub.4-7
cycloalkylthio group such as cyclobutylthio, cyclopentylthio,
cyclohexylthio and cycloheptylthio is preferred.
[0378] Examples of C.sub.1-6 alkoxy group represented by R.sup.2
are methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy,
n-pentyloxy, (1,1-dimethyl) propoxy and n-hexyloxy, among which
C.sub.3-6 alkoxy group such as n-propoxy, iso-propoxy, n-butoxy,
tert-butoxy, n-pentyloxy, (1,1-dimethyl) propoxy and n-hexyloxy is
preferred.
[0379] Examples of C.sub.3-7 cycloalkoxy group represented by
R.sup.2 are cyclopropoxy, cyclobutoxy, cyclopentyloxy,
cyclohexyloxy and cycloheptyloxy, among which C.sub.4-7 cycloalkoxy
group such as cyclobutoxy, cyclopentyloxy, cyclohexyloxy and
cycloheptyloxy is preferred.
[0380] R.sup.2 is preferably C.sub.1-6 alkyl group, more preferably
C.sub.4-6 alkyl group such as n-butyl, iso-butyl, (1-methyl)
propyl, tert-butyl, n-pentyl, (2-methyl) butyl, (3-methyl) butyl,
neopentyl and n-hexyl, with greater preference is given to
tert-butyl and neopentyl.
[0381] Above mentioned .alpha.-amino acid represented by
--NH--CHR.sup.2--CO-- may be any isomer, whether D, L or DL, with
preference given to D-isomer.
[0382] With respect to general formula [I], the parent amino acid
for the L-.alpha.-amino acid residue represented by C is
exemplified by commonly known L-.alpha.-amino acids such as
glycine, L-alanine, L-valine, L-norvaline, L-leucine, L-isoleucine,
L-tert-leucine, L-norleucine, L-methionine, L-2-aminobutyric acid,
L-serine, L-threonine, L-phenylalanine, L-aspartic acid, L-glutamic
acid, L-asparagine, L-glutamine, L-lysine, L-tryptophan,
L-arginine, L-tyrosine and L-proline, with preference given to
L-leucine, L-norleucine and L-tryptophan. The .alpha.-amino group
of these L-.alpha.-amino acids may be replaced by a C.sub.1-6 alkyl
group (e.g., methyl, ethyl, n-propyl or tert-butyl). Such
L-.alpha.-amino acids include L-N-methylleucine,
L-N-methylnorleucine and L-N(.alpha.)-methyltryptophan.
[0383] With respect to general formula [I], the parent amino acid
for the D-.alpha.-amino acid residue having an aromatic cyclic
group represented by E is exemplified by D-.alpha.-amino acids
having an aromatic cyclic group in the side chain thereof. Examples
of such amino acids include D-tryptophan, D-5-methyltryptophan,
D-phenylalanine, D-tyrosine, D-1-naphthylalanine,
D-2-naphthylalanine, D-3-benzothienylalanine, D-4-biphenylalanine
and D-pentamethyl phenylalanine, with preference given to
D-tryptophan and D-5-methyltryptophan. D-tryptophan is more
preferred. The .alpha.-amino group of these D-.alpha.-amino acids
having an aromatic ring may be replaced by a C.sub.1-6 alkyl group
(e.g., methyl, ethyl, n-propyl or tert-butyl). The amino group of
the indole ring of D-tryptophan may be replaced by a hydrocarbon
group such as a C.sub.1-6 alkyl (e.g., methyl, ethyl, n-propyl or
tert-butyl), C.sub.3-8 cycloalkyl (e.g., cyclopentyl or
cyclohexyl), C.sub.6-12 aryl (e.g., phenyl or 4-methylphenyl) or
C.sub.7-15 aralkyl (e.g., benzyl or phenethyl) or by an acyl group
such as a C.sub.1-6 aliphatic acyl (e.g., formyl, acetyl or
propionyl), C.sub.4-9 alicyclic acyl (e.g., cyclopentanecarbonyl or
cyclohexanecarbonyl), C.sub.7-15 arylacyl (e.g., benzoyl or
4-methylbenzoyl), C.sub.8-16 aralkylacyl (e.g., phenylacetyl,
2-phenylpropionyl, 3-phenylpropionyl or diphenylacetyl) or
C.sub.1-6 alkoxycarbonyl (e.g., methoxycarbonyl or ethoxycarbonyl).
Such .alpha.-amino acids include D-N(.alpha.)-methyltryptophan,
D-N-methylphenylalanine, D-N-methyltyrosine,
D-N.sup.in-methyltryptophan, D-N.sup.in-ethyltryptophan,
D-N.sup.in-formyltryptophan and D-N.sup.in-acetyltryptophan,
D-N.sup.in-methyltryptophan, D-N.sup.in-formyltryptophan and
D-N.sup.in-acetyltryptophan are preferred.
[0384] E preferably represents Trp (N.sup.in-R.sup.3), wherein
R.sup.3 represents a hydrogen atom, C.sub.1-6 alkyl group,
C.sub.3-7 cycloalkyl group, --COR.sup.4 (R.sup.4 represents a
hydrogen atom, C.sub.1-6 alkyl group, C.sub.6-15 aryl group or
C.sub.6-15 aryl-C.sub.1-3 alkyl group), --COOR.sup.5 (R.sup.5
represents C.sub.1-6 alkyl group, C.sub.6-15 aryl group or
C.sub.6-15 aryl-C.sub.1-3 alkyl group) or --CONHR.sup.6 (R.sup.6
represents a hydrogen atom, C.sub.1-6 alkyl group, C.sub.6-15 aryl
group or C.sub.6-15 aryl-C.sub.1-3 alkyl group) and R.sup.3 is
directly combined with N atom of indole group in tryptophan
residue.
[0385] Examples of C.sub.1-6 alkyl group represented by R.sup.3 are
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, (1-methyl)
propyl, tert-butyl, n-pentyl, (2-methyl) butyl, (3-methyl) butyl,
neopentyl, n-hexyl, (2,2-dimethyl) butyl and (3,3-dimethyl) butyl,
among which C.sub.1-3 alkyl group such as methyl, ethyl, n-propyl
and iso-propyl is preferred.
[0386] Examples of C.sub.3-7 cycloalkyl group represented by
R.sup.3 are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl, among which C.sub.5-7 cycloalkyl group such as
cyclopentyl, cyclohexyl and cycloheptyl is preferred.
[0387] Examples of C.sub.1-6 alkyl group represented by R.sup.4,
R.sup.5 and R.sup.6 are methyl, ethyl, n-propyl, iso-propyl,
n-butyl, iso-butyl, (1-methyl) propyl, tert-butyl, n-pentyl,
(2-methyl) butyl, (3-methyl) butyl, neopentyl, n-hexyl,
(2,2-dimethyl) butyl and (3,3-dimethyl) butyl, among which
C.sub.1-3 alkyl group such as methyl, ethyl, n-propyl and
iso-propyl is preferred.
[0388] Examples of C.sub.6-15 aryl group represented by R.sup.4,
R.sup.5 and R.sup.6 are phenyl, .alpha.-naphthyl and
.beta.-naphthyl, among which phenyl is preferred.
[0389] Examples of C.sub.6-15 aryl-C.sub.1-3 alkyl group
represented by R.sup.4, R.sup.5 and R.sup.6 are benzyl,
phenylethyl, phenylpropyl, .alpha.-naphthylmethyl,
.alpha.-naphthylethyl, .alpha.-naphthylpropyl,
.beta.-naphthylmethyl, .beta.-naphthylethyl, .beta.-naphthylpropyl,
among which C.sub.6-15 aryl-methyl group such as benzyl,
.alpha.-naphthylmethyl and .beta.-naphthylmethyl, is preferred.
[0390] Specific embodiment of --COR.sup.4 is exemplified by formyl,
acetyl, propionyl, butyryl, isobutyryl, isovaleryl, pivaloyl,
n-benzylcarbonyl, benzoyl and phenylacetyl.
[0391] Specific embodiment of --COOR.sup.5 is exemplified by
methoxycarbonyl, ethoxycarbonyl, phenoxycarbonyl and
benzyloxycarbonyl.
[0392] Specific embodiment of --CONHR.sup.6 is exemplified by
carbamoyl, methylaminocarbonyl, ethylaminocarbonyl,
n-propylaminocarbonyl, iso-propylaminocarbonyl,
n-butylaminocarbonyl, iso-butylaminocarbonyl, phenylaminocarbonyl
and benzylaminocarbonyl.
[0393] R.sup.3 is preferably a hydrogen atom and --COR.sup.4
(R.sup.4 represents a hydrogen atom, C.sub.1-6 alkyl group,
C.sub.6-15 aryl group or C.sub.6-15 aryl-C.sub.1-3 alkyl group),
with greater preference is given to a hydrogen atom, formyl and
acetyl.
[0394] In the hexapeptide represented by general formula [I], or a
salt thereof followings are preferred:
[0395] X is an the L-isomer; Y is an L-isomer; A is selected from
the group consisting of D-glutamic acid, D-aspartic acid, D-cysteic
acid and D-tetrazolylalanine residues; B is of the D-configuration;
B is selected from the group consisting of
1-aminocyclopropane-1-carboxylic acid,
1-aminocyclobutane-1-carboxylic acid,
1-aminocyclopentane-1-carboxylic acid,
1-aminocyclohexane-1-carboxylic acid and
1-aminocycloheptane-1-carb- oxylic acid; B is selected from the
group consisting of D-leucine, D-alloisoleucine, D-tert-leucine,
D-.gamma.-methyl leucine, D-phenylglycine, D-2-thienylglycine,
D-3-thienylglycine, D-2-cyclopentylglycine, D-phenylalanine,
D-2-thienylalanine, D-valine, D-2-furylglycine and D-3-furylglycine
residues; C is selected from the group consisting of L-leucine,
L-isoleucine, L-valine, L-norleucine and L-.alpha.-amino acid
residues having an aromatic group; E is selected from the group
consisting of D-tryptophan or derivatives thereof,
D-1-naphthylalanine, D-2-naphthylalanine, D-benzothienylalanine,
D-4-bisphenylalanine and D-pentamethyl phenylalanine residues; the
D-tryptophan derivative is selected from the group consisting of
D-N.sup.in-methyltryptophan, D-N.sup.in-formyltryptophan and
D-N.sup.in-acetyltryptophan residues; More preferred ones are
followings. A is a D-aspartic acid residue; X is a tryptophan,
L-(.beta.-4-phenylpiperazinamido)aspartic acid,
L-[.beta.-4-(2-methoxyphe- nyl)piperazinamid]aspartic acid,
L-N(.delta.)-phenylacetylornithine (.delta. is a superscript, the
same applies below), L-(N.sup.4-[indol-3-yl]acetyl)ornithine,
L-(4-benzyloxy)proline, L-(N.sup.5-benzyl)glutamine or
L-(N(.delta.)-[indol-3-yl]ethyl)asparagine residue; Y is an
L-leucine, L-aspartic acid or L-O-benzylserine residue; B is a
D-leucine, D-.gamma. methyl leucine, D-2-thienylglycine or
D-3-thienylglycine residue; C is selected from the group consisting
of L-leucine, L-phenylalanine and L-tryptophan residues; and E is a
D-tryptophan residue.
[0396] The anti-endothelin substance in the present invention is
preferably the peptide (I) described in European Patent Publication
No. 528312 and Japanese Patent Application No. 278722/1993.
[0397] Most preferably, the anti-endothelin substance is a peptide
shown below.
[0398] (1) cyclo[-D-Asp-Asp(R1')-Asp-D-Thg(2)-Leu-D-Trp-],
[0399] (2)
cyclo[-D-Asp(OC.sub.2H.sub.5)-Asp(R1')-Asp(OC.sub.2H.sub.5)-D-T-
hg(2)-Leu-D-Trp-]
[0400] (3) cyclo[-D-Asp-Asp(B7)-Asp-D.gamma.MeLeu-Leu-D-Trp-]
[0401] wherein Asp represents aspartic acid; Asp(R1') represents
aspartic acid .beta.-4-phenylpiperazinamide; Thg(2) represents
2-thienylglycine; Leu represents leucine; Trp represents
tryptophan; Asp(B7) represents aspartic acid
.beta.-4-(2-methoxyphenyl)piperazinamide; .gamma.MeLeu represents
.gamma.-methylleucine.
[0402] The above-described anti-endothelin substance, peptides in
particular, may be used in the form of salts, preferably
pharmacologically acceptable salts. Such salts may be organic or
inorganic. Examples of the inorganic salts include salts with bases
such as alkali metals (e.g., sodium and potassium), and polyvalent
metals such as alkaline earth metals (e.g., calcium and magnesium),
zinc, copper and aluminium and salts with inorganic acids (e.g.,
hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid
and phosphoric acid). Examples of organic salts include salts with
organic acids such as carboxylic acid (e.g., formic acid, acetic
acid, trifluoroacetic acid and maleic acid), organic sulfonic acid
(methanesulfonic acid, benzenesulfonic acid and toluenesulfonic
acid) and amino acids (e.g., arginine, aspartic acid and glutamic
acid), ammonium salts and salts with organic bases such as
tert-amine (e.g., trimethylamine, triethylamine, pyridine,
picoline, dicyclohexylamine and N-N'-dibenzylethylenediamine). When
the anti-endothelin substance has an acidic group such as carboxyl,
sodium salts and salts with arginine are preferred. When the
anti-endothelin substance has a basic group such as amino,
hydrochlorides and acetates are preferred.
[0403] The above-described salts may be in the form of complexes.
Examples of the complexes are complexes with alkali metals (e.g.,
sodium and potassium) and polyvalent metals such as alkaline earth
metals (e.g., calcium and magnesium), zinc, copper and aluminium.
The complexes are preferably complexes with polyvalent metals such
as alkaline earth metals (e.g., calcium and magnesium), zinc,
copper and aluminium, with greater preference given to a zinc
complex.
[0404] The peptide represented by the general formula: 22
[0405] wherein A' represents a D-acidic-.alpha.-amino acid residue
which is esterified with an alkyl group and other symbols have the
same meanings as defined above, or a salt thereof, among the
peptide [I], or ester thereof, or salt thereof, is novel.
[0406] In the D-acidic-.alpha.-amino acid residue which is
esterified with an alkyl, represented by A', the alkyl group are
exemplified by methyl, ethyl, n-propyl, iso-propyl, n-butyl,
iso-butyl, tert-butyl, n-pentyl and n-hexyl, among which C.sub.1-3
alkyl group such as methyl, ethyl, n-propyl and iso-propyl is
preferred.
[0407] The peptide [II] or salt thereof is produced by subjecting
the peptide [I] or salt thereof to a per se known esterification
with an alkyl group.
[0408] When sustained-release preparations are produced by using
the peptide [II] or salt thereof, thus obtained sustained-release
preparations exhibit both suppression of initial burst of drug and
constant release of drug.
[0409] The zinc salt of a peptide represented by the general
formula [I] is novel.
[0410] The zinc salt of a peptide [I] is produced by mixing the
peptide [I] or a water-soluble salt (e.g.: sodium salt) thereof
with a water-soluble zinc salt in water. The zinc salt of peptide
[I] precipitated is isolated by centrifugation and the like. Thus
obtained precipitate is dispersed in distilled water and
centrifuged again. These operations are repeated to give a purified
zinc salt of peptide [I]. The purified zinc salt of peptide [I] is
subjected to drying such as vacuum drying and lyophilizing. The
mixing ratio (peptide [I]/water-soluble zinc salt) (mol ratio) is
about 10/1 to 1/10, preferably about 5/1 to 1/5. The concentration
of these in water is within solubility of each and not lower than
solubility of a produced complex. The above-mentioned water-soluble
zinc salts are exemplified by inorganic acid zinc salts such as
zinc halogenide (e.g., zinc chloride, zinc bromide, zinc iodide,
zinc fluoride), zinc sulfate, zinc nitrate and zinc thiocyanate and
organic acid zinc salts such as aliphatic carboxylic acid zinc
salts (e.g., zinc acetate, zinc glycolate, zinc lactate, zinc
tartrate) and aromatic acid zinc salts (e.g., zinc benzoate, zinc
salicylate, zinc phenolsulfate). The water-soluble zinc salts are
preferably aliphatic carboxylic acid zinc salts, with greater
preference given to zinc acetate.
[0411] When sustained-release preparations are produced by using
the zinc salt of the peptide [I], thus obtained sustained-release
preparations exhibit both suppression of initial burst of drug and
constant release of drug. The sustained-release preparations are
high in drug content.
[0412] In the present invention, examples of means which can be
used for sustained retention of the anti-endothelin substance in
the living body are injectable sustained-release preparations
(e.g., microcapsules and microspheres) using a biodegradable
polymer, and indwellable preparations (shaped as needles, for
example). Also available are electrically driven pumps or osmotic
pressure pumps (Alzet etc.) capable of sustained release of a given
amount of anti-endothelin substance. Other examples include
preparations for non-invasive administration at such sites as the
skin (percutaneous preparations), mucosa (transnasal preparations,
transvaginal preparations etc.) and digestive tract (oral
preparations, rectal suppositories etc.). The sustained-release
preparation mentioned herein may be any preparation, as long as the
pharmaceutical action is sustained for at least 24 hours after a
single administration or an almost constant effective blood
concentration lasts for at least 24 hours, with preference given to
a preparation capable of sustaining the pharmaceutical action or an
effective blood concentration for at least 72 hours after a single
administration. Although an effective blood concentration may be
sustained by increasing the frequency of administration in the case
of oral preparations of short duration of action, increased
frequency of administration is inconvenient for the patient and the
degree of certainty is low. Microcapsular preparations using a
biodegradable polymer are preferred because they are easy to
administer and long in duration of action after administration. The
anti-endothelin substance incorporated in the microcapsular
preparation is preferably an endothelin antagonist. Although the
amount of endothelin antagonist added varies depending on the
activity thereof, target disease, duration of effect and other
factors, the endothelin antagonist is used at normally about 0.001
to 50% (w/w), preferably about 0.01 to 30% (w/w), and more
preferably about 0.1 to 20% (w/w), relative to the base
biodegradable polymer.
[0413] Examples of biodegradable polymers include aliphatic
polyesters (e.g., polymers, copolymers or mixtures thereof produced
from one or more of .alpha.-hydroxycarboxylic acids such as
glycolic acid, lactic acid and hydroxybutyric acid,
hydroxydicarboxylic acids such as malic acid, hydroxytricarboxylic
acids such as citric acid and others), poly-.alpha.-cyanoacrylic
acid esters, polyamino acids (e.g., poly-.gamma.-benzyl-L-glutamic
acid) and maleic anhydride copolymers (e.g., styrene-maleic acid
copolymers). These may be used as a mixture. Here, the type of
polymerization may be random, block or graft.
[0414] The biodegradable polymer is preferably an aliphatic
polyester (e.g., a polymer, copolymer or mixture thereof produced
from one or more .alpha.-hydroxycarboxylic acids as glycolic acid,
lactic acid and hydroxybutyric acid, hydroxydicarboxylic acids such
as malic acid, hydroxytricarboxylic acids such as citric acid, and
others).
[0415] Examples of the above-described copolymers include
copolymers of glycolic acid and other .alpha.-hydroxy acids, the
.alpha.-hydroxy acid being preferably lactic acid, 2-hydroxybutyric
acid or the like. Although the .alpha.-hydroxycarboxylic acid may
be a D-, L- or D,L-isomer, it is preferable that the ratio of the
D-isomer/L-isomer (mol %) fall within the range from about 75/25 to
25/75. More preferably, the .alpha.-hydroxycarboxylic acid is a
hydroxycarboxylic acid wherein the ratio of D-isomer/L-isomer (mol
%) falls within the range from about 60/40 to 40/60.
[0416] With respect to the copolymer of glycolic acid and
2-hydroxybutyric acid, it is preferable that glycolic acid account
for about 10 to 75 mol % and 2-hydroxybutyric acid account for the
remaining portion. More preferably, glycolic acid accounts for
about 20 to 75 mol %, still more preferably about 30 to 70 mol %.
The glycolic acid copolymer has a weight-average molecular weight
of about 2,000 to 50,000, preferably about 3,000 to 40,000, more
preferably about 8,000 to 25,000. The dispersity of the glycolic
acid copolymer (weight-average molecular weight/number-average
molecular weight) is preferably about 1.2 to 4.0. Greater
preference is given to a copolymer wherein the dispersity is about
1.5 to 3.5. The present glycolic acid copolymer can be produced by
a known process such as the method described in Japanese Patent
Unexamined Publication No. 28521/1986. It is preferable that the
copolymer be produced by catalyst-free dehydration polymerization
condensation.
[0417] The above-described glycolic acid copolymer may be used in a
mixture with polylactic acid. Although the polylactic acid may be a
D-isomer, L-isomer or a mixture thereof, it is preferable that the
ratio of the D-isomer/L-isomer (mol %) fall within the range from
about 75/25 to 20/80. More preferred is a polylactic acid wherein
the ratio of the D-isomer/L-isomer (mol %) falls within the range
from about 60/40 to 25/75, with greater preference given to a
polylactic acid wherein the ratio of the D-isomer/L-isomer (mol %)
falls within the range from about 55/45 to 25/75. The polylactic
acid preferably has a weight-average molecular weight of about
1,500 to 30,000. More preferred is a polylactic acid wherein the
weight-average molecular weight falls within the range from about
2,000 to 20,000, with greater preference given to a polylactic acid
wherein the weight-average molecular weight falls within the range
from about 3,000 to 15,000. Also, the dispersity of the polylactic
acid is preferably about 1.2 to 4.0, more preferably about 1.5 to
3.5.
[0418] For producing polylactic acid, two methods are known:
ring-opening polymerization of lactide, a dimer of lactic acid, and
dehydration polymerization condensation of lactic acid. For
obtaining a polylactic acid of relatively low molecular weight for
the present invention, direct dehydration polymerization
condensation of lactic acid is preferred. This method is, for
example, described in Japanese Patent Unexamined Publication No.
28521/1986.
[0419] The present glycolic acid copolymer and polylactic acid are
used over the mixing ratio range of, for example, from about 10/90
to 90/10 (% by weight), preferably from about 20/80 to 80/20, more
preferably from about 30/70 to 70/30.
[0420] In the case of a copolymer of glycolic acid and lactic acid,
the content ratio (lactic acid/glycolic acid) (mol %) is preferably
about 100/0 to 40/60, more preferably about 90/10 to 45/55. The
weight-average molecular weight of the copolymer of glycolic acid
and lactic acid is preferably about 4,000 to 25,000, more
preferably about 5,000 to 20,000.
[0421] The dispersity of the copolymer of glycolic acid and lactic
acid (weight-average molecular weight/number average molecular
weight) is preferably from about 1.2 to 4.0, more preferably from
about 1.5 to 3.5. The copolymer of glycolic acid and lactic acid
can be produced by a known method, such as the method described in
Japanese Patent Unexamined Publication No. 28521/1986. The
copolymer is preferably produced by catalyst-free dehydration
polymerization condensation.
[0422] In the present invention, the aliphatic polyester produced
by catalyst-free dehydration polymerization condensation usually
has a terminal carboxyl group.
[0423] More preferably, the biodegradable polymer is an aliphatic
polyester (e.g., a polymer, copolymer or mixture thereof produced
from one or more .alpha.-hydroxycarboxylic acids such as glycolic
acid, lactic acid and hydroxybutyric acid, hydroxydicarboxylic
acids such as malic acid, hydroxytricarboxylic acids such as citric
acid, and others) as having a terminal carboxyl group.
[0424] A biodegradable polymer having a terminal carboxyl group is
a polymer in which the number-average molecular weights by GPC
determination and that by end-group determination almost agree.
[0425] To quantitate terminal free carboxyl groups, about 1 to 3 g
of the biodegradable polymer is dissolved in a mixed solvent of
acetone (25 ml) and methanol (5 ml), and the solution is quickly
titrated with a 0.05 N alcoholic solution of potassium hydroxide
while stirring at room temperature with phenolphthalein as an
indicator to determine the terminal carboxyl group content; the
number-average molecular weight is calculated from the following
equation:
Number-average molecular weight by end-group determination=20,000
A/B
[0426] where A is the weight mass (g) of the biodegradable polymer,
and B is the amount (ml) of the 0.05 N alcoholic solution of
potassium hydroxide added until the titration end point is
reached.
[0427] This value is hereinafter referred to as number-average
molecular weight by end-group determination.
[0428] For example, in the case of a polymer having a terminal
carboxyl group, produced from one or more .alpha.-hydroxy acids by
catalyst-free dehydration polymerization condensation, the
number-average molecular weight by GPC determination and the
number-average molecular weight by end-group determination almost
agree with each other. On the other hand, in the case of a polymer
having no terminal carboxyl groups and which is synthesized from a
cyclic dimer by ring-opening polymerization using a catalyst, the
number-average molecular weight by end-group determination is
significantly higher than that by GPC determination. This
difference makes it possible to clearly differentiate a polymer
having a terminal carboxyl group from a polymer having no terminal
carboxyl group.
[0429] While the number-average molecular weight by end-group
determination is an absolute value, that by GPC determination is a
relative value that varies depending on various analytical
conditions (e.g., kind of mobile phase, kind of column, reference
substance, slice width, baseline etc.); it is therefore difficult
to have an absolute numerical representation of the latter.
However, the description that the number-average molecular weights
by GPC determination and end-group determination "almost agree"
here denotes that the latter falls within the range from about 0.5
to 2 times, preferably from about 0.8 to 1.5 times, the former.
Also, the description that the number-average molecular weight by
end-group determination is "significantly higher" than the
number-average molecular weight by GPC determination here denotes
that the former is about 2 times or more greater than the
latter.
[0430] In the present invention, preference is given to a polymer
wherein the number-average molecular weights by GPC determination
and by end-group determination almost agree.
[0431] Regarding weight-average molecular weights and
number-average molecular weights by GPC determination, the present
specification holds that the former is based on polystyrene
obtained by gel permeation chromatography (GPC) with 9 polystyrenes
as reference substances with weight-average molecular weights of
120,000, 52,000, 22,000, 9,200, 5,050, 2,950, 1,050, 580 and 162,
respectively. Measurements were taken using a GPC column KF804Lx2
(produced by Showa Denko) and an RI monitor L-3300 (produced by
Hitachi, Ltd.), with chloroform as a mobile phase.
[0432] The dispersity is calculated by the formula: (weight-average
molecular weight/number-average molecular weight)
[0433] The sustained-release preparation of the present invention
can, for example, be produced from a w/o emulsion with a solution
containing an anti-endothelin substance as an internal aqueous
phase and a solution containing a biodegradable polymer as an oil
phase. This is achieved by known methods, including aqueous drying,
phase separation, spray drying and modifications thereof.
[0434] The solvent used in the oil phase for the above-mentioned
methods is preferably an organic solvent which dissolves
biodegradable polymers and which has a boiling point not higher
than 120.degree. C. Such solvents include halogenated hydrocarbons
(e.g., dichloromethane, chloroform and carbon tetrachloride),
alcohols (e.g., ethanol and methanol) and acetonitrile. These may
be used in combination. The solvent is preferably dichloromethane,
acetonitrile or the like.
[0435] When the anti-endothelin substance for the present invention
has a carboxyl group, its water solubility is low because it is
often acidic; to increase its pharmaceutical solubility, it is
often used in the form of an organic or inorganic salt. Such
organic or inorganic salts are preferably alkali metal salts (e.g.,
sodium salt and potassium salt), with preference given to sodium
salt. In order to include a pharmacologically necessary amount of
drug, it is necessary to prepare a solution of very high
concentration, however, since the volume of the aqueous phase for
preparing the above w/o emulsion is usually very small. In such
case, when the drug is low in water solubility, though soluble in
water, it can fail to be completely dissolved, resulting in uneven
mixing in preparing the emulsion. By dissolving the anti-endothelin
substance along with an organic basic substance, a uniform solution
of the anti-endothelin substance can be prepared that is soluble in
water but low in solubility. Also, the addition of an organic basic
substance suppresses the usually rapid initial drug release from
microcapsules produced using a biodegradable polymer, allowing
sustained release of a given amount of drug over a given period of
time. The organic basic substance is preferably a basic amino acid,
particularly arginine, histidine, lysine or the like. The organic
basic substance is further exemplified by a peptide comprising two
or more of basic amino acids such as arginyl-arginine.
[0436] As for the content ratio of the organic basic substance, the
weight ratio of the anti-endothelin substance to the organic basic
substance is normally 1:1,000 to 1,000:1, preferably 1:100 to
100:1, and more preferably 1:10 to 10:1. The weight ratio of the
biodegradable polymer to the organic basic substance is normally
1,000:1 to 5:1, preferably 500:1 to 10:1, and more preferably 100:1
to 10:1.
[0437] In the process of producing sustained-release preparations,
the addition of water-soluble polyvalent metal salts suppress the
initial burst of drug, allowing sustained release of a given amount
of drug over a given period of time and causing a high drug
content. The water-soluble polyvalent metal salts may be any one,
without limitation, as long as it is soluble in water and does not
adversely affect the living body.
[0438] The water-soluble polyvalent metal salts are preferably
polyvalent metal salts whose water solubility at normal temperature
(about 20.degree. C.) is over about 20 mg/ml, more preferably over
about 100 mg/ml.
[0439] Examples of water-soluble polyvalent metal salts are
polyvalent metal salts with inorganic acids and that with organic
acids. Polyvalent metals are exemplified by alkaline earth metal
(e.g., calcium, magnesium), zinc (II), iron (II, III), copper (II),
tin (II, IV) and aluminium (II, III). Inorganic acids are
exemplified by hydrohalogenic acid (e.g., hydrochloric acid,
hydrobromic acid, hydroiodic acid, hydrofluoric acid), sulfuric
acid, nitric acid and thiocyanic acid. Organic acids are
exemplified by aliphatic carboxylic acid (e.g., acetic acid,
glycolic acid, lactic acid, tartaric acid) and aromatic acid (e.g.,
benzoic acid, salycylic acid, phenolsulfonic acid). The
water-soluble polyvalent metal salts are preferably water-soluble
zinc salts. The water-soluble zinc salts are exemplified by
inorganic acid zinc salts such as zinc halogenide (e.g., zinc
chloride, zinc bromide, zinc iodide, zinc fluoride), zinc sulfate,
zinc nitrate and zinc thiocyanate and organic acid zinc salts such
as aliphatic carboxylic acid zinc salts (e.g., zinc acetate, zinc
glycolate, zinc lactate, zinc tartrate) and aromatic acid zinc
salts (e.g., zinc benzoate, zinc salicylate, zinc phenolsulfate).
The water-soluble zinc salts are preferably aliphatic carboxylic
acid zinc salts, with greater preference given to zinc acetate.
[0440] As for the content ratio of the water-soluble polyvalent
metal salts, the weight ratio of the anti-endothelin substance to
the water-soluble polyvalent metal salts is preferably 1:100 to
100:1, more preferably 1:10 to 10:1. The weight ratio of the
biodegradable polymer to the water-soluble polyvalent metal salts
is preferably 1,000:1 to 1:1, more preferably 100:1 to 2:1.
[0441] In the present invention, the anti-endothelin substance may
be dissolved or suspended directly in an organic solvent solution
of the biodegradable polymer. The anti-endothelin substance may be
soluble or insoluble in the organic solvent. The anti-endothelin
substance is sometimes soluble in the solution of the biodegradable
polymer in the organic solvent, even when the anti-endothelin
substance is insoluble in the organic solvent. Any organic solvent
is acceptable, as long as it is substantially immiscible with water
and dissolves the biodegradable polymer, and the resulting polymer
solution dissolves the anti-endothelin substance. The organic
solvent preferably has a water solubility not higher than 3% at
normal temperature (20.degree. C.). Also, the boiling point of the
organic solvent is preferably not higher than 120.degree. C.
Example organic solvents include halogenated hydrocarbons (e.g.,
dichloromethane, chloroform, chloroethane, trichloroethane and
carbon tetrachloride), ethers (e.g., isopropyl ether), fatty acid
esters (e.g., butyl acetate) and aromatic hydrocarbons (e.g.,
benzene, toluene and xylene). These may be used in combination in
appropriate ratios. The organic solvent is preferably
dichloromethane. Dissolution of the anti-endothelin substance means
that no anti-endothelin substance remains undissolved in the
resulting solution, as examined by macroscopic observation at
normal temperature (20.degree. C.).
[0442] The sustained-release preparation of the present invention
is preferably produced by, for example, the method of
microcapsulation (or modification thereof) based on aqueous drying
or phase separation as described below.
[0443] (i) Aqueous Drying Method (w/o/w Method)
[0444] An anti-endothelin substance is dissolved in water. The
anti-endothelin substance concentration in the aqueous solution is,
for example, about 0.1 to 500% (w/v), preferably about 1 to 400%
(w/v), and more preferably about 10 to 300% (w/v). To the aqueous
solution, an organic basic substance, preferably a basic amino acid
(e.g., arginine) or a peptide comprising two or more of basic amino
acids (e.g., arginyl-arginine) may be added. The concentration of
the organic basic substance used for this purpose in the aqueous
solution is about 0.01 to 500% (w/v), preferably about 0.1 to 400%
(w/v), and more preferably about 1 to 300% (w/v). To the aqueous
solution, water-soluble polyvalent metals may be added in the same
manner as the organic basic substance. To the aqueous solution may
be added pH regulators (e.g., acetic acid, hydrochloric acid and
sodium hydroxide), stabilizers (e.g., serum albumin and gelatin),
preservatives (paraoxybenzoic acids) and other additives. The
aqueous solution thus obtained is emulsified and dispersed in an
organic solvent solution of a biodegradable polymer or copolymer
synthesized from .alpha.-hydroxycarboxylic acid to yield a w/o
emulsion. Although the biodegradable polymer concentration in the
organic solvent solution varies depending on the molecular weight
of the biodegradable polymer and the kind of organic solvent, it is
selected over the range from about 0.01 to 80% (w/w), preferably
about 0.1 to 70% (w/w), and more preferably about 1 to 60%
(w/w).
[0445] The ratio of the aqueous solution and the organic solvent
solution of the biodegradable polymer is normally 1:1,000 (v/v) to
1:1 (v/v), preferably 1:100 (v/v) to 1:5 (v/v), and more preferably
1:50 (v/v) to 1:5 (v/v). This emulsification is achieved by known
methods of dispersion using a turbine type mechanical stirrer,
homogenizer etc.
[0446] The w/o emulsion thus prepared is added to an aqueous phase
to form a w/o/w emulsion, followed by evaporation of the solvent in
the oil phase, to yield microcapsules. The volume of the aqueous
phase is chosen over the range normally from about 1 to 10,000
times, preferably from about 2 to 5,000 times, and more preferably
from about 5 to 2,000 times the volume of the oil phase.
[0447] In addition to the above additives, an emulsifier may be
added to the aqueous phase. The emulsifier may be any one, as long
as it is capable of forming a stable o/w emulsion. Examples of such
emulsifiers include anionic surfactants, nonionic surfactants,
polyoxyethylene castor oil derivatives, polyvinylpyrrolidone,
polyvinyl alcohol, carboxymethyl cellulose, lecithin, gelatin and
hyaluronic acid. These may be used singly or in combination. The
concentration of emulsifier used may be chosen as appropriate over
the range normally from about 0.001 to 20% (w/w), preferably from
about 0.01 to 10% (w/w), and more preferably from about 0.05 to 5%
(w/w).
[0448] The microcapsules thus obtained are collected by
centrifugation or filtration, after which they are repeatedly
washed with distilled water in several cycles to remove the free
anti-endothelin substance, emulsifier etc. adhering to the
microcapsule surface, again dispersed in distilled water etc. and
then lyophilized. Where necessary, the microcapsules are heated
under reduced pressure to further remove the water and organic
solvent. Preferably, this removal is carried out at a heating rate
of 10 to 20.degree. C. per minute at a temperature higher by at
least 5.degree. C. than the intermediate glass transition point of
the biodegradable polymer, as determined using a differential
scanning calorimeter, usually within 1 weeks or 2 or 3 days, more
preferably within 24 hours, after the microcapsules have reached a
given temperature.
[0449] (ii) Aqueous Drying Method (o/w Method)
[0450] An anti-endothelin substance is added to an organic solvent
solution of a biodegradable polymer to a ratio by weight as defined
above, to prepare an organic solvent solution or suspension
containing both the anti-endothelin substance and the biodegradable
polymer. In this operation, the biodegradable polymer concentration
in the organic solvent solution varies depending on the molecular
weight of the biodegradable polymer and the kind of the organic
solvent, it is chosen over the range normally from about 0.01 to
80% (w/w), preferably from about 0.1 to 70% (w/w), and more
preferably from about 1 to 60% (w/w). To the organic solvent
solution or suspension, water-soluble polyvalent salts may
added.
[0451] The organic solvent solution or suspension thus prepared is
added to an aqueous phase to form an o/w emulsion, followed by
evaporation of the solvent in the oil phase, to yield
microcapsules. The volume of the aqueous phase is chosen over the
range normally from about 1 to 10,000 times, preferably from about
2 to 5,000 times, and more preferably from about 5 to 2,000 times
the volume of the oil phase.
[0452] In addition to the above additives, an emulsifier may be
added to the aqueous phase. The emulsifier may be any one, as long
as it is capable of forming a stable o/w emulsion. Examples of such
emulsifiers include anionic surfactants, nonionic surfactants,
polyoxyethylene castor oil derivatives, polyvinylpyrrolidone,
polyvinyl alcohol, carboxymethyl cellulose, lecithin, gelatin and
hyaluronic acid. These may be used singly or in combination. The
concentration of the emulsifier used may be chosen as appropriate
over the range normally from about 0.001 to 20% (w/w), preferably
from about 0.01 to 10% (w/w), and more preferably from about 0.05
to 5% (w/w).
[0453] The microcapsules thus obtained are collected by
centrifugation or filtration, after which they are repeatedly
washed with distilled water in several cycles to remove the free
anti-endothelin substance, emulsifier etc. adhering to the
microcapsule surface, and again dispersed in distilled water etc.
and then lyophilized. Where necessary, the microcapsules are then
heated under reduced pressure to further remove water and organic
solvent. Preferably, this removal is achieved at a heating rate of
10 to 20.degree. C. per minute at a temperature higher by at least
5.degree. C. than the intermediate glass transition point of the
biodegradable polymer, as determined using a differential scanning
calorimeter, usually within 1 weeks or 2 or 3 days, more preferably
within 24 hours after the microcapsules have reached a given
temperature.
[0454] (iii) Phase Separation Method
[0455] In producing microcapsules by the phase separation method, a
coacervating agent is gradually added to the above-described w/o
emulsion or organic solvent solution during stirring, to separate
and solidify the biodegradable polymer. The coacervating agent is
added in an amount by volume about 0.01 to 1,000 times, preferably
about 0.05 to 500 times, and more preferably about 0.1 to 200 times
the volume of the w/o emulsion or organic solvent solution.
[0456] Any coacervating agent is acceptable, as long as it is a
polymer, mineral oil or vegetable oil compound that is miscible in
the solvent for the biodegradable polymer and which does not
dissolve the polymer. Example coacervating agents include silicon
oil, sesame oil, soybean oil, corn oil, cotton seed oil, coconut
oil, linseed oil, mineral oil, n-hexane and n-heptane. These may be
used in combination.
[0457] The microcapsules thus obtained are collected by filtration,
after which they are repeatedly washed with heptane etc. to remove
the coacervating agent. The free drug and solvent are removed in
the same manner as in the aqueous drying method.
[0458] Solvent removal can be achieved by known methods, including
the method in which the solvent is evaporated under normal or
gradually reduced pressure during stirring using a propeller
stirrer or magnetic stirrer, and the method in which the solvent is
evaporated while adjusting the degree of vacuum using a rotary
evaporator etc.
[0459] In production by the aqueous drying method or coacervation
method, an antiflocculant may be added to prevent grain
flocculation. The antiflocculant is exemplified by water-soluble
polysaccharides such as mannitol, lactose, glucose and starches
(e.g., corn starch), proteins such as glycine, fibrin and collagen
and inorganic salts such as sodium chloride and sodium hydrogen
phosphate.
[0460] In producing microcapsules by the spray drying method, a w/o
emulsion or organic solvent solution containing the above-described
anti-endothelin substance and biodegradable polymer is sprayed via
a nozzle into the drying chamber of a spray drier to volatilize the
organic solvent in the fine droplets in a very short time to yield
fine microcapsules. The nozzle is exemplified by the double-fluid
nozzle, pressure nozzle and rotary disc nozzle. Where desired, to
prevent microcapsule flocculation, an aqueous solution of the
above-described antiflocculant may be effectively sprayed via
another nozzle simultaneously with spraying of the w/o emulsion or
organic solvent solution containing the anti-endothelin substance
and biodegradable polymer.
[0461] The microcapsules thus obtained may have their water and
organic solvent removed at increased temperature under reduced
pressure as necessary.
[0462] The above-described microcapsules can be administered as
such or in the form of various dosage forms of non-oral
preparations (e.g., intramuscular, subcutaneous or visceral
injections or indwellable preparations, nasal, rectal or uterine
transmucosal preparations) or oral preparations (e.g., capsules
such as hard capsules and soft capsules), or solid preparations
such as granules and powders or liquid preparations such as syrups,
emulsions and suspensions.
[0463] In addition to the above-described dosage forms of
microcapsules, the w/o emulsion or organic solvent solution
containing an anti-endothelin substance and biodegradable polymer
can be shaped in rods, needles, pellets, films and other forms and
administered as intramuscular, subcutaneous or visceral injections
or indwellable preparations, nasal, rectal or uterine transmucosal
preparations, oral preparations (e.g., capsules such as hard
capsules and soft capsules), solid preparations such as granules
and powders, and liquid preparations such as syrups, emulsions and
suspensions.
[0464] The injectable preparation of the present invention can be
produced by known methods. The injectable preparation is produced
by, for example, suspending the above-described sustained-release
preparation of microcapsules etc. in water, along with a dispersing
agent (e.g., surfactants such as Tween 80 and HCO-60, and
polysaccharides such as carboxymethyl cellulose and sodium
alginate), a preservative (e.g., methyl paraben and propyl
paraben), an isotonizing agent (e.g., sodium chloride, mannitol,
sorbitol and glucose), to yield an aqueous suspension, or by
dispersion in a vegetable oil such as sesame oil or corn oil or
middle-chain fatty acid triglyceride (e.g., Migriol 812) to yield
an oily suspension. The particle size of the sustained-release
preparation is chosen over the range from about 0.1 to 300 .mu.m,
for instance, as long as the requirements concerning the degree of
dispersion and needle passage are met, when the sustained-release
preparation is used as an injectable suspension. Preferably, the
particle size falls within the range from about 1 to 150 .mu.m,
more preferably from about 2 to 100 .mu.m. A sustained-release
preparation can be prepared as a sterile preparation without
limitation by the method in which the entire production process is
sterile, the method in which a gamma ray is used as a sterilant,
and the method in which an antiseptic is added.
[0465] With low toxicity, the sustained-release preparation of the
present invention can be safely used in mammals (e.g., humans,
bovines, swines, dogs, cats, mice, rats and rabbits).
[0466] The sustained-release preparation of the present invention
is used to treat or prevent endothelin-associated diseases,
particularly chronic ones. Such diseases include cardiac/cerebral
circulatory diseases, renal diseases, hypertension (e.g., pulmonary
hypertension), asthma, inflammation, arthritis, hepatic cancer,
cirrhosis and chronic complications in diabetes mellitus. The
sustained-release preparation of the present invention is used to
treat or prevent arteriosclerosis, diabetic nephropathy, diabetic
myocarditis and diabetic retinopathy, in particular.
[0467] Varying depending on type, content and dosage form of the
active ingredient anti-endothelin substance, duration of
anti-endothelin substance release, target disease (e.g., diabetic
nephropathy), subject animal and other factors, the dose of the
sustained-release preparation may be set at levels such that the
anti-endothelin substance is effective. The dose per administration
of the active ingredient anti-endothelin substance is chosen as
appropriate over the range from about 0.01 to 100 mg/kg body weight
for each adult when the preparation is a 1-month preparation. More
preferably, the dose may be chosen as appropriate over the range
from about 0.05 to 50 mg/kg body weight.
[0468] The dose per administration of the sustained-release
preparation is chosen as appropriate over the range from about 0.1
to 1,000 mg/kg body weight for each adult. More preferably, the
dose may be chosen as appropriate over the range from about 0.5 to
500 mg/kg body weight. Dosing frequency can be chosen as
appropriate, e.g., once weekly, once every several weeks, once
monthly or once every several months, depending on type, content
and dosage form of the active ingredient anti-endothelin substance,
duration of anti-endothelin substance release, subject disease,
subject animal and other factors.
[0469] The preparation of the present invention may be used in
combination with other drugs, specifically conventional therapeutic
drugs for diabetic nephropathy, such as hypotensive drugs. Although
the preparation of the present invention may be stored at normal
temperatures or cold places, it is preferable to store it at a cold
place. Normal temperatures and cold places mentioned herein are as
defined by the Pharmacopoeia of Japan.
[0470] The present invention is hereinafter described in more
detail by means of the following working examples and experimental
examples, which are not to be construed as limitative.
REFERENCE EXAMPLE 1
[0471] Synthesis of
cyclo[-D-Asp-Asp(B7)-Asp-D-.gamma.-MeLeu-Leu-D-Trp-dis- odium
Salt
[0472] 4.4 g of cyclo[-D-Asp-Asp(B7)-Asp-D-.gamma.MeLeu-Leu-D-Trp-]
(hereinafter referred to briefly as peptide B) was dissolved in 50
ml of methanol and concentrated. The concentrate was again
dissolved in 50 ml of methanol and subjected to ice cooling. To
thus obtained solution 0.1 N NaOH solution (46.4 ml) was added
dropwise, and the pH of the solution was adjusted to 7-8 by further
addition of 0.1 N NaOH solution. The resulting solution was
concentrated. The concentrate was lyophilized after addition of
distilled water. Peptide B disodium salt (Yield 4.5 g)
[0473] Elemental analysis:
[0474] As
C.sub.47H.sub.61N.sub.9O.sub.11Na.sub.2.CF.sub.3CO.sub.2Na.0.5CH-
.sub.3CO.sub.2Na.3H.sub.2O
[0475] Calculated: C, 49.18; H, 5.65; N, 10.32 Found: C, 49.08; H,
5.50; N, 10.33.
EXAMPLE 1
[0476] 51 mg of the disodium salt of the cyclic peptide
cyclo[-D-Asp-Asp(R1')-Asp-D-Thg(2)-Leu-D-Trp-]
[0477] wherein Asp represents aspartic acid; Asp(R1') represents
aspartic acid .beta.-4-phenylpiperazinamide; Thg(2) represents
2-thienylglycine; Leu represents leucine; Trp represents
tryptophan, described in European Patent Publication No. 528312 and
49 mg of L-arginine (Wako Pure Chemical) were dissolved in 300 ul
of distilled water. This solution was added to a solution of 1.92 g
of a lactic acid-glycolic acid copolymer (lactic acid/glycolic
acid=75/25 mol %, GPC weight-average molecular weight 14,000, GPC
number-average molecular weight 2,000, number-average molecular
weight by end-group determination 2,200, produced by Wako Pure
Chemical Industry, Lot No. 920729) in 2 ml of dichloromethane, and
the mixture was stirred using a homogenizer (Polytron) to yield a
w/o emulsion. After cooling to 17.degree. C., the emulsion was
injected to 1,000 ml of a 0.1% (w/w) aqueous solution of polyvinyl
alcohol (EG-40, produced by The Nippon Synthetic Chemical Industry,
Co., Ltd.), previously adjusted to 16.degree. C., followed by
stirring in a turbine homomixer at 7,000 rpm to yield a w/o/w
emulsion, which was then stirred at room temperature for 3 hours to
volatilize the dichloromethane and solidify the oil phase, which
was then collected via centrifugation at 2,000 rpm using a
centrifuge (05PR-22, Hitachi, Ltd.). The precipitate was again
dispersed in distilled water, centrifuged and washed to remove the
free drug etc. After the collected microcapsules were re-dispersed
in a small amount of distilled water, 0.3 g of D-mannitol was
added, and the dispersion was lyophilized to yield powdery
microcapsules.
EXAMPLE 2
[0478] About 39 mg of microcapsules as obtained in Example 1 was
dispersed in 1.95 ml of a dispersant for injection (distilled water
containing 2.5 mg of carboxymethyl cellulose, 0.5 mg of polysorbate
80 and 25 mg of mannitol, all dissolved therein) to yield an
injectable preparation.
EXAMPLE 3
[0479] 3.6 g of lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=75125 mol %, GPC weight-average molecular weight
15,038, GPC number-average molecular weight 5,195, produced by Wako
Pure Chemical Industry) was dissolved in 6.6 g (5 ml) of
dichloromethane. To this solution was added a solution of peptide A
disodium salt (250 mg) and L-arginine (100 mg) in 0.5 ml of
distilled water, and the mixture was stirred for about 30 seconds
using a homogenizer (Polytron) to yield a w/o emulsion. The
emulsion was injected to 800 ml of a 0.1% (w/w) aqueous solution of
polyvinyl alcohol (EG-40, produced by The Nippon Synthetic Chemical
Industry, Co., Ltd.), previously adjusted to 18.degree. C.,
followed by stirring in a turbine homomixer at 6,000 rpm to yield a
w/o/w emulsion, which was then stirred at a room temperature for 3
hours to volatile the dichloromethane and solidify the oil phase,
which was then collected via centrifugation at 2,000 rpm using a
centrifuge (05PR-22, Hitachi, Ltd.). The precipitate was again
dispersed in distilled water, centrifuged and washed to remove the
free drug etc. After 100 mg of D-mannitol was added to the
collected microcapsules, the microcapsules were re-dispersed in a
small amount of distilled water, and the dispersion was lyophilized
to yield powdery microcapsules.
[0480] The microcapsules thus obtained were homogenized and
extracted in 0.1 M ammonium acetate solution containing 30% (v/v)
acetonitrile for 3 hours, and then assayed by HPLC (high
performance liquid chromatography).
[0481] As the result, the content of peptide A disodium salt was
5.2 mg per 100 mg of microcapsules.
EXAMPLE 4
[0482] Powdery microcapsules were obtained in the same manner as
Example 3, except that L-arginyl-arginine (Kokusan Chemical Works
Ltd.) was substituted for L-arginine.
[0483] The content of peptide A disodium salt was 7.4 mg per 100 mg
of microcapsules.
EXAMPLE 5
[0484] Synthesis of
cyclo(-D-Asp(OC.sub.2H.sub.5)-Asp(R1')-Asp(OC.sub.2H.s-
ub.5)-D-Thg(2)-Leu-D-Trp-]
[0485] 10 ml of ethanol was cooled to -10.degree. C. in a dry
ice-acetone bath, and 2.6 ml of thionyl chloride was added in a
small amount. After 5 minutes, 1.0 g of peptide A disodium salt was
added to the mixture and stirred at room temperature. After 2
hours, ethanol and excess thionyl chloride was removed under
reduced pressure to give an oily substance. The oily substance was
dissolved in a small amount of ethanol, and again the solvent was
removed under reduced pressure. This operation was repeated three
times and a small amount of diethylether was added to give 1.05 g
of titled compound. The result of analysis of peptide A
diethylester was described below.
[0486] 1) Mass spectrometry (LSIMS method):
[0487] [M+H].sup.+=984 (theoretical value=984)
[0488] [M+Na].sup.+=1,006 (theoretical value=1,006)
[0489] 2) Elemental analysis: As
C.sub.49H.sub.61N.sub.9O.sub.11S.2NaCl.2H- .sub.2O.HCl
[0490] Calculated: C, 50.20; H, 5.67; N, 10.75
[0491] Found: C, 50.35; H, 5.75; N, 10.81.
EXAMPLE 6
[0492] 0.5 g of lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=50/50 ml %, GPC weight-average molecular weight
5,900, GPC number-average molecular weight 2,600, produced by Wako
Pure Chemical Industry) was dissolved in 6.6 g (5 ml) of
dichloromethane. To this solution was added 0.15 g of peptide A
diethylester which was obtained in Example 5, and the mixture was
stirred for about 30 seconds using a homogenizer (Polytron) to
yield a s/o emulsion. The emulsion was injected to 400 ml of a 0.1%
(w/w) aqueous solution of polyvinyl alcohol (EG-40, produced by The
Nippon Synthetic Chemical Industry, Co., Ltd.), previously adjusted
to 18.degree. C., followed by stirring in a turbine homomixer at
6,000 rpm to yield a s/o/w emulsion, which was then stirred at a
room temperature for 3 hours to volatile the dichloromethane and
solidify the oil phase, which was then collected via centrifugation
at 2,000 rpm using a centrifuge (05PR-22, Hitachi, Ltd.). The
precipitate was again dispersed in distilled water, centrifuged and
washed to remove the free drug etc. After 50 mg of D-mannitol was
added to the collected microcapsules, the microcapsules were
re-dispersed in a small amount of distilled water, and the
dispersion was lyophilized to yield powdery microcapsules.
[0493] The microcapsules thus obtained were homogenized and
extracted in 0.1 M phosphate buffered solution containing 50%
acetinitrile for 3 hours, and then assayed by HPLC (high
performance liquid chromatography).
[0494] As the result, the content of peptide A diethylester was
25.2 mg per 100 mg of microcapsules.
EXAMPLE 7
[0495] 3.2 g of peptide A disodium salt and 7.28 g of zinc acetate
di-hydrate were each dissolved in 160 ml of distilled water, and
thus obtained two solutions were mixed together. This mixture was
stayed at 4.degree. C. for a day, and then centrifuged at 3,000 rpm
using a centrifuge (05PR-22, Hitachi, Ltd.). Thus obtained
precipitate was again dispersed in distilled water, centrifuged and
washed to remove the free drug etc. After small amount of distilled
water was added to the collected precipitate to re-disperse the
precipitate, the dispersion was lyophilized to yield a crude
peptide A zinc salt as a 2.81 g of dried powder.
[0496] The dried powder thus obtained were homogenized and
extracted in 50 mM EDTA solution containing 30% (v/v) acetonitrile
for 3 hours, and then assayed by HPLC (high performance liquid
chromatography).
[0497] As the result, the content of peptide A in dried powder was
80.7% (w/w).
EXAMPLE 8
[0498] 0.97 g of lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=75/25 mol %, GPC weight-average molecular weight
15,038, GPC number-average molecuar weight 5,195, produced by Wako
Pure Chemical Industry) was dissolved in 13.2 g (10 ml) of
dichloromethane. To this solution was added the crude peptide A
zinc salt (300 mg) which was obtained in Example 7, and the mixture
was stirred for about 30 seconds using a homogenizer (Polytron) to
yield a s/o emulsion. The emulsion was injected to 400 ml of a 0.1%
(w/w) aqueous solution of polyvinyl alcohol (EG-40, produced by The
Nippon Synthetic Chemical Industry, Co., Ltd.), previously adjusted
to 18.degree. C., followed by stirring in a turbine homomixer at
6,000 rpm to yield a s/o/w emulsion, which was then stirred at a
room temperature for 3 hours to volatile the dichloromethane and
solidify the oil phase, which was then collected via centrifugation
at 2,000 rpm using a centrifuge (05PR-22, Hitachi, Ltd.). The
precipitate was again dispersed in distilled water, centrifuged and
washed to remove the free drug etc. After 50 mg of D-mannitol was
added to the collected microcapsules, the microcapsules were
re-dispersed in a small amount of distilled water, and the
dispersion was lyophilized to yield powdery microcapsules.
[0499] The microcapsules thus obtained were homogenized and
extracted in 50 mM EDTA (ethylenediaminetetraacetic acid) solution
containing 30% (v/v) acetonitrile for 3 hours, and then assayed by
HPLC (high performance liquid chromatography).
[0500] As the result, the content of a crude peptide A zinc salt in
terms of peptide A disodium salt was 21.2 mg per 100 mg of
microcapsules.
EXAMPLE 9
[0501] Powdery microcapsules were obtained in the same manner as
Example 3, except that peptide B disodium salt was substituted for
peptide A disodium salt.
[0502] The content of peptide B disodium salt was 5.2 mg per 100 mg
of microcapsules.
EXAMPLE 10
[0503] 1.2 g of lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=75/25 mol %, GPC weight-average molecular weight
13,585, GPC number-average molecular weight 4,413, produced by Wako
Pure Chemical Industry) was dissolved in 26.4 g (20 ml) of
dichloromethane. To this solution was added a solution of peptide A
disodium salt (400 mg) and zinc acetate di-hydrate (400 mg) in 1.7
ml of distilled water, and the mixture was stirred for about 30
seconds using a homogenizer (Polytron) to yield a w/o emulsion. The
emulsion was injected to 800 ml of a 0.1% (w/w) aqueous solution of
polyvinyl alcohol (EG-40, produced by The Nippon Synthetic Chemical
Industry, Co., Ltd.), previously adjusted to 18.degree. C.,
followed by stirring in a turbine homomixer at 6,000 rpm to yield a
w/o/w emulsion, which was then stirred at a room temperature for 3
hours to volatile the dichloromethane and solidify the oil phase,
which was then collected via centrifugation at 2,000 rpm using a
centrifuge (05PR-22, Hitachi, Ltd.). The precipitate was again
dispersed in distilled water, centrifuged and washed to remove the
free drug etc. After 50 mg of D-mannitol was added to the collected
microcapsules, the microcapsules were re-dispersed in a small
amount of distilled water, and the dispersion was lyophilized to
yield powdery microcapsules.
[0504] The microcapsules thus obtained were homogenized and
extracted in 50 mM EDTA solution containing 30% (v/v) acetonitrile
for 3 hours, and then assayed by HPLC (high performance liquid
chromatography).
[0505] As the result, the content of peptide A disodium salt was 12
mg per 100 mg of microcapsules.
EXAMPLE 11
[0506] 1.4 g of lactic acid-glycolic acid copolymer (lactic
acid/glycolic acid=75/25 mol %, GPC weight-average molecular weight
13,585, GPC number-average molecular weight 4,413, produced by Wako
Pure Chemical Industry) was dissolved in 6.6 g (5 ml) of
dichloromethane. To this solution was added peptide A disodium salt
(437 mg) and zinc acetate di-hydrate (467 mg), and the mixture was
stirred for about 30 seconds using a homogenizer (Polytron) to
yield a s/o emulsion. The emulsion was injected to 800 ml of a 0.1%
(w/w) aqueous solution of polyvinyl alcohol (EG-40, produced by The
Nippon Synthetic Chemical Industry, Co., Ltd.), previously adjusted
to 18.degree. C., followed by stirring in a turbine homomixer at
6,000 rpm to yield a s/o/w emulsion, which was then stirred at a
room temperature for 3 hours to volatile the dichloromethane and
solidify the oil phase, which was then collected via centrifugation
at 2,000 rpm using a centrifuge (05PR-22, Hitachi, Ltd.). The
precipitate was again dispersed in distilled water, centrifuged and
washed to remove the free drug etc. After 50 mg of D-mannitol was
added to the collected microcapsules, the microcapsules were
re-dispersed in a small amount of distilled water, and the
dispersion was lyophilized to yield powdery microcapsules.
[0507] The microcapsules thus obtained were homogenized and
extracted in 50 mM EDTA solution containing 30% (v/v) acetonitrile
for 3 hours, and then assayed by HPLC (high performance liquid
chromatography).
[0508] As the result, the content of peptide A disodium salt was
12.2 mg per 100 mg of microcapsules.
COMPARATIVE EXAMPLE 1
[0509] Powdery microcapsules were obtained in the same manner as
Example 3, except that peptide A disodium salt was not used.
COMPARATIVE EXAMPLE 2
[0510] 1.6 g of peptide A disodium salt and 3.65 g of zinc acetate
were each dissolved in 80 ml of distilled water, and thus obtained
two solutions were mixed together. This mixture was centrifuged at
3,000 rpm using a centrifuge (05PR-22, Hitachi, Ltd.). Thus
obtained precipitate was again dispersed in distilled water,
centrifuged and washed to remove the free drug etc. After a small
amount of distilled water was added to the collected precipitate to
re-disperse the precipitate, the dispersion was lyophilized to
yield a crude peptide A zinc salt as 1.23 g of a dried powder.
EXPERIMENTAL EXAMPLE 1
[0511] An injectable preparation as obtained in Example 2 was
subcutaneously administered to the back of 8-week-old male SD rats.
After administration, rats were killed at given intervals and the
microcapsules remaining at the administration site were taken out
and assayed for drug content. This procedure was repeated to obtain
the time course of drug release from the microcapsules given to the
live body. The results are shown in FIG. 1. The drug content in the
microcapsules given to the live body decreased over a period of 1
month or more, demonstrating that the anti-endothelin substance
could be sustained in the live body.
EXPERIMENTAL EXAMPLE 2
[0512] About 100 mg of microcapsules as obtained in Example 1 were
dispersed in 2.5 ml of a dispersant for injection (distilled water
containing 2.5 mg of carboxymethyl cellulose, 0.5 mg of polysorbate
80 and 25 mg of mannitol, all dissolved therein). The resulting
dispersion was subcutaneously administered to the back of
13-week-old male Wistar fatty rats. The male Wistar fatty rat, a
line of rat which genetically develops obesity and hyperglycemia,
is characterized by increased leakage of protein and albumin in
urine with the development of hyperglycemia. The results of urinary
protein and albumin assays in a control group receiving no
microcapsules and an administration group receiving the
microcapsule are given in Table 1.
1TABLE 1 Urinary Albumin (mg/day, mean) Weeks after administration
0 2 4 6 8 Control group 9 -- 32 -- 37 Administration group 6 -- 18
-- 23 Urinary Protein (mg/day, mean) Weeks after administration 0 2
4 6 8 Control group 97 98 83 112 132 Administration group 98 68 68
64 92
[0513] As seen in Table 1, during the period of about 6 weeks after
microcapsule administration, smaller amounts of protein and albumin
were excreted in the urine, in comparison with the initial values
and control values. These results demonstrate that urinary protein
and albumin excretion, a symptom of diabetic nephropathy, were
suppressed during the period when the endothelin antagonist was
sustained in the live body as shown in Experimental Example 1,
suggesting the utility of the present invention as a therapy for
diabetic nephropathy.
EXPERIMENTAL EXAMPLE 3
[0514] About 190 mg of microcapsules as obtained in Example 3 were
dispersed in 1.5 ml of dispersant for injection (distilled water
containing 7.5 mg of carboxymethyl cellulose, 1.5 mg of polysorbate
80 and 75 mg of mannitol, all dissolved therein). The resulting
dispersion was subcutaneously administered to the back of
8-week-old male Wistar fatty rats using 18 G needles (the dosage of
peptide A disodium salt per one rat was about 10 mg). The same
administration was conducted once a month for 3 months. As a
control, microcapsules containing no peptide A disodium salt as
obtained in Comparative Example 1 were subcutaneously administered
to the back of 8-week-old male Wistar fatty rats.
[0515] At regular intervals after administration, excreted urine
was sampled and urinary albumin was assayed. As seen in Table 2, 9
and 12 weeks after the administration, the urinary albumin
excretion in an administration group receiving the microcapsules of
Example 2 was suppressed, compared with that in a control
group.
2TABLE 2 Urinary Albumin (mg/day, mean) Weeks after administration
0 9 12 Control group 2 .+-. 1 32 .+-. 8 45 .+-. 10 Administration
group 3 .+-. 1 17 .+-. 4 26 .+-. 11
EXPERIMENTAL EXAMPLE 4
[0516] 6-week-old male Wistar rats anaesthetized with pentobarbital
were innoculated with 25 mg of deoxycorticosterone after surgical
removal of left-hand side kidney. The rats were allowed to drink 1%
(w/v) of saline solution freely for 3 weeks. The microcapsules as
obtained in Example 3 were dispersed in a dispersant for injection
(2.5 mg of carboxymethyl cellulose, 0.5 mg of polysorbate 80 and 25
mg of mannitol, all dissolved in 0.5 ml of distilled water) to get
100 mg/ml of peptide A disodium salt in the resulting dispersion.
The dispersion was subcutaneously administered to the back of the
rats using 18 G needles (the dosage of peptide A disodium salt was
100 mg/kg). As a control, microcapsules containing no peptide A
disodium salt as obtained in Comparative Example 1 were
subcutaneously administered to the back of 6-week-old male Wistar
rats which were treated in the same manner.
[0517] As the results, systolic pressure in an administration group
receiving the microcapsules of Example 3 began to decrease at one
week after administration, and was kept lower by about 28 and 25
mmHg compared with that in a control group each until 2 and 4 weeks
after administration. These results demonstrate that the sustained
release of anti-endothelin substance makes it possible to keep
blood pressure low.
EXPERIMENTAL EXAMPLE 5
[0518] 5-week-old male Wistar rats surgically innoculated with Mini
Osmotic Pump (Alzet Model 2002, produced by Alza) containing 45 mg
of peptide A disodium salt were subcutaneously administered with
monocrotaline (100 mg/kg). The Mini Osmotic Pump were replaced
after 2 weeks. The release rate of peptide A disodium salt from the
Mini Osmotic Pump was 2.5 mg/rat/day, calculated from the remaining
amount of peptide A disodium salt in the removed Mini Osmotic Pump.
As a control, Mini Osmotic Pump containing no peptide A disodium
salt were surgically innoculated in 5-week-old male Wistar rats,
and the rats were treated in the same manner. At 4 weeks after
monocrotaline administration, chests of the rats anaesthetized with
pentobarbital were surgically opened under artificial respiration,
and the pressure of right ventricles was monitored via an inserted
catheter after a steady stae was achieved.
[0519] As the results, in the group treated with peptide A disodium
salt, elevation of right ventricle pressure was moderately
suppressed (lower by 26 mmHg compared with a control group). In
addition, hypertrophy of right ventricle was not significant (lower
by 0.23 mg tissue/g body weight) compared with the control group.
These results demonstrate that the sustained presence of endothelin
antagonist in blood is effective enough to improve the pathology of
pulmonary hypertension, and that sustained release preparation of
anti-endothelin substance is useful in the treatment of pulmonary
hypertension.
EXPERIMENTAL EXAMPLE 6
[0520] About 30 mg of the microcapsules as obtained in Example 6
were dispersed in 0.5 ml of a dispersant for injection (distilled
water containing 2.5 mg of carboxymethyl cellulose, 0.5 mg of
polysorbate 80 and 25 mg of mannitol, all dissolved therein). The
resulting dispersion was subcutaneously administered to the back of
9-week-old SD rats using 20 G needles (the dosage of peptide A
diethylester per one rat was about 12.6 mg). At regular intervals
after administration, blood was gathered from rats tails and the
concentration of peptide A diethylester in serum was assayed by EIA
(Enzyme immunoassay). As seen in Table 3 almost constant blood
concentration was kept for 2 weeks.
3TABLE 3 peptide A diethylester in serum (ng/ml) Days after
administration 1 7 4 Administration group 17.8 19.7 12.5
EXPERIMENTAL EXAMPLE 7
[0521] About 50 mg of the microcapsules as obtained in Example 8
were dispersed in 0.5 ml of a dispersant for injection (distilled
water containing 2.5 mg of carboxymethyl cellulose, 0.5 mg of
polysorbate 80 and 25 mg of mannitol, all dissolved therein). The
resulting dispersion was subcutaneously administered to the back of
6-week-old SD rats using 20 G needles (the dosage of a crude
peptide A zinc salt per one rat was about 10 mg in terms of peptide
A disodium salt). At regular intervals after administration, blood
was gathered from rats tails and the concentration of peptide A in
serum was assayed by EIA. The results are given in Table 4. The
amount of a peptide A zinc salt in the table is calculated in terms
of peptide A disodium salt.
4TABLE 4 peptide A zinc salt in serum (ng/ml) Days after
administration 1 7 14 21 Administration group 5.09 6.50 10.18
11.23
[0522] As seen in Table 4, an almost constant blood concentration
was kept for 3 weeks in an administration group receiving the
preparation of Example 7. As a control, a crude peptide A zinc salt
as obtained in Comparative Example 2 was dispersed in the
dispersant for injection and was subcutaneously administered to
rats (the dosage of a crude peptide A zinc salt per one rat was
about 10 mg in terms of peptide A disodium salt), then peptide A in
serum was decreased to be undetectable 3 days after the
administration.
EXPERIMENTAL EXAMPLE 8
[0523] About 70 mg of the microcapsules as obtained in Example 10
were dispersed in 0.5 ml of a dispersant for injection (distilled
water containing 2.5 mg of carboxymethyl cellulose, 0.5 mg of
polysorbate 80 and 25 mg of mannitol, all dissolved therein). The
resulting dispersion was subcutaneously administered to the back of
6-week-old SD rats using 20 G needles (the dosage of peptide A
disodium salt per one rat was about 10 mg). At regular intervals
after administration, blood was gathered from rats tails and the
concentration of peptide A disodium salt in serum was assayed by
EIA. The results are given in Table 5.
5TABLE 5 peptide A disodium salt in serum (ng/ml) Days after
administration 1 7 14 21 Administration group 11.12 26.77 8.37
5.74
[0524] As seen in Table 5, an almost constant blood concentration
was kept for 2 weeks in an administration group receiving the
preparation of Example 10.
EXPERIMENTAL EXAMPLE 9
[0525] About 70 mg of the microcapsules as obtained in Example 11
were dispersed in 0.5 ml of a dispersant for injection (distilled
water containing 2.5 mg of carboxymethyl cellulose, 0.5 mg of
polysorbate 80 and 25 mg of mannitol, all dissolved therein). The
resulting dispersion was subcutaneously administered to the back of
6-week-old SD rats using 20 G needles (the dosage of peptide A
disodium salt per one rat was about 10 mg). At regular intervals
after administration, blood was gathered from rats tails and the
concentration of peptide A disodium salt in serum was assayed by
EIA. The results are given in Table 6.
6TABLE 6 peptide A disodium salt in serum (ng/ml) Days after
administration 1 7 14 Administration group 5.79 8.99 10.91
[0526] As seen in Table 6, an almost constant blood concentration
was kept for 2 weeks in an administration group receiving the
preparation of Example 11.
[0527] The sustained-release preparation of the present invention
sustainedly releases an anti-endothelin substance, serving well in
the treatment of endothelin-associated diseases, particularly
chronic complications in diabetes mellitus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0528] FIG. 1 shows the changes over time in percent drug retention
in microcapsules of a sustained-release preparation given to rats,
obtained at the site of administration in Experimental Example
1.
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