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US4732891A
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Placenta-derived anticoagulating substance, process for preparing same and anticoagulant comprising same as an effective component
Assignee: Kowa Co Ltd
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Filing: 1986-09-19
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Granted: 1988-03-22
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Expires: 2006-09-19
Abstract
Abstract Novel human placenta-derived anticoagulating substances having the following properties: (1) molecular weight of 34,000+/-2,000 determined by SDA-polyacrylamide gel electrphoresis under reduced state; (2) isoelectric point of 4.7+/-0.1 determined by isoelectric column electrophoresis using an ampholite; (3) stability inactivated by heat treatment at 50 DEG C. to 30 minutes, stable at a pH of 4-10, and stable in plasma at 37 DEG C. for 30 minutes; (4) activity capable of prolonging a recalcification time, a prothrombin time, and an activated partial thromboplastin time; and (5) the existence of several amino acids including aspartic acid, threonine, serine, and so on; are prepared by homogenizing human placenta, subjecting the resulting homogenate to centrifugal separation, extracting an anticoagulating substance from the residue or from a microsome fraction contained in the supernatant with a surface active agent and/or a chelating agent, and purifying and isolating the substance from the extract. The anticoagulating substances have a high anticoagulating activity and exhibit a higher activity in the state where the tissue thromboplastin activity is exacerbated or in the state of exacerbating the coagulation. Hence, the anticoagulant containing the substance as an effective component has reduced side effects and is thus harmless.
Inventors
- 👤 Masahiro Maki
- 👤 Hideo Tani
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 18:18
CPC Classifications
A; A61; A61K; A61K35/00; A61K35/12; A61K35/48; A61K35/50; C; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K14/4701; C07K14/4721; A61P; A61P7/00; A61P7/02; A61K38/00; G; G03; G03C; G03C1/00; G03C1/005; G03C1/04; G03C1/047; G03C2001/0471; Y; Y10; Y10S; Y10S530/00; Y10S530/827; Y10S530/842; Y10S530/85; Y10S530/851
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
165 words
A human placenta-derived anticoagulating substance substantially free from glucose having the following properties: (1) a molecular weight, as determined by SDSpolyacrylamide gel electrophoresis, reduced state, of 34,000±2,000; (2) an isoelectric point, as determined by isoelectric column electrophoresis using an ampholite, of 4.7±0.1; (3) a stability such that said substance is (a) inactivated by heat treatment at 50° C. for 30 minutes, (b) stable at a pH of 4 to 10, and (c) stable in plasma at 37° C. for 30 minutes; (4) an activity such that said substance is (a) capable of prolonging a recalcification (b) capable of prolonging a prothrombin time, (c) capable of prolonging an activated partial thromboplastin time; and said substance has (5) an amino acid composition consisting essentially of in mole percent: ______________________________________
Aspartic acid
9.8
Threonine
7.0
Serine 6.3
Glutamic acid
13.3
Proline 2.0
Glycine 7.5
Alanine 8.0
1/2 Cystine
0.3
Valine 4.6
Methionine
1.8
Isoleucine
5.3
Leucine 11.8
Tyrosine 3.6
Phenylalanine
4.2
Histidine
1.4
Lysine 7.2
Arginine 6.2.
______________________________________
Claim 2
Dependent Claim
165 words
A human placenta-derived anticoagulating substance substantially free from glucose having the following properties: (1) a molecular weight, as determined by SDSpolyacrylamide gel electrophoresis, reduced state, of 34,000±2,000; (2) an isoelectric point, as determined by isoelectric column electrophoresis using an ampholite, of 4.7±0.1; (3) a stability such that said substance is (a) inactivated by heat treatment at 50° C. for 30 minutes, (b) stable at a pH of 4 to 10, and (c) stable in plasma at 37° C. for 30 minutes; (4) an activity such that said substance is (a) capable of prolonging a recalcification (b) capable of prolonging a prothrombin time, (c) capable of prolonging an activated partial thromboplastin time; and said substance has (5) an amino acid composition consisting essentially of in mole percent: ______________________________________
Aspartic acid
9.8
Threonine
7.0
Serine 6.3
Glutamic acid
13.3
Proline 2.0
Glycine 7.5
Alanine 8.0
1/2 Cystine
0.3
Valine 4.6
Methionine
1.8
Isoleucine
5.3
Leucine 11.8
Tyrosine 3.6
Phenylalanine
4.2
Histidine
1.4
Lysine 7.2
Arginine 6.2.
______________________________________
Claim 3
Dependent Claim
16 words
An anticoagulant composition comprising an effective amount of the human placenta-derived anticoagulating substance of claim 1.
Claim 4
Dependent Claim
16 words
The anticoagulant composition according to claim 2, which is in the form of a freeze-dried composition.
Claim 5
Dependent Claim
24 words
The anticoagulant composition according to claim 2, which further comprises one or more stabilizers selected from the group consisting of albumin, gelatin and mannitol.
Claim 6
Dependent Claim
29 words
The anticoagulant composition according to claim 2, wherein said effective amount is such that daily dosages in the range of from 10 μg to 10 mg/kg can be provided.
Claim 7
Dependent Claim
74 words
A process for producing the human placenta-derived anticoagulating substance according to claim 1, which comprises: (a) homogenizing human placenta, (b) subjecting the resulting homogenate to centrifugal separation, to form a supernatant and a sediment, (c) extracting an anticoagulating substance from the residue or from a microsome fraction contained in the supernatant with a surface active agent or a chelating agent or a mixture thereof, and (d) purifying and isolating the substance from the extract.
Claim 8
Dependent Claim
58 words
The process according to claim 6, which further comprises, after subjecting the resulting homogenate to centrifugal separation but prior to said extraction, subjecting the formed supernatant to ultracentrifugal separation at 50,000 to 100,000 x g to form a residue and a microsome fraction, which are separated and each washed with buffer, then combined together for said extraction step.
Claim 9
Dependent Claim
19 words
The process according to claim 6, which further comprises after said extraction, subjecting said extract to ammonium sulfate fractionation.
Claim 10
Dependent Claim
62 words
The process according to claim 8, wherein said ammonium sulfate factionation comprises: (a) adding solid ammonium sulfate to the extract until reaching a 35% saturation concentration, then subjecting a formed supernatant to centrifugal separation, and then (b) adding solid ammonium sulfate to the supernatant until reaching a 85% saturation concentration, then obtaining the ammonium sulfate fraction as a precipitate by centrifugal separation.
Claim 11
Dependent Claim
29 words
A method of retarding the coagulation of blood in a mammal, which comprises administering to said mammal and effective amount of the human-placenta derived anticoagulating substance of claim 1.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-03-22 | 1991-09-21 | 1992-03-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-03-22 | 1995-09-21 | 1996-03-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-03-22 | 1999-09-21 | 2000-03-22 | $7700.00 | Window Closed / Lapsed |