Patent US4945086A successfully refreshed from Google Patents.
US4945086A
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Smooth muscle cell growth inhibitor
Assignee: Leland Stanford Junior University
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Filing: 1988-05-03
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Granted: 1990-07-31
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Expires: 2008-05-03
Abstract
Abstract An epithelium-derived inhibitor of the growth of smooth muscle cells is disclosed along with methods for purifying this substance. As initially isolated, the inhibitor comprises a heparan sulfate proteoglycan having a buoyant density of less than 1.4 g/ml which releases a glycosaminoglycan chain having a molecular weight of about 55,000 to 75,000 on protease cleavage. Growth inhibiting activity is found in the glycosaminoglycan chain and in glycosaminoglycan fragments derived therefrom. Use of a dialyzable detergent in purification steps greatly aids in the handling and purification of the inhibitor. The inhibitor can be used in a variety of techniques for inhibiting the growth of smooth muscle cells, both in vivo and in vitro.
Inventors
- 👤 William E. Benitz
- 👤 Merton Bernfield
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 21:59
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K14/4701; C07K14/4702; C07K14/4703; A; A61; A61P; A61P43/00; A61P9/00; A61P9/10; C12; C12P; C12P19/00; C12P19/26; A61K; A61K38/00
Claims Analysis
Total Claims: 33
Claim 1
Independent Claim
68 words
A smooth muscle cell growth inhibitor, comprising a substantially pure glycosaminoglycan having smooth muscle cell growth inhibition activity and being isolatable from an endothelial-cell-conditioned medium as part of a heparan sulfate proteoglycan having molecular weight of about 10 5 to 10 7 and a buoyant density of less than 1.4 g/ml which release a glycosaminoglycan chain having a molecular weight of about 55,000 to 75,000 on protease cleavage.
Claim 2
Dependent Claim
68 words
A smooth muscle cell growth inhibitor, comprising a substantially pure glycosaminoglycan having smooth muscle cell growth inhibition activity and being isolatable from an endothelial-cell-conditioned medium as part of a heparan sulfate proteoglycan having molecular weight of about 10 5 to 10 7 and a buoyant density of less than 1.4 g/ml which release a glycosaminoglycan chain having a molecular weight of about 55,000 to 75,000 on protease cleavage.
Claim 3
Dependent Claim
30 words
The inhibitor of claim 1, wherein said glycosaminoglycan comprises at least 90 weight % of molecules having a molecular weight of at least 1000 in a composition containing said inhibitor.
Claim 4
Dependent Claim
25 words
The inhibitor of claim 1, wherein said endothelial-cell-conditioned media contains cells consisting essentially of human, procaine, bovine, sheep, rabbit, rat, mouse, or monkey endothelial cells.
Claim 5
Dependent Claim
39 words
The inhibitor of claim 1, wherein said glycosaminoglycan chain has a molecular weight of about 60,000-70,000 and said proteoglycan has a buoyant density of about 1.3 g/ml and a molecular weight of about 0.8×10 6 to 1.2×10 6 .
Claim 6
Dependent Claim
17 words
The inhibitor of claim 1, wherein said glycosaminoglycan has a molecular weight of about 55,000 to 75,000.
Claim 7
Dependent Claim
17 words
The inhibitor of claim 1, wherein said glycosaminoglycan has a molecular weight of about 1,000 to 55,000.
Claim 8
Dependent Claim
28 words
The inhibitor of claim 1, wherein said proteoglycan binds specifically with an antibody having specific binding affinity for a protein epitope of a basement membrane heparan sulfate proteoglycan.
Claim 9
Dependent Claim
17 words
The inhibitor of claim 7, wherein said basement membrane heparan sulfate proteoglycan is produced by EHS tumor.
Claim 10
Dependent Claim
22 words
The inhibitor of claim 1, wherein said activity is retained when said inhibitor is contacted with chondroitin ABC lyase under cleavage conditions.
Claim 11
Dependent Claim
38 words
The inhibitor of claim 1, wherein the inhibitor is soluble as an intact protoglycan in said medium in the presence of albumin or serum, and no endoglycosidic cleavage is required to release soluble glycosaminoglycan chains from endothelial cells.
Claim 12
Dependent Claim
62 words
A smooth muscle cell growth inhibitor, comprising a substantially pure heparan sulfate proteoglycan isolatable from an endothelial-cell-conditioned medium and having a molecular weight of about 10 5 to about 10 7 , a buoyant density of less than 1.4 g/ml, glycosaminoglycan chains with an average molecular weight of about 55,000 to 75,000 on protease cleavage, and smooth muscle cell growth inhibition activity.
Claim 13
Dependent Claim
62 words
A smooth muscle cell growth inhibitor, comprising a substantially pure heparan sulfate proteoglycan isolatable from an endothelial-cell-conditioned medium and having a molecular weight of about 10 5 to about 10 7 , a buoyant density of less than 1.4 g/ml, glycosaminoglycan chains with an average molecular weight of about 55,000 to 75,000 on protease cleavage, and smooth muscle cell growth inhibition activity.
Claim 14
Dependent Claim
16 words
The inhibitor of claim 11, wherein said proteoglycan has a buoyant density of about 1.3 g/ml.
Claim 15
Dependent Claim
14 words
The inhibitor of claim 11, wherein said molecular weight is about 10 6 .
Claim 16
Dependent Claim
24 words
The inhibitor of claim 11, wherein said proteoglycan comprises a proteinaceous epitope recognized by an antibody specific for a basement membrane heparan sulfate proteoglycan.
Claim 17
Dependent Claim
60 words
In a method of purifying a proteoglycan smooth muscle cell growth inhibitor from a liquid medium containing said inhibitor as biochemically synthesized by endothelial cells, an improvement which comprises: including a dialyzable detergent in said medium prior to additions of reagents which induce dissociative Or chaotropic conditions, and maintaining said detergent in the presence of said proteoglycan during said purifying.
Claim 18
Dependent Claim
60 words
In a method of purifying a proteoglycan smooth muscle cell growth inhibitor from a liquid medium containing said inhibitor as biochemically synthesized by endothelial cells, an improvement which comprises: including a dialyzable detergent in said medium prior to additions of reagents which induce dissociative Or chaotropic conditions, and maintaining said detergent in the presence of said proteoglycan during said purifying.
Claim 19
Dependent Claim
15 words
The method of claim 15, wherein said purifying comprises gel filtration of an endothelium-conditioned medium.
Claim 20
Dependent Claim
12 words
The method of claim 15, wherein said purifying comprises density gradient centrifugation.
Claim 21
Independent Claim
49 words
A method of purifying a smooth muscle growth inhibitor, which comprises: separating endothelial cells in a growth medium from said medium to provide an endothelium-conditioned medium; adding a dialyzable detergent to said conditioned medium; and separating a heparan sulfate proteoglycan from other macromolecules present in said detergent-added conditioned medium.
Claim 22
Independent Claim
49 words
A method of purifying a smooth muscle growth inhibitor, which comprises: separating endothelial cells in a growth medium from said medium to provide an endothelium-conditioned medium; adding a dialyzable detergent to said conditioned medium; and separating a heparan sulfate proteoglycan from other macromolecules present in said detergent-added conditioned medium.
Claim 23
Dependent Claim
17 words
The method of claim 18, wherein said separating comprises ion exchange, gel filtration, or density gradient centrifugation.
Claim 24
Dependent Claim
27 words
The method of claim 19, wherein said separating comprises density gradient centrifugation and collection of a fraction at a buoyant density of about 1.25 to 1.35 g/ml.
Claim 25
Dependent Claim
21 words
The method of claim 18, comprising gel filtration to provide a sample characterized by a molecular weight of at least 500,000.
Claim 26
Dependent Claim
25 words
The method of claim 21, further comprising density gradient centrifugation and collection of a fraction at a buoyant density of about 1.25 to 1.35 g/ml.
Claim 27
Dependent Claim
29 words
The method of claim 18, wherein said detergent is dialyzable through a semipermeable membrane have a molecular weight cutoff of 500,000 or less from a solution containing said inhibitor.
Claim 28
Dependent Claim
13 words
The method of claim 18, wherein said detergent is octylglucoside or sodium deoxycholate.
Claim 29
Dependent Claim
31 words
A method of inhibiting growth of smooth muscle cells, which comprises adding to a growth medium containing said cells an effective growth-inhibiting amount of a glycosaminoglycan or proteoglycan of claim 1.
Claim 30
Dependent Claim
24 words
The method of claim 25, wherein said amount provides a concentration of from 20 to 1000 ng/ml in a liquid medium contacting said cells.
Claim 31
Dependent Claim
17 words
The method of claim 25, wherein said growth medium is an artery or vein of a mammal.
Claim 32
Dependent Claim
25 words
The method of claim 27, wherein said adding is by intravenous injection and said amount provides a serum concentration of from 50 to 500 ng/ml.
Claim 33
Dependent Claim
17 words
The method of claim 25, wherein said growth medium is an in vitro tissue- or cell-culture medium.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1993-07-30 | 1994-01-29 | 1994-07-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-07-30 | 1998-01-29 | 1998-07-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-07-30 | 2002-01-29 | 2002-07-31 | $7700.00 | Window Closed / Lapsed |