US5677276A
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US
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Immobilization of peptides to hyaluronate
Assignee: Sanford Burnham Prebys Medical Discovery Institute
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Filing: 1995-06-05
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Granted: 1997-10-14
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Expires: 2015-06-05
Abstract
Abstract The present invention provides novel conjugates of a synthetic polypeptide containing RGD or (dR)GD and a biodegradable polymer, hyaluronate. The conjugates are prepared by any one of three different methods provided by the present invention: (1) an epoxide method (2) a sodium periodate method, and (3) a tresyl chloride method. The conjugates prepared by these methods are useful to aid in wound healing and tissue regeneration by providing a temporary matrix for tissue repair. The invention also provides novel RGD-peptides.
Inventors
- 👤 Kenneth T. Dickerson
- 👤 James R. Glass
- 👤 Lin-Shu Liu
- 👤 James W. Polarek
- 👤 William S. Craig
- 👤 Daniel G. Mullen
- 👤 Soan Cheng
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 02:22
CPC Classifications
C; C07; C07K; C07K1/00; C07K1/107; C07K1/1072; C07K1/1077; C07K14/00; C07K14/001; C07K14/435; C07K14/78; A; A61; A61K; A61K38/00
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
72 words
A composition comprising cross-linked hyaluronate (HA) polymer and a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, wherein said HA and said peptide are coupled with a multifunctional epoxide linked to the sugar backbone of HA.
Claim 2
Dependent Claim
72 words
A composition comprising cross-linked hyaluronate (HA) polymer and a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, wherein said HA and said peptide are coupled with a multifunctional epoxide linked to the sugar backbone of HA.
Claim 3
Dependent Claim
19 words
The composition of claim 1, wherein the multifunctional epoxide is linked to HA via at least one ether bond.
Claim 4
Dependent Claim
19 words
The composition of claim 2, wherein the multifunctional epoxide is linked to HA via more than one ether bond.
Claim 5
Dependent Claim
17 words
The composition of claim 1, wherein the multifunctional epoxide is linked to HA via an ester bond.
Claim 6
Dependent Claim
23 words
The composition of claim 1, wherein the multifunctional epoxide is linked to HA via at least one ether bond and an ester bond.
Claim 7
Dependent Claim
32 words
The composition of claim 1, wherein the multifunctional epoxide is selected from the group consisting of butanediol diglycidyl ether (BDDE) , polyglycerolpolyglycidyl ether (PGPGE) , pentaerythriolpolyglycidyl ether (PEPGE) and diglycerolpolyglycidyl ether (DGPGE).
Claim 8
Dependent Claim
11 words
The composition of claim 6, wherein the multifunctional epoxide is BDDE.
Claim 9
Dependent Claim
31 words
The composition of claim 1, wherein said peptide contains at least three additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg.
Claim 10
Dependent Claim
17 words
The composition of claim 1, wherein at least two of said additional amino acids are in sequence.
Claim 11
Dependent Claim
21 words
The composition of claim 1 wherein the size of said peptide is equal to or less than 10 amino acid residues.
Claim 12
Dependent Claim
179 words
The composition of claim 1, wherein said peptide is selected from the group consisting of: G R R R R R G R G D I P T NH2 (SEQ ID No. 2); G R R R R R G G R G D I P T NH2 (SEQ ID No. 3) ; G R R R R G G G R G D I P T NH2 (SEQ ID No. 4); Ac R R R G G R G D I P K NH2 (SEQ ID No. 6); G R R R G G R G D I P NH2 (SEQ ID NO. 7); G R R R G G G R G D I P T NH2 (SEQ ID No. 8); G R R R G G R G D I P T NH2 (SEQ ID No. 9); R R R R G R G D I P T NH2 (SEQ ID NO. 10); R R R G R G D I P T NH2 (SEQ ID NO. 11); and polypeptides containing any of the above sequences.
Claim 13
Independent Claim
82 words
A method for preparing cross-linked HA polymer and a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, comprising reacting a solution of HA with a multifunctional epoxide in the presence of said peptide so as to simultaneously cross-link HA and couple said peptide to said HA.
Claim 14
Independent Claim
82 words
A method for preparing cross-linked HA polymer and a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, comprising reacting a solution of HA with a multifunctional epoxide in the presence of said peptide so as to simultaneously cross-link HA and couple said peptide to said HA.
Claim 15
Dependent Claim
11 words
The method of claim 12, wherein the multifunctional epoxide is BDDE.
Claim 16
Dependent Claim
9 words
A composition produced by the method of claim 12.
Claim 17
Dependent Claim
16 words
A method of treating a wound, comprising placing the composition of claim 1 into a wound.
Claim 18
Dependent Claim
36 words
The method of claim 15, wherein said wound is selected from the group consisting of a severe burn, a skin graft donor site, a decubitus ulcer, a diabetic ulcer, a surgical incision, and a keloid-forming wound.
Claim 19
Dependent Claim
22 words
A method of inducing tissue regeneration comprising placing the composition of claim 1 on the area where said tissue regeneration is desired.
Claim 20
Dependent Claim
75 words
A composition comprising HA polymer and a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, wherein said HA and said peptide are coupled with tresyl chloride and said peptide is linked to HA via more than one methylene bridge.
Claim 21
Dependent Claim
75 words
A composition comprising HA polymer and a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, wherein said HA and said peptide are coupled with tresyl chloride and said peptide is linked to HA via more than one methylene bridge.
Claim 22
Dependent Claim
11 words
The composition of claim 18, wherein the HA polymer is cross-linked.
Claim 23
Dependent Claim
18 words
The composition of claim 18, further comprising a linear spacer arm between said HA polymer and said peptide.
Claim 24
Dependent Claim
12 words
The composition of claim 20, wherein the linear spacer arm is 6-amino-1-hexanol.
Claim 25
Dependent Claim
31 words
The composition of claim 18, wherein said peptide contains at least three additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg.
Claim 26
Dependent Claim
17 words
The composition of claim 18, wherein at least two of said additional amino acids are in sequence.
Claim 27
Dependent Claim
21 words
The composition of claim 18 wherein the size of said peptide is equal to or less than 10 amino acid residues.
Claim 28
Dependent Claim
202 words
The composition of claim 18, wherein said peptide is selected from the group consisting of: G R R R R R G G G R G D I P NH2 (SEQ ID No. 12); Ac G R R R R R G G G R G D I P K NH2 (SEQ ID No. 13); G R R R R R G R G D I P T NH2 (SEQ ID No. 2); G R R R R R G G R G D I P T NH2 (SEQ ID No. 3); G R R R R G G G R G D I P T NH2 (SEQ ID No. 4); Ac R R R G G R G D I P K NH2 (SEQ ID No. 6); G R R R G G R G D I P NH2 (SEQ ID No. 7); G R R R G G G R G D I P T NH2 (SEQ ID NO. 8); G R R R G G R G D I P T NH2 (SEQ ID No. 9); Ac R R G R G D I P K NH2 (SEQ ID No. 14); and polypeptides containing any of the above sequences.
Claim 29
Independent Claim
120 words
A method for preparing HA polymer coupled to a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, comprising a. coupling a linear spacer arm to HA prior to coupling said peptide, said linear spacer arm having more than one methylene; b. activating said HA with tresyl chloride to obtain tresylate-HA; and c. coupling the primary amine of the peptide with the tresylate-HA to couple the peptide on the HA, wherein the HA polymer and said peptide are linked via more than one methylene bridge.
Claim 30
Independent Claim
120 words
A method for preparing HA polymer coupled to a peptide having cell attachment promoting activity containing the amino acid sequence Y-Gly-Asp, wherein Y is Arg or D-Arg, said peptide further containing at least two additional amino acids independently selected from the group consisting of Arg, D-Arg, Lys, D-Lys, Orn, D-Orn, L-HomoArg, and D-HomoArg, comprising a. coupling a linear spacer arm to HA prior to coupling said peptide, said linear spacer arm having more than one methylene; b. activating said HA with tresyl chloride to obtain tresylate-HA; and c. coupling the primary amine of the peptide with the tresylate-HA to couple the peptide on the HA, wherein the HA polymer and said peptide are linked via more than one methylene bridge.
Claim 31
Dependent Claim
12 words
The method of claim 26, further comprising cross-linking the HA with BDDE.
Claim 32
Dependent Claim
18 words
The method of claim 27, wherein the cross-linking step is done prior to activating HA with tresyl chloride.
Claim 33
Dependent Claim
12 words
The method of claim 26, wherein the linear spacer arm is 6-amino-1-hexanol.
Claim 34
Dependent Claim
9 words
A composition produced by the method of claim 26.
Claim 35
Dependent Claim
16 words
A method of treating a wound, comprising placing the composition of claim 18 into a wound.
Claim 36
Dependent Claim
36 words
The method of claim 31, wherein said wound is selected from the group consisting of a severe burn, a skin graft donor site, a decubitus ulcer, a diabetic ulcer, a surgical incision, and a keloid-forming wound.
Claim 37
Dependent Claim
22 words
A method of inducing tissue regeneration comprising placing the composition of claim 18 on the area where said tissue regeneration is desired.
Claim 38
Dependent Claim
309 words
A peptide having cell attachment activity comprising one of the following sequences: G R R R R R G R G D I P T NH2 (SEQ ID No. 2); G R R R R R G G R G D I P T NH2 (SEQ ID No. 3) ; G R R R R G G G R G D I P T NH2 (SEQ ID No. 4); Ac R R R G G R G D I P K NH2 (SEQ ID No. 6); G R R R G G R G D I P NH2 (SEQ ID No. 7); G R R R G G G R G D I P T NH2 (SEQ ID No. 8); G R R R G G R G D I P T NH2 (SEQ ID No. 9); R R R R G R G D I P T NH2 (SEQ ID No. 10); R R R G R G D I P T NH2 (SEQ ID No. 11); G R R R R R G G G R G D I P NH2 (SEQ ID No. 12); Ac G R R R R R G G G R G D I P K NH2 (SEQ ID No. 13); Ac R R G R G D I P K NH2 (SEQ ID No. 14); G R G D S P A S S K K K K (SEQ ID No. 15); G R R G R R G R G D I P T NH2 (SEQ ID No. 18); G R R G R R G R G D T NH2 (SEQ ID No. 19); G R R G R R G R G D I T NH2 (SEQ ID No. 20); and R R G R R G R G D I P T NH2 (SEQ ID No. 21).
Claim 39
Dependent Claim
309 words
A peptide having cell attachment activity comprising one of the following sequences: G R R R R R G R G D I P T NH2 (SEQ ID No. 2); G R R R R R G G R G D I P T NH2 (SEQ ID No. 3) ; G R R R R G G G R G D I P T NH2 (SEQ ID No. 4); Ac R R R G G R G D I P K NH2 (SEQ ID No. 6); G R R R G G R G D I P NH2 (SEQ ID No. 7); G R R R G G G R G D I P T NH2 (SEQ ID No. 8); G R R R G G R G D I P T NH2 (SEQ ID No. 9); R R R R G R G D I P T NH2 (SEQ ID No. 10); R R R G R G D I P T NH2 (SEQ ID No. 11); G R R R R R G G G R G D I P NH2 (SEQ ID No. 12); Ac G R R R R R G G G R G D I P K NH2 (SEQ ID No. 13); Ac R R G R G D I P K NH2 (SEQ ID No. 14); G R G D S P A S S K K K K (SEQ ID No. 15); G R R G R R G R G D I P T NH2 (SEQ ID No. 18); G R R G R R G R G D T NH2 (SEQ ID No. 19); G R R G R R G R G D I T NH2 (SEQ ID No. 20); and R R G R R G R G D I P T NH2 (SEQ ID No. 21).
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