U.S. patent application number 09/883138 was filed with the patent office on 2002-04-11 for compositions and systems for forming crosslinked biomaterials and associated methods of preparation and use.
Invention is credited to DeLustro, Frank A., Trollsas, Olof Mikael, Wallace, Donald G..
Application Number | 20020042473 09/883138 |
Document ID | / |
Family ID | 25382057 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020042473 |
Kind Code |
A1 |
Trollsas, Olof Mikael ; et
al. |
April 11, 2002 |
Compositions and systems for forming crosslinked biomaterials and
associated methods of preparation and use
Abstract
Crosslinkable compositions are provided that readily crosslink
in situ to provide biocompatible, nonimmunogenic crosslinked
biomaterials. The compositions contain at least three
biocompatible, nonimmunogenic components having reactive functional
groups thereon, with the functional groups selected so as to enable
inter-reaction between the components, i.e., crosslinking. In a
preferred embodiment, a first component is polynucleophilic, a
second component is polyelectrophilic, and at least one third
component contains one or more functional groups reactive with the
nucleophilic moieties one the first or second component. At least
one of the components is a polyfunctional hydrophilic polymer; the
other components may also comprise hydrophilic polymers, or they
may be low molecular weight, typically hydrophobic, crosslinkers.
Methods for preparing and using the compositions are also provided.
Exemplary uses include tissue augmentation, biologically active
agent delivery, bioadhesion, and prevention of adhesions following
surgery or injury.
Inventors: |
Trollsas, Olof Mikael; (Los
Gatos, CA) ; Wallace, Donald G.; (Menlo Park, CA)
; DeLustro, Frank A.; (Belmont, CA) |
Correspondence
Address: |
REED & ASSOCIATES
800 MENLO AVENUE
SUITE 210
MENLO PARK
CA
94025
US
|
Family ID: |
25382057 |
Appl. No.: |
09/883138 |
Filed: |
June 15, 2001 |
Related U.S. Patent Documents
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09883138 |
Jun 15, 2001 |
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09733739 |
Dec 8, 2000 |
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6323278 |
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09733739 |
Dec 8, 2000 |
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09302852 |
Apr 30, 1999 |
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6166130 |
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09302852 |
Apr 30, 1999 |
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09229851 |
Jan 13, 1999 |
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6051648 |
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09229851 |
Jan 13, 1999 |
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08769806 |
Dec 18, 1996 |
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5874500 |
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08769806 |
Dec 18, 1996 |
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08573799 |
Dec 18, 1995 |
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08769806 |
Dec 18, 1996 |
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09649337 |
Aug 28, 2000 |
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60151273 |
Aug 27, 1999 |
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Current U.S.
Class: |
525/54.1 |
Current CPC
Class: |
A61L 27/26 20130101;
C08L 63/00 20130101; C09J 171/02 20130101; C08G 75/26 20130101;
C08L 53/00 20130101; A61L 24/08 20130101; C08L 51/003 20130101;
C08H 1/06 20130101; A61L 24/046 20130101; A61L 31/041 20130101;
C08G 75/045 20130101; C08H 1/00 20130101; A61L 27/54 20130101; C08L
2203/02 20130101; C08L 71/02 20130101; C08G 2650/50 20130101; C08L
71/02 20130101; C08L 81/02 20130101; A61L 24/046 20130101; C08B
37/0075 20130101; C08G 65/329 20130101; C09J 153/00 20130101; C08F
283/04 20130101; C09J 171/02 20130101; C09J 151/003 20130101; C08F
283/06 20130101; A61L 31/041 20130101; A61L 27/26 20130101; A61L
24/043 20130101; C08F 283/00 20130101; A61L 27/34 20130101; A61L
2300/00 20130101; A61L 27/34 20130101; C08L 71/00 20130101; C08L
71/02 20130101; C08L 63/00 20130101; C08L 71/02 20130101; A61L
24/043 20130101; A61L 31/16 20130101; A61L 24/08 20130101; C08L
71/02 20130101; C08L 71/02 20130101; C08L 71/02 20130101; C08L 5/16
20130101 |
Class at
Publication: |
525/54.1 |
International
Class: |
C08H 001/00 |
Claims
1. A crosslinkable composition comprised of: (a) a first
crosslinkable component A having m nucleophilic groups, wherein
m.gtoreq.2; (b) a second crosslinkable component B having n
electrophilic groups capable of reaction with the m nucleophilic
groups to form covalent bonds, wherein n.gtoreq.2 and m+n>4; and
(c) a third crosslinkable component C having at least one
functional group selected from (i) nucleophilic groups capable of
reacting with the electrophilic groups of component B and (ii)
electrophilic groups capable of reacting with the nucleophilic
groups of component A, wherein the total number of functional
groups on component C is represented by p, such that m+n+p>5
wherein each of components A, B and C is biocompatible and
nonimmunogenic, and at least one of components A, B and C is
comprised of a hydrophilic polymer, and crosslinking of the
composition results in a biocompatible, nonimmunogenic, crosslinked
matrix.
2. The composition of claim 1, wherein p.gtoreq.2.
3. The composition of claim 1, wherein the m nucleophilic groups
are identical.
4. The composition of claim 2, wherein the m nucleophilic groups
are identical.
5. The composition of claim 1, wherein at least two of the m
nucleophilic groups are different.
6. The composition of claim 1, wherein the n electrophilic groups
are identical.
7. The composition of claim 4, wherein the n electrophilic groups
are identical.
8. The composition of claim 1, wherein at least two of the n
electrophilic groups are different.
9. The composition of claim 1, wherein the at least one functional
group on component C is nucleophilic.
10. The composition of claim 4, wherein the functional groups on
component C are nucleophilic.
11. The composition of claim 10, wherein the functional groups on
component C are the same as the m nucleophilic groups on component
A.
12. The composition of claim 10, wherein at least one of the
functional groups on component C is different than the m
nucleophilic groups on component A.
13. The composition of claim 1, wherein the at least one functional
group on component C is electrophilic.
14. The composition of claim 13, wherein the functional groups on
component C are electrophilic.
15. The composition of claim 14, wherein the functional groups on
component C are the same as the n electrophilic groups on component
B.
16. The composition of claim 14, wherein at least one functional
group on component C is different than the n electrophilic groups
on component B.
17. The composition of claim 1, wherein component A has the
structural formula (I), component B has the structural formula
(II), and component C has the structural formula (III)
R.sub.1(--[Q.sup.1].sub.q--X).sub.m
(I)R.sup.2(--[Q.sup.2].sub.r--Y).sub.n
(II)R.sup.3(--[Q.sup.3].sub.s--Fn- ).sub.p (III)wherein: R.sup.1,
R.sup.2 and R.sup.3 are independently selected from the group
consisting of C.sub.2 to C.sub.14 hydrocarbyl,
heteroatom-containing C.sub.2 to C.sub.14 hydrocarbyl, hydrophilic
polymers, and hydrophobic polymers, providing that at least one of
R.sup.1, R.sup.2 and R.sup.3 is a hydrophilic polymer; X represents
one of the m nucleophilic groups of component A; Y represents one
of the n electrophilic groups of component B; Fn represents a
functional group on component C; Q.sup.1, Q.sup.2 and Q.sup.3 are
linking groups; q, r and s are independently zero or 1; and m, n
and p are as defined previously.
18. The composition of claim 17, wherein at least one of R.sup.1,
R.sup.2 and R.sup.3 is a synthetic hydrophilic polymer.
19. The composition of claim 17, wherein: (a) R.sup.1 is a first
synthetic hydrophilic polymer; (b) R.sup.2 is selected from the
group consisting of (i) a second synthetic hydrophilic polymer that
may or may not be the same as R.sup.1 and (ii) C.sub.2 to C.sub.14
hydrocarbyl groups containing zero to 2 heteroatoms selected from
N, O and S; and (c) R.sup.3 is selected from the group consisting
of (i) a third synthetic hydrophilic polymer that may or may not be
the same as R.sup.1 or R.sup.2 and (ii) C.sub.2 to C.sub.14
hydrocarbyl groups containing zero to 2 heteroatoms selected from
N, O, S and combinations thereof.
20. The composition of claim 18, wherein the synthetic hydrophilic
polymer is of a linear, branched, dendrimeric, hyperbranched, or
star polymer.
21. The composition of claim 19, wherein the synthetic hydrophilic
polymer is selected from the group consisting of: polyalkylene
oxides; polyglycerols; poly(oxyalkylene)-substituted polyols;
polyacrylic acid and analogs thereof; polymaleic acid;
polyacrylamides; poly(olefinic alcohol)s; poly(N-vinyl lactams);
polyoxazolines; polyvinylamines; and copolymers thereof.
22. The composition of claim 21, wherein the synthetic hydrophilic
polymers is a polyalkylene oxide or polyglycerol.
23. The composition of 22, wherein the synthetic hydrophilic
polymer is a polyalkylene oxide is selected from the group
consisting of polyethylene glycol and poly(ethylene
oxide)-poly(propylene oxide) copolymers.
24. The composition of claim 23, wherein the polyalkylene oxide is
polyethylene glycol.
25. The composition of claim 21, wherein the synthetic hydrophilic
polymer is a poly(oxyalkylene)-substituted diol or polyol.
26. The composition of claim 25, wherein the synthetic hydrophilic
polymer is selected from the group consisting of
mono-poly(oxyalkylene)-substitut- ed propylene glycol,
di-(polyoxyalkylene)-substituted propylene glycol,
mono-poly(oxyalkylene)-substituted trimethylene glycol,
di-(polyoxyalkylene)-substituted trimethylene glycol,
mono-poly(oxyalkylene)-substituted glycerol,
di-(polyoxyalkylene)-substit- uted glycerol, and
tri-(polyoxyalkylene)-substituted glycerol.
27. The composition of claim 21, wherein the synthetic hydrophilic
polymer is selected from the group consisting of poly(acrylic acid)
and analogs and copolymers thereof.
28. The composition of claim 27, wherein the synthetic hydrophilic
polymer is selected from the group consisting of poly(acrylic
acid), poly(methacrylic acid), poly(hydroxyethylmethacrylate),
poly(hydroxyethylacrylate), poly(methylalkylsulfoxide acrylates),
poly(methylalkylsulfoxide methacrylates), and copolymers
thereof.
29. The composition of claim 21, wherein the synthetic hydrophilic
polymer is polymaleic acid.
30. The composition of claim 21, wherein the synthetic hydrophilic
polymer is a polyacrylamide.
31. The composition of claim 30, wherein the synthetic hydrophilic
polymer is selected from the group consisting of polyacrylamide,
poly(methacrylamide), poly(dimethylacrylamide),
poly(N-isopropylacrylamid- e), and copolymers thereof.
32. The composition of claim 21, wherein the synthetic hydrophilic
polymer is a poly(olefinic alcohol).
33. The composition of claim 32, wherein the poly(olefinic alcohol)
is polyvinyl alcohol or a copolymer thereof.
34. The composition of claim 21, wherein the synthetic hydrophilic
polymer is a poly(N-vinyl lactam).
35. The composition of claim 34, wherein the poly(N-vinyl lactam)
is selected from the group consisting of poly(vinyl pyrrolidone),
poly(vinyl caprolactam), and copolymers thereof.
36. The composition of claim 19, wherein at least one of R.sup.2
and R.sup.3 is C.sub.2 to C.sub.14 hydrocarbyl containing zero to 2
heteroatoms selected from N, O and S.
37. The composition of claim 36, wherein at least one of R.sup.2
and R.sup.3 is C.sub.2 to C.sub.14 hydrocarbyl.
38. The composition of claim 19, wherein r, s and t are zero.
39. The composition of claim 19, wherein at least one of r, s and t
is 1.
40. The composition of claim 19, wherein one or more of Q.sub.1,
Q.sup.2 and Q.sup.3 contains at least one biodegradable
linkage.
41. The composition of claim 40, wherein the biodegradable linkage
is a hydrolyzable linkage.
42. The composition of claim 40, wherein the biodegradable linkage
is an enzymatically cleavable linkage.
43. The composition of claim 41, wherein the biodegradable linkage
is an enzymatically hydrolyzable linkage.
44. The composition of claim 1, wherein the nucleophilic groups on
component A and any nucleophilic groups on component C are selected
from the group consisting of --NH.sub.2, --NHR.sup.4,
--N(R.sup.4).sub.2, --SH, --OH, --COOH, --C.sub.6H.sub.4--OH,
--PH.sub.2, --PHR.sup.1, --P(R.sup.5).sub.2, --NH--NH.sub.2,
--CO--NH--NH.sub.2, and --C.sub.5H.sub.4N, wherein R.sup.4 and
R.sup.5 are C.sub.1-C.sub.12 hydrocarbyl.
45. The composition of claim 44, wherein the nucleophilic groups
are selected from --NH.sub.2 and --NHR.sup.4 where R.sup.4 is lower
hydrocarbyl.
46. The composition of claim 45, wherein the electrophilic groups
on component B and any electrophilic groups on component C are
amino-reactive groups.
47. The composition of claim 46, wherein the amino-reactive groups
contain an electrophilically reactive carbonyl group susceptible to
nucleophilic attack by a primary or secondary amine.
48. The composition of claim 47, wherein the amino-reactive groups
are carboxylic acid esters.
49. The composition of claim 47, wherein the amino-reactive groups
are carboxylic acids or aldehydes.
50. The composition of claim 46, wherein the amino-reactive groups
are selected from the group consisting of succinimidyl ester,
sulfosuccinimidyl ester, maleimido, epoxy, isocyanato,
thioisocyanato, and ethenesulfonyl.
51. The composition of claim 44, wherein the nucleophilic groups
are sulfhydryl groups.
52. The composition of claim 51, wherein the electrophilic groups
on component B and any electrophilic groups on component C are
sulfhydryl-reactive groups.
53. The composition of claim 52, wherein the sulfhydryl-reactive
groups are selected so as to form a thioester, thioether, or
disulfide linkage upon reaction with the sulfhydryl groups.
54. The composition of claim 52, wherein the sulfhydryl-reactive
groups contain an electrophilically reactive carbonyl group
susceptible to nucleophilic attack by sulfhydryl group.
55. The composition of claim 54, wherein the sulfhydryl-reactive
groups are carboxylic acid esters.
56. The composition of claim 54, wherein the amino-reactive groups
are carboxylic acids or aldehydes.
57. The composition of claim 52, wherein the sulfhydryl-reactive
groups have the structure --S--S--Ar where Ar is a substituted or
unsubstituted nitrogen-containing heteroaromatic moiety or a
non-heterocyclic aromatic group substituted with an
electron-withdrawing moiety.
58. The composition of claim 52, wherein the sulfhydryl-reactive
groups are selected from the group consisting of succinimidyl
ester, sulfosuccinimidyl ester, maleimido, epoxy, and
ethenesulfonyl.
59. The composition of claim 1, further including at least one
additional crosslinkable component D having at least one functional
group selected from nucleophilic groups and electrophilic groups,
and the total number of functional groups on component D is
represented by q, such that q.gtoreq.1.
60. The composition of claim 48, wherein q.gtoreq.2.
61. A crosslinkable composition comprising a plurality of
biocompatible, non-immunogenic reactive compounds each comprised of
a molecular core having at least one functional group attached
thereto through a direct covalent bond or through a linking group,
wherein under reaction-enabling conditions each reactive compound
is capable of substantially immediate covalent reaction with at
least one other of the plurality of reactive compounds by virtue of
the at least one functional group, and further wherein: each
molecular core is selected from the group consisting of synthetic
hydrophilic polymers, naturally occurring hydrophilic polymers,
hydrophobic polymers, and C.sub.2-C.sub.14 hydrocarbyl groups
containing zero to 2 heteroatoms selected from N, O, S and
combinations thereof; at least one of the molecular cores is a
synthetic hydrophilic polymer; and at least two of the molecular
cores contain at least two functional groups.
62. A crosslinkable composition comprising a plurality of
biocompatible, non-immunogenic reactive compounds each comprised of
a molecular core having at least two functional groups covalently
attached thereto, wherein under reaction-enabling conditions each
reactive compound is capable of substantially immediate covalent
reaction with at least one other of the plurality of reactive
compounds by virtue of the at least two functional groups, and
further wherein: each molecular core is selected from the group
consisting of synthetic hydrophilic polymers and C.sub.2-C.sub.14
hydrocarbyl groups containing zero to 2 heteroatoms selected from
N, O and combinations thereof; and at least one of the molecular
cores is a synthetic hydrophilic polymer.
63. A crosslinkable composition comprising a plurality of
biocompatible, non-immunogenic reactive compounds each comprised
of: a molecular core selected from the group consisting of
synthetic hydrophilic polymers and C.sub.2-C.sub.14 hydrocarbyl
groups containing zero to 2 heteroatoms selected from N, O, S and
combinations thereof, with the proviso that at least one of the
reactive compounds has a molecular core composed of a synthetic
hydrophilic polymer; at least two functional groups attached to
each molecular core through a direct covalent bond or through a
linking group, wherein the functional groups of at least one of the
reactive compounds are hydroxyl or sulfhydryl groups and the
functional groups of at least one other of the reactive compounds
are electrophilic groups capable of undergoing reaction with the
hydroxyl or sulfhydryl groups to form covalent bonds, such that
upon admixture of the composition with an aqueous base, a
biocompatible, non-immunogenic crosslinked material is formed.
64. A crosslinkable composition comprising at least three
biocompatible, non-immunogenic reactive compounds, wherein a first
of said reactive compounds is comprised of a synthetic hydrophilic
polymer having at least two functional groups attached thereto, a
second of said reactive compounds is comprised of a
C.sub.2-C.sub.14 hydrocarbyl group containing zero to 2 heteroatoms
selected from N, O, S and combinations thereof, with at least two
functional groups attached thereto, and a third of said reactive
compounds is comprised of a naturally occurring hydrophilic polymer
with at least two functional groups attached thereto, wherein the
functional groups of at least one of the reactive compounds are
hydroxyl or sulfhydryl groups and the functional groups of at least
one other of the reactive compounds are electrophilic groups
capable of undergoing reaction with the hydroxyl or sulfhydryl
groups to form a covalent bond, such that upon admixture of the
composition with an aqueous base, a biocompatible, non-immunogenic
crosslinked material is formed.
65. A crosslinkable composition comprising at least three
biocompatible, non-immunogenic compounds each capable of reacting
with at least one other of said compounds upon admixture of the
composition with an aqueous medium to form a covalently crosslinked
material, wherein: a first compound comprises a synthetic
hydrophilic polymer having at least two primary amino groups
attached thereto; a second compound comprises a synthetic
hydrophilic polymer having at least two amine-reactive
electrophilic groups attached thereto; and a third compound
comprises a C.sub.2-C.sub.14 hydrocarbyl group containing zero to 2
heteroatoms selected from N, O, S and combinations thereof, and
substituted with at least one functional group capable of
undergoing reaction with the primary amino groups or the
amine-reactive electrophilic groups.
66. A crosslinkable composition comprising at least three
biocompatible, non-immunogenic compounds each capable of reacting
with at least one other of said compounds upon admixture of the
composition with an aqueous medium to form a covalently crosslinked
material, wherein: a first compound comprises a synthetic
hydrophilic polymer having at least two sulfhydryl groups attached
thereto; a second compound comprises a synthetic hydrophilic
polymer having at least two sulfhydryl-reactive electrophilic
groups attached thereto; and a third compound comprises a
C.sub.2-C.sub.14 hydrocarbyl group containing zero to 2 heteroatoms
selected from N, O and combinations thereof, and substituted with
at least one functional group capable of undergoing reaction with
the sulfhydryl groups or the sulfhydryl-reactive groups.
67. A crosslinked composition prepared by admixing the composition
of claim 1 with an aqueous solution, with the proviso that if the
nucleophilic groups on component A or the functional groups on
component C are hydroxyl or thiol groups, the aqueous solution
contains a base.
68. The composition of claim 67, wherein the base is a
non-nucleophilic base.
69. The composition of claim 67, further including a
therapeutically effective amount of a biologically active
agent.
70. The composition of claim 69, wherein the biologically active
agent is selected from the group consisting of: enzymes, receptor
antagonists, receptor agonists, hormones, growth factors,
autogeneous bone marrow, antibiotics, antimicrobial agents,
antibodies, cells and genes.
71. The composition of claim 70, wherein the biologically active
agent is a growth factor or a derivative, analog or fragment
thereof.
72. The composition of claim 70, wherein the biologically active
agent is a cell.
73. The composition of claim 70, wherein the biologically active
agent is a gene.
74. A crosslinkable system comprised of (a) a first crosslinkable
component A having m nucleophilic groups, wherein m.gtoreq.2; (b) a
second crosslinkable component B having n electrophilic groups
capable of reaction with the m nucleophilic groups to form covalent
bonds, wherein n.gtoreq.2 and m+n>4; and (c) at least one
additional crosslinkable component C having at least one functional
group selected from (i) nucleophilic groups capable of reacting
with the electrophilic groups of component B and (ii) electrophilic
groups capable of reacting with the nucleophilic groups of
component A, wherein the total number of functional groups on
component C is represented by p, such that m+n+p>5, wherein each
crosslinkable component is biocompatible, nonimmunogenic, and
physically segregated from each other crosslinkable component, and
at least one of the crosslinkable components A, B and C is
comprised of a hydrophilic polymer, and crosslinking of the
composition results in a biocompatible, nonimmunogenic, crosslinked
matrix.
75. The crosslinkable system of claim 74, wherein component A is
contained in a sterile aqueous medium.
76. A method for effecting the nonsurgical attachment of a first
surface to a second surface, comprising the steps of: providing the
crosslinkable system of claim 74; admixing the crosslinkable
components in a sterile aqueous medium to provide a mixture and
initiate crosslinking, and, immediately thereafter, applying the
mixture to the first surface; and contacting the first surface with
a second surface to effect adhesion therebetween.
77. The method of claim 76, wherein one of the first and second
surfaces is a native tissue surface and the other of the first and
second surfaces is selected from a non-native tissue surface and
the surface of a synthetic implant.
78. The method of claim 76, wherein the first and second surfaces
are native tissue surfaces.
79. A method for effecting the augmentation of tissue within the
body of a mammalian subject, comprising the steps of: providing the
crosslinkable system of claim 74; administering the components of
the crosslinkable system to a tissue site in need of augmentation;
and allowing the components to crosslink in situ to provide tissue
augmentation.
80. The method of claim 79, wherein the components of the
crosslinkable system are admixed prior to administration to the
tissue site.
81. The method of claim 79, wherein the components are separately
administered to the tissue site.
82. The method of claim 79, wherein the tissue is soft tissue.
83. The method of claim 79, wherein the tissue is hard tissue.
84. A method for preventing the formation of adhesions following
surgery or injury, comprising the steps of: providing the
crosslinkable system of claim 74; admixing the crosslinkable
components in a sterile aqueous medium to provide a mixture and
initiate crosslinking, and, immediately thereafter, applying the
mixture to a tissue comprising, surrounding, or adjacent to a wound
before substantial crosslinking has occurred; and allowing the
components to crosslink in situ.
85. The method of claim 84, further comprising effecting surgical
closure of the wound.
86. A crosslinkable composition comprised of (a) at least one first
component composed of branched polyglycerol containing two or more
nucleophilic groups; and (b) at least one second component
functionalized to contain two or more electrophilic groups capable
of reaction with the nucleophilic groups to form covalent bonds,
wherein upon admixture of the components in an aqueous medium, the
composition crosslinks to provide a biocompatible, non-immunogenic,
crosslinked material.
Description
BACKGROUND OF THE INVENTION
[0001] U.S. Pat. No. 5,162,430, issued Nov. 10, 1992, to Rhee et
al., and commonly owned by the assignee of the present invention,
discloses collagen-synthetic polymer conjugates prepared by
covalently binding collagen to synthetic hydrophilic polymers such
as various derivatives of polyethylene glycol.
[0002] Commonly owned U.S. Pat. No. 5,324,775, issued Jun. 28,
1994, to Rhee et al., discloses various insert, naturally
occurring, biocompatible polymers (such as polysaccharides)
covalently bound to synthetic, non-immunogenic, hydrophilic
polyethylene glycol polymers.
[0003] Commonly owned U.S. Pat. No. 5,328,955, issued Jul. 12,
1994, to Rhee et al., discloses various activated forms of
polyethylene glycol and various linkages which can be used to
produce collagen-synthetic polymer conjugates having a range of
physical and chemical properties.
[0004] Commonly owned, copending U.S. application Ser. No.
08/403,358, filed Mar. 14, 1995, a European counterpart of which
was published as EP 96102366, discloses a crosslinked biomaterial
composition that is prepared using a hydrophobic crosslinking
agent, or a mixture of hydrophilic and hydrophobic crosslinking
agents. Preferred hydrophobic crosslinking agents include any
hydrophobic polymer that contains, or can be chemically derivatized
to contain, two or more succinimidyl groups.
[0005] Commonly owned, copending U.S. application Ser. No.
08/403,360, filed Mar. 14, 1995, issued Mar. 13, 1996 as U.S. Pat.
No. 5,580,923 to Yeung et al., discloses a composition useful in
the prevention of surgical adhesions comprising a substrate
material and an anti-adhesion binding agent; where the substrate
material preferably comprises collagen and the binding agent
preferably comprises at least one tissue-reactive functional group
and at least one substrate-reactive functional group.
[0006] Commonly owned, U.S. application Ser. No. 08/476,825, filed
Jun. 7, 1995, issued Mar. 25, 1997 as U.S. Pat. No. 5,614,587 to
Rhee et al., discloses bioadhesive compositions comprising collagen
crosslinked using a multifunctionally activated synthetic
hydrophilic polymer, as well as methods of using such compositions
to effect adhesion between a first surface and a second surface,
wherein at least one of the first and second surfaces is preferably
a native tissue surface.
[0007] Japanese patent publication No. 07090241 discloses a
composition used for temporary adhesion of a lens material to a
support, to mount the material on a machining device, comprising a
mixture of polyethylene glycol, having an average molecular weight
in the range of 1000-5000, and poly-N-vinylpyrrolidone, having an
average molecular weight in the range of 30,000-200,000.
[0008] West and Hubbell, Biomaterials (1995) 16:1153-1156, disclose
the prevention of post-operative adhesions using a photopolymerized
polyethylene glycol-co-lactic acid diacrylate hydrogel and a
physically crosslinked polyethylene glycol-co-polypropylene glycol
hydrogel, Poloxamer 407.RTM..
[0009] Each publication cited above and is incorporated herein by
reference to describe and disclose the subject matter for which it
is cited.
[0010] The invention is directed to a versatile biocompatible
composition not previously disclosed or envisioned by those in the
biomaterial field. The composition is comprised of a crosslinkable
matrix that may be readily crosslinked upon admixture with an
aqueous medium to provide a crosslinked composition having a
variety of uses, e.g., as a bioadhesive, a drug delivery platform,
an implant coating, etc. All components of the composition are
biocompatible and nonimmunogenic, and do not leave any toxic,
inflammatory or immunogenic reaction products at the site of
administration. Preferably, the composition is not subject to
enzymatic cleavage by matrix metalloproteinases such as
collagenase, and is therefore not readily degradable in vivo.
Further, the composition may be readily tailored, in terms of the
selection and quantity of each component, to enhance certain
properties, e.g., compression strength, swellability, tack,
hydrophilicity, optical clarity, and the like.
SUMMARY OF THE INVENTION
[0011] Accordingly, in one aspect of the invention, a composition
is provided that is readily crosslinkable, either in situ or ex
situ, to give a biocompatible, nonimmunogenic crosslinked matrix
having utility in a host of different contexts, e.g., in
bioadhesion, biologically active agent delivery, tissue
augmentation, and other applications. The composition is comprised
of:
[0012] (a) a first crosslinkable component A having m nucleophilic
groups, wherein m.gtoreq.2;
[0013] (b) a second crosslinkable component B having n
electrophilic groups capable of reaction with the m nucleophilic
groups to form covalent bonds, wherein n.gtoreq.2 and m+n>4;
and
[0014] (c) a third crosslinkable component C having at least one
functional group selected from (i) nucleophilic groups capable of
reacting with the electrophilic groups of component B and (ii)
electrophilic groups capable of reacting with the nucleophilic
groups of component A,
[0015] wherein each of components A, B and C is biocompatible and
nonimmunogenic, at least one of components A, B and C is a
hydrophilic polymer, and admixture of components A, B and C in an
aqueous medium results in crosslinking of the composition to give a
biocompatible, nonimmnunogenic, crosslinked matrix.
[0016] Each of the components may be polymeric, in which case at
least two components are generally although not necessarily
composed of a purely synthetic polymer rather than a naturally
occurring or semi-synthetic polymer, wherein "semi-synthetic"
refers to a chemically modified naturally occurring polymer.
Alternatively, one or two of components A, B and C (but not all
three) may be a low molecular weight crosslinking agent, typically
an agent comprised of a hydrocarbyl moiety containing 2 to 14
carbon atoms and at least two functional groups, i.e., nucleophilic
or electrophilic groups, depending on the component. For
convenience, the term "polynucleophilic" will be used herein to
refer to a compound having two or more nucleophilic moieties, and
the term "polyelectrophilic" will be used to refer to a compound
having two or more electrophilic moieties.
[0017] In another aspect of the invention, a crosslinkable
composition is provided that comprises a plurality of
biocompatible, non-immunogenic reactive compounds each composed of
a molecular core having at least one functional group attached
thereto (i.e., through a direct covalent bond or through a linking
group), wherein under reaction-enabling conditions each reactive
compound is capable of substantially immediate covalent reaction
with at least one other of the plurality of reactive compounds by
virtue of the at least one functional group. At least two of the
reactive compounds contain two or more functional groups to enable
crosslinking, and for preparation of highly crosslinked structures,
all of the reactive components contain two or more reactive
functional groups. Each molecular core is selected from the group
consisting of synthetic hydrophilic polymers, naturally occurring
hydrophilic polymers, hydrophobic polymers, and C.sub.2-C.sub.14
hydrocarbyl groups containing zero to 2 heteroatoms selected from
N, O, S and combinations thereof, with the proviso that at least
one of the molecular cores is a synthetic hydrophilic polymer.
Preferably, each molecular core is selected from the group
consisting of synthetic hydrophilic polymers and C.sub.2-C.sub.14
hydrocarbyl groups containing zero to 2 heteroatoms selected from
N, O and combinations thereof.
[0018] In a related aspect of the invention, a crosslinkable
composition is provided that comprises at least three
biocompatible, non-immunogenic reactive compounds, wherein a first
reactive compound is composed of a synthetic hydrophilic polymer
having at least two functional groups attached thereto, a second
reactive compound is comprised of a C.sub.2-C.sub.14 hydrocarbyl
group containing zero to 2 heteroatoms selected from N, O, S and
combinations thereof, with at least two functional groups attached
thereto, and a third reactive compound is comprised of a naturally
occurring hydrophilic polymer with at least two functional groups
attached thereto. The functional groups of at least one of the
reactive compounds are hydroxyl or sulfhydryl groups and the
functional groups of at least one other of the reactive compounds
are electrophilic groups capable of undergoing reaction with the
hydroxyl or sulfhydryl groups to form a covalent bond, such that
upon admixture of the composition with an aqueous base, a
biocompatible, non-immunogenic crosslinked material is formed.
[0019] In another aspect of the invention, a biocompatible,
nonimmunogenic, crosslinked matrix is provided by allowing the
components of the crosslinkable composition to crosslink under
appropriate reaction conditions. As will be discussed in detail
infra, suitable reaction conditions involve admixture of all
components in an aqueous medium. With certain types of nucleophilic
groups, e.g., sulfhydryl and hydroxyl groups, it is preferred that
the aqueous medium contain a base, which serves to increase the
nucleophilic reactivity of such groups. Preferred bases are
generally, although not necessarily, non-nucleophilic.
[0020] In other aspects of the invention, methods for preparing and
using the aforementioned compositions also provided. Methods of
using the compositions encompassed by the present invention include
drug delivery methods, use in bioadhesion, delivery of cells and
genes, tissue augmentation, prevention of adhesions following
surgery or injury, and implant coating. Other methods of use are
also within the scope of the invention, as will be described
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1 to 15 schematically illustrate reaction of various
polyelectrophilic components with polyamino-substituted
polyethylene glycol (PEG) as a representative polynucleophile. In
FIGS. 1-10, the polyelectrophilic components are composed of a
pentaerythritol core with each of the four hydroxyl groups
substituted with PEG, and with each PEG branch terminated with a
reactive electrophilic group. In FIGS. 14-18, the polyelectrophilic
components are composed of low molecular weight, hydrophobic
molecular cores difunctionalized with succinimidyl esters.
[0022] FIG. 16 provides in graph form the tensile test results
obtained in Example 10.
[0023] FIG. 17 schematically illustrates devices that are useful
for measuring tensile strength.
[0024] FIG. 18 illustrates the formation of an amide-linked
conjugate resulting from reaction of succinimidyl-glutaryl-PEG with
amino-PEG.
[0025] FIG. 19 illustrates the formation of a thioester-linked PEG
conjugate resulting from reaction of succinimidyl-PEG with
thiol-PEG.
[0026] FIG. 20 depicts a device that is useful to test burst
strength of a collagen membrane.
[0027] FIG. 21 illustrates a device (a pressurized carotid artery
model) that is useful to test burst strength of a repaired artery
slit defect.
DETAILED DESCRIPTION OF THE INVENTION
[0028] I. Definitions and Nomenclature
[0029] Before describing the present invention in detail, it is to
be understood that unless otherwise indicated this invention is not
limited to particular compositional forms, crosslinkable
components, crosslinking techniques, or methods of use, as such may
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0030] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, "a crosslinkable component" refers not only to a
single crosslinkable component but also to a combination of two or
more different crosslinkable component, "a hydrophilic polymer"
refers to a combination of hydrophilic polymers as well as to a
single hydrophilic polymer, and the like.
[0031] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by one of ordinary
skill in the art to which the invention pertains. Although any
methods and materials similar or equivalent to those described
herein may be useful in the practice or testing of the present
invention, preferred methods and materials are described below. All
patents, patent applications and other publications mentioned
herein are incorporated herein by reference. Specific terminology
of particular importance to the description of the present
invention is defined below.
[0032] The term "crosslinked" herein refers to a composition
containing intermolecular crosslinks and optionally intramolecular
crosslinks as well, arising from the formation of covalent bonds.
Covalent bonding between two crosslinkable components may be
direct, in which case an atom in one component is directly bound to
an atom in the other component, or it may be indirect, through a
linking group. A crosslinked matrix may, in addition to covalent
bonds, also include intermolecular and/or intramolecular
noncovalent bonds such as hydrogen bonds and electrostatic (ionic)
bonds. The term "crosslinkable" refers to a component or compound
that is capable of undergoing reaction to form a crosslinked
composition.
[0033] The terms "nucleophile" and "nucleophilic" refer to a
functional group that is electron rich, has an unshared pair of
electrons acting as a reactive site, and reacts with a positively
charged or electron-deficient site, generally present on another
molecule.
[0034] The terms "electrophile" and "electrophilic" refer to a
functional group that is susceptible to nucleophilic attack, i.e.,
susceptible to reaction with an incoming nucleophilic group.
Electrophilic groups herein are positively charged or
electron-deficient, typically electron-deficient.
[0035] The term "activated" refers to a modification of an existing
functional group to generate or introduce a new reactive functional
group from the prior existing functional group, wherein the new
reactive functional group is capable of undergoing reaction with
another functional group to form a covalent bond. For example, a
component containing carboxylic acid (--COOH) groups can be
activated by reaction with N-hydroxysuccinimide or
N-hydroxysulfosuccinimide using known procedures, to form an
activated carboxylate (which is a reactive electrophilic group),
i.e., an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide
ester, respectively. In another example, carboxylic acid groups can
be activated by reaction with an acyl halide, e.g., an acyl
chloride, again using known procedures, to provide an activated
electrophilic group in the form of an anhydride.
[0036] The terms "hydrophilic" and "hydrophobic" are generally
defined in terms of a partition coefficient P, which is the ratio
of the equilibrium concentration of a compound in an organic phase
to that in an aqueous phase. A hydrophilic compound has a log P
value less than 1.0, typically less than about -0.5, where P is the
partition coefficient of the compound between octanol and water,
while hydrophobic compounds will generally have a log P greater
than about 3.0, typically greater than about 5.0. Preferred
crosslinkable components herein are hydrophilic, although as long
as the crosslinkable composition as a whole contains at least one
hydrophilic component, crosslinkable hydrophobic components may
also be present.
[0037] The term "polymer" is not only used in the conventional
sense to refer to molecules composed of repeating monomer units,
including homopolymers, block copolymers, random copolymers, and
graft copolymers, but is also used, as indicated in parent
application Ser. No. 09/733, 739, to refer to polyfunctional small
molecules that do not contain repeating monomer units but are
"polymeric" in the sense of being "polyfunctional," i.e.,
containing two or more functional groups. Accordingly, it will be
appreciated that when the term "polymer" is used, difunctional and
polyfunctional small molecules are included. Such moieties include,
by way of example: the difunctional electrophiles disuccinimidyl
suberate (DSS), bis(sulfosuccinimidyl) suberate (BS.sup.3),
dithiobis(succinimidylpropionate) (DSP),
bis(2-succinimidooxy-carbonyloxy) ethyl sulfone (BSOCOES),
3,3'-dithiobis(sulfosuccinimidylpropionate (DTSSP); and the di- and
polyfunctional nucleophiles ethylenediamine
(H.sub.2N--CH.sub.2--CH.sub.2- --NH.sub.2), tetramethylene diamine
(H.sub.2N--[CH.sub.2].sub.4--NH.sub.2)- , pentamethylene diamine
(cadaverine) (H.sub.2N--[CH.sub.2].sub.5--NH.sub.- 2),
hexamethylene diamine (H.sub.2N--[CH.sub.2].sub.6--NH.sub.2),
bos(2-aminoethyl)amine (HN--[CH.sub.2--CH.sub.2--NH.sub.2].sub.2),
and tris (2-aminoethyl)amine
(N--[CH.sub.2--CH.sub.2--NH.sub.2].sub.3). All suitable polymers
herein are nontoxic, non-inflammatory and nonimmunogenic, and will
preferably be essentially nondegradable in vivo over a period of at
least several months.
[0038] The term "synthetic" to refer to various polymers herein is
intended to mean "chemically synthesized." Therefore, a synthetic
polymer in the present compositions may have a molecular structure
that is identical to a naturally occurring polymer, but the
polymerper se, as incorporated in the compositions of the
invention, has been chemically synthesized in the laboratory or
industrially. "Synthetic" polymers also include semi-synthetic
polymers, i.e., naturally occurring polymers, obtained from a
natural source, that have been chemically modified in some way.
Generally, however, the synthetic polymers herein are purely
synthetic, i.e., they are neither semi-synthetic nor have a
structure that is identical to that of a naturally occurring
polymer.
[0039] The term "synthetic hydrophilic polymer" as used herein
refers to a synthetic polymer composed of molecular segments that
render the polymer as a whole "hydrophilic," as defined above.
Preferred polymers are highly pure or are purified to a highly pure
state such that the polymer is or is treated to become
pharmaceutically pure. Most hydrophilic polymers can be rendered
water soluble by incorporating a sufficient number of oxygen (or
less frequently nitrogen) atoms available for forming hydrogen
bonds in aqueous solutions. Hydrophilic polymers useful herein
include, but are not limited to: polyalkylene oxides, particularly
polyethylene glycol and poly(ethylene oxide)-poly(propylene oxide)
copolymers, including block and random copolymers; polyols such as
glycerol, polyglycerol (particularly highly branched polyglycerol),
propylene glycol and trimethylene glycol substituted with one or
more polyalkylene oxides, e.g., mono-, di- and tri-polyoxyethylated
glycerol, mono- and di-polyoxyethylated propylene glycol, and mono-
and di-polyoxyethylated trimethylene glycol; polyoxyethylated
sorbitol, polyoxyethylated glucose; acrylic acid polymers and
analogs and copolymers thereof, such as polyacrylic acid per se,
polymethacrylic acid, poly(hydroxyethylmethacryl- ate),
poly(hydroxyethylacrylate), poly(methylalkylsulfoxide
methacrylate), poly(methylalkylsulfoxide acrylate) and copolymers
of any of the foregoing, and/or with additional acrylate species
such as aminoethyl acrylate and mono-2-(acryloxy)-ethyl succinate;
polymaleic acid; poly(acrylamides) such as polyacrylamide per se,
poly(methacrylamide), poly(dimethylacrylamide), and
poly(N-isopropyl-acrylamide); poly(olefinic alcohol)s such as
poly(vinyl alcohol); poly(N-vinyl lactams) such as poly(vinyl
pyrrolidone), poly(N-vinyl caprolactain), and copolymers thereof;
polyoxazolines, including poly(methyloxazoline) and
poly(ethyloxazoline); and polyvinylamines.
[0040] Hydrophobic polymers, including low molecular weight
polyfunctional species, can also be used in the crosslinkable
compositions of the invention. Hydrophobic polymers preferably
contain, or can be derivatized to contain, two or more
electrophilic groups, such as succinimidyl groups, most preferably,
two, three, or four electrophilic groups. Generally, "hydrophobic
polymers" herein contain a relatively small proportion of oxygen
and/or nitrogen atoms. Preferred hydrophobic polymers for use in
the invention generally have a carbon chain that is no longer than
about 14 carbons. Polymers having carbon chains substantially
longer than 14 carbons generally have very poor solubility in
aqueous solutions and, as such, have very long reaction times when
mixed with aqueous solutions of synthetic polymers containing
multiple nucleophilic groups.
[0041] The term "collagen" as used herein refers to all forms of
collagen, including those, which have been processed or otherwise
modified. Preferred collagens are treated to remove the immunogenic
telopeptide regions ("atelopeptide collagen"), are soluble, and may
be in fibrillar or non-fibrillar form. Type I collagen is best
suited to most applications involving bone or cartilage repair.
However, other forms of collagen are also useful in the practice of
the invention, and are not excluded from consideration here.
Collagen crosslinked using heat, radiation, or chemical agents such
as glutaraldehyde may also be used to form particularly rigid
crosslinked compositions. Collagen crosslinked using glutaraldehyde
or other (nonpolymer) linking agents is typically referred to
herein as "GAX" while collagen crosslinked using heat and/or
radiation is termed "HRX." Collagen used in connection with the
preferred embodiments of the invention is in a pharmaceutically
pure form such that it can be incorporated into a human body for
the intended purpose.
[0042] Those of ordinary skill in the art will appreciate that
synthetic polymers such as polyethylene glycol cannot be prepared
practically to have exact molecular weights, and that the term
"molecular weight" as used herein refers to the weight average
molecular weight of a number of molecules in any given sample, as
commonly used in the art. Thus, a sample of PEG 2,000 might contain
a statistical mixture of polymer molecules ranging in weight from,
for example, 1,500 to 2,500 daltons with one molecule differing
slightly from the next over a range. Specification of a range of
molecular weights indicates that the average molecular weight may
be any value between the limits specified, and may include
molecules outside those limits. Thus, a molecular weight range of
about 800 to about 20,000 indicates an average molecular weight of
at least about 800, ranging up to about 20 kDa.
[0043] The term "cytokine" is used to describe biologically active
molecules including growth factors and active peptides, which aid
in healing or regrowth of normal tissue. The function of cytokines
is two-fold: 1) they can incite local cells to produce new collagen
or tissue, or 2) they can attract cells to the site in need of
correction. As such, cytokines serve to encourage "biological
anchoring" of the collagen implant within the host tissue. As
previously described, the cytokines can either be admixed with the
collagen-polymer conjugate or chemically coupled to the conjugate.
For example, one 30 may incorporate cytokines such as epidermal
growth factor (EGF), transforming growth factor (TGF)-.alpha.,
TGF-.beta. (including any combination of TGF-.beta.3s),
TGF-.beta.1, TGF-.beta.32, platelet derived growth factor (PDGF-AA,
PDGF-AB, PDGF-BB), acidic fibroblast growth factor (FGF), basic
FGF, connective tissue activating peptides (CTAP),
.beta.-thromboglobulin, insulin-like growth factors, tumor necrosis
factors (TNF), interleukins, colony stimulating factors (CSFs),
erythropoietin (EPO), nerve growth factor (NGF), interferons (IFN)
bone morphogenic protein (BMP), osteogenic factors, and the like.
Incorporation of cytokines, and appropriate combinations of
cytokines can facilitate the regrowth and remodeling of the implant
into normal bone tissue, or may be used in the treatment of
wounds.
[0044] The term "effective amount" refers to the amount of
composition required in order to obtain the effect desired. Thus, a
"tissue growth-promoting amount" of a composition refers to the
amount needed in order to stimulate tissue growth to a detectable
degree. Tissue, in this context, includes connective tissue, bone,
cartilage, epidermis and dermis, blood, and other tissues. The
actual amount that is determined to be an effective amount will
vary depending on factors such as the size, condition, sex and age
of the patient and can be more readily determined by the
caregiver.
[0045] The term "solid implant" refers to any solid object which is
designed for insertion and use within the body, and includes bone
and cartilage implants (e.g., artificial joints, retaining pins,
cranial plates, and the like, of metal, plastic and/or other
materials), breast implants (e.g., silicone gel envelopes, foam
forms, and the like), catheters and cannulas intended for long-term
use (beyond about three days) in place, artificial organs and
vessels (e.g., artificial hearts, pancreases, kidneys, blood
vessels, and the like), drug delivery devices (including monolithic
implants, pumps and controlled release devices such as Alzet.RTM.
minipumps, steroid pellets for anabolic growth or contraception,
and the like), sutures for dermal or internal use, periodontal
membranes, ophthalmic shields, corneal lenticules, and the
like.
[0046] The term "suitable fibrous material" as used herein, refers
to a fibrous material which is substantially insoluble in water,
non-immunogenic, biocompatible, and immiscible with the
crosslinkable compositions of the invention. The fibrous material
may comprise any of a variety of materials having these
characteristics and may be combined with crosslinkable compositions
herein in order to form and/or provide structural integrity to
various implants or devices used in connection with medical and
pharmaceutical uses. For example, the crosslinkable compositions of
the invention can be coated on the "suitable fibrous material,"
which can then be wrapped around a bone to provide structural
integrity to the bone. Thus, the "suitable fibrous material" is
useful in forming the "solid implants" of the invention.
[0047] The term "in situ" as used herein means at the site of
administration. Thus, the injectable reaction mixture compositions
are injected or otherwise applied to a specific site within a
patient's body, e.g., a site in need of augmentation, and allowed
to crosslink at the site of injection. Suitable sites will
generally be intradermal or subcutaneous regions for augmenting
dermal support, at a bone fracture site for bone repair, within
sphincter tissue for sphincter augmentation (e.g., for restoration
of continence), within a wound or suture, to promote tissue
regrowth; and within or adjacent to vessel anastomoses, to promote
vessel regrowth.
[0048] The term "aqueous medium" includes solutions, suspensions,
dispersions, colloids, and the like containing water.
[0049] The term "substantially immediately" means within less than
five minutes, preferably within less than two minutes, and the term
"immediately" means within less than one minute, preferably within
less than 30 seconds.
[0050] The terms "active agent," and "biologically active agent"
are used interchangeably herein to refer to a chemical material or
compound suitable for administration to a patient and that induces
a desired effect. The terms include agents that are therapeutically
effective as well as prophylactically effective. Also included are
derivatives and analogs of those compounds or classes of compounds
specifically mentioned that also induce the desired effect.
[0051] The term "hydrogel" is used in the conventional sense to
refer to water-swellable polymeric matrices that can absorb a
substantial amount of water to form elastic gels, wherein
"matrices" are three-dimensional networks of macromolecules held
together by covalent or noncovalent crosslinks. Upon placement in
an aqueous environment, dry hydrogels swell to the extent allowed
by the degree of cross-linking.
[0052] With regard to nomenclature pertinent to molecular
structures, the following definitions apply:
[0053] The term "alkyl" as used herein refers to a branched or
unbranched saturated hydrocarbon group typically although not
necessarily containing 1 to about 24 carbon atoms, such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl,
decyl, and the like, as well as cycloalkyl groups such as
cyclopentyl, cyclohexyl and the like. Generally, although again not
necessarily, alkyl groups herein contain 1 to about 12 carbon
atoms. The term "lower alkyl" intends an alkyl group of one to six
carbon atoms, preferably one to four carbon atoms. "Substituted
alkyl" refers to alkyl substituted with one or more substituent
groups. "Alkylene," "lower alkylene" and "substituted alkylene"
refer to divalent alkyl, lower alkyl, and substituted alkyl groups,
respectively.
[0054] The term "aryl" as used herein, and unless otherwise
specified, refers to an aromatic substituent containing a single
aromatic ring or multiple aromatic rings that are fused together,
linked covalently, or linked to a common group such as a methylene
or ethylene moiety. The common linking group may also be a carbonyl
as in benzophenone, an oxygen atom as in diphenylether, or a
nitrogen atom as in diphenylamine. Preferred aryl groups contain
one aromatic ring or two fused or linked aromatic rings, e.g.,
phenyl, naphthyl, biphenyl, diphenylether, diphenylamine,
benzophenone, and the like. "Substituted aryl" refers to an aryl
moiety substituted with one or more substituent groups, and the
terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl
in which at least one carbon atom is replaced with a heteroatom.
The terms "arylene" and "substituted arylene" refer to divalent
aryl and substituted aryl groups as just defined.
[0055] The term "heteroatom-containing" as in a
"heteroatom-containing hydrocarbyl group" refers to a molecule or
molecular fragment in which one or more carbon atoms is replaced
with an atom other than carbon, e.g., nitrogen, oxygen, sulfur,
phosphorus or silicon.
[0056] "Hydrocarbyl" refers to univalent hydrocarbyl radicals
containing 1 to about 30 carbon atoms, preferably 1 to about 24
carbon atoms, most preferably 1 to about 12 carbon atoms, including
branched or unbranched, saturated or unsaturated species, such as
alkyl groups, alkenyl groups, aryl groups, and the like. The term
"lower hydrocarbyl" intends a hydrocarbyl group of one to six
carbon atoms, preferably one to four carbon atoms. The term
"hydrocarbylene" intends a divalent hydrocarbyl moiety containing 1
to about 30 carbon atoms, preferably 1 to about 24 carbon atoms,
most preferably 1 to about 12 carbon atoms, including branched or
unbranched, saturated or unsaturated species, or the like. The term
"lower hydrocarbylene" intends a hydrocarbylene group of one to six
carbon atoms, preferably one to four carbon atoms. "Substituted
hydrocarbyl" refers to hydrocarbyl substituted with one or more
substituent groups, and the terms "heteroatom-containing
hydrocarbyl" and "heterohydrocarbyl" refer to hydrocarbyl in which
at least one carbon atom is replaced with a heteroatom. Similarly,
"substituted hydrocarbylene" refers to hydrocarbylene substituted
with one or more substituent groups, and the terms
"heteroatom-containing hydrocarbylene" and "heterohydrocarbylene"
refer to hydrocarbylene in which at least one carbon atom is
replaced with a heteroatom. If not otherwise indicated,
"hydrocarbyl" indicates unsubstituted hydrocarbyl, substituted
hydrocarbyl, heteroatom-containing hydrocarbyl, and substituted
heteroatom-containing hydrocarbyl. Unless otherwise indicated, the
terms "hydrocarbyl" and "hydrocarbylene" include substituted
hydrocarbyl and substituted hydrocarbylene, heteroatom-containing
hydrocarbyl and heteroatom-containing hydrocarbylene, and
substituted heteroatom-containing hydrocarbyl and substituted
heteroatom-containing hydrocarbylene, respectively.
[0057] By "substituted" as in "substituted hydrocarbyl,"
"substituted alkyl," and the like, as alluded to in some of the
aforementioned definitions, is meant that in the hydrocarbyl,
alkyl, or other moiety, at least one hydrogen atom bound to a
carbon atom is replaced with one or more substituents that are
functional groups such as alkoxy, hydroxy, halo, nitro, and the
like. Unless otherwise indicated, it is to be understood that
specified molecular segments can be substituted with one or more
substituents that do not compromise a compound's utility. For
example, "succinimidyl" is intended to include unsubstituted
succinimidyl as well as sulfosuccinimidyl and other succinimidyl
groups substituted on a ring carbon atom, e.g., with alkoxy
substituents, polyether substituents, or the like.
[0058] II. The Crosslinkable Composition
[0059] In accordance with the present invention, a crosslinkable
polymer composition is provided that contains a minimum of three
components, each of which participates in a reaction that results
in a crosslinked matrix. The components of the crosslinkable
composition are selected so that crosslinking gives rise to a
biocompatible, nonimmunogenic matrix useful in a variety of
contexts, including adhesion, biologically active agent delivery,
tissue augmentation, and other applications. The crosslinkable
composition of the invention is comprised of at least three
crosslinkable components: a first component, component A, which has
m nucleophilic groups, wherein m.gtoreq.2; a second component,
component B, which has n electrophilic groups capable of reaction
with the m nucleophilic groups, wherein n.gtoreq.2 and m+n>4;
and a third component, component C, which has at least one
functional group that is either electrophilic and capable of
reaction with the nucleophilic groups of component A, or
nucleophilic and capable of reaction with the electrophilic groups
of component B. Thus, the total number of functional groups present
on components A, B and C in combination is >5; that is, the
total functional groups given by m+n+p must be >5, where p is
the number of functional groups on component C and, as indicated,
is .gtoreq.1. Each of the components is biocompatible and
nonimmunogenic, and at least one component is comprised of a
hydrophilic polymer. For those compositions in which a higher
degree of crosslinking is required, e.g., when a less swellable
biomaterial is desirable or increased compressive strength is
necessary, p should be .gtoreq.2. Also, as will be appreciated, the
crosslinkable composition may contain additional components D, E,
F, etc., having one or more reactive nucleophilic or electrophilic
groups and thereby participate in formation of the crosslinked
biomaterial via covalent bonding to other components.
[0060] The m nucleophilic groups on component A may all be the
same, or, alternatively, A may contain two or more different
nucleophilic groups. Similarly, the n electrophilic groups on
component B may all be the same, or two or more different
electrophilic groups may be present. The functional group(s) on
component C, if nucleophilic, may or may not be the same as the
nucleophilic groups on component A, and, conversely, if
electrophilic, the functional group(s) on component C may or may
not be the same as the electrophilic groups on component B.
[0061] Accordingly, the components may be represented by the
structural formulae
R.sup.1(--[Q.sup.1].sub.q--X).sub.m (component A), (I)
R.sup.2(--[Q.sup.2].sub.r--Y).sub.n (component B), and (II)
R.sup.3(--[Q.sup.3].sub.s--Fn).sub.p (component C), (III)
[0062] wherein:
[0063] R.sup.1, R.sup.2 and R.sup.3 are independently selected from
the group consisting of C.sub.2 to C.sub.14 hydrocarbyl,
heteroatom-containing C.sub.2 to C.sub.14 hydrocarbyl, hydrophilic
polymers, and hydrophobic polymers, providing that at least one of
R.sup.1, R.sup.2 and R.sup.3 is a hydrophilic polymer, preferably a
synthetic hydrophilic polymer;
[0064] X represents one of the m nucleophilic groups of component
A, and the various X moieties on A may be the same or
different;
[0065] Y represents one of the n electrophilic groups of component
B, and the various Y moieties on A may be the same or
different;
[0066] Fn represents a functional group on component C;
[0067] Q.sup.1, Q.sup.2 and Q.sup.3 are linking groups;
[0068] m.gtoreq.2, n.gtoreq.2, m+n is>4, p.gtoreq.1, and q, r
and s are independently zero or 1.
[0069] A. Reactive Groups
[0070] X may be virtually any nucleophilic group, so long as
reaction can occur with the electrophilic group Y and also with Fn
when Fn is electrophilic. Analogously, Y may be virtually any
electrophilic group, so long as reaction can take place with X and
also with Fn when Fn is nucleophilic. The only limitation is a
practical one, in that reaction between X and Y, X and Fn.sub.EL
(where Fn.sub.EL indicates an electrophilic Fn group), and Y and
Fn.sub.NU, should be fairly rapid and take place automatically upon
admixture with an aqueous medium, without need for heat or
potentially toxic or non-biodegradable reaction catalysts or other
chemical reagents. It is also preferred although not essential that
reaction occur without need for ultraviolet or other radiation.
Ideally, the reactions between X and Y, and between either X and
Fn.sub.EL or Y and Fn.sub.NU, should be complete in under 60
minutes, preferably under 30 minutes. Most preferably, the reaction
occurs in about 5 to 15 minutes or less. Examples of nucleophilic
groups suitable as X or Fn.sub.NU include, but are not limited to,
--NH.sub.2, --NHR.sup.4, --N(R.sup.4).sub.2, --SH, --OH, --COOH,
--C.sub.6H.sub.4--OH, --PH.sub.2, --PHR.sup.5,
----P(R.sup.5).sub.2, --NH--NH.sub.2, --CO--NH--NH.sub.2,
--C.sub.5H.sub.4N, etc. wherein R.sup.4 and R.sup.5 are
hydrocarbyl, typically alkyl or monocyclic aryl, preferably alkyl,
and most preferably lower alkyl. Organometallic moieties are also
useful nucleophilic groups for the purposes of the invention,
particularly those that act as carbanion donors. Organometallic
nucleophiles are not, however, preferred. Examples of
organometallic moieties include: Grignard functionalities
--R.sup.6MgHal wherein R.sup.6 is a carbon atom (substituted or
unsubstituted), and Hal is halo, typically bromo, iodo or chloro,
preferably bromo; and lithium-containing functionalities, typically
alkyllithium groups; sodium-containing functionalities.
[0071] It will be appreciated by those of ordinary skill in the art
that certain nucleophilic groups must be activated with a base so
as to be capable of reaction with an electrophile. For example,
when there are nucleophilic sulfhydryl and hydroxyl groups in the
crosslinkable composition, the composition must be admixed with an
aqueous base in order to remove a proton and provide an --S.sup.-
or --O.sup.- species to enable reaction with an electrophile.
Unless it is desirable for the base to participate in the
crosslinking reaction, a nonnucleophilic base is preferred. In some
embodiments, the base may be present as a component of a buffer
solution. Suitable bases and corresponding crosslinking reactions
are described infra in Section III.
[0072] The selection of electrophilic groups provided within the
crosslinkable composition, i.e., on component B and on component C
when Fn is electrophilic, must be made so that reaction is possible
with the specific nucleophilic groups. Thus, when the X moieties
are amino groups, the Y and any Fn.sub.EL groups are selected so as
to react with amino groups. Analogously, when the X moieties are
sulfhydryl moieties, the corresponding electrophilic groups are
sulfhydryl-reactive groups, and the like.
[0073] By way of example, when X is amino (generally although not
necessarily primary amino), the electrophilic groups present on Y
and Fn.sub.EL are amino reactive groups such as, but not limited
to: (1) carboxylic acid esters, including cyclic esters and
"activated" esters; (2) acid chloride groups (--CO--Cl); (3)
anhydrides (--(CO)--O--(CO)--R); (4) ketones and aldehydes,
including .alpha.,.beta.-unsaturated aldehydes and ketones such as
--CH.dbd.CH--CH.dbd.O and --CH.dbd.CH--C(CH.sub.3).db- d.O; (5)
halides; (6) isocyanate (--N.dbd.C.dbd.O); (7) isothiocyanate
(--N.dbd.C.dbd.S); (8) epoxides; (9) activated hydroxyl groups
(e.g., activated with conventional activating agents such as
carbonyldiimidazole or sulfonyl chloride); and (10) olefins,
including conjugated olefins, such as ethenesulfonyl
(--SO.sub.2CH.dbd.CH.sub.2) and analogous functional groups,
including acrylate (--CO.sub.2--C.dbd.CH.sub.2), methacrylate
(--CO.sub.2--C(CH.sub.3).dbd.CH.sub.2)), ethyl acrylate
(--CO.sub.2--C(CH.sub.2CH.sub.3).dbd.CH.sub.2), and ethyleneimino
(--CH.dbd.CH--C.dbd.NH). Since a carboxylic acid group per se is
not susceptible to reaction with a nucleophilic amine, components
containing carboxylic acid groups must be activated so as to be
amine-reactive. Activation may be accomplished in a variety of
ways, but often involves reaction with a suitable
hydroxyl-containing compound in the presence of a dehydrating agent
such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU).
For example, a carboxylic acid can be reacted with an
alkoxy-substituted N-hydroxysuccinimide or
N-hydroxysulfosuccinimide in the presence of DCC to form reactive
electrophilic groups, the N-hydroxysuccinimnide ester and the
N-hydroxysulfosuccinimide ester, respectively. Carboxylic acids may
also be activated by reaction with an acyl halide such as an acyl
chloride (e.g., acetyl chloride), to provide a reactive anhydride
group. In a further example, a carboxylic acid may be converted to
an acid chloride group using, e.g., thionyl chloride or an acyl
chloride capable of an exchange reaction. Specific reagents and
procedures used to carry out such activation reactions will be
known to those of ordinary skill in the art and are described in
the pertinent texts and literature.
[0074] Analogously, when X is sulfhydryl, the electrophilic groups
present on Y and Fn.sub.EL are groups that react with a sulthydryl
moiety. Such reactive groups include those that form thioester
linkages upon reaction with a sulfhydryl group, such as those
described in applicants' PCT Publication No. WO 00/62827 to Wallace
et al. As explained in detail therein, such "sulfhydryl reactive"
groups include, but are not limited to: mixed anhydrides; ester
derivatives of phosphorus; ester derivatives of p-nitrophenol,
p-nitrothiophenol and pentafluorophenol; esters of substituted
hydroxylamines, including N-hydroxyphthalimide esters,
N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, and
N-hydroxyglutarimide esters; esters of 1-hydroxybenzotriazole;
3-hydroxy-3,4-dihydro-benzotriazin-4-one;
3-hydroxy-3,4-dihydro-quinazoli- ne-4-one; carbonylimidazole
derivatives; acid chlorides; ketenes; and isocyanates. With these
sulfhlydryl reactive groups, auxiliary reagents can also be used to
facilitate bond formation, e.g.,
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide can be used to
facilitate coupling of sulfhydryl groups to carboxyl-containing
groups.
[0075] In addition to the sulfhydryl reactive groups that form
thioester linkages, various other sulfhydryl reactive
functionalities can be utilized that form other types of linkages.
For example, compounds that contain methyl imidate derivatives form
imido-thioester linkages with sulfhydryl groups. Alternatively,
sulfhydryl reactive groups can be employed that form disulfide
bonds with sulthydryl groups; such groups generally have the
structure --S--S--Ar where Ar is a substituted or unsubstituted
nitrogen-containing heteroaromatic moiety or a non-heterocyclic
aromatic group substituted with an electron-withdrawing moiety,
such that Ar may be, for example, 4-pyridinyl, o-nitrophenyl,
m-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2-nitro-4-benzoic
acid, 2-nitro-4-pyridinyl, etc. In such instances, auxiliary
reagents, i.e., mild oxidizing agents such as hydrogen peroxide,
can be used to facilitate disulfide bond formation.
[0076] Yet another class of sulfhydryl reactive groups forms
thioether bonds with sulfhydryl groups. Such groups include, inter
alia, maleimido, substituted maleimido, haloalkyl, epoxy, imino,
and aziridino, as well as olefins (including conjugated olefins)
such as ethenesulfonyl, etheneimino, acrylate, methacrylate, and
.alpha.,.beta.-unsaturated aldehydes and ketones.
[0077] When X is --OH, the electrophilic functional groups on the
remaining component(s) must react with hydroxyl groups. The
hydroxyl group may be activated as described above with respect to
carboxylic acid groups, or it may react directly in the presence of
base with a sufficiently reactive electrophile such as an epoxide
group, an aziridine group, an acyl halide, an anhydride.
[0078] When X is an organometallic nucleophile such as a Grignard
functionality or an alkyllithium group, suitable electrophilic
functional groups for reaction therewith are those containing
carbonyl groups, including, by way of example, ketones and
aldehydes.
[0079] It will also be appreciated that certain functional groups
can react as nucleophiles or as electrophiles, depending on the
selected reaction partner and/or the reaction conditions. For
example, a carboxylic acid group can act as a nucleophile in the
presence of a fairly strong base, but generally acts as an
electrophile allowing nucleophilic attack at the carbonyl carbon
and concomitant replacement of the hydroxyl group with the incoming
nucleophile.
[0080] The covalent linkages in the crosslinked structure that
result upon covalent binding of specific nucleophilic components to
specific electrophilic components in the crosslinkable composition
include, solely by way of example, the following (the optional
linking groups Q.sup.1, Q.sup.2 and Q.sup.3 are omitted for
clarity):
1TABLE 1 REPRESENTATIVE NUCLEOPHILIC REPRESENTATIVE COMPONENT
ELECTROPHILIC COMPONENT (A, FN.sub.NU) (B, FN.sub.EL) RESULTING
LINKAGE R.sup.1-NH.sub.2 R.sup.2-O--(CO)--O--N(COCH.sub.2)
R.sup.1-NH--(CO)--O-R.sup.2 (succinimidyl carbonate terminus)
R.sup.1-SH R.sup.2-O--(CO)--O--N(COCH.sub.2)
R.sup.1-S--(CO)--O-R.sup.- 2 R.sup.1-OH
R.sup.2-O--(CO)--O--N(COCH.sub.2) R.sup.1-O--(CO)-R.sup.2
R.sup.1-NH.sub.2 R.sup.2-O(CO)--CH.dbd.CH.- sub.2
R.sup.1-NH--CH.sub.2CH.sub.2--(CO)--O-R.sup.2 (acrylate terminus)
R.sup.1-SH R.sup.2-O--(CO)--CH.dbd.CH.sub.2
R.sup.1-S--CH.sub.2CH.sub.2--(CO)--O-R.sup.2 R.sup.1-OH
R.sup.2-O--(CO)--CH.dbd.CH.sub.2
R.sup.1-O--CH.sub.2CH.sub.2--(CO)--O-R.s- up.2 R.sup.1-NH.sub.2
R.sup.2-O(CO)--(CH.sub.2).sub.3--CO.sub.2--N(- COCH.sub.2)
R.sup.1-NH--(CO)--(CH.sub.2).sub.3--(CO)--O-R.sup.2 (succinimidyl
glutarate terminus) R.sup.1-SH
R.sup.2-O(CO)--(CH.sub.2).sub.3--CO.sub.2--N(COCH.sub.2)
R.sup.1-S--(CO)--(CH.sub.2).sub.3--(CO)-OR.sup.2 R.sup.1-OH
R.sup.2-O(CO)--(CH.sub.2).sub.3--CO.sub.2--N(COCH.sub.2)
R.sup.1-O--(CO)--(CH.sub.2).sub.3--(CO)-OR.sup.2 R.sup.1-NH.sub.2
R.sup.2-O--CH.sub.2--CO.sub.2--N(COCH.sub.2)
R.sup.1-NH--(CO)--CH.sub.2-O- R.sup.2 (succinimidyl acetate
terminus) R.sup.1-SH R.sup.2-O--CH.sub.2--CO.sub.2--N(COCH.sub.2)
R.sup.1-S--(CO)--CH.sub.2-OR- .sup.2 R.sup.1-OH
R.sup.2-O--CH.sub.2--CO.sub.2--N(COCH.sub.2)
R.sup.1-O--(CO)--CH.sub.2-OR.sup.2 R.sup.1-NH.sub.2
R.sup.2-O--NH(CO)--(CH.sub.2).sub.2--CO.sub.2--
R.sup.1-NH--(CO)--(CH.sub- .2).sub.2--(CO)--NH--OR.sup.2
N(COCH.sub.2) (succinimidyl succinamide terminus) R.sup.1-SH
R.sup.2-O--NH(CO)--(CH.sub.2).sub- .2--CO.sub.2--
R.sup.1-S--(CO)--(CH.sub.2).sub.2--(CO)--NH--OR.sup.2 N(COCH.sub.2)
R.sup.1-OH R.sup.2-O--NH(CO)--(CH.sub.2).sub.2--CO- .sub.2--
R.sup.1-O--(CO)--(CH.sub.2).sub.2--(CO)--NH--OR.sup.2 N(COCH.sub.2)
R.sup.1-NH.sub.2 R.sup.2-O--(CH.sub.2).sub.2--CHO
R.sup.1-NH--(CO)--(CH.sub.2).sub.2-OR.sup.2 (propionaldehyde
terminus) R.sup.1-NH.sub.2 1 R.sup.1-NH--CH.sub.2--CH(OH)-
--CH.sub.2-OR.sup.2 and
R.sup.1-N[CH.sub.2--CH(OH)--CH.sub.2-OR.sup.2].sub- .2 (glycidyl
ether terminus) R.sup.1-NH.sub.2
R.sup.2-O--(CH.sub.2).sub.2--N.dbd.C.dbd.O
R.sup.1-NH--(CO)--NH--CH.sub.2- -OR.sup.2 (isocyanate terminus)
R.sup.1-NH.sub.2 R.sup.2-SO.sub.2--CH.dbd.CH.sub.2
R.sup.1-NH--CH.sub.2CH.sub.2--SO.sub.2-- R.sup.2 (vinyl sulfone
terminus) R.sup.1-SH R.sup.2-SO.sub.2--CH.dbd.CH.sub.2
R.sup.1-S--CH.sub.2CH.sub.2--SO.sub.2-R- .sup.2
[0081] B. Linking Groups
[0082] The functional groups X, Y and Fn may be directly attached
to the compound core (R.sup.1, R.sup.2 or R.sup.3, respectively),
or they may be indirectly attached through a linking group, with
longer linking groups also termed "chain extenders." In structural
formulae (I), (II) and (III),
R.sup.1(--[Q.sup.1].sub.qX).sub.m (component A) (I)
R.sup.2(--[Q.sup.2].sub.r--Y).sub.n (component B) (II)
R.sup.3(--[Q.sup.3].sub.s--Fn).sub.p (component C) (III)
[0083] the optional linking groups are represented by Q.sup.1,
Q.sup.2 and Q.sup.3, wherein the linking groups are present when q,
r and s are equal to 1 (with R, X, Y, Fn, m n and p as defined
previously).
[0084] Suitable linking groups are well known in the art. See, for
example, International Patent Publication No. WO 97/22371. Linking
groups are useful to avoid steric hindrance problems that are
sometimes associated with the formation of direct linkages between
molecules. Linking groups may additionally be used to link several
multifunctionally activated compounds together to make larger
molecules. In a preferred embodiment, a linking group can be used
to alter the degradative properties of the compositions after
administration and resultant gel formation. For example, linking
groups can be incorporated into components A, B or C to promote
hydrolysis, to discourage hydrolysis, or to provide a site for
enzymatic degradation.
[0085] Examples of linking groups that provide hydrolyzable sites,
include, inter alia: ester linkages; anhydride linkages, such as
obtained by incorporation of glutarate and succinate; ortho ester
linkages; ortho carbonate linkages such as trimethylene carbonate;
amide linkages; phosphoester linkages; a-hydroxy acid linkages,
such as may be obtained by incorporation of lactic acid and
glycolic acid; lactone-based linkages, such as may be obtained by
incorporation of caprolactone, valerolactone, .gamma.-butyrolactone
and p-dioxanone; and amide linkages such as in a dimeric,
oligomeric, or poly(amino acid) segment. Examples of non-degradable
linking groups include succinimide, propionic acid and
carboxymethylate linkages. See, for example, PCT WO 99/07417.
Examples of enzymatically degradable linkages include
Leu-Gly-Pro-Ala, which is degraded by collagenase; and Gly-Pro-Lys,
which is degraded by plasmin.
[0086] Linking groups can also enhance or suppress the reactivity
of the various nucleophilic and electrophilic groups. For example,
electron-withdrawing groups within one or two carbons of a
sulfhydryl group would be expected to diminish its effectiveness in
coupling, due to a lowering of nucleophilicity. Carbon-carbon
double bonds and carbonyl groups will also have such an effect.
Conversely, electron-withdrawing groups adjacent to a carbonyl
group (e.g., the reactive carbonyl of
glutaryl-N-hydroxysuccinimidyl) would increase the reactivity of
the carbonyl carbon with respect to an incoming nucleophile. By
contrast, sterically bulky groups in the vicinity of a functional
group can be used to diminish reactivity and thus coupling rate as
a result of steric hindrance.
[0087] By way of example, particular linking groups and
corresponding component structure are indicated in Table 2:
2 TABLE 2 LINKING GROUP COMPONENT STRUCTURE --O--(CH.sub.2).sub.n--
Component A: R.sup.1--O--(CH.sub.2).sub.n--X Component B:
R.sup.2--O--(CH.sub.2).sub.n--Y Component C:
R.sup.3--O--(CH.sub.2).sub.n--Z --S--(CH.sub.2).sub.n-- Component
A: R.sup.1--S--(CH.sub.2).sub.n--X Component B:
R.sup.2--S--(CH.sub.2).sub.n--Y Component C:
R.sup.3--S--(CH.sub.2).sub.n--Z --NH--(CH.sub.2).sub.n-- Component
A: R.sup.1--NH--(CH.sub.2).sub.n--X Component B:
R.sup.2--NH--(CH.sub.2).sub.n--Y Component C:
R.sup.3--NH--(CH.sub.2).sub.n--Z --O--(CO)--NH--(CH.sub.2).sub.-
n-- Component A: R.sup.1--O--(CO)--NH--(CH.sub.2).sub.n--X
Component B: R.sup.2--O--(CO)--NH--(CH.sub.2).sub.n--Y Component C:
R.sup.3--O--(CO)--NH--(CH.sub.2).sub.n--Z
--NH--(CO)--O--(CH.sub.2).sub.n-- Component A:
R.sup.1--NH--(CO)--O--(CH.sub.2).sub.n--X Component B:
R.sup.2--NH--(CO)--O--(CH.sub.2).sub.n--Y Component C:
R.sup.3--NH--(CO)--O--(CH.sub.2).sub.n--Z --O--(CO)--(CH.sub.2).s-
ub.n-- Component A: R.sup.1--O--(CO)--(CH.sub.2).sub.n--X Component
B: R.sup.2--O--(CO)--(CH.sub.2).sub.n--Y Component C:
R.sup.3--O--(CO)--(CH.sub.2).sub.n--Z --(CO)--O--(CH.sub.2).sub.n--
Component A: R.sup.1--(CO)--O--(CH.sub.2).sub.n--X Component B:
R.sup.2--(CO)--O--(CH.sub.2).sub.n--Y Component C:
R.sup.3--(CO)--O--(CH.sub.2).sub.n--Z --O--(CO)--O--(CH.sub.2).su-
b.n-- Component A: R.sup.1--O--(CO)--O--(CH.sub.2).sub.n--X
Component B: R.sup.2--O--(CO)--O--(CH.sub.2).sub.n--Y Component C:
R.sup.3--O--(CO)--O--(CH.sub.2).sub.n--Z --O--(CO)--CHR.sup.7--
Component A: R.sup.1--O--(CO)--CHR.sup.7-- -X Component B:
R.sup.2--O--(CO)--CHR.sup.7--Y Component C:
R.sup.3--O--(CO)--CHR.sup.7--Z --O--R.sup.8--(CO)--NH-- Component
A: R.sup.1--O--R.sup.8--(CO)-- -NH--X Component B:
R.sup.2--O--R.sup.8--(CO)--NH--Y Component C:
R.sup.3--O--R.sup.8--(CO)--NH--Z
[0088] In the table, n is generally in the range of 1 to about 10,
R.sup.7 is generally hydrocarbyl, typically alkyl or aryl,
preferably alkyl, and most preferably lower alkyl, and R.sup.8 is
hydrocarbylene, heteroatom-containing hydrocarbylene, substituted
hydrocarbylene, or substituted heteroatom-containing
hydrocarbylene) typically alkylene or arylene (again, optionally
substituted and/or containing a heteroatom), preferably lower
alkylene (e.g., methylene, ethylene, n-propylene, n-butylene,
etc.), phenylene, or amidoalkylene (e.g.,
--(CO)--NH--CH.sub.2).
[0089] Other general principles that should be considered with
respect to linking groups are as follows: If higher molecular
weight components are to be used, they preferably have
biodegradable linkages as described above, so that fragments larger
than 20,000 mol. wt. are not generated during resorption in the
body. In addition, to promote water miscibility and/or solubility,
it may be desired to add sufficient electric charge or
hydrophilicity. Hydrophilic groups can be easily introduced using
known chemical synthesis, so long as they do not give rise to
unwanted swelling or an undesirable decrease in compressive
strength. In particular, polyalkoxy segments may weaken gel
strength.
[0090] C. The Component Core
[0091] The "core" of each crosslinkable component is comprised of
the molecular structure to which the nucleophilic or electrophilic
groups are bound. Using the formulae (I)
R.sup.1--[Q.sup.1].sub.q--X).sub.m, for component A, (II)
R.sup.2(--[Q.sup.2].sub.r--Y).sub.n for component B, and (III)
R.sup.3(--[Q.sup.3].sub.5--Fn).sub.p for component C, the "core"
groups are R.sup.1, R.sup.2 and R.sup.3. Each molecular core of the
reactive components of the crosslinkable composition is generally
selected from synthetic and naturally occurring hydrophilic
polymers, hydrophobic polymers, and C.sub.2--C.sub.14 hydrocarbyl
groups zero to 2 heteroatoms selected from N, O and S, with the
proviso that at least one of the crosslinkable components A, B and
C comprises a molecular core of a synthetic hydrophilic polymer. In
a preferred embodiment, at least two of A, B and C comprises a
molecular core of a synthetic hydrophilic polymer.
[0092] 1. Hydrophilic Polymers and "Activation" Thereof
[0093] A "hydrophilic polymer" as used herein refers to a synthetic
polymer having an average molecular weight and composition
effective to render the polymer "hydrophilic" as defined in Part
(I) of this section. Synthetic hydrophilic polymers useful herein
include, but are not limited to: polyalkylene oxides, particularly
polyethylene glycol and poly(ethylene oxide)-poly(propylene oxide)
copolymers, including block and random copolymers; polyols such as
glycerol, polyglycerol (particularly highly branched polyglycerol),
propylene glycol and trimethylene glycol substituted with one or
more polyalkylene oxides, e.g., mono-, di- and tri-polyoxyethylated
glycerol, mono- and di-polyoxyethylated propylene glycol, and mono-
and di-polyoxyethylated trimethylene glycol; polyoxyethylated
sorbitol, polyoxyethylated glucose; acrylic acid polymers and
analogs and copolymers thereof, such as polyacrylic acid per se,
polymethacrylic acid, poly(hydroxyethyl-methacry- late),
poly(hydroxyethylacrylate), poly(methylalkylsulfoxide
methacrylate), poly(methylalkylsulfoxide acrylate) and copolymers
of any of the foregoing, and/or with additional acrylate species
such as aminoethyl acrylate and mono-2-(acryloxy)-ethyl succinate;
polymaleic acid; poly(acrylamides) such as polyacrylamide per se,
poly(methacrylamide), poly(dimethylacrylamide), and
poly(N-isopropylacrylamide); poly(olefinic alcohol)s such as
poly(vinyl alcohol); poly(N-vinyl lactams) such as poly(vinyl
pyrrolidone), poly(N-vinyl caprolactam), and copolymers thereof;
polyoxazolines, including poly(methyloxazoline) and
poly(ethyloxazoline); and polyvinylamines. It must be emphasized
that the aforementioned list of polymers is not exhaustive, and a
variety of other synthetic hydrophilic polymers may be used, as
will be appreciated by those skilled in the art.
[0094] The synthetic hydrophilic polymer may be a homopolymer, a
block copolymer, a random copolymer, or a graft copolymer. In
addition, the polymer may be linear or branched, and if branched,
may be minimally to highly branched, dendrimeric, hyperbranched, or
a star polymer. The polymer may include biodegradable segments and
blocks, either distributed throughout the polymer's molecular
structure or present as a single block, as in a block copolymer.
Biodegradable segments are those that degrade so as to break
covalent bonds. Typically, biodegradable segments are segments that
are hydrolyzed in the presence of water and/or enzymatically
cleaved in situ. Biodegradable segments may be composed of small
molecular segments such as ester linkages, anhydride linkages,
ortho ester linkages, ortho carbonate linkages, amide linkages,
phosphonate linkages, etc. Larger biodegradable "blocks" will
generally be composed of oligomeric or polymeric segments
incorporated within the hydrophilic polymer. Illustrative
oligomeric and polymeric segments that are biodegradable include,
by way of example, poly(amino acid) segments, poly(orthoester)
segments, poly(orthocarbonate) segments, and the like.
[0095] Other suitable synthetic hydrophilic polymers include
chemically synthesized polypeptides, particularly polynucleophilic
polypeptides that have been synthesized to incorporate amino acids
containing primary amino groups (such as lysine) and/or amino acids
containing thiol groups (such as cysteine). Poly(lysine), a
synthetically produced polymer of the amino acid lysine (145 MW),
is particularly preferred. Poly(lysine)s have been prepared having
anywhere from 6 to about 4,000 primary amino groups, corresponding
to molecular weights of about 870 to about 580,000. Poly(lysine)s
for use in the present invention preferably have a molecular weight
within the range of about 1,000 to about 300,000, more preferably
within the range of about 5,000 to about 100,000, and most
preferably, within the range of about 8,000 to about 15,000.
Poly(lysine)s of varying molecular weights are commercially
available from Peninsula Laboratories, Inc. (Belmont, Calif.).
[0096] The synthetic hydrophilic polymer may be a homopolymer, a
block copolymer, a random copolymer, or a graft copolymer. In
addition, the polymer may be linear or branched, and if branched,
may be minimally to highly branched, dendrimeric, hyperbranched, or
a star polymer. The polymer may include biodegradable segments and
blocks, either distributed throughout the polymer's molecular
structure or present as a single block, as in a block copolymer.
Biodegradable segments are those that degrade so as to break
covalent bonds. Typically, biodegradable segments are segments that
are hydrolyzed in the presence of water and/or enzymatically
cleaved in situ. Biodegradable segments may be composed of small
molecular segments such as ester linkages, anhydride linkages,
ortho ester linkages, ortho carbonate linkages, amide linkages,
phosphonate linkages, etc. Larger biodegradable "blocks" will
generally be composed of oligomeric or polymeric segments
incorporated within the hydrophilic polymer. Illustrative
oligomeric and polymeric segments that are biodegradable include,
by way of example, poly(amino acid) segments, poly(orthoester)
segments, poly(orthocarbonate) segments, and the like.
[0097] Although a variety of different synthetic hydrophilic
polymers can be used in the present compositions, as indicated
above, preferred synthetic hydrophilic polymers are polyethylene
glycol (PEG) and polyglycerol (PG), particularly highly branched
polyglycerol. Various forms of PEG are extensively used in the
modification of biologically active molecules because PEG lacks
toxicity, antigenicity, and immunogenicity (i.e., is
biocompatible), can be formulated so as to have a wide range of
solubilities, and does not typically interfere with the enzymatic
activities and/or conformations of peptides. A particularly
preferred synthetic hydrophilic polymer for certain applications is
a polyethylene glycol (PEG) having a molecular weight within the
range of about 100 to about 100,000 mol. wt., although for highly
branched PEG, far higher molecular weight polymers can be
employed--up to 1,000,000 or more--providing that biodegradable
sites are incorporated ensuring that all degradation products will
have a molecular weight of less than about 30,000. For most PEGs,
however, the preferred molecular weight is about 1,000 to about
20,000 mol. wt., more preferably within the range of about 7,500 to
about 20,000 mol. wt. Most preferably, the polyethylene glycol has
a molecular weight of approximately 10,000 mol. wt.
[0098] Naturally occurring hydrophilic polymers include, but are
not limited to: proteins such as collagen, fibronectin, albumins,
globulins, fibrinogen, fibrin and thrombin, with collagen
particularly preferred; carboxylated polysaccharides such as
polymannuronic acid and polygalacturonic acid; aminated
polysaccharides, particularly the glycosaminoglycans, e.g.,
hyaluronic acid, chitin, chondroitin sulfate A, B, or C, keratin
sulfate, keratosulfate and heparin; and activated polysaccharides
such as dextran and starch derivatives. Collagen and
glycosaminoglycans are preferred naturally occurring hydrophilic
polymers for use herein.
[0099] In general, collagen from any source may be used in the
compositions of the invention; for example, collagen may be
extracted and purified from human or other mammalian source, such
as bovine or porcine corium and human placenta, or may be
recombinantly or otherwise produced. The preparation of purified,
substantially non-antigenic collagen in solution from bovine skin
is well known in the art. Commonly owned U.S. Pat. No. 5,428,022,
issued Jun. 27, 1995 to Palefsky et al., discloses methods of
extracting and purifying collagen from the human placenta. Commonly
owned U.S. Pat. No. 5,667,839, issued Sep. 16, 1997 to Berg,
discloses methods of producing recombinant human collagen in the
milk of transgenic animals, including transgenic cows. The term
"collagen" or "collagen material" as used herein refers to all
forms of collagen, including those that have been processed or
otherwise modified.
[0100] Collagen of any type, including, but not limited to, types
I, II, III, IV, or any combination thereof, may be used in the
compositions of the invention, although type I is generally
preferred. Either atelopeptide or telopeptide-containing collagen
may be used; however, when collagen from a source, such as bovine
collagen, is used, atelopeptide collagen is generally preferred,
because of its reduced immunogenicity compared to
telopeptide-containing collagen.
[0101] Collagen that has not been previously crosslinked by methods
such as heat, irradiation, or chemical crosslinking agents is
preferred for use in the compositions of the invention, although
previously crosslinked collagen may be used. Non-crosslinked
atelopeptide fibrillar collagen is commercially available from
Cohesion Corporation (Palo Alto, Calif.) at collagen concentrations
of 35 mg/ml and 65 mg/mil under the trademarks Zyderm.RTM. I
Collagen and Zyderm.RTM. II Collagen, respectively.
Glutaraldehyde-crosslinked atelopeptide fibrillar collagen is
commercially available from Cohesion Corporation at a collagen
concentration of 35 mg/ml under the trademark Zyplast.RTM..
[0102] Collagens for use in the present invention are generally,
although not necessarily, in aqueous suspension at a concentration
between about 20 mg/ml to about 120 mg/ml, preferably between about
30 mg/ml to about 90 mg/ml.
[0103] Although intact collagen is preferred, denatured collagen,
commonly known as gelatin, can also be used in the compositions of
the invention. Gelatin may have the added benefit of being
degradable faster than collagen.
[0104] Because of its tacky consistency, nonfibrillar collagen is
generally preferred for use in compositions of the invention that
are intended for use as bioadhesives. The term "nonfibrillar
collagen" refers to any modified or unmodified collagen material
that is in substantially nonfibrillar form at pH 7, as indicated by
optical clarity of an aqueous suspension of the collagen.
[0105] Collagen that is already in nonfibrillar form may be used in
the compositions of the invention. As used herein, the term
"nonfibrillar collagen" is intended to encompass collagen types
that are nonfibrillar in native form, as well as collagens that
have been chemically modified such that they are in nonfibrillar
form at or around neutral pH. Collagen types that are nonfibrillar
(or microfibrillar) in native form include types IV, VI, and
VII.
[0106] Chemically modified collagens that are in nonfibrillar form
at neutral pH include succinylated collagen and methylated
collagen, both of which can be prepared according to the methods
described in U.S. Pat. No. 4,164,559, issued Aug. 14, 1979, to
Miyata et al., which is hereby incorporated by reference in its
entirety. Due to its inherent tackiness, methylated collagen is
particularly preferred for use in bioadhesive compositions, as
disclosed in commonly owned U.S. Pat. No. 5,614,587 to Rhee et
al.
[0107] Collagens for use in the crosslinkable compositions of the
present invention may start out in fibrillar form, then rendered
nonfibrillar by the addition of one or more fiber disassembly
agent. The fiber disassembly agent must be present in an amount
sufficient to render the collagen substantially nonfibrillar at pH
7, as described above. Fiber disassembly agents for use in the
present invention include, without limitation, various
biocompatible alcohols, amino acids, inorganic salts, and
carbohydrates, with biocompatible alcohols being particularly
preferred. Preferred biocompatible alcohols include glycerol and
propylene glycol. Non-biocompatible alcohols, such as ethanol,
methanol, and isopropanol, are not preferred for use in the present
invention, due to their potentially deleterious effects on the body
of the patient receiving them. Preferred amino acids include
arginine. Preferred inorganic salts include sodium chloride and
potassium chloride. Although carbohydrates, such as various sugars
including sucrose, may be used in the practice of the present
invention, they are not as preferred as other types of fiber
disassembly agents because they can have cytotoxic effects in
vivo.
[0108] Because it is opaque and less tacky than nonfibillar
collagen, fibrillar collagen is less preferred for use in
bioadhesive compositions. However, as disclosed in commonly owned,
U.S. application Ser. No. 08/476,825, fibrillar collagen, or
mixtures of nonfibrillar and fibrillar collagen, may be preferred
for use in adhesive compositions intended for long-term persistence
in vivo, if optical clarity is not a requirement.
[0109] For those compositions intended to be used in tissue
augmentation, fibrillar collagen is preferred because it tends to
form stronger crosslinked gels having greater long-term persistency
in vivo than those prepared using nonfibrillar collagen.
[0110] Any of the hydrophilic polymers herein must contain, or be
activated to contain, functional groups, i.e., nucleophilic or
electrophilic groups, which enable crosslinking. Activation of PEG
is discussed below; it is to be understood, however, that the
following discussion is for purposes of illustration and analogous
techniques may be employed with other polymers.
[0111] With respect to PEG, first of all, various functionalized
polyethylene glycols have been used effectively in fields such as
protein modification (see Abuchowski et al., Enzymes as Drugs, John
Wiley & Sons: New York, N.Y. (1981) pp. 367-383; and Dreborg et
al., Crit. Rev. Therap. Drug Carrier Syst. (1990) 6:315), peptide
chemistry (see Mutter et al., The Peptides, Academic: New York,
N.Y. 2:285-332; and Zalipsky et al., Int. J. Peptide Protein Res.
(1987) 30:740), and the synthesis of polymeric drugs (see Zalipsky
et al., Eur. Polym. J. (1983) 19:1177; and Ouchi et al., J.
Macromol. Sci. Chem. (1987) A24:1011).
[0112] Activated forms of PEG, including multifunctionally
activated PEG, are commercially available, and are also easily
prepared using known methods. For example, see Chapter 22 of
Poly(ethylene Glycol) Chemistry: Biotechnical and Biomedical
Applications, J. Milton Harris, ed., Plenum Press, NY (1992); and
Shearwater Polymers, Inc. Catalog, Polyethylene Glycol Derivatives,
Huntsville, Ala. (1997-1998).
[0113] Structures for some specific, tetrafunctionally activated
forms of PEG are shown in FIGS. 1 to 10, as are generalized
reaction products obtained by reacting the activated PEGs with
multi-amino PEGs, i.e., a PEG with two or more primary amino
groups. The activated PEGs illustrated have a pentaerythritol
(2,2-bis(hydroxymethyl)-1,3-propanediol) core. Such activated PEGs,
as will be appreciated by those in the art, are readily prepared by
conversion of the exposed hydroxyl groups in the PEGylated polyol
(i.e., the terminal hydroxyl groups on the PEG chains) to
carboxylic acid groups (typically by reaction with an anhydride in
the presence of a nitrogenous base), followed by esterification
with N-hydroxysuccinimide, N-hydroxysulfosuccinimide, or the like,
to give the polyfunctionally activated PEG.
[0114] FIG. 1 shows the reaction of tetrafunctionally activated PEG
succinimidyl glutarate, referred to herein as "SG-PEG," with
multi-amino PEG, and the reaction product obtained thereby.
[0115] Another activated form of PEG is PEG succinimidyl propionate
("SE-PEG"). The structural formula for tetrafunctionally activated
SE-PEG and the reaction product obtained upon reaction with
multi-amino PEG are shown in FIG. 2.
[0116] Analogous activated forms of PEG are PEG succinimidyl
butylate and PEG succinimidyl acetate, the structures of which are
shown in FIGS. 3 and 4, respectively, along with the reaction
products obtained upon reaction with multi-amino PEG. SE-PEG, PEG
succinimidyl butylate, and PEG succinimidyl acetate are sometimes
referred to as "PEG succinimidyl" (PEG--S); see U.S. Pat. No.
5,328,955 to Rhee et al.
[0117] Another functionally activated form of PEG is referred to as
"PEG succinimidyl succinamide" (SSA-PEG). The structural formula
for the tetrafunctionally activated form of this compound and the
reaction product obtained by reacting it with multi-amino PEG are
shown in FIG. 5. In the structure of FIG. 5, an ethylene
(--CH.sub.2CH.sub.2--) group is shown adjacent to the succinimidyl
ester; it is to be understood, however, that as with the PEG
succinimidyl compounds, related structures containing a methylene
linkage, an n-propylene linkage, or the like, are also
possible.
[0118] Yet another activated form of PEG is PEG succininidyl
carbonate (SC-PEG). The structural formula of tetrafunctionally
activated SC-PEG and the conjugate formed by reacting it with
multi-amino PEG are shown in FIG. 6.
[0119] PEG can also be derivatized to form functionally activated
PEG propionaldehyde (A-PEG), the tetrafunctionally activated form
of which is shown in FIG. 7, as is the conjugate formed by the
reaction of A-PEG with multi-amino PEG.
[0120] Yet another form of activated polyethylene glycol is
functionally activated PEG glycidyl ether (E-PEG), of which the
tetrafunctionally activated compound is shown in FIG. 8, as is the
conjugate formed by reacting such with multi-amino PEG.
[0121] Another activated derivative of polyethylene glycol is
functionally activated PEG-isocyanate (I-PEG), which is shown in
FIG. 9, along with the conjugate formed by reacting such with
multi-amino PEG.
[0122] Another activated derivative of polyethylene glycol is
functionally activated PEG-vinylsulfone (V-PEG), which is shown in
FIG. 10, along with the conjugate formed by reacting such with
multi-amino PEG.
[0123] Activation with succinimidyl groups to convert terminal
hydroxyl groups to reactive esters is one technique for preparing a
synthetic hydrophilic polymer with electrophilic moieties suitable
for reaction with nucleophiles such as primary amines, thiols, and
hydroxyl groups. Other activating agents for hydroxyl groups
include carbonyldiimidazole and sulfonyl chloride. However, as
discussed in part (B) of this section, a wide variety of
electrophilic groups may be advantageously employed for reaction
with corresponding nucleophiles. Examples of such electrophilic
groups include acid chloride groups; anhydrides, ketones,
aldehydes, isocyanate, isothiocyanate, epoxides, and olefins,
including conjugated olefins such as ethenesulfonyl
(--SO.sub.2CH.dbd.CH.sub.2) and analogous functional groups.
[0124] Hydrophilic di- or poly-nucleophilic polymers of the present
composition are exemplified in FIGS. 1 -10 by multi-amino PEG.
Various forms of multi-amino PEG are commercially available from
Shearwater Polymers (Huntsville, Ala.) and from Texaco Chemical
Company (Houston, Tex.) under the name "Jeffamine". Multi-amino
PEGs useful in the present invention include Texaco's Jeffamine
diamines ("D" series) and triamines ("T" series), which contain two
and three primary amino groups per molecule. Analogous
poly(sulfhydryl) PEGs are also available from Shearwater Polymers,
e.g., in the form of pentaerythritol poly(ethylene glycol) ether
tetra-sulfhydryl (molecular weight 10,000).
[0125] 2. Hydrophobic Polymers
[0126] The crosslinkable compositions of the invention can also
include hydrophobic polymers, although for most uses hydrophilic
polymers are preferred. Polylactic acid and polyglycolic acid are
examples of two hydrophobic polymers that can be used. With other
hydrophobic polymers, only short-chain oligomers should be used,
containing at most about 14 carbon atoms, to avoid
solubility-related problems during reaction.
[0127] 3. Low Molecular Weight Components
[0128] As indicated above, the molecular core of one or two of the
crosslinkable components can also be a low molecular weight
compound, i.e., a C.sub.2-C.sub.14 hydrocarbyl group containing
zero to 2 heteroatoms selected from N, O, S and combinations
thereof. Such a molecular core can be substituted with nucleophilic
groups or with electrophilic groups.
[0129] When the low molecular weight molecular core is substituted
with primary amino groups, the component may be, for example,
ethylenediamine (H.sub.2N--CH.sub.2CH.sub.2--NH.sub.2),
tetramethylenediamine (H.sub.2N--(CH.sub.4)--NH.sub.2),
pentamethylenediamine (cadaverine)
(H.sub.2N--(CH.sub.5)--NH.sub.2), hexamethylenediamine
(H.sub.2N--(CH.sub.6)--NH.sub.2), bis(2-aminoethyl)amine
(HN--[CH.sub.2CH.sub.2--NH.sub.2].sub.2), or
tris(2-aminoethyl)amine
(N--[CH.sub.2CH.sub.2--NH.sub.2].sub.3).
[0130] Low molecular weight diols and polyols include
trimethylolpropane, di(trimethylol propane), pentaerythritol, and
diglycerol, all of which require activation with a base in order to
facilitate their reaction as nucleophiles. Such diols and polyols
may also be functionalized to provide di- and poly-carboxylic
acids, functional groups that are, as noted earlier herein, also
useful as nucleophiles under certain conditions. Polyacids for use
in the present compositions include, without limitation,
trimethylolpropane-based tricarboxylic acid, di(trimethylol
propane)-based tetracarboxylic acid, heptanedioic acid, octanedioic
acid (suberic acid), and hexadecanedioic acid (thapsic acid), all
of which are commercially available and/or readily synthesized
using known techniques.
[0131] Low molecular weight di- and poly-electrophiles include, for
example, disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)
suberate (BS.sub.3), dithiobis(succinimidylpropionate) (DSP),
bis(2-succinimidooxycarbonyloxy) ethyl sulfone (BSOCOES), and
3,3'-dithiobis(sulfosuccinimidylpropionate (DTSPP), and their
analogs and derivatives. The aforementioned compounds are
commercially available from Pierce (Rockford, Ill.). Such di- and
poly-electrophiles can also be synthesized from di- and polyacids,
for example by reaction with an appropriate molar amount of
N-hydroxysuccinimide in the presence of DCC. Polyols such as
trimethylolpropane and di(trimethylol propane) can be converted to
carboxylic acid form using various known techniques, then further
derivatized by reaction with NHS in the presence of DCC to produce
trifunctionally and tetrafunctionally activated polymers.
[0132] D. Storage and Handling
[0133] Because crosslinkable components containing electrophilic
groups react with water, the electrophilic component or components
are generally stored and used in sterile, dry form to prevent
hydrolysis. Processes for preparing synthetic hydrophilic polymers
containing multiple electrophilic groups in sterile, dry form are
set forth in commonly assigned U.S. Pat. No. 5,643,464 to Rhee et
al. For example, the dry synthetic polymer may be compression
molded into a thin sheet or membrane, which can then be sterilized
using gamma or, preferably, e-beam irradiation. The resulting dry
membrane or sheet can be cut to the desired size or chopped into
smaller size particulates.
[0134] Components containing multiple nucleophilic groups are
generally not water-reactive and can therefore be stored either dry
or in aqueous solution. If stored as a dry, particulate, solid, the
various components of the crosslinkable composition may be blended
and stored in a single container. Admixture of all components with
water, saline, or other aqueous media should not occur until
immediately prior to use.
[0135] In an alternative embodiment, both components can be mixed
together in a single aqueous medium in which they are both
unreactive, i.e. such as in a low pH buffer. Thereafter, they can
be sprayed onto the targeted tissue site along with a high pH
buffer, after which they will rapidly react and form a gel.
[0136] Suitable liquid media for storage of crosslinkable
compositions include aqueous buffer solutions such as monobasic
sodium phosphate/dibasic sodium phosphate, sodium carbonate/sodium
bicarbonate, glutamate or acetate, at a concentration of 0.5 to 300
mM. In general, a sulfhydryl-reactive component such as PEG
substituted with maleimido groups or succinimidyl esters is
prepared in water or a dilute buffer, with a pH of between around 5
to 6. Buffers with pKs between about 8 and 10.5 for preparing a
polysulfhydryl component such as sulfhydryl-PEG are useful to
achieve fast gelation time of compositions containing mixtures of
sulfhydryl-PEG and SG-PEG. These include carbonate, borate and
AMPSO
(3--[(1,1-dimethyl-2-hydroxyethyl)amino]2-hydroxy-propane-sulfonic
acid). In contrast, using a combination of maleimidyl PEG and
sulfhydryl-PEG, a pH of around 5 to 9 is preferred for the liquid
medium used to prepare the sulfhydryl PEG. A particularly preferred
composition for hemostatic applications to actively bleeding tissue
sites comprises a mixture of maleimidyl and succinimidyl PEG as the
first component, and sulfhydryl PEG as the second component. Such
compositions produce gels with enhanced biodegradability and
superior gel times when compared to compositions having only
maleimidyl PEG or succinimicyl PEG alone.
[0137] E. Other Components of the Crosslinkable Composition
[0138] In order to enhance matrix strength, it may be generally
desirable to add a "tensile strength enhancer" to the composition.
Such tensile strength enhancers preferably comprise micron-size,
preferably 5 to 40 microns in diameter and 20 to 5000 microns in
length, high tensile strength fibers, usually with glass transition
temperatures well above 37.degree. C.
[0139] Suitable tensile strength enhancers for use in the present
invention include, inter alia, collagen fibers, polyglycolide and
polylactide fibers, as well as other organic tensile strength
enhancers and inorganic tensile strength enhancers. A particularly
useful tensile strength enhancer is Vicryl.RTM.
(polyglycolide:polylactide, 90:10) The use of tensile strength
enhancers, which are part of the broader category of "fillers," are
well known. For example, silicone gums, when cross-linked with
peroxides, are weak gels a with tensile strength on the order of
only about 34 N/cm.sup.2. When suitably compounded with reinforcing
fillers, the tensile strength of these gums may increase as much as
fifty-fold. Lichtenwalner, H. K. and Sprung, M. N., in Mark, H. F.,
Gaylord, N. G., and Bikales, N. M., Eds., Encyclopedia of Polymer
Science and Technology, Vol. 12, p. 535, John Wiley, New York,
1970. Suitable tensile strength enhancers are those that have
inherent high tensile strength and also can interact by covalent or
non-covalent bonds with the polymerized gel network. The tensile
strength enhancer should bond to the gel, either mechanically or
covalently, in order to provide tensile support. Tensile strengths
of polyglycolide resorbable sutures are approximately 89,000
N/cm.sup.2; that of collagen fibers is 5000-10,000 N/cm.sup.2
(Hayashi, T., in Biomedical Applic. of Polym. Mater., Tsuruta, T.
et al., Eds., CRC Press, Boca Raton, Fla., 1993).
[0140] The crosslinkable compositions can also be prepared to
contain various imaging agents such as iodine or barium sulfate, or
fluorine, in order to aid visualization of the compositions after
administration via X-ray or .sup.19F-MRI, respectively.
[0141] For use in tissue adhesion as discussed below, it may also
be desirable to incorporate proteins such as albumin, fibrin or
fibrinogen into the crosslinked polymer composition to promote
cellular adhesion.
[0142] In addition, the introduction of hydrocolloids such as
carboxymethylcellulose may promote tissue adhesion and/or
swellability.
[0143] III. Crosslinking
[0144] Any number of crosslinking techniques may be used to effect
crosslinking of the aforementioned compositions. Generally,
however, components A, B and C are selected such that crosslinking
occurs fairly rapidly upon admixture of all components of the
crosslinkable composition with an aqueous medium.
[0145] For crosslinking compositions in which one or more
components contain hydroxyl and/or thiol groups as nucleophilic
moieties, the aqueous medium with which the crosslinking
composition (or components thereof) are admixed should contain a
basic reagent that is effective to increase the nucleophilic
reactivity of the hydroxyl and/or thiol group (and thus the rate of
the nucleophile-electrophile reactions) but that is preferably
non-nucleophilic so as to avoid reaction with any electrophilic
groups present. A catalytic amount of base can be used, and/or a
base-containing buffer. In an alternative but less preferred
embodiment, a reactive base can be used that participates as a
reactant in the crosslinking reaction.
[0146] In general, the combined concentration of all crosslinkable
components in the aqueous admixture will be in the range of about 1
to 50 wt. %, generally about 2 to 40 wt. %. However, a preferred
concentration of the crosslinkable composition in the aqueous
medium--as well as the preferred concentration of each
crosslinkable component therein--will depend on a number of
factors, including the type of component, its molecular weight, and
the end use of the composition. For example, use of higher
concentrations of the crosslinkable components, or using highly
functionalized components, will result in the formation of a more
tightly crosslinked network, producing a stiffer, more robust gel.
As such, compositions intended for use in tissue augmentation will
generally employ concentrations of crosslinkable components that
fall toward the higher end of the preferred concentration range.
Compositions intended for use as bioadhesives or in adhesion
prevention do not need to be as firm and may therefore contain
lower concentrations of the crosslinkable components. The
appropriate concentration of each crosslinkable component can
easily be optimized to achieve a desired gelation time and gel
strength using routine experimentation.
[0147] IV. Administration and Use
[0148] The compositions of the present invention may be
administered before, during or after crosslinking. Certain uses,
which are discussed in greater detail below, such as tissue
augmentation, may require the compositions to be crosslinked before
administration, whereas other applications, such as tissue
adhesion, require the compositions to be administered before
crosslinking has reached "equilibrium." The point at which
crosslinking has reached equilibrium is defined herein as the point
at which the composition no longer feels tacky or sticky to the
touch.
[0149] The compositions of the present invention are generally
delivered to the site of administration in such a way that the
individual components of the composition come into contact with one
another for the first time at the site of administration, or
immediately preceding administration. Thus, the compositions of the
present invention are preferably delivered to the site of
administration using an apparatus that allows the components to be
delivered separately. Such delivery systems usually involve a
multi-compartment spray device. Alternatively, the components can
be delivered separately using any type of controllable extrusion
system, or they can be delivered manually in the form of separate
pastes, liquids or dry powders, and mixed together manually at the
site of administration. Many devices that are adapted for delivery
of multi-component tissue sealants/hemostatic agents are well known
in the art and can also be used in the practice of the present
invention.
[0150] Yet another way of delivering the compositions of the
present invention is to prepare the reactive components in inactive
form as either a liquid or powder. Such compositions can then be
activated after application to the tissue site, or immediately
beforehand, by applying an activator. In one embodiment, the
activator is a buffer solution having a pH that will activate the
composition once mixed therewith. Still another way of delivering
the compositions is to prepare preformed sheets, and apply the
sheets as such to the site of administration.
[0151] The crosslinkable compositions of the present invention can
be used in a variety of different applications. In general, the
present compositions can be adapted for use in any tissue
engineering application where synthetic gel matrices are currently
being utilized. For example, the compositions of the present
invention are useful as tissue sealants, in tissue augmentation, in
tissue repair, as hemostatic agents, in preventing tissue
adhesions, in providing surface modifications, and in
drug/cell/gene delivery applications. One of skill in the art can
easily determine the appropriate administration protocol to use
with any particular composition having a known gel strength and
gelation time. A more detailed description of several specific
applications is given below:
[0152] Tissue Sealants and Adhesives: In a preferred application,
the compositions described herein can be used for medical
conditions that require a coating or sealing layer to prevent the
leakage of gases, liquid or solids. The method entails applying
both components to the damaged tissue or organ to seal 1) vascular
and or other tissues or organs to stop or minimize the flow of
blood; 2) thoracic tissue to stop or minimize the leakage of air;
3) gastrointestinal tract or pancreatic tissue to stop or minimize
the leakage of fecal or tissue contents; 4) bladder or ureters to
stop or minimize the leakage of urine; 5) dura to stop or minimize
the leakage of CSF; and 6) skin or serosal tissue to stop the
leakage of serosal fluid. These compositions may also be used to
adhere tissues together such as small vessels, nerves or dermal
tissue. The material can be used 1) by applying it to the surface
of one tissue and then a second tissue may be rapidly pressed
against the first tissue or 2) by bringing the tissues in close
juxtaposition and then applying the material. In addition, the
compositions can be used to fill spaces in soft and hard tissues
that are created by disease or surgery.
[0153] Biologically Active Agent Delivery: The crosslinked
compositions of the invention may also be used for localized
delivery of various drugs and other biologically active agents.
Biologically active agents such as growth factors may be delivered
from the composition to a local tissue site in order to facilitate
tissue healing and regeneration.
[0154] The term "biologically active agent" refers to an organic
molecule that exerts biological effects in vivo. Examples of
biologically active agents include, without limitation, enzymes,
receptor antagonists or agonists, hormones, growth factors,
autogenous bone marrow, antibiotics, antimicrobial agents and
antibodies. The term "biologically active agent" is also intended
to encompass various cell types and genes that can be incorporated
into the compositions of the invention.
[0155] Preferred biologically active agents for use in the
compositions of the present invention are cytokines, such as
transforming growth factors (TGFs), fibroblast growth factors
(FGFs), platelet derived growth factors (PDGFs), epidermal growth
factors (EGFs), connective tissue activated peptides (CTAPs),
osteogenic factors, and biologically active analogs, fragments, and
derivatives of such growth factors. Members of the transforming
growth factor (TGF) supergene family, which are multifunctional
regulatory proteins, are particularly preferred. Members of the TGF
supergene family include the beta transforming growth factors (for
example, TGF-.beta. 1, TGF-.beta.2, TGF-.beta.3); bone
morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4,
BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors
(for example, fibroblast growth factor (FGF), epidermal growth
factor (EGF), platelet-derived growth factor (PDGF), insulin-like
growth factor (IGF)); Inhibins (for example, Inhibin A, Inhibin B);
growth differentiating factors (for example, GDF-1); and Activins
(for example, Activin A, Activin B, Activin AB). Growth factors can
be isolated from native or natural sources, such as from mammalian
cells, or can be prepared synthetically, such as by recombinant DNA
techniques or by various chemical processes. In addition, analogs,
fragments, or derivatives of these factors can be used, provided
that they exhibit at least some of the biological activity of the
native molecule. For example, analogs can be prepared by expression
of genes altered by site-specific mutagenesis or other genetic
engineering techniques.
[0156] Biologically active agents may be incorporated into the
crosslinked synthetic polymer composition by admixture.
Alternatively, the agents may be incorporated into the crosslinked
polymer matrix by binding these agents to the functional groups on
the synthetic polymers. Processes for covalently binding
biologically active agents such as growth factors using
functionally activated polyethylene glycols are described in
commonly assigned U.S. Pat. No. 5,162,430, issued Nov. 10, 1992, to
Rhee et al. Such compositions preferably include linkages that can
be easily biodegraded, for example as a result of enzymatic
degradation, resulting in the release of the active agent into the
target tissue, where it will exert its desired therapeutic
effect.
[0157] A simple method for incorporating biologically active agents
containing nucleophilic groups into the crosslinked polymer
composition involves mixing the active agent with a
polyelectrophilic component prior to addition of the
polynucleophilic component.
[0158] By varying the relative molar amounts of the different
components of the crosslinkable composition, it is possible to
alter the net charge of the resulting crosslinked polymer
composition, in order to prepare a matrix for the delivery of a
charged compound such as a protein or ionizable drug. As such, the
delivery of charged proteins or drugs, which would normally diffuse
rapidly out of a neutral carrier matrix, can be controlled.
[0159] For example, if a molar excess of a polynucleophilic
component is used, the resulting matrix has a net positive charge
and can be used to ionically bind and deliver negatively charged
compounds. Examples of negatively charged compounds that can be
delivered from these matrices include various drugs, cells,
proteins, and polysaccharides. Negatively charged collagens, such
as succinylated collagen, and glycosaminoglycan derivatives such as
sodium hyaluronate, keratan sulfate, keratosulfate, sodium
chondroitin sulfate A, sodium dermatan sulfate B, sodium
chondroitin sulfate C, heparin, esterified chondroitin sulfate C,
and esterified heparin, can be effectively incorporated into the
crosslinked polymer matrix as described above.
[0160] If a molar excess of a polyelectrophilic component is used,
the resulting matrix has a net negative charge and can be used to
ionically bind and deliver positively charged compounds. Examples
of positively charged compounds that can be delivered from these
matrices include various drugs, cells, proteins, and
polysaccharides. Positively charged collagens, such as methylated
collagen, and glycosaminoglycan derivatives such as esterified
deacetylated hyaluronic acid, esterified deacetylated desulfated
chondroitin sulfate A, esterified deacetylated desulfated
chondroitin sulfate C, deacetylated desulfated keratan sulfate,
deacetylated desulfated keratosulfate, esterified desulfated
heparin, and chitosan, can be effectively incorporated into the
crosslinked polymer matrix as described above.
[0161] Delivery of cells and genes: The crosslinked polymer
compositions of the present invention can also be used to deliver
various types of living cells or genes to a desired site of
administration in order to form new tissue. The term "genes" as
used herein is intended to encompass genetic material from natural
sources, synthetic nucleic acids, DNA, antisense-DNA and RNA.
[0162] When used to deliver cells, for example, mesenchymal stem
cells can be delivered to produce cells of the same type as the
tissue into which they are delivered. Mesenchymal stem cells are
not differentiated and therefore can differentiate to form various
types of new cells due to the presence of an active agent or the
effects (chemical, physical, etc.) of the local tissue environment.
Examples of mesenchymal stem cells include osteoblasts,
chondrocytes, and fibroblasts. Osteoblasts can be delivered to the
site of a bone defect to produce new bone; chondrocytes can be
delivered to the site of a cartilage defect to produce new
cartilage; fibroblasts can be delivered to produce collagen
wherever new connective tissue is needed; neurectodermal cells can
be delivered to form new nerve tissue; epithelial cells can be
delivered to form new epithelial tissues, such as liver, pancreas,
etc.
[0163] The cells or genes may be either allogeneic or xenogeneic in
origin. For example, the compositions can be used to deliver cells
or genes from other species that have been genetically modified.
Because the compositions of the invention are not easily degraded
in vivo, cells and genes entrapped within the crosslinked polymer
compositions will be isolated from the patient's own cells and, as
such, will not provoke an immune response in the patient. In order
to entrap the cells or genes within a crosslinked polymer matrix,
the cells or genes are pre-mixed with the polynucleophilic
component(s), and then the polyelectrophilic component(s) are added
to the mixture to form a crosslinked matrix, thereby entrapping the
cells or genes within the matrix. Alternatively, the initial
pre-mixing may be carried out with the polyelectrophilic
component(s), followed by subsequent addition of the
polynucleophilic component(s).
[0164] As discussed above for biologically active agents, when used
to deliver cells or genes, the synthetic polymers preferably also
contain biodegradable groups to aid in controlled release of the
cells or genes at the intended site of delivery.
[0165] Bioadhesives: As used herein, the terms "bioadhesive",
"biological adhesive", and "surgical adhesive" are used
interchangeably to refer to biocompatible compositions capable of
effecting temporary or permanent attachment between the surfaces of
two native tissues, or between a native tissue surface and either a
non-native tissue surface or a surface of a synthetic implant.
[0166] In a general method for effecting the attachment of a first
surface to a second surface, the crosslinkable composition is
applied to a first surface, which is then contacted with a second
surface to effect adhesion therebetween. Preferably, all reactive
components of the crosslinkable composition are first mixed to
initiate crosslinking, then delivered to the first surface before
substantial crosslinking has occurred. The first surface is then
contacted with the second surface, preferably immediately, to
effect adhesion. At least one of the first and second surfaces is
preferably a native tissue surface.
[0167] The two surfaces may be held together manually, or using
other appropriate means, while the crosslinking reaction is
proceeding to completion. Crosslinking is typically sufficiently
complete for adhesion to occur within about 5 to 60 minutes after
mixing of the first and second synthetic polymers. However, the
time required for complete crosslinking to occur is dependent on a
number of factors, including the type and molecular weight of each
reactive component, the degree of functionalization, and the
concentration of the crosslinkable composition (i.e., higher
concentrations result in faster crosslinking times).
[0168] At least one of the first and second surfaces is preferably
a native tissue surface. As used herein, the term "native tissue"
refers to biological tissues that are native to the body of the
patient being treated. As used herein, the term "native tissue" is
intended to include biological tissues that have been elevated or
removed from one part of the body of a patient for implantation to
another part of the body of the same patient (such as bone
autografts, skin flap autografts, etc.). For example, the
compositions of the invention can be used to adhere a piece of skin
from one part of a patient's body to another part of the body, as
in the case of a burn victim.
[0169] The other surface may be a native tissue surface, a
non-native tissue surface, or a surface of a synthetic implant. As
used herein, the term "non-native tissue" refers to biological
tissues that have been removed from the body of a donor patient
(who may be of the same species or of a different species than the
recipient patient) for implantation into the body of a recipient
patient (e.g., tissue and organ transplants). For example, the
crosslinkable polymer compositions of the present invention can be
used to adhere a donor cornea to the eye of a recipient
patient.
[0170] As used herein, the term "synthetic implant" refers to any
biocompatible material intended for implantation into the body of a
patient not encompassed by the above definitions for native tissue
and non-native tissue. Synthetic implants include, for example,
artificial blood vessels, heart valves, artificial organs, bone
prostheses, implantable lenticules, vascular grafts, stents, and
stent/graft combinations, etc.
[0171] Ophthalmic Applications: Because of their optical clarity,
the crosslinked polymer compositions of the invention are
particularly well suited for use in ophthalmic applications. For
example, a synthetic lenticule for correction of vision can be
attached to the Bowman's layer of the cornea of a patient's eye
using the methods of the present invention. As disclosed in
commonly assigned U.S. Pat. No. 5,565,519, issued Oct. 15, 1996 to
Rhee et al., a chemically modified collagen (such as succinylated
or methylated collagen) that is in substantially nonfibrillar form
at pH 7 can be crosslinked using a synthetic hydrophilic polymer,
then molded into a desired lenticular shape and allowed to complete
crosslinking. The resulting crosslinked collagen lenticule can then
be attached to the Bowman's layer of a de-epithelialized cornea of
a patient's eye using the methods of the present invention. By
applying the reaction mixture comprising the first and second
synthetic polymers to the anterior surface of the cornea, then
contacting the anterior surface of the cornea with the posterior
surface of the lenticule before substantial crosslinking has
occurred, electrophilic groups on the second synthetic polymer will
also covalently bind to collagen molecules in both the corneal
tissue and the lenticule to firmly anchor the lenticule in place.
Alternatively, the reaction mixture can be applied first to the
posterior surface of the lenticule, which is then contacted with
the anterior surface of the cornea.
[0172] The compositions of the present invention are also suitable
for use in vitreous replacement.
[0173] Tissue Augmentation: The crosslinkable compositions of the
invention can also be used for augmentation of soft or hard tissue
within the body of a mammalian subject. As such, they may be better
than currently marketed collagen-based materials for soft tissue
augmentation, because they are less immunogenic and more
persistent. Examples of soft tissue augmentation applications
include sphincter (e.g., urinary, anal, esophageal) augmentation
and the treatment of rhytids and scars. Examples of hard tissue
augmentation applications include the repair and/or replacement of
bone and/or cartilaginous tissue.
[0174] The compositions of the invention are particularly suited
for use as a replacement material for synovial fluid in
osteoarthritic joints, serving to reduce joint pain and improve
joint function by restoring a soft hydrogel network in the joint.
The crosslinked compositions can also be used as a replacement
material for the nucleus pulposus of a damaged intervertebral disk.
The nucleus pulposus of the damaged disk is first removed, and the
crosslinkable composition is then injected or otherwise introduced
into the center of the disk. The composition may either be
crosslinked prior to introduction into the disk, or allowed to
crosslink in situ.
[0175] In a general method for effecting augmentation of tissue
within the body of a mammalian subject, the reactive components of
the crosslinkable composition are injected simultaneously to a
tissue site in need of augmentation through a small-gauge (e.g.,
25-32 gauge) needle. Once inside the patient's body, the
nucleophilic groups on the polynucleophilic component(s) and the
electrophilic groups on the polyelectrophilic component(s) react
with each other to form a crosslinked polymer network in situ.
Electrophilic groups on the polyelectrophilic component(s) may also
react with primary amino groups on lysine residues of collagen
molecules within the patient's own tissue, providing for
"biological anchoring" of the compositions with the host
tissue.
[0176] Adhesion Prevention: Another use of the crosslinkable
compositions of the invention is to coat tissues in order to
prevent the formation of adhesions following surgery or injury to
internal tissues or organs. In a general method for coating tissues
to prevent the formation of adhesions following surgery, the
reactive components are mixed and a thin layer of the reaction
mixture is then applied to the tissues comprising, surrounding,
and/or adjacent to the surgical site before substantial
crosslinking has occurred. Application of the reaction mixture to
the tissue site may be by extrusion, brushing, spraying (as
described above), or by any other convenient means.
[0177] Following application of the reaction mixture to the
surgical site, crosslinking is allowed to continue in situ prior to
closure of the surgical incision. Once crosslinking has reached
equilibrium, tissues that are brought into contact with the coated
tissues will not adhere thereto. The surgical site can then be
closed using conventional means (sutures, etc.).
[0178] In general, compositions that achieve complete crosslinking
within a relatively short period of time (i.e., 5-15 minutes
following admixture of the reactive components) are preferred for
use in the prevention of surgical adhesions, so that the surgical
site may be closed relatively soon after completion of the surgical
procedure.
[0179] Coating Material for Synthetic Implants: Another use of the
crosslinked polymer compositions of the invention is as a coating
material for synthetic implants. In a general method for coating a
surface of a synthetic implant, the reactive components of the
crosslinkable composition are mixed with an aqueous medium, and a
thin layer of the reaction mixture is then applied to a surface of
the implant before substantial crosslinking has occurred. In order
to minimize cellular and fibrous reaction to the coated implant,
the reaction mixture is preferably prepared to have a net neutral
charge. Application of the reaction mixture to the implant surface
may be by extrusion, brushing, spraying (as described above), or by
any other convenient means. Following application of the reaction
mixture to the implant surface, crosslinking is allowed to continue
until complete crosslinking has been achieved.
[0180] Although this method can be used to coat the surface of any
type of synthetic implant, it is particularly useful for implants
where reduced thrombogenicity is an important consideration, such
as artificial blood vessels and heart valves, vascular grafts,
vascular stents, and stent/graft combinations. The method may also
be used to coat implantable surgical membranes (e.g., monofilament
polypropylene) or meshes (e.g., for use in hernia repair). Breast
implants may also be coated using the above method in order to
minimize capsular contracture.
[0181] The compositions of the present invention may also be used
to coat lenticules, which are made from either naturally occurring
or synthetic polymers.
[0182] Treatment of Aneurysm: The crosslinkable compositions of the
invention can be extruded or molded in the shape of a string or
coil, then dehydrated. The resulting dehydrated string or coil can
be delivered via catheter to the site of a vascular malformation,
such as an aneurysm, for the purpose of vascular occlusion and,
ultimately, repair of the malformation. The dehydrated string or
coil can be delivered in a compact size and will rehydrate inside
the blood vessel, swelling several times in size compared to its
dehydrated state, while maintaining its original shape.
[0183] Other Uses: As discussed in commonly assigned U.S. Pat. No.
5,752,974, issued May 19, 1998 to Rhee et al., the crosslinkable
polymer compositions of the invention can be used to block or fill
various lumens and voids in the body of a mammalian subject. The
compositions can also be used as biosealants to seal fissures or
crevices within a tissue or structure (such as a vessel), or
junctures between adjacent tissues or structures, to prevent
leakage of blood or other biological fluids.
[0184] The compositions can also be used as a large space-filling
device for organ displacement in a body cavity during surgical or
radiation procedures, for example, to protect the intestines during
a planned course of radiation to the pelvis.
[0185] The compositions of the invention can also be coated onto
the interior surface of a physiological lumen, such as a blood
vessel or Fallopian tube, thereby serving as a sealant to prevent
restenosis of the lumen following medical treatment, such as, for
example, balloon catheterization to remove arterial plaque deposits
from the interior surface of a blood vessel, or removal of scar
tissue or endometrial tissue from the interior of a Fallopian tube.
A thin layer of the reaction mixture is preferably applied to the
interior surface of the vessel (for example, via catheter)
immediately following mixing of the first and second synthetic
polymers. Because the compositions of the invention are not readily
degradable in vivo, the potential for restenosis due to degradation
of the coating is minimized. The use of crosslinked polymer
compositions having a net neutral charge further minimizes the
potential for restenosis.
[0186] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, the foregoing description is intended to illustrate and
not limit the scope of the invention. Other aspects, advantages and
modifications will be apparent to those skilled in the art to which
the invention pertains. All patents, patent applications, patent
publications, journal articles and other references cited herein
are incorporated by reference in their entireties.
[0187] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the compounds of the invention,
and are not intended to limit the scope of what the inventors
regard as their invention. Efforts have been made to ensure
accuracy with respect to numbers (e.g., amounts, temperature, etc.)
but some errors and deviations should be accounted for. Unless
indicated otherwise, parts are parts by weight, temperature is in
.degree. C. and pressure is at or near atmospheric.
EXAMPLE 1
Preparation of Crosslinked Compositions from Di-amino PEG
[0188] 0.15 grams of di-amino PEG (3400 MW, obtained from
Shearwater Polymers, Huntsville, Ala.) in 250 .mu.l of water was
mixed with 0.1 g of trifunctionally activated SC-PEG (5000 MW, also
obtained from Shearwater Polymers) using syringe-to-syringe mixing.
The reaction mixture was extruded onto a petri dish and formed a
soft gel at room temperature.
[0189] 0.15 gram of di-amino PEG in 250 .mu.l of water was mixed
with 0.1 g of tetrafunctionally activated SE-PEG (also from
Shearwater Polymers) using syringe-to-syringe mixing. The reaction
mixture was extruded onto a petri dish and formed a soft gel at
room temperature.
EXAMPLE 2
Preparation of Crosslinked Compositions from Di-amino PEG
[0190] The following stock solutions of various di-amino PEGs were
prepared:
[0191] Ten (10) grams of Jeffamine ED-2001 (obtained from Texaco
Chemical Company, Houston, Tex.) was dissolved in 9 ml of
water.
[0192] Ten (10) grams of Jeffamine ED-4000 (also obtained from
Texaco Chemical Company) was dissolved in 9 ml of water.
[0193] 0.1 grams of di-amino PEG (3400 MW, obtained from Shearwater
Polymers, Huntsville, Ala.) was dissolved in 300 .mu.l of
water.
[0194] Each of the three di-amino PEG solutions prepared above was
mixed with aqueous solutions of trifunctionally activated SC-PEG
(TSC-PEG, 5000 MW, also obtained from Shearwater Polymers) as set
forth in Table 3, below.
3TABLE 3 Preparation of Crosslinked Polymer Compositions Di-amino
PEG TSC-PEG + Aqueous Solvent 50 .mu.l 0 mg + 50 .mu.l water 50
.mu.l 10 mg + 50 .mu.l PBS 50 .mu.l 10 mg + 100 .mu.l PBS 250 .mu.l
50 mg + 500 .mu.l PBS
[0195] The solutions of di-amino PEG and TSC-PEG were mixed using
syringe-to-syringe mixing. Each of the materials was extruded from
the syringe and allowed to set for 1 hour at 37.degree. C. Each of
the materials formed a gel. In general, the gels became softer with
increasing water content; the gels containing the least amount of
aqueous solvent (water or PBS) were firmest.
EXAMPLE 3
Characterization of Crosslinked Multi-amino PEG Compositions
[0196] Fifty (50) milligrams of tetra-amino PEG (10,000 MW,
obtained from Shearwater Polymers, Huntsville, Ala.) in 0.5 ml PBS
was mixed, using syringe-to-syringe mixing, with 50 mg of
tetrafunctionally activated SE-PEG ("tetra SE-PEG", 10,000 MW, also
obtained from Shearwater Polymers) in 0.5 ml PBS or trifunctionally
activated SC-PEG ("tri SC-PEG", 5000 MW, also obtained from
Shearwater Polymers) in 0.5 ml PBS.
[0197] Syringes containing each of the two mixtures were incubated
at 37.degree. C. for approximately 16 hours. Both compositions
formed elastic gels. The gels were pushed out of the syringes and
sliced into 5-mm thick disks having a diameter of 5 mm, for use in
compression and swellability testing, as described below.
[0198] Compression force versus displacement for the two gels was
measured in the Instron Universal Tester, Model 4202, at a
compression rate of 2 mm per minute, using disks of the two gels
prepared as described above. Compression force (in Newtons) versus
gel displacement (in millimeters) is shown in FIGS. 1 and 2 for
gels prepared using the tetra SE-PEG and tri SC-PEG,
respectively.
[0199] Under compression forces as high as 30-35 Newtons, the gels
did not break, but remained elastic.
[0200] Disks of each of the two gels, prepared as described above,
were weighed and the dimensions (diameter and length) measured. The
disks were then immersed in PBS and incubated at 37.degree. C.
After 3 days incubation, the disks were removed from the PBS,
weighed, and measured. Results of swellability testing are shown in
Table 4, below.
4TABLE 4 Swellability Testing of Crosslinked Multi-amino PEG
Compositions Gel Weight Dimensions (in mm) (in grams)
(diameter/thickness) Crosslinking Before After Before After Agent
Swelling Swelling Swelling Swelling Tetra SE-PEG 0.116 0.310
5.0/5.0 7.1/8.1 Tri SC-PEG 0.131 0.287 5.0/6.0 6.4/8.5
[0201] As shown above, the gels swelled two to three times in
weight, as well as swelling an average of about 50% in both
diameter and thickness.
EXAMPLE 4
Preparation of Crosslinked Poly(lysine) Compositions
[0202] Ten (10) milligrams of poly-L-lysine hydrobromide (8,000 MW,
obtained from Peninsula Laboratories, Belmont, Calif.) in 0.1 ml
phosphate buffer (0.2M, pH=6.6) was mixed with 10 mg of
tetrafunctionally activated SE-PEG (10,000 MW, obtained from
Shearwater Polymers, Huntsville, Ala. in 0.1 ml PBS. The
composition formed a soft gel almost immediately.
EXAMPLE 5
Preparation and Mechanical Testing of Crosslinked Multi-amino PEG
Compositions
[0203] Gels comprising tetra-amino PEG (10,000 MW, obtained from
Shearwater Polymers, Huntsville, Ala.) and 1-4% (by weight) of
tetrafunctionally activated SE-PEG ("tetra SE-PEG", 10,000 MW, also
obtained from Shearwater Polymers) were prepared by mixing the
tetra-amino PEG (at a concentration of 25 mg/ml in water) with the
tetra SE-PEG (in PBS) in a petri dish. The resulting tetra-amino
PEG/SE-PEG mixtures were incubated for 16 hours at 37.degree.
C.
[0204] The mixture containing 1% SE-PEG did not form a gel due to
the low SE-PEG concentration. The mixture containing 2% SE-PEG
formed a gel at some point during the 16-hour incubation period.
The mixtures containing 3 and 4% SE-PEG formed gels within
approximately 4-6 minutes of mixing. The gel containing 2% SE-PEG
was readily extrudable through a 30-gauge needle; the gel
containing 3% SE-PEG could be extruded through a 27-gauge
needle.
[0205] The effect of elevated temperature on gel formation was
evaluated. Gels comprising tetra-amino PEG and 2.5% (by weight)
tetra SE-PEG were prepared and incubated at temperatures of
37.degree. C. and 40.degree.-50.degree. C. Elevated temperature was
found to have a marked effect on gelation time: the tetra-amino
PEG/SE-PEG mixture incubated at 37.degree. C. formed a gel within
approximately 20-25 minutes, whereas mixtures incubated at
40.degree.-50.degree. C. formed gels within approximately 5
minutes. Both gels were extrudable through a 27-gauge needle.
[0206] The effect of pH on gel formation was evaluated. Gels
comprising tetra-amino PEG and 2.5% (by weight) tetra SE-PEG were
prepared as set forth in Table 5 below.
5TABLE 5 Effect of pH on Gel Formation of Tetra-amino PEG/Tetra
SE-PEG Formulations pH of Tetra- pH of Tetra pH of Resulting
Gelation Gelation amino PEG SE-PEG Mixture Time Temp. 10 4.1 6.9
10-15 45.degree. C. minutes 10 7.0 7.2 <5 45.degree. C.
minutes
[0207] Extrudability through a 27-gauge needle was evaluated for
gels comprising tetra-amino PEG and 1-3% (by weight) tetra SE-PEG.
The gels were contained within 1-cc syringes. The force required to
depress the syringe plunger at a rate of 5 centimeters per minute
was measured using the Instron Universal Tester, Model 4202.
Results of extrusion testing are presented in Table 6, below.
6TABLE 6 Extrusion of Tetra-amino PEG/Tetra SB-PEG Gels Through a
27-Gauge Needle Concentration of SE-PEG (by weight) Extrusion Force
(N) 1.5-2% 10-11 2-2.5% 52 2.5-3% 88
[0208] Extrusion forces of 100N or less are considered acceptable
for manual injection without the aid of a syringe assist
device.
[0209] Tensile strength (i.e., elasticity) of 3 mm thick gels
comprising tetra-amino PEG and 5, 5, and 10% (by weight) tetra
SE-PEG was measured using the Instron Universal Tester, Model 4202.
Gels of varying initial lengths were stretched at a rate of 10
millimeters per minute. Length of each gel, strain at failure
(change in length as a percentage of the initial length), and force
at failure are set forth in Table 7, below.
7TABLE 7 Tensile Strength of Tetra-amino PEG/Tetra SE-PEG Gels
SE-PEG Conc. Initial Length (wt. %) (cm) Strain at Failure Force at
Failure (N) 10 1.4 139% 0.4 10 1.9 99% 0.5 10 2.5 78% 0.5 5 1.3
111% 0.2 5 1.3 99% 0.2 5 1.6 94% 0.2 2.5 1.0 237% <0.1 2.5 1.5
187% <0.1 2.5 1.7 129% <0.1
[0210] Gels containing 5 and 10% tetra SE-PEG approximately doubled
in length prior to breaking. Gels containing 2.5% SE-PEG
approximately tripled in length prior to breaking, but were
considerably weaker (i.e., lower force at failure) than the more
highly crosslinked gels.
EXAMPLE 6
Effect of pH on Gel Formation of Tetra-amino PEG/Tetra SE-PEG
Formulations
[0211] Gels comprising various concentrations of tetra-amino PEG
and tetra SE-PEG at Ph 6, 7, and 8 were prepared in petri dishes.
Following mixing of the tetra-amino PEG and tetra SE-PEG, the
dishes were tilted repeatedly; the gelation time was considered to
be the point at which the formulation ceased to flow. The effect of
pH on gelation time of the various tetra-amino PEG/tetra SE-PEG
formulation at room temperature is shown in Table 8 below.
8TABLE 8 Effect of pH on Gel Formation of Tetra-amino PEG/Tetra
SE-PEG Formulations Tetra-amino PEG Tetra SE-PEG Conc. (mg/ml)
Conc. (mg/ml) pH Gelation Time 20 20 6 >90.9 min. 20 20 7 20.0
min. 20 20 8 14 min. 50 50 6 24.0 min. 50 50 7 3.5 min. 50 50 8
10.0 sec. 100 100 6 9.0 min. 100 100 7 47.0 sec. 100 100 8 10.0
sec. 200 200 6 2.0 min. 200 200 7 9.0 sec. 200 200 8 5.0 sec.
[0212] The time required for gel formation decreased with
increasing pH and increasing tri-amino PEG and tetra SE-PEG
concentrations.
EXAMPLE 7
Culturing of Cells in Crosslinked Multi-amino PEG Matrix
[0213] Thirty (30) milligrams of tetra-amino PEG (10,000 MW,
obtained from Shearwater Polymers, Huntsville, Ala.) was dissolved
in 0.6 ml PBS, then sterile filtered. Thirty (30) milligrams of
tetrafunctionally activated SE-PEG ("tetra SE-PEG", 10,000 MW, also
obtained from Shearwater Polymers) was dissolved in 0.6 of PBS,
then sterile filtered.
[0214] The solutions of tetra-amino PEG and tetra SE-PEG were mixed
together with a pellet containing human skin fibroblast ("HSF")
cells (CRL #1885, passage 4, obtained from American Tissue Type
Culture Collection, Rockville, Md.). Two hundred fifty (250)
microliters of the resulting cell-containing tetra-amino PEG/tetra
SE-PEG (PEG-PEG) solution was dispensed into each of two wells on a
48-well culture plate and allowed to gel for approximately 5
minutes at room temperature. One (1) milliliter of Dulbecco
Modified Eagle's Media (supplemented with 10% fetal bovine serum,
L-glutamine, penicillin-streptomycin, and non-essential amino
acids) was added to each of the two wells. The concentration of
cells was approximately 3.times. 10.sup.5 cells per milliliter of
tetra-amino PEG/tetra SE-PEG solution, or 7.5.times. 10.sup.5 cells
per well.
[0215] To prepare a control, a pellet of HSF cells were suspended
in 1.2 ml of complete media. Two hundred fifty (250) microliters of
the control mixture was dispensed into each of three wells on the
same 48-well culture plate as used above. Each well was estimated
to contain approximately 7.5.times. 10.sup. 5 cells. Each well was
given fresh media every other day.
[0216] Initially, the cell-containing tetra-amino PEG/tetra SE-PEG
gels were clear and the cells were found to be densely populated
and spheroidal in morphology, indicating that there was little
adhesion between the cells and the PEG/PEG gel (the cells would
normally assume a flattened, spindle-shaped morphology when adhered
to a substrate, such as to the treated plastic of the tissue
culture plates). After three 3 days incubation at 37.degree. C.,
the media in the wells containing the PEG/PEG gels was found to
have lightened in color (Dulbecco Modified Eagle's Media is
normally red in color), indicating a pH change in the media. This
indicated that the cells were alive and feeding. At 7 days
incubation at 37.degree. C., the cells were still spheroidal in
morphology (indicating lack of adhesion to the gel) and the media
had lightened even further, indicating that the cells were still
viable and continued to feed.
[0217] On day 7, the contents of each well were placed in a 10%
formalin solution for histological evaluation. According to
histological evaluation, an estimated 75% of the cells in the wells
containing the PEG/PEG gels appeared to be alive, but did not
appear to be reproducing.
[0218] The results of the experiment indicate that HSF cells are
viable in the tetra-amino PEG/tetra SE-PEG crosslinked gels, but
did not seem to adhere to the gel and did not appear to reproduce
while entrapped within the gel matrix. As described above,
adherence or non-adherence of cells to a substrate material can
influence the cells' morphology. In certain types of cells,
cellular morphology can, in turn, influence certain cellular
functions. Therefore, non-adherence of the cells to the PEG-PEG gel
matrix may be an advantage in the delivery of particular cell types
whose function is influenced by cell morphology. For example, the
ability of cartilage cells to produce extracellular matrix
materials is influenced by cellular morphology: when the cells are
in the flattened, spindle-shaped configuration, the cells are in
reproductive mode; when the cells are in the spheroidal
configuration, reproduction stops, and the cells begin to produce
extracellular matrix components.
[0219] Because the PEG-PEG gels are not readily degraded in vivo,
the gels may be particularly useful in cell delivery applications
where it is desirable that the cells remain entrapped within the
matrix for extended periods of time.
EXAMPLE 8
Preparation of a Penta-erythritol-Based Tissue Sealant
Composition
[0220] Penta-erythritol tetrakis (3-mercapto-proprionate), mol. wt.
489 ("PESH--P," obtained from Aldrich Chemical Company, Milwaukee,
Wis.), 1.08 g, and penta-erythritol tetra-acrylate, mol. wt. 352
("PETA," also obtained from Aldrich), 1.0 g, are mixed together in
the presence of 5 to 10 ,g of a polyoxypropylene triamine ("T403,"
Jeffamine, Texaco Chemical Co., Houston, Tex.), which serves as a
base. All reactive species are liquids. The PESH-P and PETA are not
miscible in water. Accordingly, PETA is warmed to about 40.degree.
C. to form a liquid prior to mixing with PESH-P and T403. Within 1
to 5 minutes after mixing, depending on the level of T403, gelation
begins. The gel is allowed to cure for several hours, followed by
one hour of hydration at 37.degree. C. Thereafter, the tensile
strength of the gel is 0.88.+-.0.3 MPa. When such gels are left in
physiological saline, pH 6.7, they are stable for more than 40
days, and only swell about 20%. Burst strength data shows only
moderate adhesion to hide grindate. This would be expected, since
there is no chemical bonding of sulfhydryl or acrylate to protein
using PETA-P/PESH mixtures. In three tests of burst strength, burst
pressures of 20-40 mm Hg were observed.
EXAMPLE 9
Tensile Strength Evaluation
[0221] Materials and Methods: Penta-erythritol polyethylene glycol
ether tetrathiol, 10,000 mol. wt. ("COH.sub.206"), penta-erythritol
polyethylene glycol ether tetra succinimidyl-glutarate, 10,000 mol.
wt. ("COH102"), and penta-erythritol polyethylene glycol ether
tetra amino, 10,000 mol. wt. ("COH204"), were purchased from
Shearwater Polymers, Inc. (Huntsville, Ala.) Cyanoacrylate,
"Superglue," was purchased over the counter. Gelatin, 70-100 Bloom,
was purchased from Sigma (Saint Louis, Mo.) Sulfoethylene glycol
bis succinimidyl succinate ("S-EGS"), dimethyl suberimidate
("DMS"), and dissuccinimidyl glutarate ("DSG" ) were purchased from
Pierce Chemical Company, Rockford, Ill. Polyethylene glycol ("PEG")
200 mol. wt. di-acrylate ("PEG-di-acrylate"); PEG, 1,000 mol. wt.
di-methacrylate ("PEG-di-methacrylate"); and 2-hydroxy-ethyl
methacrylate ("HEMA") were purchased from Polysciences, Inc.,
Warrington, Pa. Polypropylene ("PPO"), 540 mol. wt. di-acrylate
("PPO-di-acrylate"); PPO, 230 mol. wt. bis-2-aminopropyl ether
("PPO-di-amino 230"); PPO, 2,000 mol. wt. bis-2-aminopropyl ether
("PPO-di-amino 2,000"); polytetrahydrofuran bis (3-aminopropyl)
("PTMO"), 350 mol. wt. ("PTMO 350"); PTMO, 1,100 mol. wt. ("PTMO
1,100"); PESH-P, 489 mol. wt.; PETA, 352 mol. wt.; and potassium
meta-bisulfite were purchased from Aldrich Chemical Company,
Milwaukee, Wis.
[0222] Ammonium persulfate was purchased from Biorad, Inc.,
Richmond, Calif. Methylated collagen was prepared from purified
bovine corium collagen, following a method modified from U.S. Pat.
No. 4,164,559 (see Example 11).
[0223] Gel preparation:
[0224] a. COH102/COH206: 100 mg COH102 were dissolved in 400 10.5
mM sodium phosphate, pH 6.0. 100 mg COH206 were dissolved in 400
.mu.l 300 mM sodium phosphate, pH 7.5. The two solutions were mixed
in a beaker and poured into a mold of approximately
8.times.0.5.times.0.5 cm. Gelation occurred in 2-3 minutes. The
sample was left at room temperature until dry. The dried matrix was
removed from the mold, and hydrated at 37.degree. C. for one hour
prior to the tensile strength test.
[0225] b. COH102/COH204: The sample was prepared as described in
a., except that the COH204 was substituted for COH206.
[0226] c. PETA/PESH-P: The sample was prepared as described in
Example 8.
[0227] d. Gelatin gels: 20% gelatin in sodium phosphate/sodium
carbonate buffer, pH 9.6, was mixed with different compounds as
indicated below and described in a., assuming 10-20 moles of active
amino per mole of gelatin, and using stoichiometric levels of the
other compound.
[0228] e. COH102/PPO-di-amino 2,000/PEG-di-acrylate: 615 mg COH102
was dissolved in 923 .mu.l ethanol, and mixed with 246 .mu.l
PPO-di-amino 2,000 and 246 .mu.l PEG-di-acrylate as described in
(a).
[0229] f. PETA/PPO-di-amino 230/PPO-di-amino 2,000: 500 .mu.l PETA,
630 .mu.l PPO-di-amino 230 and 150 .mu.l PPO-di-amino 2,000 are
mixed together as described in a.
[0230] g. COH102/PTMO: The gel was prepared as described in e, with
PTMO 1,100 substituted for the PPO-di-amino 2,000.
[0231] h. Cyanoacrylate: The glue was extruded onto water and
immediately hardened.
[0232] i. HEMA: 1.3 ml HEMA and 64 .mu.l PEG-di-acrylate were
dissolved in 600 .mu.l of 150 mM sodium phosphate buffer, pH 7.4,
and mixed with 40 mg ammonium persulfate in 100 .mu.l water. The
mixture was heated to 60-80.degree. C. for 4 hours.
[0233] j. COH102/COH206/methylated collagen: 25 mg methylated
collagen, 100 mg COH102, and 100 mg COH206 were dissolved in I ml
0.5 mM sodium phosphate, pH 6.
[0234] Tensile Strength Measurements:
[0235] The ends of the dried gels were secured, and then the
central regions of all samples were rehydrated for approximately 1
hour in physiological saline buffer, pH 6.7 at 37.degree. C. prior
to the test. Then, the matrices were extended to the point of
breakage in an Instron Model 4202 test apparatus (Instron, Inc.,
Canton, Mass.) that was fitted with a 100 N load cell. The peak
load was recorded and converted into ultimate stress using the
measured cross-section of the sample at the break point. Data were
also plotted as stress v. strain, using strain=.DELTA.L/L.sub.0,
where .DELTA.L is the extension, and L.sub.0 is the original sample
length. Tensile strength measurements were as follows:
9 Tensile Strength Material (N/cm.sup.2) HEMA >393 Cyanoacrylate
385 PETA/PESH-P 78 (n = 10) PETA/PTMO-di-amino 350/1,100 26 (n = 2)
PETA/PTMO-di-amino 1,100 34 PETA/PPO-di-amino 230/2,000 36 (n = 2)
PESH-P/PPO-di-acrylate 20 COH102/COH206/methylated collagen 37 (n =
3) COH102/PPO-di-amino 2,000/PEG-di-acrylate 200 10 (n = 2)
COH102/PTMO-di-amino 4 (n = 2) COH102/T403 5 COH206/PEG-di-acrylate
8 COH/206/PEG-di-methacrylate/PEG-diacryla- te 4
COH206/PEG-di-methacrylate 26 Gelatin/DMS 6 Gelatin/S-EGS 6 (n = 2)
Gelatin/PETA 5 Gelatin/DSS/T403 3 COH102/COH206 20% 5 (n = 4)
COH102/COH206 10% 10
EXAMPLE 10
[0236] High-strength adhesives based on COH102 and COH206 and a
comparison with adhesives prepared from PETA, PESH-P
(penta-erythritol tetrakis (3-mercapto-proprionate)), and
GLYC-20HS:
[0237] Summary:
[0238] Several types of gels were investigated as potential suture
replacement formulations. Gels based on penta-erythritol
derivatives exhibited high cohesive, but poor adhesive strength.
Gels based on a 3-armed succinimidyl glycerol-PEG exhibited low
cohesive strength, but good adhesive strength. Gels based on 60%
aqueous (w/v) COH102/COH206, to which various fibrous materials
were added, such as fibrous insoluble collagen, polyglycolide
sutures and glass wool, exhibited both good cohesive and adhesive
strengths.
[0239] High strength medical adhesives are of interest as
suture-replacements in closure of surgical incisions. In
particular, gels formed from PETA and PESH-P were shown to have
tensile strengths about 10.times. greater than those formed from
20% (w/v) solutions of COH102 and COH206. When PETA-PESH-P gels
were supplemented with fibrous or particulate polymers, gels with
even higher tensile strengths were observed.
[0240] This experiment describes the adhesive properties of
PETA/PESH-P and related gels, as well as both adhesive and tensile
properties of a formulation of COH102 and COH206 at 60% (w/v), to
which collagen and other polymers are added. Also described are
properties of gels formed from a 3-arm glycerol succinimide (NOF
Corp., Japan) and the above reagents.
[0241] Materials and methods:
[0242] PETA, PESH-P, and penta-erythritol tetrakis (3
mercaptoacetate) (PESH-A), polyethylene, surface activated 180
particle size, and polybutadiene, epoxy functionalized, epoxy E.W.
260, were purchased from Aldrich Chemical Co., Milwaukee, Wis.
GLYC-20HS (poly-oxyethylene glyceryl ether) succinimidyl succinate
2600 mw), DEPA-1 OH (poly-oxyethylene bis-amine 1040 mw) were
obtained from NOF Corporation, Japan. COH102 and COH206 were
reagent grade from Shearwater Polymers, Huntsville, Ala.
Polyethylene-co-acrylate-succinimidate (PE-AC-S) was synthesized
from a polyethylene-co-acrylate (approx. mol. wt. 400K with 5%
acrylate) purchased from Aldrich Chemical Company, Milwaukee, Wis.
Kensey-Nash insoluble collagen (Semed F) was purchased from
Kensey-Nash Corporation, Exton, Pa. Collagen Matrix, Inc, Franklin
Lakes, N.J., supplied a second type of insoluble collagen. Prolene
7-0 sutures were manufactured by Ethicon Corporation. Coarse
fibered collagen sheets were cut from the same coarse fibered
bovine corium collagen as that used for the burst test as described
in Prior, J. J., Wallace, D. G., Harner, A., and Powers, N., "A
sprayable hemostat containing fibrillar collagen, bovine thrombin,
and autologous plasma", Ann. Thor. Surg. 68, 479-485 (1999). These
collagen sheets served as a tissue model for further studies.
Smaller fiber collagen was prepared from re-precipitated
pepsin-digested bovine corium collagen manufactured by Collagen
Aesthetics, Inc., Palo Alto, Calif. Glass wool was purchased from
VWR Corporation. Poly-glycolide sutures, non-coated ("Dexon S")
were from Davis and Geck.
[0243] Gel formation for tensile strength measurements is described
above in Example 1. For burst tests, the apparatus used is
described in Wallace et al., supra. Approximately 1 ml of total
formula was sprayed or spread by spatula onto the coarse fibered
collagen sheet substrate and allowed to set. Water pressure was
applied after the formulation had reached the texture of a
relatively firm rubbery solid (no longer tacky), and the pressure
to rupture the seal was recorded as mm Hg.
[0244] 60% gels of COH102 and COH206 were prepared as follows:
COH102 was dissolved at 60% (w/v) in S-buffer (0.5 mM sodium
phosphate, pH 6.0) and COH206 was dissolved also at 60% in 300 mM
sodium phosphate at pH 7.5 or 8.9; or in 117 mM sodium phosphate,
183 mM sodium carbonate, pH 9.6 ("PC buffer"). In some cases the
above ratio of phosphate and carbonate were altered to give pH 9.44
for a faster set time. The pH used in each case was determined by
the rate of gelation desired. Various additives were added to such
a base formulation; e.g., Kensey-Nash and smaller fiber size
collagen was added at 28 mg/ml of final gel; glass wool was added
at 25 mg/ml; and polyglycolide sutures, at 40 mg/ml.
[0245] Results and Discussion:
[0246] The results are discussed below and shown in Tables 9, 10
and 11 that follow. A tensile strength of >60 N/cm.sup.2 is
considered to be "strong". A burst strength of >50 mm Hg is
considered to be "good adhesion".
[0247] Gels of PETA and PESH-P had shown good tensile strengths
(Example 8). However, when they were tested for adhesion to a
hydrated simulated tissue (coarse fibered collagen sheets) in the
burst test, they exhibited poor adhesion (<50 mm Hg burst
pressure). As shown below in Table 9, the formulation was then
modified to contain water soluble GLYC-20HS and DEPA-10H, or the
pair COH102 and COH206 (which alone in aqueous media gave good
adhesion to the collagen sheets). These materials had good tensile
strength (manual evaluation), but again poor adhesion to the
collagen sheets. The gel formed from GLYC-20HS and DEPA-10H also
had poor adhesion when no water was present in the formula. A
different result may be observed when these reagents are dissolved
in aqueous buffers, since they are water soluble.
[0248] However, when GLYC-20HS was the major component by mass, the
gels were weak but exhibited good adhesion in the burst test. Using
these particular combinations of components, it appeared that one
could achieve either high tensile strength or high adhesive
bonding, but not both.
10TABLE 9 Tensile Strength and Burst Strength of Gels Prepared with
NOF 3-arm Glycerol Succinimide Tensile Strength Burst Strength
Material (N/cm.sup.2) (mmHg) PETA 500 mg >60 23 PESH-P 540 .mu.l
T403 5 ul GLYC-20HS 50 mg >60 14.5 PETA 500 mg PESHP 540 .mu.l
DEPA-10H 9 mg PESH-A 216 ul >60 11 PETA 240 mg GLYC-20HS 40 mg
PETA 400 mg >60 15 COH102 100 mg PESH-P 440 .mu.l COH206 100 mg
DEPA-10H 8 mg GLYC-20HS 640 mg -- 25 DEPA-10H mg GLYC-20HS 400 mg
<30 >120 PESH-A 36 .mu.l T403 10 .mu.l GLYC-20HS 400 mg
<30 166, 194 PETA 50 mg PESH-A 72 .mu.l T403 20 .mu.l GLYC-20HS
200 mg <30 55 T403 19 .mu.l PESH-P 18 .mu.l
[0249] The ability of a succinimidyl-derivatized polyethylene
(PE-AC-S) to act as an effective tensile strength enhancer for
PETA-PESH-P gels and for COH102/206 gels was also assessed (Table
10). This material did not increase the tensile strength of these
gels, perhaps because it was not an extended filament, i.e. its
aspect ratio was not high enough.
11TABLE 10 Polyethylene-co-acrylate-succinimide ("PE-AC-S") as a
Tensile Strength Enhancer Material Tensile Strength (N/cm.sup.2)
PETA 400 mg 80 PESH-P 432 .mu.l (same as control with no PE-AC-S)
T403 8 .mu.l PE-AC-S 20 mg COH102 38 COH206 (60%) (weaker than
control with no PE-AC-S) +KN collagen (28 mg/ml) +PE-AC-S (40
mg/ml)
[0250] Table 11 also summarizes results with COH102 and COH206 plus
Kensey-Nash fibrillar collagen, which exhibited an enhanced tensile
strength over 20% and 60% (w/v) gels of COH102/206 alone.
Furthermore, the COH102/COH206/collagen formulation possessed good
adhesive bonding to the collagen sheets. Other additives, such as
hide grindate and Prolene 7-0 sutures also enhanced the gel
strength over controls. Some fillers, such as small fiber collagen,
polyethylene, and polybutadiene, did not exhibit tensile strength
enhancing properties. Finally, some fillers or combinations
thereof, such as glass wool and insoluble collagen plus
poly-glycolide sutures, exhibited a significant enhancement of
tensile strength, exceeding that seen with cyanoacrylate (385
N/cm.sup.2) (Example 1). Limited burst strength data were
collected, but they confirm that all these COH102/206 (60%)
formulations are highly adhesive to collagen surfaces, and thus
would be expected to adhere to tissues as well.
[0251] As shown in Table 11, the P-HEMA hydrogel is described in
Santin, M., et al., "Synthesis and characterization of a new
interpenetrated poly (2-hydroxyethylmethacrylate)-gelatin composite
polymer", Biomaterials 17, 1459-1467; and the
gelatin-PEG-di-acrylate is described in Nakayama, Y., and Matsuda,
T., "Photocurable surgical tissue adhesive glues composed of
photoreactive gelatin and poly(ethylene glycol) diacrylate", J.
Biomed. Biomat. Res. (Appl. Biomater.) 48, 511-521 (1999).
12TABLE 11 Tensile Strength and Burst Strength Tests Burst Material
Tensile Strength (N/cm.sup.2) Strength (mm Hg) COH102/206 2-12
100-200 20% GEL GELATIN- 3 PEG-DI- ACRYLATE** pHEMA 5-16 151 .+-.
34 HYDROGEL* PETA- 50-170 14,23 PESH-P PETA- 140-200 PESH-P + KN
collagen COH102/206 123 .+-. 39 268,216 (60%) + KNcollagen (n = 7)
COH102/206 180 (60%) + KNcollagen +7-0 Prolene sutures COH102/206
197,78 (60%) + hide grindate 94 COH102/206 27 (60%) no filler
COH102/206 27 (60%) + Small fiber collagen COH102/206 58 (60%) +
7-0 Prolene sutures coated COH102/206 14 (60%) + polyethylene
COH102/206 58,30 (60%) + polyethylene 28,21 + poly-butadiene
COH102/206 745 156 (60%) + glass wool 161 COH102/206 531 (60%) + KN
collagen + 718 376 Dexon S sutures COH102/206 718 376 (60%) +
Collagen Matrix collagen + Dexon S sutures
[0252] FIG. 15 depicts the tensile test of COH102/206 (60%) plus 28
mg/ml Collagen Matrix collagen plus 40 mg/ml cut pieces of Dexon S
uncoated polyglycolide sutures (4-0). The measured tensile strength
was higher than 700 N/cm.sup.2, and the measurement was interrupted
when the sample began slipping out of the testing device (downward
slope.)
EXAMPLE 11
[0253] COH102/206/methylated collagen plus the fibrous fillers
glass wool or Vicryl:
[0254] Materials:
[0255] a. Methylated collagen
[0256] Methylated collagen was prepared by a modification of the
procedure of Miyata et al, U.S. Pat. No 4,164,559. A dispersion (3%
w/v) of bovine pepsinized reconstituted collagen in 0.02M sodium
phosphate, 0.13M NaCl, pH 7.2 (prepared by the method of McPherson
et al., Collagen Rel. Res. 5, 119-135, 1985) was extruded onto a
glass surface in a thin layer and dried at room temperature.
Methanolic HCl was prepared by adding 104 g of anhydrous sodium
sulfate and 10.7 ml of conc. HCl to 1300 ml of anhydrous methanol
and allowed to stand tightly capped for 2 days. The dried collagen
was cut into 1.times.5 cm strips and added to the methanolic HCl
(200 ml methanolic HCl: 1 g dry collagen) in a sealed vessel and
gently shaken at 20.degree. C. for 3 days. The methanolic HCl was
carefully decanted off and the collagen was filtered on a sintered
glass funnel to remove traces of methanol. Complete methanol
removal was completed under vacuum overnight. The methylated
collagen was re-solubilized in distilled water, and the pH was
adjusted to 4 to 6. The amount of water was calculated to achieve a
final protein concentration of about 31 mg/ml. Samples of
solubilized methylated collagen at lower protein concentrations
were re-concentrated by brief lyophilization to remove water.
Solubilized methylated collagen was a completely transparent
material, free of fibers or opalescence, having a viscous, gel-like
consistency. Preparations which still contained hazy or insoluble
components (due to incomplete methylation of the collagen)
performed poorly in adhesive formulations, producing gels that
swelled too much and exhibited poor bond strength.
[0257] b. Adhesive without filler
[0258] For 0.5 ml of adhesive, 50 mg of dry powdered COH102
(4-armed tetra-glutarylsuccinimidyl polyethylene glycol, 10K) and
50 mg of dry powdered COH206 (4-armed tetra-thiol polyethylene
glycol, 10K) were mixed with 400 mg of methylated collagen at 31 mg
proteinlml, pH 4. Both PEG components dissolved in the aqueous
solution of collagen, yielding a transparent, viscous fluid. The
solution was spread on the tissue site with a spatula; it flowed
very little under the force of gravity. To cure the adhesive, 20-50
.mu.l of a buffer (either 134 mM sodium phosphate, 166 mM sodium
carbonate, pH 8.9; or PC buffer, pH 9.6) was added to the surface.
The buffer did not dilute the gel, but slowly soaked in. In 3-5
min, the surface of the gel was noticeably hardened.
[0259] For studies of bond strength under hydrated conditions, the
gel plus substrate was allowed to cure for 20 min on the bench,
then immersed in 50 mM sodium phosphate, 130 mM sodium chloride, pH
6.7, at 37 deg. C. for 2 hours or longer. Testing of bond strength
was performed on a tensile apparatus.
[0260] c. Adhesive with filler
[0261] Vicryl is a copolymer of glycolic acid and lactic acid
(90:10) sold as an implantable mesh by Ethicon Corporation
(Polyglactin 910; Sommerville, N.J.).
[0262] To the methylated collagen was added 19 mg of Vicryl threads
1-2 cm long which had been unraveled from implantable Vicryl mesh.
In some cases, Vicryl fibers as short as 0.3 cm were also used. The
threads and the viscous gel were blended, and then the PEG
components were added, as described above. Application to the
tissue site and curing were as above. Other fillers and their
respective amounts added to 0.5 ml of adhesive were: glass wool, 9
mg; fibrous collagen (Semed F collagen, Kensey-Nash Corporation) 8
mg; Dexon S (poly glycolide lactide sutures, "4-0"), 10 pieces 1 cm
long; elastin fibers (bovine neck ligament, 0.25 to 10 mm, Elastin
Products Co., Inc, Owensville, Mo.), 40 mg; stainless steel fibers
(Bekaert Fibre Technologies, Marietta, Ga.), 14-28 mg (Fibers were
washed with water or 1N HCl to remove a polyvinylalcohol coating);
polylactide/glycolide microparticles, prepared from
polylactide/glycolide (65:35, 40-75,000 mol. wt., Aldrich Chemical
Co., microparticles 2-4 .mu.m in diameter prepared by the method of
Zheng, J., and Hornsby, P. J., Biotechnol. Progr. 15, 763-767
(1999), 25 mg.
[0263] d. Adhesive with methylated collagen replaced by another
agent
[0264] Various long-chain molecules were tested, such as hyaluronic
acid (rooster comb, Sigma Chemical Co., St. Louis, Mo.), chitosan
(Sigma), and polylysine (Sigma). For hyaluronic acid, the formula
was: COH102, 50 mg, COH206, 50 mg, Vicryl, 14 mg, and 400 .mu.l of
hyaluronic acid, 2% (w/v) in water, pH adjusted to 4; for chitosan,
the same formula, with 400 .mu.l of 1% chitosan (w/v) in water, pH
4-5. For polylysine, COH102, 40 mg, COH206, 30 mg, dissolved
together in 50 .mu.l water; polylysine hydrobromide, 330K, 40 mg
dissolved in 60 ul water; the two solutions were mixed together,
and 7 mg Vicryl fibrils were added. In addition,
polylactide/glycolide particles, prepared as above, were tested as
a replacement for methylated collagen; 16.5 mg of particles were
suspended in 300 .mu.l of water and mixed with 50 mg COH102, 50 mg
COH206, and 14 mg Vicryl. All gels were cured with pH 9.6 buffer
overlay, as described above.
[0265] e. Adhesive without filler and without methylated
collagen
[0266] COH102 was dissolved in water at 20% (w/v); COH.sub.206 was
dissolved at 20% in pH 8.9 buffer. The two solutions were rapidly
mixed and extruded onto the site. Gelation occurred in .about.40
sec.
[0267] Mechanical tests:
[0268] Bond strength of the adhesive formulations were applied to
three types of tissue or tissue surrogates depicted in FIG. 17.
Collagen membranes (FIG. 17a; sausage casings; The SausageMaker,
Inc., Buffalo, N.Y.) were washed with isopropyl alcohol and water
to remove lipid and salt impurities, and dried. Bonding of
membranes with a 1-3 mm overlap and a 1 cm width was performed by
spreading the adhesive over the top of the sheets. Adhesive was
allowed to cure 20 min on the bench and then immersed for 30 mn to
2 hours at 37.degree. C. before pulling apart in an Instron model
4202 test apparatus (Canton, Mass.), using a 100N load cell.
Bonding of porcine carotid arteries (10b, Pelfreeze, Rogers, Ark.)
was also performed in an end-to-end geometry. Cut carotid artery
segments were abutted (4-6 mm diameter) and spread with adhesive;
no stay sutures were applied. Incubation and testing were the same
as described for the collagen membranes.
[0269] For bonding of cowhide strips (10c), de-haired calf skin
pieces were purchased from Spear Products, Inc., Quakertown, Pa.
Pieces were nearly uniform in thickness, 2-3 mm. Strips 0.4 cm wide
were cut from the hide pieces, using a single-edged razor blade.
Cut strips were abutted end to end and bonded by spreading 0.25 ml
of "CT003" adhesive or a few drops of cyanoacrylate. Incubation and
testing were the same as described for the collagen membranes.
Table 12 below shows that COH102/COH206/methylated collagen, when
filled with glass wool (Formula c), was superior in bonding
strength to unfilled Formulas a and b when tested on collagen
membranes. In fact, the bonding strength was comparable to that
obtained with a commercial cyanoacrylate adhesive (Table 9). A
medical grade cyanoacrylate (Dermabond) formed even stronger bonds
with collagen membranes (5.2.+-.1.9 N force for 7
determinations).
13TABLE 12 Bonding Performance with and without Methylated Collagen
and a Fibrous Filler Formula Bond Strength (N Force) n
COH102/206(20%) 1.6 .+-. 1.1 3 COH102/206/methylated 1.7 .+-. 1.0 4
collagen COH102/206/methylated >2.8 .+-. 0.6* 6 collagen/glass
wool *Collagen membrane tore, but sealant bond was still
intact.
[0270]
14TABLE 13 Bond Strength of Cyanoacrylate (Krazy Glue, Elmer's
Products) on Three Different Tissue Substrates Substrate Bond
Strength (N Force) Cowhide strips 10.9, 16.2 Porcine carotid artery
2.0, 3.8 Collagen membrane 3.0 .+-. 1.0 (n = 5)
[0271] Table 14 below presents data on the addition of a different
filler, Vicryl threads, to the COH102/206/ methylated collagen.
With substrates such as cowhide or carotid artery, the substrate
did not tear, and the bond strength values were representative for
the strength of the adhesive bond itself. Typically these bonds
failed adhesively, that is, the tensile strength of the adhesive
gel itself remained intact and was not the limiting factor. The
bond strengths observed in Saline at 37.degree. C. again were
comparable to those seen with cyanoacrylate for bonding the same
set of tissue substrates (Table 13).
15TABLE 14 Bond Strength of COH102/206/methylated Collagen with
Vicryl Threads as a Filler on Three Different Tissue Substrates
Incubation Time (Hrs.) Bond Strength (N Force) Substrate* 2 6.6,
5.6 Cowhide 17 6.3, 5.5 Cowhide 2 4.3, 2.2, Porcine 2.8, 5.1
Carotid Artery 2 >5.9, 3.9 Collagen Membrane *cowffide strips,
0.5 cm wide, porcine carotid artery, 0.3-0.5 cm diameter, collagen
membrane: sausage casing, 0.2 mm thick, 1 cm width.
[0272] Effect of different fillers:
[0273] Table 15 presents results of various filler materials.
Testing was performed on cowhide strips, immersed for 2 hours in
saline at 37.degree. C. It appeared that filamentous materials were
more effective than spheroidal particles. Bonding of the filler to
the gel is very important for improvement of strength.
Collagen-polyethylene glycol filaments were waxy and did not adhere
to the gel; thus, despite their high aspect ratios, they were not
effective fillers.
16TABLE 15 Effect of Different Fillers on Bond Strength of
COH102/206/methylated Collagen Bond Strength Material (N Force)
Vicryl 4.7, 7.4 Vicryl, washed with ethanol 7.2, 7.8 Vicryl,
treated with ethanol, then washed with 30% 8.3, 9.1 hydrogen
peroxide Surgical silk sutures 1-2 cm long, 30-50 u diameter 2.5,
3.8 Surgical silk sutures, unraveled to finer threads, 5.0, 6.5
washed with chloroform Fibrous collagen (Semed F, Kensy-Nash)
adjusted to 1.3, 2.8 pH 4; 0.5 to 1 mm long, .about.50 u diameter
Gelatin particles, cross-linked by heat, .about.100 u 0.6, 0.8
diameter, polygonal Hydroxyapatite particles, 0.5 to 1 mm diam. 0.7
polygonal Collagen-polyethylene glycol conjugate filament 0.8, 1.7
.about.50 u diameter, 1 cm long Stainless steel fibers 8 u
diameter, 4 mm long 4.8, 6.9 Elastin fibers 0.25 to 10 mm long 3.9,
4.0 Polylactide/glycolide particles, 2-4 u diameter 1.1, 1.1
[0274] Effect of replacing methylated collagen with other polymeric
molecules:
[0275] Table 16 shows that none of the tested materials gave bond
strengths comparable to the formula containing methylated
collagen
17TABLE 16 Replacement of Methylated Collagen by Other Molecules
Material Bond Strength (N Force) Hyaluronic acid 1.2, 1.3 Chitosan
2.1, 1.7 Polylysine 2.0
[0276] Effect of cross-linking bond:
[0277] Table 17 below shows that when the gel was formed from other
types of cross-linking reactions, the adhesion and bond strength
was affected when tested on cowhides after incubation at 37.degree.
C. Material 1 was formed from COH206 and hydrogen peroxide, which
oxidizes adjacent sulfhydryl groups to a disulfide bond. A gel
forms rapidly, and the gel can be supplemented with methylated
collagen and Vicryl; however, after several hours in saline buffer,
the gel becomes very weak; the Vicryl fibers are easily pulled out.
Material 2 utilized the reaction of sulfhydryl groups from COH206
with the double bond of a 4-arm vinyl sulfone derivative of PEG
(10K, Shearwater Polymers; FIG. 10). The presumed reaction, a
Michael-type addition, formed a thio-ether bond. Such gels had
adequate tensile strength but poor adhesion to the cowhide after
incubation in saline. Materials 3 and 4 contained COH204 (4-armed,
tetra-functional amino PEG, 10K, Shearwater Polymers); the amino
functionality presumably reacted with the succinimidyl ester of
COH102 to form an amide linkage (FIG. 18). These gels were
comparable in performance to those formed from COH102 and COH206.
(For proper reaction in the presence of methylated collagen, the
COH204 had to be titrated to pH 2-4 during the mixing of reagents;
on addition of curing buffer, its pH was increased, permitting the
reaction of the amino group). It appeared that the presence of the
succinimidyl ester was important for achieving the highest adhesion
to the tissue substrate and for good tensile strength of the gel.
Other groups that react with amines, such as aldehydes (aldehydes
conjugated to multi-armed PEG), are also anticipated to be
effective adhesive-forming reagents.
18TABLE 17 Bond Strengths of Various Functionalized PEGs Filled
with Vicryl Threads Material Strength Incubation Time (Hrs.) Bond
(N Force) COH206/Methylated 17 0.32, 0.20
Collagen/Vicryl/H.sub.2O.sub.2 COH206/4arm vinyl 2 2.2, 1.5 2
sulfone PEG/Metylated Collagen/Vicryl threads COH102/206/204/ 2 6.4
Methylated Collagen/ Vicryl threads COH102/204/ 4 3.6, 6.4
Methylated collagen/ Vicryl threads COH102/206/ 2 6.6, 5.6
Methylated collagen/ Vicryl threads
[0278] Persistence of the bond under hydrated conditions:
[0279] Table 18 shows that the adhesives formed from COH102,
COH206, and also COH204 form bonds using cowhide that persist for
long times immersed in saline buffer at 37.degree. C. Such
stringent hydrated conditions simulate the in vivo environment.
Bond weakening was observed after more than 100 hours of hydration.
The weakening of bond strength was thought to be due to hydrolysis
of carboxyl-ester and thioester (FIG. 19) network linkages. COH102
is a glutaryl-succinimidyl ester; even after reaction with the
terminal carboxyl of the succinimidyl ester, there remains a
carboxyl ester linking the glutaryl moiety to the main PEG chain;
this bond, as well as the thio-ester bond, could hydrolyze.
19TABLE 18 Bond Performance Under Long Hydration Times Material
Incubation Time (Hrs.) Bond Strength (N Force) C0H102/206/204/ 2
6.4 Methylated collagen/ 66 2.6, 4.1 Vicryl threads 70 3.0 137
0.70, 2.6 140 1.1, 0.4 COH102/204/ 4 3.6, 6.4 Methylated collagen/
64 7.0, 5.1 Vicryl threads 136 3.8, 2.7 234 2.7, 1.7 COH102/206/ 2
6.6, 5.6 Methylated collagen/ 17 6.3, 5.5 Vicryl threads 69 0.63,
0.90, 3.4, 5.4 93 2.4, 5.4 140 3.2, 2.9 235 >2.4, 3.7
[0280] Related formulas with lower weight compounds bearing
succinimidyl ester and amino or thiol reactive groups:
[0281] Table 19 presents bond strengths on cowhide strips of lower
molecular weight PEG derivatives as adhesives, again supplemented
with methylated collagen and Vicryl. GLYC-20HS is a tri-functional
succinimidyl-succinate of a 3-armed PEG built from a glycerol core,
2600 mol wt., NOF Corporation, Japan. COH201 is a tetra-amino,
4-armed PEG, 2000 mol. wt., Shearwater Polymers. The polymers were
Vicryl filling appeared to have a small effect on bond strength.
The following proportions were used: Methylated Collagen, 500 .mu.l
(22 mg/ml in water 2707-30B); GLYC-20HS, 48 mg; COH201, 60 .mu.l of
60% solution in water, titrated to pH 1-2 with 6M HCl; Vicryl
threads, 26 mg.
20TABLE 19 Low Molecular Weight Analogues to COH102 and COH206 Bond
Strength Materials Incubation Time (Hrs.) (N Force) GLYC-20HS/ 2
2.3, 0.64 COH201/Methyated Collagen GLYC-20HS/ 5 2.3, 3.3
COH201/Methylated Collagen/Vicryl threads
[0282] Burst tests on collagen disks and on slit defects in carotid
arteries:
[0283] Performance of adhesives intended for use in surgical
applications is often measured by their ability to seal fluid
leaks. Two types of leaks, or fluid pressure tests were
employed:
[0284] a. The burst test on a collagen disk
[0285] Using the device depicted in FIG. 20, collagen mat was
mounted on a brass platform and secured with a second brass ring
threaded to the first. The lower brass platform was perforated and
connected to a line filled with water. Water was driven by a
syringe pump at 5 ml/min. A shunt line led to a pressure gauge. The
test collagen mat was also perforated (2 mm diameter hole). The
adhesive preparation (approx. 0.5 ml) was applied to the mat,
covering the perforation. The adhesive was allowed to cure 3 min
(or longer, if necessary to effect cure to a firm rubber), then
water pressure was applied. The pressure necessary to rupture the
seal was recorded. For cyanoacrylate, a small (4.times.4 mm) piece
of collagen mat was glued to the lower perforated mat.
[0286] b. Slit defect on carotid artery
[0287] The pressurized carotid artery model is illustrated in FIG.
21. A porcine artery (Pel Freeze Biologicals, Rogers, Ark.) was
connected to a water line. Water was driven by a peristaltic pump.
The end of the line had a flow restricter placed on it so that
pressures up to 10 psi and more could be imposed on the line by
increasing the pump speed. First the intact artery was placed in
the system and subjected to water pressure, to assure that it would
sustain desired pressures without leaking. Sections of artery
devoid of side branches were preferred; leaking branches sometimes
were clamped off to stop leaks. Slits approximately 2 mm long were
cut transversely in the artery at four sites on a circumference.
The cut artery then simulated an anastomosis to which stay sutures
had been applied. The cut sites were then glued all around in an
attempt to seal them. Buffer (134 mM sodium acid phospate and 166
mM sodium carbonate, pH 8.9) was applied to the artery tissue just
before the glue was applied. The glue mass was further irrigated
with a few drops of this buffer to cure the gel. After 8 min cure
time, the glued joint was subjected to water pressure. Pressure was
increased at 1 psi increments and held at each pressure for 1
minute before increasing further. A leak was scored as positive if
it was dripping faster than 1 drop every 10 seconds.
[0288] Table 20 shows the burst strengths of COH102/206/methylated
collagen/Vicryl on holes of varying diameters (on collagen
membranes at 8 min cure time; cured with pH 8.9 buffer; 0.5 ml
sample spread over hole with spatula). A hole with a diameter of 5
mm is the largest defect one might contemplate in a surgical
application, since stay sutures would be used to close the largest
defects, and the largest interval between such sutures was
estimated to be 5 mm. Even with such large holes, the adhesive was
able to sustain pressures near or above the maximum expected in
hypertensive patients, i.e., 4 psi. The third data entry emphasizes
the need to have good gel curing at the interface of gel and
collagen disk. The addition of curing buffer to this surface prior
to application improves the short-term bonding.
21TABLE 20 Burst Strength of COH102/206/methylated Collagen/Vicryl
Diameter of Orifice (mm) Burst Pressure (PSI)* 2 >3.0, 7.4, 4.6
5 3.1, 5.5, 5.3 5 1.0.sup.+ *1 PSI = 0.68 N/CM.sup.2 = 51 mmHg 4
PSI = 2.7 N/CM.sup.2 = 204 mHg .sup.+Membrane not pre-treated with
a drop of pH 8.9 buffer
[0289] Table 21 presents data on closing large slit defects in
carotid arteries (4.times.2 mm slits cut on 4-6 mm diameter
artery). The COH102/206/methylated collagen/Vicryl formula was
comparable to cyanoacrylate in performance. It should be noted that
poorer results are seen on thinner arteries that stretch more under
pressure.
22TABLE 21 Burst Strength Test on Porcine Carotid Artery Material
Burst Pressure (PSI) Cure Time (min.) COH102/206/ 4.3 .+-. 2.0 (n =
5) 8 Methylated Collagen/ 8.0 .+-. 4.0 (n = 3) 30 Vicryl
Cyanoacrylate 2.7 .+-. 3.6 (n = 6) 8 (Elmer's Products)
Cyanoacrylate 5.5 .+-. 5.2 (n = 4) 8 (Dermabond)
EXAMPLE 12
[0290] 0.40 g (0.04 mmol) of COH206 (4-armed thiol of PEG,
penta-erythritol core, MW 10K) and 0.21 g (0.053 mmol) of
trimethylolpropane tris(3-mercaptopropionate) were dissolved in 0.2
g of H.sub.2O. The mixture of these two thiols was deprotonated by
adding 0.5 mg of T403 (polyoxypropylene triamine). Upon mixing the
solution with 0.112 g (0.16 mmol) of poly (ethylene glycol)
diacrylate (MW 700) a gel was formed within 2 minutes.
* * * * *