U.S. patent application number 10/373269 was filed with the patent office on 2004-02-05 for biocompatible crosslinked polymers.
This patent application is currently assigned to Incept. Invention is credited to Edelman, Peter G., Pathak, Chandrashekhar P., Sawhney, Amarpreet S..
Application Number | 20040023842 10/373269 |
Document ID | / |
Family ID | 22335273 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023842 |
Kind Code |
A1 |
Pathak, Chandrashekhar P. ;
et al. |
February 5, 2004 |
Biocompatible crosslinked polymers
Abstract
Biocompatible crosslinked polymers, and methods for their
preparation and use, are disclosed in which the biocompatible
crosslinked polymers are formed from water soluble precursors
having electrophilic and nucleophilic groups capable of reacting
and crosslinking in situ. Methods for making the resulting
biocompatible crosslinked polymers biodegradable or not are
provided, as are methods for controlling the rate of degradation.
The crosslinking reactions may be carried out in situ on organs or
tissues or outside the body. Applications for such biocompatible
crosslinked polymers and their precursors include controlled
delivery of drugs, prevention of post-operative adhesions, coating
of medical devices such as vascular grafts, wound dressings and
surgical sealants.
Inventors: |
Pathak, Chandrashekhar P.;
(Austin, TX) ; Sawhney, Amarpreet S.; (Lexington,
MA) ; Edelman, Peter G.; (Franklin, MA) |
Correspondence
Address: |
Patterson, Thuente, Skaar & Christensen, P.A.
4800 IDS Center
80 South 8th Street
Minneapolis
MN
55402-2100
US
|
Assignee: |
Incept
|
Family ID: |
22335273 |
Appl. No.: |
10/373269 |
Filed: |
February 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10373269 |
Feb 24, 2003 |
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09454900 |
Dec 3, 1999 |
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6566406 |
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60110849 |
Dec 4, 1998 |
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Current U.S.
Class: |
514/1 |
Current CPC
Class: |
A61L 27/50 20130101;
A61K 47/34 20130101; A61P 19/02 20180101; A61K 31/74 20130101; A61L
27/58 20130101; A61L 31/06 20130101; A61K 47/46 20130101; A61P
41/00 20180101; A61P 3/10 20180101; A61L 31/145 20130101; A61K
9/0024 20130101; A61L 31/148 20130101; A61P 9/10 20180101; A61P
29/00 20180101; C08G 63/08 20130101; C08G 63/912 20130101; A61L
27/52 20130101; A61P 35/00 20180101; A61L 27/18 20130101; A61P
43/00 20180101 |
Class at
Publication: |
514/1 |
International
Class: |
A61K 031/00 |
Claims
1. Method of making a biocompatible degradable hydrogel to treat a
medical condition comprising: identifying a medical condition for
treatment by use of a hydrogel formed in situ in a patient and
essentially fully degradable in a patient in less than about 180
days; and selecting precursors to form the hydrogel for treatment
of the medical condition, the selection of the precursors
comprising: selecting a first biocompatible precursor having a
water solubility of at least 1 gram per 100 milliliters and at
least two electrophilic functional groups; selecting a second
biocompatible precursor comprising at least two nucleophilic amine
functional groups; selecting one of (i), (ii), or (iii), wherein
(i) the first precursor is selected have only one or two
hydrolytically degradable ester bonds per every electrophilic
functional group on the precursor, or (ii) the second precursor is
selected to have only one or two hydrolytically degradable ester
bonds per every nucleophilic functional group on the precursor, or
(iii) selecting both (i) and (ii); and selecting the electrophilic
and nucleophilic groups to form covalent bonds that are reaction
products of the electrophilic and nucleophilic groups, wherein
essentially every ester bond in the hydrogel is separated from
other ester bonds in the hydrogel by at least three covalent bonds
when the hydrogel is formed.
2. The method of claim 1 wherein the medical condition is adhesion
prevention.
3. The method of claim 1 wherein the medical condition is tissue
adhesion.
4. The method of claim 1 wherein the medical condition is drug
delivery.
5. The method of claim 1 wherein the medical condition is wound
covering.
6. The method of claim 1 wherein the medical condition is tissue
sealing.
7. The method of claim 1 wherein the medical condition is tissue
coating.
8. The method of claim 1 wherein the solids concentration of the
hydrogel ranges from 8.5% to 20% w/w.
9. The method of claim 1 wherein the second precursor has a
molecular weight of less than about 1000.
10. The method of claim 1 wherein the first precursor comprises
carboxymethyl-hydroxybutyrate-N-hydroxysuccinimidyl polyethylene
glycol.
11. The method of claim 1 wherein the first precursor comprises
succinimidyl glutarate.
12. The method of claim 1 wherein the electrophilic functional
groups of the first precursor comprise n-hydroxysuccinimide
ester.
13. The method of claim 1 wherein the electrophilic functional
groups of the first precursor comprise a member of the group
consisting of carbonyldiimidazole, sulfonyl chloride, aryl halides,
sulfosuccinimide ester, epoxide, aldehyde, maleimides and
imidoester.
14. The method of claim 1 wherein the second precursor consists
essentially of a member of the group consisting of lysine,
dilysine, trilysine, and tetalysine.
15. The method of claim 1 wherein at least one of the precursors is
selected to further comprise a chemical group having the formula
(CH.sub.2CH.sub.2O).sub.n.
16. The method of claim 1 wherein the second precursor comprises a
lysine.
17. The method of claim 1 wherein the hydrogel is essentially fully
degradable in a patient in less than about 90 days.
18. The method of claim 1 wherein the hydrogel is essentially fully
degradable in a patient in less than about 45 days.
19. A method of making a biocompatible readily degradable hydrogel
comprising: providing a first biocompatible precursor having a
water solubility of at least 1 gram per 100 milliliters, at least
two electrophilic functional groups, and no more than two
hydrolytically degradable ester bonds per electrophilic functional
group; providing a second biocompatible precursor comprising at
least two nucleophilic functional groups and no more than two
hydrolytically degradable ester bonds per nucleophilic functional
group; wherein at least one of the precursors is selected to have
one or two hydrolytically degradable ester bonds per electrophilic
functional group; selecting the nucleophilic groups and the
electrophilic groups to form a hydrogel essentially fully
degradable in vivo in less than about 180 days, wherein the
nucleophilic groups are amines; and mixing the precursors to form a
covalently crosslinked hydrogel, wherein essentially every ester
bond in the hydrogel is separated from other ester bonds in the
hydrogel by at least three covalent bonds when the hydrogel is
formed.
20. The method of claim 19 wherein the solids concentration of the
hydrogel ranges from 8.5% to 20% w/w.
21. The method of claim 19 wherein the first precursor comprises
carboxymethyl-hydroxybutyrate-N-hydroxysuccinimidyl polyethylene
glycol.
22. The method of claim 19 wherein the second precursor has a
molecular weight of less than about 1000.
23. The method of claim 22 wherein the second precursor consists
essentially of a member of the group consisting of lysine,
dilysine, trilysine, and tetralysine.
24. The method of claim 19 wherein the first precursor comprises
succinimidyl glutarate.
25. The method of claim 24 wherein the second precursor consists
essentially of a member of the group consisting of lysine,
dilysine, trilysine, and tetralysine.
26. The method of claim 19 wherein the electrophilic functional
groups of the first precursor comprise n-hydroxysuccinimide
ester.
27. The method of claim 19 wherein the electrophilic functional
groups of the first precursor comprise a member of the group
consisting of carbonyldiimidazole, sulfonyl chloride, aryl halides,
sulfosuccinimide ester, epoxide, aldehyde, maleimides and
imidoester.
28. The method of claim 27 wherein the second precursor consists
essentially of a member of the group consisting of lysine,
dilysine, trilysine, and tetralysine.
29. The method of claim 19 wherein the second precursor comprises a
lysine.
30. The method of claim 19 wherein at least one of the precursors
is selected to further comprise a chemical group having the formula
(CH.sub.2CH.sub.2O).sub.n.
31. The method of claim 19 wherein the hydrogel is degradable vivo
in less than about 90 days.
32. The method of claim 19 wherein the hydrogel is degradable in
vivo in less than about 45 days.
33. A biocompatible degradable material in a product, the material
comprising: a hydrogel comprising a first biocompatible precursor
crosslinked with a second biocompatible precursor, wherein the
first precursor, before crosslinking, comprises polyethylene glycol
and a member of the group consisting of succinimidyl glutarate,
carboxymethyl-hydroxybutyrate-N-hydroxysuccinimide,
carbonyldiimidazole, sulfonyl chloride, aryl halides,
sulfosuccinimide ester, epoxide, aldehyde, maleimides and
imidoester, wherein the second biocompatible precursor, before
crosslinking, is a member of the group consisting of lysine,
dilysine, trilysine, and tetralysine, wherein the hydrogel forms a
product essentially fully degradable in less than about 180 days,
and the product is a member of the group consisting of adhesion
prevention, tissue adhesion, drug delivery, wound covering, tissue
sealing, and tissue coating.
34. A biocompatible readily degradable material in a product, the
material comprising: a hydrogel comprising a first biocompatible
precursor crosslinked with a second biocompatible precursor,
wherein the first precursor, before crosslinking, comprises
succinimidyl glutarate, wherein the second biocompatible precursor,
before crosslinking, consists essentially of a member of the group
consisting of lysine, dilysine, trilysine, and tetralysine, wherein
the hydrogel forms a product essentially fully degradable in less
than about 180 days, and the product is a member of the group
consisting of adhesion prevention, tissue adhesion, drug delivery,
wound covering, tissue sealing, and tissue coating.
35. A biocompatible readily degradable hydrogel article comprising:
a hydrogel article comprising a first biocompatible precursor
crosslinked with a second biocompatible precursor, wherein the
first precursor, before crosslinking, comprises at least two amines
and no more than one hydrolytically degradable ester group per
amine, wherein the second precursor, before crosslinking, comprises
at least two electrophiles and no more than one hydrolytically
degradable ester group per electrophile, wherein at least one of
the precursors has at least one hydrolytically degradable ester,
and essentially every ester bond in the hydrogel article is
separated from other ester bonds in the hydrogel by at least three
covalent bonds when the hydrogel is formed, with the ester bonds
being hydrolytically degradable so that the hydrogel article is
essentially fully hydrolytically degradable in physiological
conditions in less than about 180 days and the hydrogel article is
a member of the group consisting of an adhesion prevention hydrogel
article, a tissue adhesion hydrogel article, a drug delivery
hydrogel article, a wound covering hydrogel article, a tissue
sealing hydrogel article, and a tissue coating hydrogel
article.
36. The hydrogel article of claim 35 wherein the hydrogel article
is essentially fully hydrolytically degradable in physiological
conditions in less than about 90 days.
37. The hydrogel article of claim 35 wherein the hydrogel article
is essentially fully hydrolytically degradable in physiological
conditions in less than about 45 days.
38. The hydrogel article of claim 35 wherein the hydrogel article
is essentially fully hydrolytically degradable in physiological
conditions in less than about 10 days.
39. The hydrogel article of claim 35 wherein the solids
concentration of the hydrogel ranges from 8.5% to 20% w/w.
40. The hydrogel article of claim 35 wherein the first precursor
has a molecular weight of less than about 1000.
41. The hydrogel article of claim 35 wherein the second precursor
comprises carboxymethyl-hydroxybutyrate-N-hydroxysuccinimidyl
polyethylene glycol.
42. The hydrogel article of claim 35 wherein the second precursor
comprises succinimidyl glutarate.
43. The hydrogel article of claim 35 wherein the electrophilic
functional groups of the second precursor comprise
n-hydroxysuccinimide ester.
44. The hydrogel article of claim 35 wherein the electrophilic
functional groups of the second precursor comprise a member of the
group consisting of carbonyldiimidazole, sulfonyl chloride, aryl
halides, sulfosuccinimide ester, epoxide, aldehyde, maleimides and
imidoester.
45. The hydrogel article of claim 35 wherein the first precursor
consists essentially of a member of the group consisting of lysine,
dilysine, trilysine, and tetralysine.
46. The hydrogel article of claim 35 wherein at least one of the
precursors is selected to further comprise a chemical group having
the formula (CH.sub.2CH.sub.2O).sub.n.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/454,900, filed Dec. 3, 1999, which claims
priority to U.S. patent application Ser. No. 60/110,849, filed Dec.
4, 1998, which patent applications are hereby incorporated by
reference herein.
FIELD OF THE INVENTION
[0002] The invention relates generally to biocompatible crosslinked
polymers, methods for preparing and using same.
BACKGROUND OF THE INVENTION
[0003] In the field of medicine there has been a growing
recognition of the benefits of using biocompatible crosslinked
polymers for the treatment of local diseases. Local diseases are
diseases that are manifested at local sites within the living
animal or human body, for example atherosclerosis, postoperative
adhesions, rheumatoid arthritis, cancer, and diabetes.
Biocompatible crosslinked polymers may be used in drug and surgical
treatments of such diseases.
[0004] Historically, many local diseases have been treated by
systemic administration of drugs. In this approach, in order to
achieve therapeutic levels of drugs at local disease sites, drugs
are delivered (via oral administration or injection) at a high
systemic concentration, often with adverse side effects. As an
alternative, biocompatible crosslinked polymers may be used as
carriers to deliver drugs to local sites within the body, thereby
reducing the need for the systemic administration of high
concentrations of drugs, while enhancing effectiveness.
[0005] Local diseases also have been treated with surgery. Many of
these surgical procedures employ devices within the body. These
devices may often be formed from or coated with biocompatible
crosslinked polymers. For example, a surgical sealant is a device
formed from biocompatible crosslinked polymers that may be used to
reduce migration of fluid from or into a tissue. For surgical
sealants, as with many other surgical procedures, it is sometimes
necessary to leave devices in the body after surgery to provide a
continuing therapeutic benefit. In such cases, it may be desired
that the implant biodegrade over time, eliminating the need for a
second surgical procedure to remove the implant after its
usefulness has ended. Regardless of whether the implant biodegrades
over time, it may also be used, as described above, to deliver
drugs to local sites within the body.
[0006] Many surgical procedures are now performed in a minimally
invasive fashion that reduces morbidity associated with the
procedure. Minimally invasive surgery ("MIS") encompasses
laparoscopic, thoracoscopic, arthroscopic, intraluminal endoscopic,
endovascular, interventional radiological, catheter-based cardiac
(such as balloon angioplasty), and like techniques. These
procedures allow mechanical access to the interior of the body with
the least possible perturbation of the patient's body.
Biocompatible crosslinked polymers may be advantageously used to
form or coat many of these MIS tools. These polymers may also be
used to form sutures, surgical clips, staples, sealants, tissue
coatings, implants and drug delivery systems.
[0007] Most of the polymers used with MIS applications are
pre-formed to a specific shape before being used in a given
application. However, such pre-formed objects have limitations in
MIS procedures because they, like other large objects, are
difficult to transport through the small access sites afforded by
MIS techniques. In addition, the shape of the pre-formed object may
not be appropriate because the target tissues where such objects
are likely to be used have a variety of shapes and sizes. To
overcome these limitations, in situ curable or gelable
biocompatible crosslinked polymer systems have been explored. The
precursors of such systems are usually liquid in nature. These
liquids are then transported to the target tissue and applied on
the target organ or tissue. The liquid flows and conforms to the
shape of the target organ. The shape of the conformed liquid is
then preserved by polymerization or a gelation reaction. This
approach has several advantages, including conformity to organ
shapes and the ability to implant large quantities of liquid using
MIS procedures.
[0008] One use of in situ curable biocompatible crosslinked
polymers in MIS procedures is to form tissue coatings so as to
prevent post-surgical adhesions. For example, J. L. Hill-West et
al., "Prevention of Postoperative Adhesions in the Rat by In Situ
Photopolymerization of Bioresorbable Hydrogel Barriers," Obstetrics
and Gynecolocy, 83(1):59 (1994) describes the use of free radical
photopolymerizable water-soluble monomers to form biocompatible
crosslinked polymers and thereby prevent post-operative adhesions
in two animal models. U.S. Pat. No. 5,410,016 to Hubbell et al.
describes the use of free radical photopolymerizable monomers to
form biocompatible crosslinked polymers, which then are used as
tissue adhesives, controlled-release carriers and as tissue
coatings for the prevention of post-operative adhesions.
Free Radical Polymerization
[0009] Many of the biocompatible crosslinked polymers previously
known used free radical polymerization of vinylic or acrylic
functionalities. For example, the Hill-West article describes the
use of free radical photopolymerizable, water soluble monomers
consisting of 8000 molecular weight ("MW") polyethylene glycol
("PEG") extended at both ends with oligomers of lactic acid and
further acrylated at both ends. The aforementioned Hubbell patent
describes the use of acetophenone derivative or eosin initiated
free radical polymerization of acrylic functionalities of
water-soluble biodegradable macromolecules. U.S. Pat. No. 4,938,763
to Dunn describes the use of benzoyl peroxide initiated free
radical polymerization of liquid prepolymers.
[0010] While free radical polymerization is useful for polymer
synthesis, several considerations limit its suitability for use in
the living animal or human body. First, the initiator which
generates free radicals normally produces several small molecules
with known or unknown toxicity. For example, one of the most
commonly used photoinitiators, 2,2-dimethoxy 2-phenylacetophenone,
generates methyl benzoate and other small compounds during the
initiation step. The safety of these initiator fragments must be
established before there can be widespread use of such systems for
human or animal use. Second, free radicals are extremely reactive
species and have life times ranging from 0.01 to 1 second during a
typical free radical polymerization reaction. Third, the free
radical polymerization, once initiated, is often uncontrollable,
frequently producing polymers with high molecular weight and broad
molecular weight distribution. Fourth, the most common
functionalities used in free radical polymerization are vinylic or
acrylic, and the vinyl/acrylic polymers produced by these
compositions do not degrade inside the body. Fifth, free radical
polymerizable monomers often need to be inhibited with a small
amount of inhibitor to prevent the premature polymerization of
vinyl functionality. The most commonly used inhibitors are phenols
(for example, hydroquinone), which are toxic and hence can be used
in only limited amounts, increasing the probability of premature
polymerization and crosslinking. Finally, free radical
polymerization is often exothermic, and the heat it generates may
cause localized burn injuries.
Electrophilic-Nucleophilic Polymerization
[0011] Other crosslinked polymers have been formed using
electrophilic-nucleophilic polymerization of polymers equipped with
either electrophilic or nucleophilic functional groups. For
example, U.S. Pat. Nos. 5,296,518 and 5,104,909 to Grasel et al.
describe the formation of crosslinked polymers from ethylene oxide
rich prepolymers, wherein a polyisocyanate or low molecular weight
diisocyanate is used as the electrophilic polymer or crosslinker,
and a polyoxyethylene based polyol with in situ generated amine
groups is used as the nucleophilic precursor. U.S. Pat. No.
5,514,379 to Weissleder et al. describes the formation of
biocompatible crosslinked polymers using polymeric precursors,
including polyethylene glycol derivatives, each having multiple
electrophilic or nucleophilic functional groups. U.S. Pat. No.
5,426,148 to Tucker describes sealant compositions based on an
electrophilic-nucleophilic polymerization reaction between
polyether acetoacetylate and polyether amine precursors. U.S. Pat.
Nos. 5,874,500 and 5,527,856 to Rhee et al. also describe
biocompatible crosslinked polymers, formed from
electrophilic-nucleophilic polymerization of polymers having
multiple electrophilic or nucleophilic functionalities.
[0012] While these electrophilic-nucleophilic polymerization
methods do not suffer from the same limitations as free radical
polymerization methods, described above, they have other
limitations stemming from their use of polymeric precursors. Mixing
can be a significant impediment to such reactions since polymeric
precursors are often of a higher viscosity and diffusion is
impeded, especially with the onset of gelation. Thus, imperfections
in the crosslinked structures and weaknesses may result.
[0013] In contrast, the use of at least one small molecule
precursor (where small molecule refers to a molecule that is not a
polymer and is typically of a molecular weight less than 2000
Daltons, or else is a polymer and is of a molecular weight of less
than 1000 Daltons) allows for diffusion of the small molecule
throughout the crosslinked structure, even after gelation, and thus
may result in superior materials. This approach has heretofore been
limited to small molecules having electrophilic end groups such as
aldehyde. For example, BioGlue, marketed by Cryolife Inc., uses a
glutaraldehyde-based electrophilic small molecule to react with a
polymeric albumin-based nucleophilic polymer.
[0014] However, the small molecule electrophile approaches that are
known suffer from several limitations. For example, glutaraldehyde
is known to be a toxic compound, and in fact is used to sterilize
tissues and can cause significant tissue toxicity. For
isocyanate-based approaches, in order for in situ polymerization to
occur without local tissue toxicity, other crosslinkers are needed.
Moreover, the prior art is silent on the subject of
biodegradability of these networks. This is important because in
many applications it is important that the materials absorb and be
cleared from the body after having served their purpose.
Visualization
[0015] As described above, advances in modern surgery provide
access to the deepest internal organs with minimally invasive
surgical devices. As also described above, biocompatible
crosslinked polymers that can be formed in situ are useful in such
surgical procedures. However, most such formulations, for example,
fibrin glue, are colorless, and the amount of material used is
typically very small, leading to a film thickness of only about
0.05 to 1 mm. The resulting colorless solution or film is therefore
difficult to visualize, especially in the typically wet and moist
surgical environment. Under laparoscopic conditions, visibility is
even more difficult due to the fact that only a two-dimensional
view of the surgical field is available on the monitor that is used
in such procedures.
[0016] The use of color in biocompatible crosslinked polymers and
precursors may therefore greatly improve their utility in a
surgical environment, especially under minimally invasive surgical
procedures. Moreover, the better visibility available with the use
of color also permits efficient use of materials with minimum
wastage.
[0017] There thus exists a need for biocompatible crosslinked
polymers that can be formed without using free radical chemistry,
that can be formed from at least one small molecule precursor that
has minimal tissue toxicity, that may be biodegradable, and that
may be colored.
SUMMARY OF THE INVENTION
[0018] It is therefore an object of the present invention to
provide biocompatible crosslinked polymers and methods for their
preparation and use, in which the biocompatible crosslinked
polymers are formed without using free radical chemistry, and are
formed using at least one non-toxic small molecule precursor.
[0019] It is another object of this invention to provide such
biocompatible crosslinked polymers and methods for their
preparation and use, in which the biocompatible crosslinked
polymers are formed from aqueous solutions, preferably under
physiological conditions.
[0020] It is still another object of this invention to provide such
biocompatible crosslinked polymers and methods for their
preparation and use, in which the biocompatible crosslinked
polymers are formed in vivo.
[0021] It is a still further object of this invention to provide
such biocompatible crosslinked polymers and methods for their
preparation and use, in which the biocompatible crosslinked
polymers are biodegradable.
[0022] Another object of this invention is to provide such
biocompatible crosslinked polymers and methods for their
preparation and use, in which the biocompatible crosslinked
polymers, their precursors, or both are colored.
[0023] Another object of this invention is to provide methods for
preparing tissue conforming, biocompatible crosslinked polymers in
a desirable form, size and shape.
[0024] Another object of this invention is to provide methods for
using biocompatible crosslinked polymers to form medically useful
devices or implants for use as surgical adhesion prevention
barriers, as implantable wound dressings, as scaffolds for cellular
growth for tissue engineering, or as surgical tissue adhesives or
sealants.
[0025] Another object of this invention is to provide methods for
using biocompatible crosslinked polymers to form medically useful
devices or implants that can release bioactive compounds in a
controlled manner for local, systemic, or targeted drug
delivery.
[0026] Another object of this invention is to provide methods and
compositions for producing composite biomaterials comprising fibers
or particulates made of biodegradable biocompatible crosslinked
polymers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 depicts electrophilic water soluble and biodegradable
crosslinkers or functional polymers, which can be crosslinked with
appropriate nucleophilic precursors.
[0028] FIG. 2 depicts nucleophilic water soluble and biodegradable
crosslinkers or functional polymers, which can be crosslinked with
appropriate electrophilic precursors.
[0029] FIG. 3 depicts electrophilic water soluble and biodegradable
crosslinkers or functional polymers, which can be crosslinked with
appropriate nucleophilic precursors, wherein either the
biodegradable linkages or the functional groups are selected so as
to make the precursor water soluble.
[0030] FIG. 4 depicts nucleophilic water soluble crosslinkers or
functional polymers, which can be crosslinked with appropriate
electrophilic precursors, and which are not biodegradable.
[0031] FIG. 5 depicts electrophilic water soluble crosslinkers or
functional polymers, which can be crosslinked with appropriate
nucleophilic precursors, and which are not biodegradable.
[0032] FIG. 6 depicts the preparation of an electrophilic water
soluble crosslinker or functional polymer using carbodiimide
("CDI") activation chemistry, its crosslinking reaction with a
nucleophilic water soluble functional polymer to form a
biocompatible crosslinked polymer product, and the hydrolysis of
that biocompatible crosslinked polymer to yield water soluble
fragments.
[0033] FIG. 7 depicts the use of sulfonyl chloride activation
chemistry to prepare an electrophilic functional polymer.
[0034] FIG. 8 depicts the preparation of an electrophilic water
soluble crosslinker or functional polymer using
N-hydroxysuccinimide ("NHS") activation chemistry, its crosslinking
reaction with a nucleophilic water soluble functional polymer to
form a biocompatible crosslinked polymer product, and the
hydrolysis of that biocompatible crosslinked polymer to yield water
soluble fragments.
[0035] FIG. 9 depicts preferred NHS esters for use in the
invention.
[0036] FIG. 10 shows the N-hydroxysulfosuccinimide ("SNHS")
activation of a tetrafunctional sugar-based water soluble synthetic
crosslinker and its crosslinking reaction with 4-arm amine
terminated polyethylene glycol to form a biocompatible crosslinked
polymer product, and the hydrolysis of that biocompatible
crosslinked polymer to yield water soluble fragments.
[0037] FIG. 11 shows the variation in gelation time with the number
of amino groups for the reaction of 4 arm 10 kDa succinimidyl
glutarate PEG ("SG-PEG") with di-, tri- or tetra-lysine.
[0038] FIG. 12 shows the variation in gelation time with the
solution age of the electrophilic functional polymer.
[0039] FIG. 13 shows the variation in gelation time with the
concentration of biocompatible crosslinked polymer precursors, and
with the solution age of the 4 arm 10 kDa
carboxymethyl-hydroxybutyrate-N-hydroxysuccinimid- yl PEG
("CM-HBA-NS") electrophilic functional polymer.
[0040] FIG. 14 shows the variation in degradation time with the
concentration of biocompatible crosslinked polymer.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The novel biocompatible crosslinked polymers of this
invention are formed from the reaction of precursors having
electrophilic and nucleophilic functional groups. The precursors
are preferably water soluble, non-toxic and biologically
acceptable.
[0042] Preferably, at least one of the precursors is a small
molecule, and is referred to as a "crosslinker". More preferably,
the crosslinker has a solubility of at least 1 g/100 mL in an
aqueous solution. Preferably, one of the other precursors is a
macromolecule, and is referred to as a "functional polymer".
Functional Groups
[0043] Each precursor is multifunctional, meaning that it comprises
two or more electrophilic or nucleophilic functional groups, such
that a nucleophilic functional group on one precursor may react
with an electrophilic functional group on another precursor to form
a covalent bond. At least one of the precursors comprises more than
two functional groups, so that, as a result of
electrophilic-nucleophilic reactions, the precursors combine to
form crosslinked polymeric products. Such reactions are referred to
as "crosslinking reactions".
[0044] Preferably, each precursor comprises only nucleophilic or
only electrophilic functional groups, so long as both nucleophilic
and electrophilic precursors are used in the crosslinking reaction.
Thus, for example, if a crosslinker has nucleophilic functional
groups such as amines, the functional polymer may have
electrophilic functional groups such as N-hydroxysuccinimides. On
the other hand, if a crosslinker has electrophilic functional
groups such as sulfosuccinimides, then the functional polymer may
have nucleophilic functional groups such as amines. Thus,
functional polymers such as proteins, poly(allyl amine), or
amine-terminated di-or multifunctional poly(ethylene glycol)
("PEG") can be used.
Water Soluble Cores
[0045] The precursors preferably have biologically inert and water
soluble cores. When the core is a polymeric region that is water
soluble, preferred polymers that may be used include: polyethers,
for example polyalkylene oxides such as polyethylene glycol
("PEG"), polyethylene oxide ("PEO"), polyethylene
oxide-co-polypropylene oxide ("PPO"), co-polyethylene oxide block
or random copolymers, and polyvinyl alcohol ("PVA"); poly(vinyl
pyrrolidinone) ("PVP"); poly(amino acids); dextran and the like.
The polyethers and more particularly poly(oxyalkylenes) or
poly(ethylene oxide) or polyethylene oxide are especially
preferred. When the core is small molecular in nature, any of a
variety of hydrophilic functionalities can be used to make the
precursor water soluble. For example, functional groups like
hydroxyl, amine, sulfonate and carboxylate, which are water
soluble, maybe used to make the precursor water soluble. In
addition, N-hydroxysuccinimide ("NHS") ester of subaric acid is
insoluble in water, but by adding a sulfonate group to the
succinimide ring, the NHS ester of subaric acid may be made water
soluble, without affecting its reactivity towards amine groups.
Biodegradable Linkages
[0046] If it is desired that the biocompatible crosslinked polymer
be biodegradable or absorbable, one or more precursors having
biodegradable linkages present in between the functional groups may
be used. The biodegradable linkage optionally also may serve as the
water soluble core of one or more of the precursors. In the
alternative, or in addition, the functional groups of the
precursors may be chosen such that the product of the reaction
between them results in a biodegradable linkage. For each approach,
biodegradable linkages may be chosen such that the resulting
biodegradable biocompatible crosslinked polymer will degrade or be
absorbed in a desired period of time. Preferably, biodegradable
linkages are selected that degrade under physiological conditions
into non-toxic products.
[0047] The biodegradable linkage may be chemically or enzymatically
hydrolyzable or absorbable. Illustrative chemically hydrolyzable
biodegradable linkages include polymers, copolymers and oligomers
of glycolide, dl-lactide, l-lactide, caprolactone, dioxanone, and
trimethylene carbonate. Illustrative enzymatically hydrolyzable
biodegradable linkages include peptidic linkages cleavable by
metalloproteinases and collagenases. Additional illustrative
biodegradable linkages include polymers and copolymers of
poly(hydroxy acid)s, poly(orthocarbonate)s, poly(anhydride)s,
poly(lactone)s, poly(aminoacid)s, poly(carbonate)s, and
poly(phosphonate)s.
Visualization Agents
[0048] Where convenient, the biocompatible crosslinked polymer or
precursor solutions (or both) may contain visualization agents to
improve their visibility during surgical procedures. Visualization
agents are especially useful when used in MIS procedures, due among
other reasons to their improved visibility on a color monitor.
[0049] Visualization agents may be selected from among any of the
various non-toxic colored substances suitable for use in medical
implantable medical devices, such as FD&C dyes 3 and 6, eosin,
methylene blue, indocyanine green, or colored dyes normally found
in synthetic surgical sutures. The preferred color is green or blue
because it has better visibility in presence of blood or on a pink
or white tissue background. Red is the least preferred color.
[0050] The visualization agent may be present in either a
crosslinker or functional polymer solution, preferably in a
functional polymer solution. The preferred colored substance may or
may not become incorporated into the biocompatible crosslinked
polymer. Preferably, however, the visualization agent does not have
a functional group capable of reacting with the crosslinker or
functional polymer.
[0051] The visualization agent may be used in small quantities,
preferably less than 1% weight/volume, more preferably less that
0.01% weight/volume and most preferably less than 0.001%
weight/volume concentration.
[0052] Additional visualization agents may be used, such as
fluorescent (e.g., green or yellow fluorescent under visible light)
compounds (e.g., fluorescein or eosin), x-ray contrast agents
(e.g., iodinated compounds) for visibility under x-ray imaging
equipment, ultrasonic contrast agents, or MRI contrast agents
(e.g., Gadolinium containing compounds).
Crosslinking Reactions
[0053] The crosslinking reactions preferably occur in aqueous
solution under physiological conditions. More preferably the
crosslinking reactions occur "in situ", meaning they occur at local
sites such as on organs or tissues in a living animal or human
body. More preferably the crosslinking reactions do not release
heat of polymerization. Preferably the crosslinking reaction
leading to gelation occurs within 10 minutes, more preferably
within 2 minutes, more preferably within one minute, and most
preferably within 30 seconds.
[0054] Certain functional groups, such as alcohols or carboxylic
acids, do not normally react with other functional groups, such as
amines, under physiological conditions (e.g., pH 7.2-11.0,
37.degree. C.). However, such functional groups can be made more
reactive by using an activating group such as N-hydroxysuccinimide.
Several methods for activating such functional groups are known in
the art. Preferred activating groups include carbonyldiimidazole,
sulfonyl chloride, aryl halides, sulfosuccinimidyl esters,
N-hydroxysuccinimidyl ester, succinimidyl ester, epoxide, aldehyde,
maleimides, imidoesters and the like. The N-hydroxysuccinimide
esters or N-hydroxysulfosuccinimide groups are the most preferred
groups for crosslinking of proteins or amine functionalized
polymers such as aminoterminated polyethylene glycol ("APEG").
[0055] FIGS. 1 to 5 illustrate various embodiments of preferred
crosslinkers and functional polymers.
[0056] FIG. 1 illustrates possible configurations of degradable
electrophilic crosslinkers or functional polymers. The
biodegradable regions are represented by (); the functional groups
are represented by (); and the inert water soluble cores are
represented by (--). For crosslinkers, the central core is a water
soluble small molecule and for functional polymers the central core
is a water soluble polymer of natural or synthetic origin.
[0057] When Structure A in FIG. 1 is a functional polymer, it is a
linear water soluble and biodegradable functional polymer,
end-capped with two functional groups (e.g., N-hydroxysuccinimide
ester or NHS, epoxide or similar reactive groups). The water
soluble core may be a polyalkylene oxide, preferably polyethylene
glycol block copolymer, and it is extended with at least one
biodegradable linkage between it and each terminal functional
group. The biodegradable linkage may be a single linkage or
copolymers or homopolymers of absorbable polymers such as
polyhydroxy acids or polylactones.
[0058] When Structure B in FIG. 1 is a functional polymer it is a
branched or star shaped biodegradable functional polymer which has
an inert polymer at the center. Its inert and water soluble core is
terminated with oligomeric biodegradable extensions, which in turn
are terminated with reactive functional groups.
[0059] When Structures C and D in FIG. 1 are functional polymers,
they are multifunctional 4 arm biodegradable functional polymers.
This polymer again has a water-soluble core at the center, which is
a 4 arm, tetrafunctional polyethylene glycol (Structure C) or block
copolymer of PEO-PPO-PEO such as Tetronic 908 (Structure D) which
is extended with by small oligomeric extensions of biodegradable
polymer to maintain water solubility and terminated with reactive
functional end-groups such as CDI or NHS.
[0060] When Structure E in FIG. 1 is a functional polymer, it is a
multifunctional star or graft type biodegradable polymer. This
polymer has a water-soluble polymer like polyethylene oxide,
polyvinyl alcohol or poly(vinyl pyrrolidinone) at the core which is
completely or partially extended with biodegradable polymer. The
biodegradable polymer is terminated with reactive end groups.
[0061] Structures A-E in FIG. 1 need not have polymeric cores and
may be small molecule crosslinkers. In that case, the core may
comprise a small molecule like ethoxylated glycerol, inositol,
trimethylolpropane etc. to form the resultant crosslinker. In
addition, Structures A-E in FIG. 1 need not have polymeric
biodegradable extensions, and the biodegradable extensions may
consist of small molecules like succinate or glutarate or
combinations of 2 or more esters, such as
glycolate/2-hydroxybutyrate or glycolate/4-hydroxyproline, etc. A
dimer or trimer of 4-hydroxyproline may be used not only to add
degradability, but also to add nucleophilic reactive sites via the
pendant primary amines which are part of the hydroxyproline
moiety.
[0062] Other variations of the core, the biodegradable linkage, and
the terminal electrophilic group in Structures A-E in FIG. 1 may be
constructed, so long as the resulting functional polymer has the
properties of low tissue toxicity, water solubility, and reactivity
with nucleophilic functional groups.
[0063] FIG. 2 illustrates various embodiments of nucleophilic
biodegradable water-soluble crosslinkers and functional polymers
suitable foe use with electrophilic functional polymers and
crosslinkers described herein. The biodegradable regions are
represented by (); the functional groups are represented by (); and
the inert water soluble cores are represented by (--). For
crosslinkers, the central core is a water soluble small molecule
and for functional polymers the central core is a water soluble
polymer of natural or synthetic origin.
[0064] When Structure F in FIG. 2 is a functional polymer, it is a
linear water-soluble biodegradable polymer terminated with reactive
functional groups like primary amine. The linear water-soluble core
is a polyalkylene oxide, preferably polyethylene glycol block
copolymer, which is extended with the biodegradable region which is
a copolymer or homopolymer of polyhydroxy acids or polylactones.
This biodegradable polymer is terminated with primary amines.
[0065] When Structure G in FIG. 2 is a functional polymer, it is a
branched or star shaped biodegradable polymer which has an inert
polymer at the center. The inert polymer is extended with single or
oligomeric biodegradable extensions which are terminated with
reactive functional groups.
[0066] When Structures H and I in FIG. 2 are functional polymers,
they are multifunctional 4 arm biodegradable polymers. These
polymers again have water-soluble cores at their center which are
either a 4 arm, tetrafunctional polyethylene glycol (Structure H)
or a block copolymer of PEO-PPO-PEO such as Tetronic 908 (Structure
I), extended with small oligomeric extensions of biodegradable
polymers to maintain water solubility, and terminated with
functional groups such as amines and thiols.
[0067] When Structure J in FIG. 2 is a functional polymer, it is a
multifunctional star or graft type biodegradable polymer. This
polymer has a water-soluble polymer like polyethylene oxide,
polyvinyl alcohol or poly(vinyl pyrrolidinone) at the core which is
completely or partially extended with biodegradable polymer. The
biodegradable polymer is terminated with reactive end groups.
[0068] Structures F-J in FIG. 2 need not have polymeric cores and
may be small molecule crosslinkers. In that case, the core may
comprise a small molecule like ethoxylated glycerol, inositol,
trimethylolpropane etc. to form the resultant crosslinker.
[0069] Other variations of the core, the biodegradable linkage, and
the terminal nucleophilic group in Structures F-J in FIG. 2 may be
constructed, so long as the resulting functional polymer has the
properties of low tissue toxicity, water solubility, and reactivity
with electrophilic functional groups.
[0070] FIG. 3 illustrates configurations of water soluble
electrophilic crosslinkers or functional polymers where the core is
biodegradable. The biodegradable regions are represented by () and
the functional groups are represented by (). The biodegradable core
is terminated with a reactive functional group that is also water
solubilizing, such a N-hydroxysulfosuccinimide ester ("SNHS") or
N-hydroxyethoxylated succinimide ester ("ENHS").
[0071] Structure K in FIG. 3 depicts a difunctional biodegradable
polymer or oligomer terminated with SNHS or ENHS. The oligomers and
polymers may be made of a poly(hydroxy acid) such as poly(lactic
acid), which is insoluble in water. However, the terminal
carboxylic acid group of these oligomers or polymers can be
activated with N-hydroxysulfosuccinimide ester ("SNHS") or
N-hydroxyethoxylated succinimide ester ("ENHS") groups. An ionic
group, like a metal salt (preferably sodium salt) of sulfonic acid,
or a nonionic group, like a polyethylene oxide on the succinimide
ring, provides water solubility while the NHS ester provides
chemical reactivity towards amines. The sulfonate groups (sodium
salts) or ethoxylated groups on the succinimide ring solubilize the
oligomer or polymer without appreciably inhibiting reactivity
towards amine groups.
[0072] Structures L-O in FIG. 3 represent multi-branched or graft
type structures with terminal SNHS or ENHS group. The cores may
comprise various non-toxic polyhydroxy compounds like sugars
(xylitol, erythritol), glycerol, trimethylolpropane, which have
been reacted with anhydrides such as succinic or glutaric
anhydrides. The resultant acid groups were then activated with SNHS
or ENHS groups to form water-soluble crosslinkers or functional
polymers.
[0073] FIG. 4 illustrates various nucleophilic functional polymers
or crosslinkers that are not biodegradable. The nucleophilic
functional groups are represented by () and the inert water soluble
cores are represented by (--). For crosslinkers, the central core
is a water soluble small molecule and for functional polymers the
central core is a water soluble polymer of natural or synthetic
origin.
[0074] When Structure P in FIG. 4 is a functional polymer it may be
a water-soluble linear polymer such as polyethylene glycol
terminated with reactive end group such as primary amines and
thiols. Such polymers are commercially available from Sigma
(Milwaukee, Wis.) and Shearwater Polymers (Huntsville, Ala.). Some
other preferred difunctional polymers are PPO-PEO-PPO block
copolymers such as Pluronic F68 terminated with amine groups.
Pluronic or Tetronic polymers are normally available with terminal
hydroxyl groups. The hydroxyl groups are converted into amine
groups by methods known in the art.
[0075] When Structures Q-T in FIG. 4 are functional polymers they
may be multifunctional graft or branch type water-soluble
copolymers with terminal amine groups.
[0076] Structures P-T in FIG. 4 need not have polymeric cores and
may be small molecule crosslinkers. In that case, the core may
comprise a small molecule like ethoxylated glycerol, inositol,
trimethylolpropane, dilysine etc. to form the resultant
crosslinker.
[0077] Other variations of the core and the terminal nucleophilic
group in Structure P-T in FIG. 4 may be employed, so long as the
properties of low tissue toxicity, water solubility, and reactivity
with electrophilic functional groups are maintained.
[0078] FIG. 5 illustrates various electrophilic functional polymers
or crosslinkers that are not biodegradable. The electrophilic
functional groups are represented by () and the inert water soluble
cores are represented by (--). For crosslinkers, the central core
is a water soluble small molecule and for functional polymers the
central core is a water soluble polymer of natural or synthetic
origin.
[0079] When Structure U is a functional polymer, it may be a
water-soluble polymer such as polyethylene glycol terminated
reactive end group such as NHS or epoxide. Such polymers are
commercially available from Sigma and Shearwater polymers. Some
other preferred polymers are PPO-PEO-PPO block copolymers such as
Pluronic F68 terminated with NHS or SNHS group. Pluronic or
Tetronic polymers are normally available with terminal hydroxyl
groups. The hydroxyl groups are converted into acid group by
reacting with succinic anhydride. The terminated acid groups are
reacted with N-hydroxysuccinimide in presence of DCC to generate
NHS activated Pluronic polymer.
[0080] When Structures V-Y are functional polymers they may be
multifunctional graft or branch type PEO or PEO block copolymers
(Tetronics) activated with terminal reactive groups such as
NHS.
[0081] Structures U-Y in FIG. 5 need not have polymeric cores and
may be small molecule crosslinkers. In that case, the core may
comprise a small molecule like ethoxylated glycerol, inositol,
trimethylolpropane, dilysine etc. to form the resultant
crosslinker.
[0082] Other variations of the core and the terminal nucleophilic
group in Structures U-Y in FIG. 5 may be employed, so long as the
properties of low tissue toxicity, water solubility, and reactivity
with electrophilic functional groups are maintained.
Preparation of Structures A-Y in FIGS. 1-5
[0083] The polymeric crosslinkers and functional polymers
illustrated as Structures A-Y in FIGS. 1 to 5 may be prepared using
variety of synthetic methods. Their preferred compositions are
described in Table 1.
1TABLE 1 Preferred Crosslinkers and Functional Polymers Structure
Brief Description Typical Example A Water soluble, linear
Polyethylene glycol difunctional or ethoxylated crosslinker or
propylene glycol functional polymer with chain extended with water
soluble core, oligolactate and extended with terminated with N-
biodegradable regions hydroxysuccinimide such as oligomers of
esters. hydroxyacids or peptide sequences which are cleavable by
enzymes and terminated with protein reactive functional groups. B
Water soluble, Ethoxylated glycerol trifunctional chain extended
with crosslinker or oligolactate and functional polymer with
terminated with N- water soluble core, hydroxysuccinimide extended
with esters biodegradable regions such as oligomers of hydroxyacids
or peptide sequences and terminated with protein reactive
functional groups C Water soluble, 4 arm polyethylene
tetrafunctional glycol, erythritol or crosslinker or
pentaerythritol chain functional polymer with extended with water
soluble core, oligolactate and extended with terminated with N-
biodegradable regions hydroxysuccinimide such as oligomers of
esters hydroxyacids or peptide sequences and terminated with
protein reactive functional groups D Water soluble, Ethoxylated
ethylene tetrafunctional diamine or crosslinker or polyethylene
oxide- functional polymer with polypropylene oxide- water soluble
core, polyethylene oxide extended with block copolymer like
biodegradable regions Tetronic 908 chain such as oligomers of
extended with hydroxyacids or peptide oligotrimethylene sequences
and carbonate and terminated with protein terminated with N-
reactive functional hydroxysuccinimide groups ester E Water
soluble, branched Low molecular weight crosslinker or polyvinyl
alcohol functional polymer with with 1% to 20% water soluble core,
hydroxyl groups extended with extended with biodegradable regions
oligolactate and such as oligomers of terminated with N-
hydroxyacids or peptide hydroxysuccinimide sequences and ester
terminated with protein reactive functional groups F Water soluble,
linear Polyethylene oxide- difunctional polypropylene oxide-
crosslinker or polyethylene oxide functional polymer with block
copolymer water soluble core, surfactant like extended with
Pluronic F68 chain biodegradable regions extended with such as
oligomers of oligolactate and hydroxyacids or peptide terminated
with amino sequences and acids such as lysine terminated with
amines, or peptide sequences carboxylic acid or that may contain
two thiols amine groups G Water soluble, Ethoxylated glycerol
trifunctional chain extended with crosslinker or oligolactate and
functional polymer with terminated with water soluble core,
aminoacid such as extended with lysine biodegradable regions such
as oligomers of hydroxyacids or peptide sequences and terminated
with amines, carboxylic acid or thiols H Water soluble, 4 arm
polyethylene tetrafunctional glycol or tetra crosslinker or
erythritol chain functional polymer with extended with water
soluble core, oligolactate and extended with terminated with
biodegradable regions aminoacid such as such as oligomers of lysine
hydroxyacids or peptide sequences and terminated with amines,
carboxylic acid or thiols I Water soluble, Ethoxylated ethylene
tetrafunctional diamine or crosslinker or polyethylene oxide-
functional polymer with polypropylene oxide- water soluble core,
polyethylene oxide extended with block copolymer like biodegradable
regions Tetronic 908 chain such as oligomers of extended with
hydroxyacids or peptide oligotrimethylene sequences and carbonate
and terminated with amines, terminated with carboxylic acid or
aminoacid such as thiols lysine J Water soluble, Low molecular
weight multifunctional or polyvinyl alcohol graft type crosslinker
with 1-20% hydroxyl or functional polymer groups extended with with
water soluble oligolactate and core, extended with terminated with
biodegradable regions aminoacid such as such as oligomers of lysine
hydroxyacids or peptide sequences and terminated with amines,
carboxylic acid or thiols K Water soluble, linear Difunctional
difunctional oligolactic acid with crosslinker or terminal carboxyl
functional polymer such groups which are as oligomers of activated
with n- hydroxyacids or peptide hydroxysulfosuccinimide sequences
which are ester or terminated with protein ethoxylated n- reactive
functional hydroxysuccinimide groups ester. L Water soluble
branched Trifunctional trifunctional oligocaprolactone crosslinker
or with terminal functional polymer such carboxyl groups which as
oligomers of are activated with n- hydroxyacids or peptide
hydroxysulfosuccinimide sequences which are ester or terminated
with protein ethoxylated n- reactive functional hydroxysuccinimide
groups ester. M Water soluble, branched Tetrafunctional
tetrafunctional oligocaprolactone crosslinker or with terminal
functional polymer such carboxyl groups which as oligomers of are
activated with n- hydroxyacids or peptide hydroxysulfosuccinimide
sequences which are ester or terminated with protein ethoxylated n-
reactive functional hydroxysuccinimide groups ester. N Water
soluble, branched Tetrafunctional tetrafunctional oligocaprolactone
crosslinker or with terminal functional polymer such carboxyl
groups which as oligomers of are activated with n- hydroxyacids or
peptide hydroxysulfosuccinimide sequences which are ester or
terminated with protein ethoxylated n- reactive functional
hydroxysuccinimide groups ester. O Water soluble, branched
Multifunctional multifunctional oligolactic acid with crosslinker
or terminal carboxyl functional polymer such groups which are as
oligomers of activated with n- hydroxyacids or peptide
hydroxysulfosuccinimide sequences which are ester or terminated
with protein ethoxylated n- reactive functional hydroxysuccinimide
groups ester. P Water soluble, linear Polyethylene glycol
difunctional with terminal amines crosslinker or groups functional
polymer terminated with amines, carboxylic acid or thiols
functional groups Q Water soluble, branched Ethoxylated glycerol
trifunctional with terminal amines crosslinker or groups functional
polymer terminated with amines, carboxylic acid or thiols as
functional group R Water soluble, branched 4 arm polyethylene
tetrafunctional glycol modified to crosslinker or produce terminal
functional polymer amine groups terminated with amines, carboxylic
acid or thiols functional groups S Water soluble, branched
Ethoxylated ethylene tetrafunctional diamine or crosslinker or
polyethylene oxide- functional polymer polypropylene oxide-
terminated with amines, polyethylene oxide carboxylic acid or block
copolymer like thiols functional Tetronic 908 modified groups to
generate terminal amine groups T Water soluble, branched
Polylysine, albumin, or graft crosslinker or polyallyl amine
functional polymer with terminal amines, carboxylic acid or thiols
functional groups U Water soluble, linear Polyethylene glycol
difunctional with n- crosslinker or hydroxysuccinimide as
functional polymer end groups terminated with protein reactive
functional groups V Water soluble branched Ethoxylated glycerol
trifunctional terminated with n- crosslinker or hydroxysuccinimide
functional polymer terminated with protein reactive functional
groups W Water soluble branched 4 arm polyethylene tetrafunctional
glycol terminated crosslinker or with n- functional polymer
hydroxysuccinimide terminated with protein esters reactive
functional groups X Water soluble branched Ethoxylated ethylene
tetrafunctional diamine or crosslinker or polyethylene oxide-
functional polymer polypropylene oxide- terminated with protein
polyethylene oxide reactive functional block copolymer like groups
Tetronic 908 with n- hydroxysuccinimide ester as end group Y Water
soluble, branched Poly(vinyl or graft polymer pyrrolidinone)-co-
crosslinker or poly(n- functional polymer with hydroxysuccinimide
protein reactive acrylate) copolymer functional groups (9:1),
molecular weight < 40000 Da
[0084] First, the biodegradable links of Structures A-J in FIGS. 1
and 2 may be composed of specific di or multifunctional synthetic
amino acid sequences which are recognized and cleaved by enzymes
such as collagenase, and may be synthesized using methods known to
those skilled in the peptide synthesis art. For example, Structures
A-E in FIG. 1 may be obtained by first using carboxyl, amine or
hydroxy terminated polyethylene glycol as a starting material for
building a suitable peptide sequence. The terminal end of the
peptide sequence is converted into a carboxylic acid by reacting
succinic anhydride with an appropriate amino acid. The acid group
generated is converted to an NHS ester by reaction with
N-hydroxysuccinimide.
[0085] The functional polymers described in FIG. 2 may be prepared
using a variety of synthetic methods. In a preferred embodiment,
the polymer shown as Structure F may be obtained by ring opening
polymerization of cyclic lactones or carbonates initiated by a
dihydroxy compound such as Pluronic F 68 in the presence of a
suitable catalyst such as stannous 2-ethylhexanoate. The molar
equivalent ratio of caprolactone to Pluronic is kept below 10 to
obtain a low molecular weight chain extension product so as to
maintain water solubility. The terminal hydroxyl groups of the
resultant copolymer are converted into amine or thiol by methods
known in the art.
[0086] In a preferred method, the hydroxyl groups of a
Pluronic-caprolactone copolymer are activated using tresyl
chloride. The activated groups are then reacted with lysine to
produce lysine terminated Pluronic-caprolactone copolymer.
Alternatively, an amine-blocked lysine derivative is reacted with
the hydroxyl groups of a Pluronic-caprolactone copolymer and then
the amine groups are regenerated using a suitable deblocking
reaction.
[0087] Structures G, H, I and J in FIG. 2 may represent
multifunctional branched or graft type copolymers having
water-soluble core extended with oligohydroxy acid polymer and
terminated with amine or thiol groups.
[0088] For example, in a preferred embodiment, the functional
polymer illustrated as Structure G in FIG. 2 is obtained by ring
opening polymerization of cyclic lactones or carbonates initiated
by a tetrahydroxy compound such as 4 arm, tetrahydroxy polyethylene
glycol (molecular weight 10,000 Da), in the presence of a suitable
catalyst such as stannous octoate. The molar equivalent ratio of
cyclic lactone or carbonate to PEG is kept below 10 to obtain a low
molecular weight extension, and to maintain water solubility
(polymers of cyclic lactones generally are not as water soluble as
PEG). Alternatively, hydroxyacid as a biodegradable link may be
attached to the PEG chain using blocking/deblocking chemistry known
in the peptide synthesis art. The terminal hydroxy groups of the
resultant copolymer are activated using a variety of reactive
groups known in the art. The CDI activation chemistry and sulfonyl
chloride activation chemistry is shown in FIGS. 6 and 7,
respectively.
[0089] The most preferred reactive groups are N-hydroxysuccinimide
esters, synthesized by any of several methods. In a preferred
method, hydroxyl groups are converted to carboxylic groups by
reacting them with anhydrides such as succinic anhydride in the
presence of tertiary amines such as pyridine or triethylamine or
dimethylaminopyridine ("DMAP"). Other anhydrides such as glutaric
anhydride, phthalic anhydride, maleic anhydride and the like may
also be used. The resultant terminal carboxyl groups are reacted
with N-hydroxysuccinimide in the presence of
dicyclohexylcarbodiimide ("DCC") to produce N-hydroxysuccinimide
ester (referred as NHS activation). The NHS activation and
crosslinking reaction scheme is shown in FIG. 8. The most preferred
N-hydroxysuccinimide esters are shown in FIG. 9.
[0090] In a preferred embodiment, the polymer shown as structure H
is obtained by ring opening polymerization of glycolide or
trimethylene carbonate initiated by a tetrahydroxy compound such as
tetrafunctional polyethylene glycol (molecular weight 2000 Da) in
the presence of a catalyst such as stannous 2-ethylhexoate. The
molar equivalent ratio of glycolide to PEG is kept from 2 to 10 to
obtain a low molecular weight extension. The terminal hydroxy
groups of the resultant copolymer are converted into amine groups
by reaction with lysine as mentioned previously. Similar
embodiments can be obtained using analogous chain extension
synthetic strategies to obtain structures F, G, I and J by starting
with the appropriate corresponding polyol.
[0091] Structures K, L, M, N, and O in FIG. 3 are made using a
variety of synthetic methods. In a preferred embodiment, the
polymer shown as Structure L in FIG. 3 is obtained by ring opening
polymerization of cyclic lactones by a trihydroxy compound such as
glycerol in the presence of a catalyst such as stannous
2-ethylhexanroate. The molar equivalent ratio of cyclic lactone to
glycerol is kept below 2, so that only low molecular weight
oligomers are obtained. The low molecular weight oligomer ester is
insoluble in water. The terminal hydroxy groups of the resultant
copolymer are activated using N-hydroxysulfosuccinimide groups.
This is achieved by converting hydroxy groups to carboxylic groups
by reacting with anhydrides such as succinic anhydride in presence
of tertiary amines. The resultant terminal carboxyl groups are
reacted with N-hydroxysulfosuccinimide or N-hydroxyethoxylated
succinimide in the presence of dicyclohexylcarbodiimide ("DCC") to
produce a sulfonated or ethoxylated NHS ester. The sulfonate or PEO
chain on the succinimide ring gives water solubility to the
oligoester.
[0092] The foregoing method generally is applied to solubilize only
low molecular weight multi-branched oligoesters, with molecular
weights below 1000. In another variation of this method, various
non-toxic polyhydroxy compounds, preferably sugars, such as
erythritol, xylitol are reacted with succinic anhydride in the
presence of a tertiary amine. The terminal carboxyl group of
succinated erythritol is esterified with N-hydroxysulfosuccinimide
(FIG. 9). Similar embodiments may be obtained using analogous
synthetic strategies to obtain structures K, and M-O by starting
with the appropriate starting materials.
[0093] Structures P-R may be synthesized by reacting the
appropriate starting material, such as a linear (P) or 2- or 3-arm
branched PEG (Q, R) with hydroxy end groups, with lysine as
mentioned previously, such that the arms of the PEG oligomers are
capped with amine end groups. Structure S may be synthesized, using
a multistep reaction, from PEG, glycerol and a diisocyanate. In the
first step a PEG diol is reacted with excess diisocyanate, such as
4,4'diphenyl methane diisocyanate ("MDI"),
methylene-bis(4-cyclohexylisocyanate) ("HMDI") or
hexamethylenediisocyana- te ("HDI"). After purification the
resultant PEG diisocyanate is added dropwise to excess glycerol or
trimethylol propane or other triol and reacted to completion. The
purified product, now having diol end groups, is again reacted with
excess diisocyanate and purified, yielding a PEG-tetraisocyanate.
This tetrafunctional PEG subsequently may be reacted with excess
PEG diols, yielding a 4 arm PEG synthesized from a PEG diol
oligomer. In the final step lysine end groups are incorporated, as
discussed previously.
[0094] Structure T may be synthesized as follows. First synthesize
a random copolymer of PEG-monoacrylate and some other acrylate or
combination of acrylates, such that the final polyacrylate is water
soluble. Other acrylates include, but are not limited to,
2-hydroxyethylacrylate, acrylic acid, and acrylamide. Conditions
may be varied to control the molecular weight as desired. In the
final step, the acrylate is reacted with lysine as discussed
previously, using an appropriate quantity to achieve the desired
degree of amination.
[0095] One method of synthesizing Structures U-Y is to use
dicyclohexylcarbodiimide coupling to a carboxylate end group. For
Structures U-W, one can react the appropriate PEG-diol, -triol or
-tetra-hydroxy starting material with excess succinic anhydride or
glutaric anhydride such that all end groups are effectively
carboxylated. Structures X and Y may be made in a manner similar to
that used for Structures S and T, except that in the last step,
instead of end capping with lysine, end capping with succinic
anhydride or glutaric anhydride is performed.
Preparation of Biocompatible Polymers
[0096] Several biocompatible crosslinked polymers may be produced
using the crosslinkers and functional polymers described in FIGS. 1
to 5. Preferred combinations of such polymers suitable for
producing such biocompatible crosslinked polymers are described in
Table 1 and Table 2. In Table 2, the crosslinker functional groups
are N-hydroxy succinimide esters and the functional polymer
functional groups are primary amines.
2TABLE 2 Biocompatible Polymers Synthesized from Crosslinkers and
Functional Polymers Of Table 1 Functional Crosslinker Polymer
Structure Structure Concentration Medium B or C H and R Molar
Borate or Equivalent; triethanol >20% W/V amine buffer, pH 7-9
A, B or C H, P, Q, R Molar Borate or and S Equivalent; triethanol
>20% W/V amine buffer, pH 7-9 Y T, H, P and Q Molar Borate or
Equivalent; triethanol >10% W/V amine buffer, pH 7-9 W, V H and
J Molar Bicarbonate Equivalent; buffer, pH 9 >10% W/V X I, J and
H Molar Borate or Equivalent; triethanol >20% W/V amine buffer,
pH 7-9
[0097] The reaction conditions for crosslinking will depend on the
nature of the functional groups. Preferred reactions are conducted
in buffered aqueous solutions at pH 5 to 12. The preferred buffers
are sodium borate buffer (pH 10) and triethanol amine buffer (pH
7). In some embodiments, organic solvents such as ethanol or
isopropanol may be added to improve the reaction speed or to adjust
the viscosity of a given formulation.
[0098] The synthetic crosslinked gels described above degrade due
to hydrolysis of the biodegradable region. The degradation of gels
containing synthetic peptide sequences will depend on the specific
enzyme and its concentration. In some cases, a specific enzyme may
be added during the crosslinking reaction to accelerate the
degradation process.
[0099] When the crosslinker and functional polymers are synthetic
(for example, when they are based on polyalkylene oxide), then it
is desirable and in some cases essential to use molar equivalent
quantities of the reactants. In some cases, molar excess
crosslinker may be added to compensate for side reactions such as
reactions due to hydrolysis of the functional group.
[0100] When choosing the crosslinker and crosslinkable polymer, at
least one of polymers must have more than 2 functional groups per
molecule and at least one degradable region, if it is desired that
the resultant biocompatible crosslinked polymer be biodegradable.
For example, the difunctional crosslinker shown as Structure A in
FIG. 1 cannot form a crosslinked network with the difunctional
polymers shown as Structure F in FIG. 2 or Structure P in FIG. 4.
Generally, it is preferred that each biocompatible crosslinked
polymer precursor have more than 2 and more preferably 4 functional
groups.
[0101] Preferred electrophilic groups are NHS, SNHS and ENHS (FIG.
9). Preferred nucleophilic groups are primary amines. The advantage
of the NHS-amine reaction is that the reaction kinetics lead to
quick gelation usually within 10 minutes, more usually within 1
minute and most usually within 10 seconds. This fast gelation is
preferred for in situ reactions on live tissue.
[0102] The NHS-amine crosslinking reaction leads to formation of
N-hydroxysuccinimide as a side product. The sulfonated or
ethoxylated forms of N-hydroxysuccinimide are preferred due to
their increased solubility in water and hence their rapid clearance
from the body. The sulfonic acid salt on the succinimide ring does
not alter the reactivity of NHS group with the primary amines.
[0103] The NHS-amine crosslinking reaction may be carried out in
aqueous solutions and in the presence of buffers. The preferred
buffers are phosphate buffer (pH 5.0-7.5), triethanolamine buffer
(pH 7.5-9.0) and borate buffer (pH 9.0-12) and sodium bicarbonate
buffer (pH 9.0-10.0).
[0104] Aqueous solutions of NHS based crosslinkers and functional
polymers preferably are made just before the crosslinking reaction
due to reaction of NHS groups with water. Longer "pot life" may be
obtained by keeping these solutions at lower pH (pH 4-5).
[0105] The crosslinking density of the resultant biocompatible
crosslinked polymer is controlled by the overall molecular weight
of the crosslinker and functional polymer and the number of
functional groups available per molecule. A lower molecular weight
between crosslinks such as 600 Da will give much higher
crosslinking density as compared to a higher molecular weight such
as 10,000 Da. Higher molecular weight functional polymers are
preferred, preferably more than 3000 Da, so as to obtain elastic
gels.
[0106] The crosslinking density also may be controlled by the
overall percent solids of the crosslinker and functional polymer
solutions. Increasing the percent solids increases the probability
that an electrophilic group will combine with a nucleophilic group
prior to inactivation by hydrolysis. Yet another method to control
crosslink density is by adjusting the stoichiometry of nucleophilic
groups to electrophilic groups. A one to one ratio leads to the
highest crosslink density.
Preparation of Biodegradable Polymers
[0107] The biodegradable crosslinkers described in FIGS. 1 and 3
may be reacted with proteins, such as albumin, other serum
proteins, or serum concentrates to generate crosslinked polymeric
networks. Briefly, aqueous solutions of the crosslinkers described
in FIG. 1 and FIG. 3 (at a concentration of 50 to 300 mg/ml) are
mixed with concentrated solutions of albumin (600 mg/ml) to produce
a crosslinked hydrogel. This reaction can be accelerated if a
buffering agent, e.g., borate buffer or triethanol amine, is added
during the crosslinking step.
[0108] The resultant crosslinked hydrogel is a semisynthetic
hydrogel whose degradation depends on the degradable segment in the
crosslinker as well as degradation of albumin by enzymes. In the
absence of any degradable enzymes, the crosslinked polymer will
degrade solely by the hydrolysis of the biodegradable segment. If
polyglycolate is used as the biodegradable segment, the crosslinked
polymer will degrade in 1-30 days depending on the crosslinking
density of the network. Similarly, a polycaprolactone based
crosslinked network will degrade in 1-8 months. The degradation
time generally varies according to the type of degradable segment
used, in the following order: polyglycolate<polylactate<pol-
ytrimethylene carbonate<polycaprolactone. Thus, it is possible
to construct a hydrogel with a desired degradation profile, from a
few days to months, using a proper degradable segment.
[0109] The hydrophobicity generated by biodegradable blocks such as
oligohydroxy acid blocks or the hydrophobicity of PPO blocks in
Pluronic or Tetronic polymers are helpful in dissolving small
organic drug molecules. Other properties which will be affected by
incorporation of biodegradable or hydrophobic blocks are: water
absorption, mechanical properties and thermosensitivity.
Methods of Using Biocompatible Polymers
[0110] The biocompatible crosslinked polymers and their precursors
described above may be used in a variety of applications, such as
components of tissue adhesives, tissue sealants, drug delivery
vehicles, wound covering agents, barriers in preventing
postoperative adhesions, and others. These and other suitable
applications are reviewed in Schlag and Redl, "Fibrin Sealant" in
Operative Surgery, volumes 1-7 (1986), which is incorporated herein
by reference.
In Situ Formation
[0111] In many applications, the biocompatible crosslinked polymers
of this invention typically will be formed "in situ" at a surgical
site in the body. The various methodologies and devices for
performing "in situ" gelation, developed for other adhesive or
sealant systems such fibrin glue or sealant applications, may be
used with the biocompatible crosslinked polymers of this invention.
Thus, in one embodiment, an aqueous solution of a freshly prepared
crosslinker (e.g., SNHS-terminated oligolactide synthesized from a
glycerol core in phosphate buffered saline ("PBS") at pH 5 to 7.2)
and a functional polymer (e.g., albumin or amine terminated
tetrafunctional polyethylene glycol at pH 10 in sodium borate) are
applied and mixed on the tissue using a double barrel syringe (one
syringe for each solution). The two solutions may be applied
simultaneously or sequentially. In some embodiments, it is
preferred to apply the precursor solutions sequentially so as to
"prime" the tissue, resulting in improved adherence of the
biocompatible crosslinked polymer to the tissue. Where the tissue
is primed, the crosslinker precursor is preferably applied to the
tissue first, followed by the functional polymer solution.
[0112] One may use specialized devices to apply the precursor
solutions, such as those described in U.S. Pat. Nos. 4,874,368;
4,631,055; 4,735,616; 4,359,049; 4,978,336; 5,116,315; 4,902,281;
4,932,942; Published Patent Cooperation Treaty Patent Application
No. WO 91/09641; and R. A. Tange, "Fibrin Sealant" in Operative
Medicine: Otolaryngology, volume 1 (1986), the disclosures of which
are herein incorporated by reference.
Drug Delivery
[0113] The subject crosslinkers, functional polymer and their
reaction products, the crosslinked materials advantageously may be
used for localized drug therapy. Biologically active agents or drug
compounds that may be added and delivered from the crosslinked
polymer or gel include: proteins, glycosaminoglycans,
carbohydrates, nucleic acid, inorganic and organic biologically
active compounds where specific biologically active agents include
but are not limited to: enzymes, antibiotics, antineoplastic
agents, local anesthetics, hormones, angiogenic agents,
anti-angiogenic agents, growth factors, antibodies,
neurotransmitters, psychoactive drugs, anticancer drugs,
chemotherapeutic drugs, drugs affecting reproductive organs, genes,
and oligonucleotides.
[0114] To prepare such crosslinked composition, the bioactive
compounds described above are mixed with the crosslinkable polymer
prior to making the aqueous solution or during the aseptic
manufacturing of the functional polymer. This mixture then is mixed
with the crosslinker to produce a crosslinked material in which the
biologically active substance is entrapped. Functional polymers
made from inert polymers like Pluronic, Tetronics or Tween.TM.
surfactants are preferred in releasing small molecule hydrophobic
drugs.
[0115] In a preferred embodiment, the active agent or agents are
present in a separate phase when crosslinker and crosslinkable
polymers are reacted to produce a crosslinked polymer network or
gel. This phase separation prevents participation of bioactive
substance in the chemical crosslinking reaction such as reaction
between NHS ester and amine group. The separate phase also helps to
modulate the release kinetics of active agent from the crosslinked
material or gel, where `separate phase` could be oil (oil-in water
emulsion), biodegradable vehicle; and the like. Biodegradable
vehicles in which the active agent may be present include:
encapsulation vehicles, such as microparticles, microspheres,
microbeads, micropellets, and the like, where the active agent is
encapsulated in a bioerodable or biodegradable polymers such as
polymers and copolymers of: poly(anhydride), poly(hydroxy acid)s,
poly(lactone)s, poly(trimethylene carbonate), poly(glycolic acid),
poly(lactic acid), poly(glycolic acid)-co-poly(glycolic acid),
poly(orthocarbonate), poly(caprolactone), crosslinked biodegradable
hydrogel networks like fibrin glue or fibrin sealant, caging and
entrapping molecules, like cyclodextrin, molecular sieves and the
like. Microspheres made from polymers and copolymers of
poly(lactone)s and poly(hydroxy acid) are particularly preferred as
biodegradable encapsulation vehicles.
[0116] In using crosslinked materials which are described herein as
drug delivery vehicles, the active agent or encapsulated active
agent may be present in solution or suspended form in crosslinker
component or functional polymer solution component. The
nucleophilic component, whether it be in the crosslinker or the
functional polymer is the preferred vehicle due to absence of
reactive groups. The functional polymer along with bioactive agent,
with or without encapsulating vehicle, is administered to the host
along with equivalent amount of crosslinker and aqueous buffers.
The chemical reaction between crosslinker and the functional
polymer solution readily takes place to form a crosslinked gel and
acts as a depot for release of the active agent to the host. Such
methods of drug delivery find use in both systemic and local
administration of an active agent.
[0117] In using the crosslinked composition for drug delivery as
mentioned above, the amount of crosslinkable polymer, crosslinker
and the dosage agent introduced in the host will necessarily depend
upon the particular drug and the condition to be treated.
Administration may be by any convenient means such as syringe,
canula, trocar, catheter and the like.
[0118] Controlled rates of drug delivery also may be obtained with
the system of the present invention by degradable, covalent
attachment of the bioactive molecules to the crosslinked hydrogel
network. The nature of the covalent attachment can be controlled to
enable control of the release rate from hours to weeks or longer.
By using a composite made from linkages with a range of hydrolysis
times, a controlled release profile may be extended for longer
durations.
Composite Biomaterials
[0119] The biocompatible crosslinked polymers of this invention
optionally may be reinforced with flexible or rigid fibers, fiber
mesh, fiber cloth and the like. The insertion of fibers improves
mechanical properties like flexibility, strength, and tear
resistance. In implantable medical applications, biodegradable
fibers, cloth, or sheets made from oxidized cellulose or
poly(hydroxy acid)s polymers like polylactic acid or polyglycolic
acid, are preferred. Such reinforced structures may be produced
using any convenient protocol known in the art.
[0120] In a preferred method, aqueous solutions of functional
polymers and crosslinkers are mixed in appropriate buffers and
proportions are added to a fiber cloth or net such as Interceed
(Ethicon Inc., New Brunswick, N.J.). The liquid mixture flows into
the interstices of the cloth and becomes crosslinked to produce a
composite hydrogel. Care is taken to ensure that the fibers or
fiber mesh are buried completely inside the crosslinked hydrogel
material. The composite structure can be washed to remove side
products such as N-hydroxysuccinimide. The fibers used are
preferably hydrophilic in nature to ensure complete wetting of the
fibers by the aqueous gelling composition.
EXAMPLES
[0121] The following non-limiting examples are intended to
illustrate the synthesis of new biocompatible crosslinked polymers
and their precursors, and their use in making several medical
products. Those skilled in the art will appreciate that
modifications can be made to these examples, drawings,
illustrations and claims that are intended to fall within the scope
the present invention.
Materials and Equipment
[0122] Polyethylene glycol was purchased form various sources such
as Shearwater Polymers, Union Carbide, Fluka and Polysciences.
Multifunctional hydroxyl and amine terminated polyethylene glycol
were purchased from Shearwater Polymers, Dow Chemicals and Texaco.
Pluronic.RTM. and Tetronic.RTM. series polyols were purchased from
BASF Corporation. DL-lactide, glycolide, caprolactone and
trimethylene carbonate was obtained from commercial sources like
Purac, DuPont, Polysciences, Aldrich, Fluka, Medisorb, Wako and
Boehringer Ingelheim. N-hydroxysulfosuccinimide was purchased from
Pierce. All other reagents, solvents were of reagent grade and were
purchased from commercial sources such as Polysciences, Fluka,
Aldrich and Sigma. Most of the reagents and solvents were purified
and dried using standard laboratory procedures such as described in
D. D. Perrin et al., Purification of Laboratory Chemicals (Pergamon
Press 1980).
General Analysis
[0123] The polymers synthesized according to these examples were
chemically analyzed using structure-determining methods such as
nuclear (proton and carbon-13) magnetic resonance spectroscopy,
infrared spectroscopy. Molecular weights were determined using high
pressure liquid chromatography and gel permeation chromatography.
Thermal characterization of the polymers, including melting point
and glass transition temperatures, were performed using
differential scanning calorimetric analysis. Aqueous solution
properties such as micelle and gel formation was determined using
fluorescence spectroscopy, UV-visible spectroscopy and laser light
scattering instruments.
[0124] In vitro degradation of the polymers was followed
gravimetrically at 37.degree. C., in an aqueous buffered medium
such as phosphate buffered saline (at pH 7.2). In vivo
biocompatibility and degradation life times was assessed by
injecting or forming a gelling formulation directly into the
peritoneal cavity of a rat or rabbit and observing its degradation
over a period of 2 days to 12 months.
[0125] Alternatively, the degradation was also assessed by
prefabricating a sterile implant, made by a process like solution
casting, then surgically implanting the implant within an animal
body. The degradation of the implant over time was monitored
gravimetrically or by chemical analysis. The biocompatibility of
the implant was assessed by standard histological techniques.
Example 1
Synthesis of a Water-soluble Difunctional, Biodegradable Functional
Polymer Based on Polyalkylene Oxide Block Copolymer
[0126] First, Polyethylene glycol-co-polycaprolactone polyol
("F68C2") was synthesized as follows:
[0127] 30 g of Pluronic F68 was dried under vacuum at 110.degree.
C. for 6 h and then mixed with 1.710 g of caprolactone and 30 mg of
stannous 2-ethylhexanoate in a glass sealing tube. The glass tube
then was sealed under nitrogen atmosphere and heated to 170.degree.
C. and maintained at this temperature for 16 h. The Pluronic
F68-caprolactone polymer was cooled and recovered by breaking the
glass sealing tube, and then further purified by several
precipitations from a toluene-hexane solvent-nonsolvent system.
[0128] The polymer then was dried in vacuum at 40.degree. C. and
used immediately in the activation reaction described below:
[0129] Reaction with succinic anhydride ("F68C2S"):
[0130] 30 g of Pluronic F68-caprolactone copolymer was dissolved in
200 ml dry N,N-dimethyl formamide ("DMF") and 0.845 g of succinic
anhydride was added to the reaction mixture. The mixture was heated
to 100.degree. C. under a nitrogen atmosphere for 16 h. The
solution then was cooled and added to 4000 ml hexane to precipitate
the carboxyl terminated polymer. It was further purified by
repeated (3 times) precipitation from a toluene-hexane
solvent-nonsolvent system. The polymer was dried under vacuum at
40.degree. C.
[0131] This polymer was immediately used in activation reaction
described below:
[0132] Activation of carboxyl groups with N-hydroxysuccinimide
("F68C2SSNHS"):
[0133] 30 g of Pluronic F68-caprolactone succinate copolymer was
dissolved in 200 ml dry DMF. The solution was cooled to 4.degree.
C. and 1.504 g of 1,3-dicyclohexyl carbodiimide ("DCC") and 1.583 g
of N-hydroxysulfosuccinimide ("SNHS") were added to the reaction
mixture. The mixture was stirred at 4.degree. C. for 6 h and then
stirred overnight at room temperature under nitrogen atmosphere.
Dicyclohexylurea was removed by filtration and the F68C2S-SNHS
derivative was isolated by removing the DMF under vacuum and
repeated precipitation using a toluene-hexane solvent-nonsolvent
system. The product was stored under nitrogen atmosphere at
-20.degree. C.
Example 2
Amine Terminated Synthetic Biodegradable Crosslinkable Polymer
[0134] Reaction of F68TMC2SSNHS with Lysine:
[0135] 3.55 g of lysine was dissolved in 200 ml 0.1M borate buffer
(pH 8.5). The mixture was cooled to 0.degree. C. in ice bath and 10
g of F68C2SSNHS were added to the mixture. The mixture was stirred
for 6 h at room temperature and lyophilized. The lyophilized powder
was dissolved in 30 ml toluene and filtered. The filtrate was added
to 4000 ml cold diethyl ether. The precipitated amine terminated
polymer was recovered by filtration and dried under vacuum. The
polymer was stored under argon at -20.degree. C.
Example 3
Synthesis of Carboxyl Terminated Oligolactic Acid Polymer Activated
with N-hydroxysulfosuccinimide
[0136] Synthesis of difunctional oligolactate with terminal
carboxyl acid end-groups activated with N-hydroxysulfosuccinimide
groups.
[0137] Part 1: Synthesis of oligomeric poly(lactic acid) with
terminal carboxyl acid groups ("PLA-S"):
[0138] In a 250 ml 3 neck flask equipped with mechanical stirrer,
nitrogen inlet and distillation condenser, 2 grams of succinic acid
and 34.1 ml 1N HCl and 3.83 g L-lactic acid, sodium salt were
charged. The flask was then immersed in a silicone oil bath
maintained at 150.degree. C. Most of the water from the reaction
mixture was removed over period of 5 hours by distillation. The
remaining water was removed by heating the reaction mixture under
vacuum at 180.degree. C. for 15 h. The reaction mixture was cooled
and lyophilized at 0.degree. C. to remove traces of water. The
product was isolated by dissolving in toluene and precipitating in
hexane. The precipitated polymer was isolated by filtration and
dried in vacuum for 48 h at 60.degree. C.
[0139] Part 2: Activation of terminal groups with
N-hydroxysulfosuccinimid- e group:
[0140] A 3 necked flask equipped with magnetic stirrer and nitrogen
inlet was charged with 2 g of PLA-S copolymer and 20 ml DMF. The
solution was cooled 4.degree. C. and 3.657 g of
N-hydroxysulfosuccinimide and 3.657 g of 1,3-dicyclohexyl
carbodiimide were added to the reaction mixture. The mixture was
stirred at 4.degree. C. for 6 h and overnight at room temperature
under nitrogen atmosphere. Dicyclohexylurea was removed by
filtration and SNHS derivative was by isolated by removing the DMF
under vacuum and repeated precipitation using toluene-hexane
solvent-nonsolvent system. The product was stored under nitrogen
atmosphere at 4.degree. C.
Example 4
Preparation of Polyethylene Glycol Based Tetrafunctional
Crosslinker
[0141] Part 1: Synthesis of tetrafunctional polyethylene
glycol-co-polyglycolate copolymer ("4PEG2KG"):
[0142] 30 grams of 4 arm polyethylene glycol, molecular weight 2000
("4PEG2K") was dried at 100.degree. C. for 16 hours prior to use.
30 grams 4PEG2K, 7.66 g of glycolide and 25 mg of stannous
2-ethylhexanoate were charged into a 3 necked flask equipped with a
Teflon coated magnetic stirring needle. The flask was then immersed
into silicone oil bath maintained at 160.degree. C. The
polymerization reaction was carried out for 16 h under nitrogen
atmosphere. At the end of the reaction, the reaction mixture was
dissolved in 100 ml toluene. The hydroxy terminated glycolate
copolymer was isolated by pouring the toluene solution in 4000 ml
cold hexane. It was further purified by repeated
dissolution-precipitation process from toluene-hexane
solvent-nonsolvent system and dried under vacuum at 60.degree. C.
It then was immediately used for end capping reaction mentioned
below:
[0143] Part 2: Conversion of hydroxyl groups into carboxylic groups
("4PEG2KGS") and SNHS ester.
[0144] 30 g of 4PEG2KG copolymer was dissolved in 150 ml dry
pyridine. 8.72 g of succinic anhydride was added to it and the
solution was refluxed for 2 h under nitrogen atmosphere. The
polymer was isolated by pouring the cold pyridine solution to 4000
ml hexane. The acid terminated polymer ("4PEG2KGS") was used in
SNHS activation reaction. Briefly, to a solution of 30 g of
4PEG2KGS in 300 ml dry methylene chloride were added 10.58 g of
SNHS and 10.05 g DCC. The reaction mixture was stirred overnight
under nitrogen atmosphere. Dicyclohexylurea was removed by
filtration. The filtrate was evaporated and the residue obtained
was redissolved in 100 ml toluene. The toluene solution was
precipitated in 2000 ml hexane. The SNHS activated polymer was
stored under nitrogen atmosphere until further use.
Example 5
Sulfonyl Chloride Activated Crosslinkers
[0145] Activation of tetrafunctional polyethylene
glycol-co-polyglycolate copolymer ("4PEG2KGS") with tresyl
chloride:
[0146] 30 g of 4PEG2KG was dissolved in 10 ml dry benzene. The
solution was cooled to 0.degree. C. and 5.92 g of triethyl amine
and 10.70 g tresyl chloride were added under nitrogen atmosphere.
After refluxing for 3 h under nitrogen atmosphere, the reaction
mixture was cooled and filtered to remove triethylamine
hydrochloride. The filtrate was poured into 3000 ml hexane to
precipitate the activated polymer. The residue was redissolved in
THF and filtered over neutral alumina to remove traces of
triethylamine hydrochloride. The polymer was recovered by adding
the THF solution to 3000 ml diethyl ether and stored under nitrogen
atmosphere.
Example 6
Synthesis of Multifunctional Oligopolycaprolactone Terminated with
SNHS
[0147] Part 1: Synthesis of polycaprolactone ("PCL1"):
[0148] 2.00 g of glycerol, 8.17 g of caprolactone and 50 mg of
stannous 2-ethylhexanoate were charged into 100 ml Pyrex pressure
sealing tube. The tube was frozen in liquid nitrogen and connected
to vacuum line for 10 minutes. The tube then was connected to argon
gas line and sealed under argon. The sealed reaction mixture then
was immersed in oil bath maintained at 160.degree. C. and
polymerization was carried out for 16 h at 160.degree. C. The
polymer was recovered by dissolving it in 30 ml toluene and
precipitating in 2000 ml cold hexane. The precipitated liquid
oligomer was recovered and dried under vacuum for 1 day at
60.degree. C.
[0149] Part 2: End-capping of PCL1 with succinic anhydride
("PCL-S"):
[0150] 10 g of PCL1 was dissolved in 150 ml dry benzene. About 50
ml of benzene was distilled to remove traces of water from the
reaction mixture. The solution was cooled to 30.degree. C. To this
warm solution, 6.67 g of triethyl amine and 7.86 g of succinic
anhydride were added. The reaction mixture was then refluxed for 6
h and concentrated by distillation under vacuum. The product was
recovered by adding the filtrate to 2000 ml cold dry hexane.
[0151] Part 3: Activation of PCL-S with SNHS:
[0152] PCLl-succinate (5.0 g) was dissolved in 10 ml of anhydrous
methylene chloride, cooled to 0.degree. C. and 7.82 g of
N-hydroxysulfosuccinimide and 7.42 N,N-dicyclohexylcarbodiimide
were added under stirring. After stirring the mixture overnight,
the precipitated dicyclohexylurea was removed by filtration and the
solution was concentrated by removing solvent. The .sup.1H-NMR
spectrum showed succinimide singlet at 2.80 ppm (2H).
Example 7
Preparation of Polyethylene glycol-co-polytrimethylene Carbonate
Copolymer Terminated with N-hydroxysuccinimide
[0153] Preparation of tetrafunctional polyethylene
glycol-co-polytrimethyl- ene carbonate copolymer
("4PEG10KTMC2")
[0154] 30 g of tetrahydroxy polyethylene glycol, molecular weight
10000, was dried under vacuum at 90-100.degree. C. in a glass
sealing tube. The tube then was cooled and transferred inside an
air bag where 2.45 g of trimethylene carbonate and 20 mg of
stannous octoate were added to the tube. The glass tube was then
sealed under vacuum and heated with stirring at 155.degree. C. and
maintained at this temperature for 16 h. The polyethylene
glycol-co-polytrimethylene carbonate polymer was cooled and
recovered by breaking the glass sealing tube. It was further
purified by several precipitations from toluene-hexane
solvent-nonsolvent system.
[0155] Part 2: Synthesis of glutarate derivative of 4PEG10KTMC2
("4PEG10KTMC2G"):
[0156] 10 g of 4PEG10KTMC was dissolved in 120 ml dry toluene.
About 50 ml of toluene was distilled to remove traces of water from
the reaction mixture. The warm solution was cooled to 60.degree. C.
To this solution, 1.23 g of triethyl amine and 1.40 g of glutaric
anhydride were added. The reaction mixture was heated to 60.degree.
C. for 1 h and filtered. The product was recovered by adding the
filtrate to 2000 ml cold dry hexane.
[0157] Part 3: Activation of terminal carboxyl groups using
N-hydroxysuccinimide ("4PEG10KTMC2GNHS"):
[0158] 30 g of 4PEG10KTMC2G was dissolved in 100 ml of dry DMF and
1.53 g of N-hydroxysuccinimide and 5 g molecular sieves 3A.degree.
were added. 1.28 g of DCC dissolved in 5 ml dry DMF was added
dropwise and the reaction mixture was kept at room temperature for
24 h under nitrogen atmosphere. The mixture was diluted with 50 ml
cold benzene and precipitated using cold hexane. The precipitate
was collected on a sintered glass filter with suction. The
dissolution and precipitation procedure was then repeated three
times, using toluene-diethyl ether as solvent-nonsolvent system and
dried under vacuum. The product was stored under nitrogen
atmosphere at -20.degree. C. until further use.
Example 8
Succinated Polyhydroxy Compounds Activated with
N-hydroxysulfosuccinimide ES
[0159] 10 g of erythritol was dissolved in 200 ml dry toluene.
About 50 ml of toluene was distilled to remove traces of water from
the erythritol. The solution was cooled to 50-60.degree. C. and 20
ml pyridine and 8.58 g of succinic anhydride were added to the
solution. The reaction mixture was then refluxed for 3 h and
unreacted pyridine and toluene were evaporated to dryness under
reduced pressure. The residue was used in activation reaction.
[0160] Part 2: Activation of ES with SNHS:
[0161] Erythritol-succinate (ES, 2.0 g) was dissolved in 10 ml of
anhydrous dimethyl formamide ("DMF"), cooled to 0.degree. C. and
3.47 g of N-hydroxysulfosuccinimide and 3.30
N,N-dicyclohexylcarbodiimide were added under stirring. After
stirring the mixture overnight, the precipitated dicyclohexylurea
was removed by filtration and the solution was concentrated by
removing solvent. It was further purified by column
chromatography.
Example 9
Preparation of Synthetic Crosslinked Biodegradable Gels
[0162] 1.57 g (0.8 mM) of 4 arm amine terminated polyethylene
glycol molecular weight 2000 was dissolved in 10 ml 0.1 M sodium
borate buffer at pH 9.5. 2 g of 4 arm SNHS activated 4PEG2KGS
polymer (molecular weight 2500) was dissolved in phosphate buffered
saline. These two solutions were mixed to produce a crosslinked
gel. In another variation of this method, the 4PEG2KGS polymer
solid was directly added to the amine terminated polymer solution
to produce a crosslinked polymer.
[0163] In another variation, a crosslinker consisting of an
equimolar solution of dilysine can be used in place of the 4 arm
PEG amine solution to form a hydrogel. Gelation was seen to occur
within 10 seconds of mixing the two solutions. Similarly, other
crosslinkers described in examples 1 to 7 may be reacted in molar
equivalent proportions with other amine terminated polymers such as
albumin or amine terminated biodegradable polymers similar to
described in Example 2. The preferred compositions for making
biodegradable hydrogels were described in Table 2. The amine
terminated polymer solution described above was added with 0.1% of
F D and C blue or indigo dye prior to crosslinking reaction. The
addition of dye allows the preparation of colored gels.
Example 10
Preparation of Composite Synthetic Crosslinked Colored
Biodegradable Gels
[0164] 3 grams of bovine serum albumin was dissolved in 3 ml of
phosphate buffered solution. Commercial sutures based on synthetic
biodegradable polymers, such as Vicryl was cut/ground into several
small pieces (size less than 1 mm) using cryogenic grinding. These
colored suture particles (approximately 100 mg) were mixed with the
albumin solution to form a suspension. 100 mg of crosslinker such
as 4PEG10KTMC2GNHS was mixed with 0.2 ml of albumin suspension.
This viscous solution then was mixed with 40 mg of triethanol amine
(buffering agent). The addition of triethanol amine gels the
solution in 60 seconds. The colored suture particles entrapped in
the crosslinked gel help to visualize the gel especially when under
laparoscopic conditions and also acts to strengthen the hydrogel as
a reinforcing agent. The suture particles in above examples can be
replaced with biodegradable microparticles loaded with drugs or
bioactive compounds.
Example 11
Formulation of SG-PEG with Di-lysine
[0165] A four arm PEG with SG end groups (Shearwater Polymers,
approx. 9,100 g/mol, 0.704 grams, 6.5.times.10.sup.-5 moles) was
dissolved in 2.96 g 0.01M pH 4.0 phosphate buffer (19.2% solids).
Di-lysine (Sigma, 347.3 g/mol, 0.03 grams, 8.7.times.10.sup.-5
moles) was dissolved in 3.64 grams Of 0.1M pH 9.5 borate buffer
(0.8% solids). On combination of the two solutions, the percent
solids was 10%. The di-lysine has 3 amine groups. The SG-PEG has 4
NHS groups. After correction for the less than 100% degree of
substitution on the SG-PEG, the formulation gives a 1:1
stoichiometry of amine groups to NHS groups.
Example 12
Formulation of SG-PEG with Tri-lysine
[0166] A four arm PEG with SG end groups (Shearwater Polymers,
approx. 9,100 g/mol, 0.675 grams, 6.2.times.10.sup.-5 moles) was
dissolved in 2.82 g 0.01M pH 4.0 phosphate buffer (19.3% solids).
Tri-lysine (Sigma, 402.5 g/mol, 0.025 grams, 6.2.times.10.sup.-5
moles) was dissolved in 3.47 grams Of 0.1M pH 9.5 borate buffer
(0.7% solids). On combination of the two solutions, the percent
solids was 10%. The tri-lysine has 4 amine groups. The SG-PEG has 4
NHS groups. After correction for the less than 100% degree of
substitution on the SG-PEG, the formulation gives a 1:1
stoichiometry of amine groups to NHS groups.
Example 13
Formulation of SG-PEG with Tetra-lysine
[0167] A four arm PEG with SG end groups (Shearwater Polymers,
approx. 9,100 g/mol, 0.640 grams, 5.9.times.10.sup.-5 moles) was
dissolved in 2.68 g 0.01M pH 4.0 phosphate buffer (19.2% solids).
Tetra-lysine (Sigma, 530.7 g/mol, 0.025 grams, 4.7.times.10.sup.-5
moles) was dissolved in 3.30 grams of 0.1M pH 9.5 borate buffer
(0.8% solids). On combination of the two solutions, the percent
solids was 10%. The tetra-lysine has 5 amine groups. The SG-PEG has
4 NHS groups. After correction for the less than 100% degree of
substitution on the SG-PEG, the formulation gives a 1:1
stoichiometry of amine groups to NHS groups.
Example 14
Gel Time Measurement
[0168] The amine solution (100 .mu.L) was aliquotted into a
100.times.13 test tube. A flea-stirbar (7.times.2 mm, Fisher
Scientific p/n 58948-976) was placed in the test tube. The test
tube was held stationary over a digital magnetic stirrer (VWR
Series 400S Stirrer) set at 300 rpm. A 1 cc tuberculin syringe
(Becton Dickinson, p/n BD309602) was filled with 100 .mu.L of the
ester solution. The syringe was inserted up to the flanges so that
the distal end was just over the amine solution. Simultaneously the
plunger was depressed and a stop watch started. When the solution
solidifies sufficiently so that the stir bar stops spinning, the
stop watch was stopped. Each solution was measured in triplicate
and the mean .+-.1 standard deviation was plotted. Results for the
formulations of examples 1, 2 and 3 are shown in FIG. 11.
Example 15
Change in Gel Time as a Function of Ester Solution Age
[0169] An important characteristic of these systems is the loss in
reactivity over time from reconstitution of the ester solution.
This loss in reactivity occurs due to hydrolysis of the
N-hydroxysuccinimidyl ester, before the activated molecule can
combine with its respective nucleophile. The loss of reactivity was
characterized by measuring the change in gel time as a function of
time from reconstitution of the NHS ester solution. The gel time
was measured at 1/2 hour intervals. The NHS ester solution was
stored at ambient conditions during this measurement. Results for
the solutions described in Examples 11, 12 and 13 are shown in FIG.
12.
Example 16
Gel Formation at Different Percent Solids From 4 Arm CM-HBA-NS PEG
and Lys-Lys
[0170] Using the gel time method described in Example 13, five
different gel compositions were made using carboxymethyl
hydroxybutyrate-hydroxysuc- cinimide end-capped 4 arm PEG (CM-HBA)
(Shearwater Polymers) and di-lysine (Sigma). The formulations are
listed below in Table 3.
3TABLE 3 Phosphate Lys-Lys Borate Conc. (%) CM-HBA (g) (g) (g) (g)
8.5 0.2469 1.264 0.01 1.5012 10 0.2904 1.2209 0.012 1.4994 12.5
0.363 1.1483 0.015 1.4964 15 0.4356 1.0757 0.018 1.4936 20 0.5808
0.9305 0.024 1.4876
[0171] The formulations were adjusted to give a 1 to 1 ratio of
electrophilic end groups on the CM-HBA (4) to nucleophilic reactive
groups on the di-lysine ("Lys-Lys") (3). The CM-HBA quantities were
dissolved in 0.01M pH 5.0 phosphate buffer. The di-lysine was
dissolved in 0.1M pH 11 borate buffer. Gel time results are shown
in FIG. 13. This data also shows that the higher percent solids
solutions also are the most stable with respect to retention of
speed of reaction.
Example 17
Degradation of Hydrogels
[0172] Hydrogel plugs made during the gel time measurements of
Example 14 were placed in approximately 25 mL 0.1M phosphate
buffered saline at pH 7.4 in 50 mL Falcon tubes and placed in a
constant temperature bath at 37.degree. C. The hydrogel plugs were
observed visually at periodic intervals and the time of gel
disappearance noted. The data are plotted in FIG. 14.
Example 18
Precursor Spray Procedure to form a 7.5% Solids Hydrogel from 4 Arm
SG and Dilysine
[0173] An ethylene oxide sterilized air assisted sprayer was used
in conjunction with aqueous solutions of polymerizable monomers.
Solution 1 consisted of a 14.4% solution of 4 arm SG (MW 10,000 Da,
purchased from Shearwater Polymers) dissolved in 0.01M phosphate
buffer at pH 4.0 and was sterile filtered (Pall Gelman syringe
filter, p/n 4905) and drawn up in a sterile 5 cc syringe. Solution
2 consisted of a 1.2% solution of a dilysine (purchased from Sigma
Chemicals) dissolved in 0.1M borate buffer at pH 11 with 0.5 mg/mL
methylene blue for visualization and was also sterile filtered and
drawn up in a sterile 5 cc syringe. These solutions, when combined
1:1 on a volumetric basis, result in a 1:1 ratio of NHS ester to
amine end group. The final % solids after combination is 7.5%. The
two syringes were individually loaded in the two separate
receptacles through a luer-lok type of linkage. Airflow from a
regulated source of compressed air (an air compressor such as those
commercially available for airbrushes) was connected to the device
using a piece of Tygon tube. On compressing the syringe plungers a
steady spray of the two liquid components was observed. When this
spray was directed to a piece of tissue (rat cecum) a hydrogel
coating was observed to form on the surface of the tissue. This
hydrogel coating was rinsed with saline (the hydrogel coating is
resistant to rinsing) and was observed to be well adherent to the
tissue surface. Within a short period of time (less than a minute)
an area of 10 cm.times.5 cm could be coated with ease.
Example 19
Precursor Spray Procedure to Form a 12.5% Solids Hydrogel From 4
Arm CM and Dilysine
[0174] A hydrogel barrier film made from 4 arm CM-HBA NS (MW 10,000
Da, purchased from Shearwater Polymers), and dilysine was similarly
prepared and sprayed as described in Example 18. In the present
example the 4 arm CM solution was made up to 24.0% solids and the
dilysine solution was made up to 1.0% solids such that on
combination in an equal volume delivery system a 1:1 ratio of NHS
to amine end groups results, giving a final % solids of 12.5%.
Example 20
Spray Application of Crosslinker and Polymer to From Crosslinked
Film
[0175] Two solutions (component A and component B) were prepared.
Component A consisted of dilysine in 0.1M borate buffer, pH 9.5.
Component B consisted of either 4 arm SG-PEG or 4 arm CM-HBA-NS in
0.01M phosphate buffer, pH 4.0. These solutions were prepared such
that the amine to ester stoichiometric ratio was 1:1 and the final
total solution concentration was 7.5% or 12.5%, respectively.
[0176] A Fibriject.TM. (Micromedics, Inc) 5 cc syringe holder and
cap was used, preloaded with 5 cc of each solution and attached to
a dual barrel atomizing sprayer. The sprayer has two hubs for the
syringes to connect to allowing the two fluids to be advanced
through two separate lumens over any preset distance. A third hub
exists for the application of the atomizing gas. Air was used in
this example. The distal tip of the sprayer contains a chamber
where the gas expands out of an introduction tube, then flows past
the two polymer solution nozzles in an annular space around each.
The gas is accelerated in the annular spaces using a flow rate
suitable for the complete atomization of the two fluid streams
(.about.2 L/min.). Two overlapping spray cones are thus formed
allowing for well mixed, thin, uniform coatings to be applied to
surfaces.
Example 21
Adhesion Prevention in Rat Cecum Model
[0177] Surgical procedure:
[0178] Male Sprague Dawley rats (250-350 grams,) were anesthetized
with an intramuscular 4 ml/kg "cocktail" of Ketamine (25 mg/ml),
Xylazine (1.3 mg/mL) and Acepromazine (0.33 mg/mL). The abdominal
area was shaved and prepped for aseptic surgery. A midline incision
was made to expose the abdominal contents. The cecum was identified
and location within the abdomen was noted. The cecum was pulled out
of the abdomen and the surface of one side was abraded using dry
sterile gauze. A technique of abrading one area by stroking the
surface 12 times with the gauze was used. The cecal arterial supply
was interrupted using bipolar coagulation along the entire surface
area of the damaged cecum.
[0179] The opposing abdominal sidewall which lays in proximity to
the damaged cecal surface was deperitonealized with a scalpel blade
and the underlying muscle layer was scraped to the point of
hemorrhaging.
[0180] The cecum was sprayed with either the SG-PEG system or the
CM-HBA-NS system using the air assisted spray method described in
the preceding example. The cecum was placed with the damaged
(ischemic area) side up opposite the damaged side wall. Active
bleeding was controlled before closing. The peritoneum and muscle
wall was closed with 3-0 nylon and the skin was closed with 4-0
silk. Rats were returned to their cages for one to two weeks at
which time evaluation of the adhesion between the side wall and
cecum was noted. The rats were killed at 10 days and the tenacity
and extent of adhesion was evaluated. The results are summarized in
Table 4.
4TABLE 4 Material Reference Rat # Applied Example Findings on Day
10 403 7.5% 4aSG Example 18 Small amount of gel with Lys-Lys
present on cecum. No w/MB adhesions from cecum to sidewall. No gel
on sidewall. 404 7.5% 4aSG Example 18 Some mesentary stuck to with
Lys-Lys cecum. No gel. No w/MB adhesions. 405 7.5% 4aSG Example 18
Small amount of gel with Lys-Lys present on cecum. Some w/MB
mesentary stuck to cecum and sidewall. Some gel between mesentary
and cecum where stuck. No adhesions. 406 12.5% 4aCM Example 19 No
gel present. No with Lys-Lys adhesions. w/MB 407 12.5% 4aCM Example
19 No gel on cecum or with Lys-Lys sidewall. No adhesions. w/MB 408
12.5% 4aCM Example 19 Rat died post-op with Lys-Lys (anesthesia
overdose). w/MB
[0181] While preferred illustrative embodiments of the invention
are described above, it will be apparent to one skilled in the art
that various changes and modifications may be made therein without
departing from the invention, and it is intended in the appended
claims to cover all such changes and modifications which fall
within the true spirit and scope of the invention.
* * * * *