U.S. patent application number 14/061272 was filed with the patent office on 2014-05-01 for polymers and methods thereof for wound healing.
This patent application is currently assigned to Rutgers, The State University of New Jersey. The applicant listed for this patent is Rutgers, The State University of New Jersey. Invention is credited to Sabrina S. Snyder, Kathryn E. Uhrich.
Application Number | 20140120057 14/061272 |
Document ID | / |
Family ID | 50547438 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120057 |
Kind Code |
A1 |
Uhrich; Kathryn E. ; et
al. |
May 1, 2014 |
POLYMERS AND METHODS THEREOF FOR WOUND HEALING
Abstract
Certain embodiments of the invention provide a copolymer having
a backbone, wherein the backbone comprises a) one or more units
that comprise a group that will yield a biologically active agent
upon hydrolysis of the backbone; and b) one or more units of
formula (II): ##STR00001## wherein y is 1 or more. Other
embodiments of the invention provide a therapeutic method for
treating a wound in an animal comprising administering to an animal
in need of such therapy, an effective amount of a copolymer as
described herein.
Inventors: |
Uhrich; Kathryn E.; (New
Brunswick, NJ) ; Snyder; Sabrina S.; (New Brunswick,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rutgers, The State University of New Jersey |
New Brunswick |
NJ |
US |
|
|
Assignee: |
Rutgers, The State University of
New Jersey
New Brunswick
NJ
|
Family ID: |
50547438 |
Appl. No.: |
14/061272 |
Filed: |
October 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61718556 |
Oct 25, 2012 |
|
|
|
Current U.S.
Class: |
424/78.37 ;
528/361 |
Current CPC
Class: |
A61K 31/765
20130101 |
Class at
Publication: |
424/78.37 ;
528/361 |
International
Class: |
A61K 31/765 20060101
A61K031/765 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The invention described herein was made with government
support under Grant Number R01DE019926 awarded by the National
Institutes of Health. The United States Government has certain
rights in the invention.
Claims
1. A copolymer having a backbone, wherein the backbone comprises a)
one or more units that comprise a group that will yield a
biologically active agent upon hydrolysis of the backbone; and b)
one or more units of formula (II): ##STR00018## wherein y is 1 or
more.
2. The copolymer of claim 1, wherein the one or more units that
comprise a group that will yield a biologically active agent upon
hydrolysis of the backbone is a polyanhydride.
3. The copolymer of claim 2, wherein the polyanhydride comprises
one or more units of formula (I) in the backbone:
--C(.dbd.O)R.sup.1-A-L-A-R.sup.1C(.dbd.O)--O (I) wherein each
R.sup.1 is a group that will provide a biologically active agent
upon hydrolysis of the polymer; each A is independently an ester or
an amide linkage; and each L is independently a linker
molecule.
4. The copolymer of claim 3, wherein A is independently an ester
linkage.
5. The copolymer of claim 3, wherein L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 1 to 25 carbon atoms, wherein one or more of the carbon atoms
is optionally replaced by (--O--), (--NR--) or phenylene, and
wherein the chain is optionally substituted on carbon with one or
more substituents selected from the group consisting of
(C.sub.1-C.sub.6)alkoxy, (C.sub.3-C.sub.6)cycloalkyl,
(C.sub.1-C.sub.6)alkanoyl, (C.sub.1-C.sub.6)alkanoyloxy,
(C.sub.1-C.sub.6)alkoxycarbonyl, (C.sub.1-C.sub.6)alkylthio, azido,
cyano, nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy,
heteroaryl, and heteroaryloxy wherein each R is independently
selected from H or (C.sub.1-C.sub.6)alkyl.
6. The copolymer of claim 3, wherein L is
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2C(Et).sub.2CH.sub.2--.
7. The copolymer of claim 1, wherein the biologically active agent
is an antimicrobial, anti-inflammatory, antioxidant, analgesic,
anticoagulant or fibrinolytic.
8. The copolymer of claim 7, wherein the biologically active agent
is an anti-inflammatory agent.
9. The copolymer of claim 8, wherein the anti-inflammatory agent is
salicylic acid.
10. The copolymer of claim 1, wherein the ratio of the a) one or
more units that comprise a group that will yield a biologically
active agent upon hydrolysis of the backbone to the b) one or more
units of formula (II), ranges from between about 5:1 to about
1:5.
11. The copolymer of claim 10, wherein the ratio ranges from 2:1 to
1:2.
12. The copolymer of claim 11, wherein the ratio is 1:1 or 2:1.
13. The copolymer of claim 1 comprising one or more units of
formula (III): ##STR00019## wherein each L is independently a
linker molecule; x is 5 or more; y is 1 or more; and z is 5 or
more.
14. The copolymer of claim 1 which has an average molecular weight
of about 10,000 daltons to about 30,000 daltons.
15. The copolymer of claim 1, further comprising a second
biologically active agent dispersed in the matrix of the
copolymer.
16. A pharmaceutical composition comprising a copolymer of claim 1
and a pharmaceutically acceptable carrier.
17. A method of making a copolymer as described in claim 1
comprising co-polymerizing (a) one or more monomer(s) that
comprises one or more units that comprise a group that will yield a
biologically active agent upon hydrolysis of the backbone; and (b)
one or more monomer(s) that comprises one or more units of formula
(II); under conditions to provide the polymer.
18. A therapeutic method for treating a wound in an animal
comprising administering to an animal in need of such therapy an
effective amount of a copolymer as described in claim 1.
19. A therapeutic method for the prevention of fibrous adhesions at
a wound site in an animal comprising administering to an animal in
need of such therapy an effective amount of a copolymer as
described in claim 1.
20. A therapeutic method for providing localized analgesia at a
wound site in an animal comprising administering to an animal in
need of such therapy an effective amount of a copolymer as
described in claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims priority from U.S. Provisional
Application No. 61/718,556, filed Oct. 25, 2012, which application
is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Fibrous adhesions are a serious complication that can arise
from trauma to the body as they can lead to chronic pain,
infertility, and intestinal obstruction. Adhesions are bands of
fibrous tissue that join two surfaces in the body, which are not
normally connected. They generally form after injury to an area
that results in increased inflammation. Surgery, trauma,
infections, radiation, and ischemia can all lead to adhesion
formation, with surgery being the most common cause. Fibrous
adhesions have an enormous impact on the healthcare system. It has
been estimated that 95% of abdominal and pelvic surgeries,
including gynecologic, result in adhesions. Adhesion-related
problems account for 6% of all hospital readmissions and 1% of all
hospitalizations in the United States. Adhesions increase surgery
time, hospital stay, complications, blood loss, morbidity, and
mortality.
[0004] In addition to improved surgical techniques, both
pharmaceuticals and physical barriers have been explored as means
to prevent adhesion formation (Tingstedt et al., Eur Surg Res 39,
259-268 (2007); Ward, et al., Journal of Surgical Research, 165(1),
91-111 (2009)). Systemic administration of such drugs at
therapeutic levels can cause undesired side effects and delay
healing after surgery. There have been some attempts to inject the
drugs into the peritoneal cavity; however, most of these have shown
little to no efficacy in laboratory testing primarily due to the
tendency for drugs placed in the peritoneal cavity to be quickly
absorbed by the mesothelium and subsequently distributed throughout
the body. Various solids, gels, and fluids have been used as
physical barriers. None of these devices have been shown
efficacious enough at reducing adhesion formation to warrant their
ubiquitous use.
[0005] Accordingly, there is a need for more efficacious treatments
for wound healing (e.g., the mitigation of pain, inflammation
and/or other complications, such as fibrous adhesions).
SUMMARY OF THE INVENTION
[0006] Certain embodiments of the invention provide a copolymer
having a backbone, wherein the backbone comprises a) one or more
units that comprise a group that will yield a biologically active
agent upon hydrolysis of the backbone; and b) one or more units of
formula (II):
##STR00002## [0007] wherein y is 1 or more.
[0008] Certain embodiments of the invention provide a
pharmaceutical composition comprising a copolymer as described
herein and a pharmaceutically acceptable carrier.
[0009] Certain embodiments of the invention provide a method of
making a copolymer as described herein comprising co-polymerizing
(a) one or more monomer(s) that comprises one or more units that
comprise a group that will yield a biologically active agent upon
hydrolysis of the backbone; and (b) one or more monomer(s) that
comprises one or more units of formula (II); under conditions to
provide the polymer.
[0010] Certain embodiments of the invention provide a method of
making a copolymer as described herein.
[0011] Certain embodiments of the invention provide a copolymer
prepared by methods described herein.
[0012] Certain embodiments of the invention provide a therapeutic
method for treating a wound in an animal comprising administering
to an animal in need of such therapy, an effective amount of a
copolymer or composition as described herein.
[0013] Certain embodiments of the invention provide a therapeutic
method for the prevention of fibrous adhesions at a wound site in
an animal comprising administering to an animal in need of such
therapy, an effective amount of a copolymer or composition as
described herein.
[0014] Certain embodiments of the invention provide a therapeutic
method for providing localized analgesia at a wound site in an
animal comprising administering to an animal in need of such
therapy, an effective amount of a copolymer or composition as
described herein.
[0015] Certain embodiments of the invention provide a method for
promoting wound healing in an animal, comprising contacting a
copolymer or composition as described herein with a wound of the
animal.
[0016] Certain embodiments of the invention provide a method for
the prevention of fibrous adhesions at a wound site in an animal,
comprising contacting a copolymer or composition as described
herein with the wound of the animal.
[0017] Certain embodiments of the invention provide a method for
providing localized analgesia at a wound site in an animal,
comprising contacting a copolymer or composition as described
herein with the wound of the animal.
[0018] Certain embodiments of the invention provide a copolymer or
composition as described herein for use in medical therapy.
[0019] Certain embodiments of the invention provide for the use of
a copolymer or composition as described herein for the manufacture
of a medicament for the treatment of a wound in an animal, such as
a human.
[0020] Certain embodiments of the invention provide a copolymer as
described herein for use in treating a wound.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1. Diagram of the steps that lead from surgical trauma
to either normal peritoneum repair or adhesion formation.
[0022] FIG. 2. Cumulative release of SA from (a) adipic-PA and (b)
diethylmalonic-PA samples with admixtures (all percentages are
w/w): (A) polymer alone, (B) 1% SA, (C) 5% SA, (D) 10% SA, (E) 1%
diacid, (F) 5% diacid, (G) 10% diacid, (H) 1% 1:1 SA/diacid, (I) 5%
1:1 SA/diacid, (J) 10% 1:1 SA/diacid.
[0023] FIG. 3. Scanning electron microscopy (SEM) image of 2:1
PLGA/adipic-PA electrospun membrane.
[0024] FIG. 4. SEM image of PA microspheres.
[0025] FIG. 5. Experimental lesion formed by electrocautery with
sutures (Rajab, et al., Journal of Surgical Research 161, 246-249
(2010)).
[0026] FIG. 6. Graph of SA release with various linkers.
[0027] FIG. 7. Possible formulation geometries: A) microspheres; B)
flexible films; and C) polymer powder dispersed within mineral
oil.
[0028] FIG. 8. PEG copolymer gel, which behaved like viscous
liquids.
[0029] FIG. 9. (A) Degradation of the SAPAE to SA and other
biocompatible molecules. (B) In vitro salicylic acid release
profile from the SAPAE indicates a linear release profile over the
critical period of adhesion formation. (C) Fibroblast viability and
proliferation is not significantly affected by 0.1 mg/mL SAPAE. (D)
0.1 mg/mL SAPAE significantly (p<0.001) decreases TNF-.alpha.
expression by LPS activated macrophages, thus demonstrating its
ability to inhibit inflammation.
[0030] FIG. 10. Synthetic scheme for the SAA diacid, SAA polymer,
and SAA:PEG copolymers.
[0031] FIG. 11. .sup.1H NMR spectra of 2:1 SAA:PEG (g* indicates
hydrogen atoms adjacent to a carboxylic acid end group, as opposed
to g which indicates hydrogen atoms adjacent to an anhydride
group).
[0032] FIG. 12. SAA:PEG copolymer M.sub.n and T.sub.g changes over
3 weeks of storage at different temperatures. Specifically,
copolymers with ratios of 1:2 (without desiccant), 1:2d (with
desiccant), 1:1 and 2:1 were analyzed at -20.degree. C., 4.degree.
C. and 25.degree. C. From left to right, each bar represents the
following time point within each grouping (e.g., grouping 1:2):
week 0, week 1, week 2 and week 3.
[0033] FIG. 13. In vitro SA release from SAA:PEG copolymers.
[0034] FIG. 14. In vitro cell viability over 72 hours for cells
exposed to SAA:PEG copolymers with ratios of 1:2 (a), 1:1 (b), and
2:1 (c) (* indicates significant decrease from DMSO control,
p<0.05). Cell viability was normalized to the DMSO control at 24
hours. Cell viability is shown, from left to right, within each
concentration group at 24 hours, 48 hours and 72 hours.
[0035] FIG. 15. TFN-.alpha. expression by macrophages exposed to
LPS and SAA:PEG copolymers (* indicates significant difference from
10 ng/mL LPS control, p<0.05). TNF-.alpha. secretion was
normalized to the LPS positive control (set to 1) and the LPS free
control (set to 0).
DETAILED DESCRIPTION
Copolymers
[0036] Certain embodiments of the invention provide a copolymer
having a backbone, wherein the backbone comprises a) one or more
units that comprise a group that will yield a biologically active
agent upon hydrolysis of the backbone; and b) one or more units of
formula (II):
##STR00003## [0037] wherein y is 1 or more. In certain embodiments,
y is about 1 to about 15. In certain embodiments, y is about 5 to
about 15. In certain embodiments, y is about 10 to about 15. In
certain embodiments, y is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14 or 15. In certain embodiments, y is about 11.
[0038] In certain embodiments, the one or more units of formula
(II) have an average molecular weight of about 100 daltons to about
750 daltons. In certain embodiments, the one or more units of
formula (II) have an average molecular weight of about 250 daltons
to about 750 daltons. In certain embodiments, the one or more units
of formula (II) have an average molecular weight of about 600
daltons.
[0039] In certain embodiments of the invention, the one or more
units that comprise a group that will yield a biologically active
agent upon hydrolysis of the backbone is a polyanhydride.
[0040] In certain embodiments of the invention, the polyanhydride
is a poly(anhydride-ester).
[0041] In certain embodiments of the invention, the polyanhydride
is a poly(anhydride-amide).
[0042] In certain embodiments, the polyanhydride comprises one or
more units of formula (I) in the backbone:
--C(.dbd.O)R.sup.1-A-L-A-R.sup.1C(.dbd.O)--O-- (I) [0043] wherein
[0044] each R.sup.1 is a group that will provide a biologically
active agent upon hydrolysis of the polymer; [0045] each A is
independently an ester or amide linkage; and [0046] each L is
independently a linker molecule.
[0047] In certain embodiments, A is independently an ester
linkage.
[0048] In certain embodiments, A is independently an amide
linkage.
[0049] In certain embodiments, the polyanhydride comprises
repeating units of formula (I) in the backbone.
[0050] In certain embodiments, the polyanhydride comprises a first
group of one or more units of formula (I) in the backbone and a
second group of one or more units of formula (I) in the backbone,
wherein the L in the first group is different than the L in the
second group.
[0051] In certain embodiments, the polyanhydride comprises a first
group of repeating units of formula (I) in the backbone and a
second group of repeating units of formula (I) in the backbone,
wherein the L in the first group is different than the L in the
second group.
[0052] In certain embodiments, the one or more groups that will
yield a biologically active compound upon hydrolysis of the
backbone has an average molecular weight of about 1,000 daltons to
about 100,000 daltons. In certain embodiments, the one or more
groups that will yield a biologically active compound upon
hydrolysis of the backbone has an average molecular weight of about
5,000 daltons to about 100,000 daltons. In certain embodiments, the
one or more groups that will yield a biologically active compound
upon hydrolysis of the backbone has an average molecular weight of
about 5,000 daltons to about 50,000 daltons. In certain
embodiments, the one or more groups that will yield a biologically
active compound upon hydrolysis of the backbone has an average
molecular weight of about 10,000 daltons to about 30,000
daltons.
[0053] In certain embodiments, the biologically active agent is an
antimicrobial, anti-inflammatory, antioxidant, analgesic,
anticoagulant or fibrinolytic.
[0054] In certain embodiments, the biologically active agent is an
anti-inflammatory agent.
[0055] In certain embodiments, the anti-inflammatory is
3-amino-4-hydroxybutyric acid, aceclofenac, alminoprofen, amfenac,
bromfenac, bumadizon, carprofen, diclofenac, diflunisal, enfenamic
acid, etodolac, fendosal, flufenamic acid, gentisic acid,
meclofenamic acid, mefenamic acid, mesalamine, niflumic acid,
olsalazine, oxaceprol, S-adenosylmethionine, salicylic acid,
salsalate, sulfasalazine or tolfenamic acid.
[0056] In certain embodiments, the anti-inflammatory agent is
salicylic acid.
[0057] In certain embodiments, the biologically active agent is an
antimicrobial.
[0058] In certain embodiments, the antimicrobial is
2-p-sulfanilyanilinoethanol, 4-sulfanilamidosalicylic acid,
acediasulfone, amoxicillin, amphotericin B, ampicillin, apalcillin,
apicycline, apramycin, aspoxicillin, aztreonam, bacitracin,
bambermycin(s), biapenem, carbenicillin, carumonam, cefadroxil,
cefamandole, cefatrizine, cefbuperazone, cefclidin, cefdinir,
cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefminox,
cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime,
cefotetan, cefotiam, cefozopran, cefpimizole, cefpiramide,
cefpirome, cefprozil, cefroxadine, ceftazidime, cefteram,
ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin,
cephalosporin C, cephradine, ciprofloxacin, clinafloxacin,
cyclacillin, diathymosulfone, enoxacin, epicillin, flomoxef,
grepafloxacin, hetacillin, imipenem, lomefloxacin, lucensomycin,
lymecycline, meropenem, moxalactam, mupirocin, nadifloxacin,
natamycin, norfloxacin, panipenem, pazufloxacin, penicillin N,
pipacycline, pipemidic acid, polymyxin, quinacillin, ritipenem,
rolitetracycline, salazosulfadimidine, sancycline, sparfloxacin,
succisulfone, sulfachrysoidine, sulfaloxic acid, teicoplanin,
temafloxacin, temocillin, tetracycline, thiostrepton, ticarcillin,
tigemonam, tosufloxacin, trovafloxacin or vancomycin.
[0059] In certain embodiments, the biologically active agent is an
antioxidant.
[0060] In certain embodiments, the antioxidant is vanillic acid,
syringic acid, ferulic acid, sinapic acid, or p-coumaric acid.
[0061] In certain embodiments, the biologically active agent is an
analgesic (e.g., salicylic acid).
[0062] In certain embodiments, the biologically active agent is an
anticoagulant.
[0063] In certain embodiments, the anticoagulant is argatroban.
[0064] In certain embodiments, the biologically active agent is a
fibrinolytic.
[0065] In certain embodiments, the fibrinolytic is:
##STR00004##
[0066] In certain embodiments, the ratio of the a) one or more
units that comprise a group that will yield a biologically active
agent upon hydrolysis of the backbone to the b) one or more units
of formula (II), ranges from between about 10:1 to about 1:10. In
certain embodiments, the ratio of the a) one or more units that
comprise a group that will yield a biologically active agent upon
hydrolysis of the backbone to the b) one or more units of formula
(II), ranges from between about 5:1 to about 1:5. In certain
embodiments, the ratio of the a) one or more units that comprise a
group that will yield a biologically active agent upon hydrolysis
of the backbone to the b) one or more units of formula (II), ranged
from between about 2:1 to about 1:2. In certain embodiments, the
ratio of the a) one or more units that comprise a group that will
yield a biologically active agent upon hydrolysis of the backbone
to the b) one or more units of formula (II), is e.g., 5:1, 4:1,
3:1, 2:1, 1:1, 1:2, 1:3, 1:2, 1:3, 1:4 or 1:5. In certain
embodiments, the ratio is 1:1 or 2:1. In certain embodiments, the
ratio is less than 2:1.
[0067] Certain embodiments of the invention provide a block
copolymer comprising a) a first block comprising a polyanhydride
having a backbone, wherein the backbone comprises one or more units
that comprise a group that will yield a biologically active agent
upon hydrolysis of the backbone, and b) a second block comprising
one or more units of formula (II):
##STR00005## [0068] wherein y is 1 or more.
[0069] In certain embodiments, the first block comprises at least
about 5 or more groups.
[0070] In certain embodiments, the second block comprises at least
about 5 or more groups.
[0071] Certain embodiments of the invention provide a copolymer as
described herein comprising one or more units of formula (III):
##STR00006##
[0072] wherein each L is independently a linker molecule; [0073] x
is 5 or more; [0074] y is 1 or more; and [0075] z is 5 or more.
[0076] In certain embodiments, y is about 1 to about 15. In certain
embodiments, y is about 5 to about 15. In certain embodiments, y is
about 10 to about 15. In certain embodiments, y is about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In certain embodiments,
y is about 11.
[0077] Certain embodiments of the invention provide a copolymer as
described herein comprising one or more units of formula (IV):
##STR00007##
[0078] wherein each L is independently a linker molecule; [0079] x
is 5 or more; [0080] y is 1 or more; and [0081] z is 5 or more.
[0082] In certain embodiments, y is about 1 to about 15. In certain
embodiments, y is about 5 to about 15. In certain embodiments, y is
about 10 to about 15. In certain embodiments, y is about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In certain embodiments,
y is about 11.
[0083] In certain embodiments, each linker molecule is selected
from a branched aliphatic, linear aliphatic, and oxygen-containing
linker molecule.
[0084] In certain embodiments, L is adipic
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--) or diethylmalonic
(--CH.sub.2C(Et).sub.2CH.sub.2--).
[0085] In certain embodiments L is adipic
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--).
[0086] In certain embodiments, L is diethylmalonic
(--CH.sub.2C(Et).sub.2CH.sub.2--).
[0087] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 1 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, and wherein the chain is optionally substituted on
carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected
from the group consisting of (C.sub.1-C.sub.6)alkoxy,
(C.sub.3-C.sub.6)cycloalkyl, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkanoyloxy, (C.sub.1-C.sub.6)alkoxycarbonyl,
(C.sub.1-C.sub.6)alkylthio, azido, cyano, nitro, halo, hydroxy,
oxo, carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein
each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0088] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 1 to 25 carbon atoms, wherein the chain is optionally
substituted on carbon with one or more (e.g. 1, 2, 3, or 4)
substituents selected from the group consisting of
(C.sub.1-C.sub.6)alkoxy, (C.sub.3-C.sub.6)cycloalkyl,
(C.sub.1-C.sub.6)alkanoyl, (C.sub.1-C.sub.6)alkanoyloxy,
(C.sub.1-C.sub.6)alkoxycarbonyl, (C.sub.1-C.sub.6)alkylthio, azido,
cyano, nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy,
heteroaryl, and heteroaryloxy.
[0089] In certain embodiments, L is a peptide.
[0090] In certain embodiments, L is an amino acid.
[0091] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 1 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, wherein each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0092] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 3 to 15 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, and wherein the chain is optionally substituted on
carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected
from the group consisting of (C.sub.1-C.sub.6)alkoxy,
(C.sub.3-C.sub.6)cycloalkyl, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkanoyloxy, (C.sub.1-C.sub.6)alkoxycarbonyl,
(C.sub.1-C.sub.6)alkylthio, azido, cyano, nitro, halo, hydroxy,
oxo, carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein
each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0093] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 3 to 15 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, wherein each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0094] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 3 to 15 carbon atoms.
[0095] In certain embodiments, L is a divalent, branched or
unbranched, hydrocarbon chain, having from 3 to 15 carbon
atoms.
[0096] In certain embodiments, L is a divalent hydrocarbon chain
having 4 carbon atoms.
[0097] In certain embodiments, L is a divalent, branched or
unbranched, hydrocarbon chain, having from 6 to 10 carbon
atoms.
[0098] In certain embodiments, L is a divalent hydrocarbon chain
having 7, 8, or 9 carbon atoms.
[0099] In certain embodiments, L is a divalent hydrocarbon chain
having 8 carbon atoms.
[0100] In certain embodiments, L is 1,4 phenylene or 1,3
phenylene.
[0101] In certain embodiments, the copolymer as described herein
and prepared in accordance with the present invention has an
average molecular weight of about 1,000 daltons to about 100,000
daltons. In certain embodiments, the copolymer has an average
molecular weight of about 5,000 daltons to about 100,000 daltons.
In certain embodiments, the copolymer has an average molecular
weight of about 5,000 daltons to about 50,000 daltons. In certain
embodiments, the copolymer has an average molecular weight of about
10,000 daltons to about 30,000 daltons.
[0102] In certain embodiments, a copolymer as described herein
further comprises a second biologically active agent dispersed in
the matrix of the copolymer.
[0103] In certain embodiments, the second biologically active agent
is the same as the biologically active agent yielded by hydrolysis
of the copolymer backbone.
[0104] In certain embodiments, the second biologically active agent
is different than the biologically active agent yielded by
hydrolysis of the copolymer backbone.
[0105] In certain embodiments, the second biologically active agent
is an antimicrobial, anti-inflammatory, antioxidant, analgesic,
anticoagulant or fibrinolytic.
[0106] In certain embodiments, the second biologically active agent
is an anti-inflammatory agent.
[0107] In certain embodiments, the anti-inflammatory agent is
salicylic acid.
[0108] In certain embodiments, the second biologically active agent
is an antioxidant.
[0109] In certain embodiments, the antioxidant is vitamin E or
melatonin.
[0110] In certain embodiments, a copolymer as described herein
further comprises a compound of formula (V) dispersed in the matrix
of the copolymer:
##STR00008##
[0111] In certain embodiments, a copolymer as described herein
further comprises a compound of formula (VI) dispersed in the
matrix of the copolymer:
##STR00009##
[0112] Certain embodiments of the invention provide a
pharmaceutical composition comprising a copolymer as described
herein and a pharmaceutically acceptable carrier.
[0113] Certain embodiments of the invention provide a medical
device comprising a copolymer as described herein.
[0114] Certain embodiments of the invention provide a medical
device comprising a copolymer as described herein and an adhesion
barrier.
[0115] In certain embodiments, the adhesion barrier is a film,
fabric or gel.
[0116] In certain embodiments, the adhesion barrier is a film.
[0117] In certain embodiments, the film is Seprafilm.
[0118] In certain embodiments, the adhesion barrier is a gel.
[0119] In certain embodiments, the gel is Intercoat.
[0120] Certain embodiments of the invention provide a method of
making a copolymer as described herein, comprising co-polymerizing
(a) one or more monomer(s) that comprises one or more units that
comprise a group that will yield a biologically active agent upon
hydrolysis of the backbone; and (b) one or more monomer(s) that
comprises one or more units of formula (II); under conditions to
provide the polymer.
[0121] Certain embodiments of the invention provide a method of
making a copolymer as described herein.
[0122] Certain embodiments of the invention provide a copolymer
prepared by methods described herein.
[0123] Certain embodiments of the invention provide a therapeutic
method for treating a wound in an animal comprising administering
to an animal in need of such therapy, an effective amount of a
copolymer or composition as described herein.
[0124] Certain embodiments of the invention provide a therapeutic
method for the prevention of fibrous adhesions at a wound site in
an animal comprising administering to an animal in need of such
therapy, an effective amount of a copolymer or composition as
described herein.
[0125] Certain embodiments of the invention provide a therapeutic
method for providing localized analgesia at a wound site in an
animal comprising administering to an animal in need of such
therapy, an effective amount of a copolymer or composition as
described herein.
[0126] Certain embodiments of the invention provide a therapeutic
method for providing localized analgesia at a wound site in an
animal comprising administering to an animal in need of such
therapy, an effective amount of a copolymer or composition as
described herein, wherein the biologically active agent is an
analgesic.
[0127] In certain embodiments of the invention, the copolymer or
composition is administered by injection.
[0128] Certain embodiments of the invention provide a method for
promoting wound healing in an animal, comprising contacting a
copolymer or composition as described herein with a wound of the
animal.
[0129] Certain embodiments of the invention provide a method for
the prevention of fibrous adhesions at a wound site in an animal,
comprising contacting a copolymer or composition as described
herein with the wound of the animal.
[0130] Certain embodiments of the invention provide a method for
providing localized analgesia at a wound site in an animal,
comprising contacting a copolymer or composition as described
herein with the wound of the animal.
[0131] Certain embodiments of the invention provide a method for
providing localized analgesia at a wound site in an animal,
comprising contacting a copolymer or composition as described
herein with the wound of the animal, wherein the biologically
active agent is an analgesic.
[0132] Certain embodiments of the invention provide a copolymer or
composition as described herein for use in medical therapy.
[0133] Certain embodiments of the invention provide for the use of
a copolymer or composition as described herein for the manufacture
of a medicament for the treatment of a wound in an animal, such as
a human.
[0134] Certain embodiments of the invention provide for the use of
a copolymer or composition as described herein for the manufacture
of a medicament for the prevention of fibrous adhesions at a wound
site in an animal, such as a human.
[0135] Certain embodiments of the invention provide for the use of
a copolymer or composition as described herein for the manufacture
of a medicament for providing localized analgesia at a wound site
in an animal, such as a human.
[0136] Certain embodiments of the invention provide a copolymer or
composition as described herein for use in treating a wound.
[0137] Certain embodiments of the invention provide a copolymer or
composition as described herein for use in preventing fibrous
adhesions at a wound site.
[0138] Certain embodiments of the invention provide a copolymer or
composition as described herein for use in providing localized
analgesia at a wound site.
[0139] In certain embodiments, the animal is a mammal.
[0140] In certain embodiments, the mammal is a human.
[0141] In another embodiment of the invention, an article of
manufacture, or "kit", containing materials useful for the
treatment of wounds, providing analgesia and/or the prevention of
fibrous adhesions described above is provided. In one embodiment,
the kit comprises a copolymer as described herein. In one
embodiment, the kit comprises a container comprising a copolymer as
described herein. In certain embodiments, the container may further
comprise a desiccant. The kit may also further comprise a label or
package insert on or associated with the container. The term
"package insert" is used to refer to instructions customarily
included in commercial packages of therapeutic products, that
contain information about the indications, usage, dosage,
administration, contraindications and/or warnings concerning the
use of such therapeutic products. Suitable containers include, for
example, bottles, vials, syringes, etc. The container may be formed
from a variety of materials such as glass or plastic.
[0142] The invention also provides processes and intermediates
disclosed herein that are useful for preparing the copolymers as
described herein (see, e.g., the Examples). The intermediates
described herein may have therapeutic activity, and therefore, may
also be used for the treatment of a wound, the prevention of
fibrous adhesions or providing localized analgesia.
Compositions Comprising a Polyanhydride and an Adhesion Barrier
[0143] Certain embodiments of the present invention provide a
composition comprising 1) a polyanhydride having a backbone,
wherein the backbone comprises one or more units that comprise a
group that will yield a biologically active agent upon hydrolysis
of the backbone; and 2) an adhesion barrier.
[0144] In certain embodiments, the biologically active agent is an
antimicrobial, anti-inflammatory, antioxidant, analgesic,
anticoagulant or fibrinolytic.
[0145] In certain embodiments, the biologically active agent is an
anti-inflammatory agent.
[0146] In certain embodiments, the anti-inflammatory is
3-amino-4-hydroxybutyric acid, aceclofenac, alminoprofen, amfenac,
bromfenac, bumadizon, carprofen, diclofenac, diflunisal, enfenamic
acid, etodolac, fendosal, flufenamic acid, gentisic acid,
meclofenamic acid, mefenamic acid, mesalamine, niflumic acid,
olsalazine, oxaceprol, S-adenosylmethionine, salicylic acid,
salsalate, sulfasalazine or tolfenamic acid.
[0147] In certain embodiments, the anti-inflammatory agent is
salicylic acid.
[0148] In certain embodiments, the biologically active agent is an
antimicrobial.
[0149] In certain embodiments, the antimicrobial is
2-p-sulfanilyanilinoethanol, 4-sulfanilamidosalicylic acid,
acediasulfone, amoxicillin, amphotericin B, ampicillin, apalcillin,
apicycline, apramycin, aspoxicillin, aztreonam, bacitracin,
bambermycin(s), biapenem, carbenicillin, carumonam, cefadroxil,
cefamandole, cefatrizine, cefbuperazone, cefclidin, cefdinir,
cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefminox,
cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime,
cefotetan, cefotiam, cefozopran, cefpimizole, cefpiramide,
cefpirome, cefprozil, cefroxadine, ceftazidime, cefteram,
ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin,
cephalosporin C, cephradine, ciprofloxacin, clinafloxacin,
cyclacillin, diathymosulfone, enoxacin, epicillin, flomoxef,
grepafloxacin, hetacillin, imipenem, lomefloxacin, lucensomycin,
lymecycline, meropenem, moxalactam, mupirocin, nadifloxacin,
natamycin, norfloxacin, panipenem, pazufloxacin, penicillin N,
pipacycline, pipemidic acid, polymyxin, quinacillin, ritipenem,
rolitetracycline, salazosulfadimidine, sancycline, sparfloxacin,
succisulfone, sulfachrysoidine, sulfaloxic acid, teicoplanin,
temafloxacin, temocillin, tetracycline, thiostrepton, ticarcillin,
tigemonam, tosufloxacin, trovafloxacin or vancomycin.
[0150] In certain embodiments, the biologically active agent is an
antioxidant.
[0151] In certain embodiments, the antioxidant is vanillic acid,
syringic acid, ferulic acid, sinapic acid, or p-coumaric acid.
[0152] In certain embodiments, the biologically active agent is an
analgesic (e.g., salicylic acid).
[0153] In certain embodiments, the biologically active agent is an
anticoagulant.
[0154] In certain embodiments, the anticoagulant is argatroban.
[0155] In certain embodiments, the biologically active agent is a
fibrinolytic.
[0156] In certain embodiments, the fibrinolytic is:
##STR00010##
[0157] In certain embodiments, the adhesion barrier is a film,
fabric, mesh, or gel.
[0158] In certain embodiments, the adhesion barrier is a film.
[0159] In certain embodiments, the film is Seprafilm.
[0160] In certain embodiments, the adhesion barrier is a gel.
[0161] In certain embodiments, the gel is Intercoat.
[0162] In certain embodiments, the polyanhydride comprises one or
more units of formula (I) in the backbone:
--C(.dbd.O)R.sup.1-A-L-A-R.sup.1C(.dbd.O)--O (I) [0163] wherein
[0164] each R.sup.1 is a group that will provide a biologically
active agent upon hydrolysis of the polymer; [0165] each A is
independently an ester or an amide linkage; and [0166] each L is
independently a linker molecule.
[0167] In certain embodiments, A is independently an ester
linkage.
[0168] In certain embodiments, A is independently an amide
linkage.
[0169] In certain embodiments each linker molecule is selected from
a branched aliphatic, linear aliphatic, and oxygen-containing
linker molecule.
[0170] In certain embodiments, L is adipic
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--) or diethylmalonic
(--CH.sub.2C(Et).sub.2CH.sub.2--).
[0171] In certain embodiments L is adipic
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--).
[0172] In certain embodiments, L is diethylmalonic
(--CH.sub.2C(Et).sub.2CH.sub.2--).
[0173] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 1 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, and wherein the chain is optionally substituted on
carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected
from the group consisting of (C.sub.1-C.sub.6)alkoxy,
(C.sub.3-C.sub.6)cycloalkyl, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkanoyloxy, (C.sub.1-C.sub.6)alkoxycarbonyl,
(C.sub.1-C.sub.6)alkylthio, azido, cyano, nitro, halo, hydroxy,
oxo, carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein
each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0174] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 1 to 25 carbon atoms, wherein the chain is optionally
substituted on carbon with one or more (e.g. 1, 2, 3, or 4)
substituents selected from the group consisting of
(C.sub.1-C.sub.6)alkoxy, (C.sub.3-C.sub.6)cycloalkyl,
(C.sub.1-C.sub.6)alkanoyl, (C.sub.1-C.sub.6)alkanoyloxy,
(C.sub.1-C.sub.6)alkoxycarbonyl, (C.sub.1-C.sub.6)alkylthio, azido,
cyano, nitro, halo, hydroxy, oxo, carboxy, aryl, aryloxy,
heteroaryl, and heteroaryloxy.
[0175] In certain embodiments, L is a peptide.
[0176] In certain embodiments, L is an amino acid.
[0177] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 1 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, wherein each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0178] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 3 to 15 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, and wherein the chain is optionally substituted on
carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected
from the group consisting of (C.sub.1-C.sub.6)alkoxy,
(C.sub.3-C.sub.6)cycloalkyl, (C.sub.1-C.sub.6)alkanoyl,
(C.sub.1-C.sub.6)alkanoyloxy, (C.sub.1-C.sub.6)alkoxycarbonyl,
(C.sub.1-C.sub.6)alkylthio, azido, cyano, nitro, halo, hydroxy,
oxo, carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein
each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0179] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 3 to 15 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4)
of the carbon atoms is optionally replaced by (--O--), (--NR--) or
phenylene, wherein each R is independently selected from H or
(C.sub.1-C.sub.6)alkyl.
[0180] In certain embodiments, L is a divalent, branched or
unbranched, saturated or unsaturated, hydrocarbon chain, having
from 3 to 15 carbon atoms.
[0181] In certain embodiments, L is a divalent, branched or
unbranched, hydrocarbon chain, having from 3 to 15 carbon
atoms.
[0182] In certain embodiments, L is a divalent hydrocarbon chain
having 4 carbon atoms.
[0183] In certain embodiments, L is a divalent, branched or
unbranched, hydrocarbon chain, having from 6 to 10 carbon
atoms.
[0184] In certain embodiments, L is a divalent hydrocarbon chain
having 7, 8, or 9 carbon atoms.
[0185] In certain embodiments, L is a divalent hydrocarbon chain
having 8 carbon atoms.
[0186] In certain embodiments, L is 1,4 phenylene or 1,3
phenylene.
[0187] In certain embodiments, the polyanhydride comprises
repeating units of formula (I) in the backbone.
[0188] In certain embodiments, polyanhydride comprises one or more
units of formula (Ia) in the backbone:
##STR00011## [0189] wherein L is a linker molecule.
[0190] In certain embodiments, the polyanhydride comprises
repeating units of formula (Ia) in the backbone.
[0191] In certain embodiments, polyanhydride comprises one or more
units of formula (Ib) in the backbone:
##STR00012## [0192] wherein L is a linker molecule.
[0193] In certain embodiments, the polyanhydride comprises
repeating units of formula (Ib) in the backbone.
[0194] In certain embodiments, the polyanhydride as described
herein further comprises a second biologically active agent
dispersed in the matrix of the polymer.
[0195] In certain embodiments, the second biologically active agent
is the same as the biologically active agent yielded by hydrolysis
of the polyanhydride backbone.
[0196] In certain embodiments, the second biologically active agent
is different than the biologically active agent yielded by
hydrolysis of the polyanhydride backbone.
[0197] In certain embodiments, the second biologically active agent
is an antimicrobial, anti-inflammatory, antioxidant, analgesic,
anticoagulant or fibrinolytic.
[0198] In certain embodiments, the second biologically active agent
is an anti-inflammatory agent.
[0199] In certain embodiments, the anti-inflammatory agent is
salicylic acid.
[0200] In certain embodiments, the second biologically active agent
is an antioxidant.
[0201] In certain embodiments, the antioxidant is vitamin E or
melatonin.
[0202] In certain embodiments, the polyanhydride as described
herein further comprises a compound of formula (V) dispersed in the
matrix of the polymer:
##STR00013##
[0203] In certain embodiments, a polymer as described herein
further comprises a compound of formula (VI) dispersed in the
matrix of the polymer:
##STR00014##
[0204] In certain embodiments, the polyanhydride is blended with a
second polymer to generate a polymer blend.
[0205] In certain embodiments, the second polymer is polyethylene
glycol (PEG), poly(lactic-co-glycolic acid) (PLGA) or poly(vinyl
pyrrolidone) (PVP).
[0206] In certain embodiments, the second polymer is PEG.
[0207] In certain embodiments, the second polymer is PLGA.
[0208] In certain embodiments, the second polymer is PVP.
[0209] In certain embodiments, the second polymer is a blend of PEG
and PLGA.
[0210] In certain embodiments, the polymer blend is electrospun to
generate electrospun nanofibers.
[0211] In certain embodiments, the electrospun nanofibers are
associated with the adhesion barrier. In certain embodiments, the
electrospun nanofibers may associated with the adhesion barrier
using water or dimethylsulfoxide (DMSO).
[0212] In certain embodiments, the polymer is formulated into
microspheres.
[0213] In certain embodiments, the microspheres are admixed with
the adhesion barrier.
[0214] Certain embodiments of the invention provide a medical
device comprising a composition as described herein.
DEFINITIONS
[0215] Unless otherwise described: halo is fluoro, chloro, bromo,
or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight
and branched groups; but reference to an individual radical such as
propyl embraces only the straight chain radical, a branched chain
isomer such as isopropyl being specifically referred to. Aryl
denotes a phenyl radical or an ortho-fused bicyclic carbocyclic
radical having about nine to ten ring atoms in which at least one
ring is aromatic. Heteroaryl encompasses a radical of a monocyclic
aromatic ring containing five or six ring atoms consisting of
carbon and one to four heteroatoms each selected from the group
consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is
absent or is H, O, (C.sub.1-C.sub.4)alkyl, phenyl or benzyl, as
well as a radical of an ortho-fused bicyclic heterocycle of about
eight to ten ring atoms comprising one to four heteroatoms each
selected from the group consisting of non-peroxide oxygen, sulfur,
and N(X).
[0216] The term "amino acid," comprises the residues of the natural
amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl,
Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in
D or L form, as well as unnatural amino acids (e.g. phosphoserine,
phosphothreonine, phosphotyrosine, hydroxyproline,
gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic
acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,
penicillamine, ornithine, citruline, .alpha.-methyl-alanine,
para-benzoylphenylalanine, phenylglycine, propargylglycine,
sarcosine, and tert-butylglycine). The term also comprises natural
and unnatural amino acids bearing a conventional amino protecting
group (e.g. acetyl or benzyloxycarbonyl), as well as natural and
unnatural amino acids protected at the carboxy terminus (e.g. as a
(C.sub.1-C.sub.6)alkyl, phenyl or benzyl ester or amide; or as an
.alpha.-methylbenzyl amide). Other suitable amino and carboxy
protecting groups are known to those skilled in the art (See for
example, T. W. Greene, Protecting Groups In Organic Synthesis;
Wiley: New York, 1981, and references cited therein). An amino acid
can be linked to the remainder of a compound of formula I through
the carboxy terminus, the amino terminus, or through any other
convenient point of attachment, such as, for example, through the
sulfur of cysteine.
[0217] Specific values listed below for radicals, substituents, and
ranges, are for illustration only; they do not exclude other
defined values or other values within defined ranges for the
radicals and substituents.
[0218] Specifically, (C.sub.1-C.sub.6)alkyl can be methyl, ethyl,
propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl,
or hexyl; (C.sub.3-C.sub.6)cycloalkyl can be cyclopropyl,
cyclobutyl, cyclopentyl, or cyclohexyl; (C.sub.1-C.sub.6)alkoxy can
be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,
sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
(C.sub.1-C.sub.6)alkanoyl can be acetyl, propanoyl or butanoyl;
(C.sub.1-C.sub.6)alkoxycarbonyl can be methoxycarbonyl,
ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,
butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl;
(C.sub.1-C.sub.6)alkylthio can be methylthio, ethylthio,
propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or
hexylthio; (C.sub.2-C.sub.6)alkanoyloxy can be acetoxy,
propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or
hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and
heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl,
isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl,
tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its
N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its
N-oxide).
[0219] As used herein, the phrases "dispersed in the matrix of the
copolymer" and "dispersed in the matrix of the polymer" mean that
an agent, such as an anti-inflammatory agent, is located within the
matrix of a copolymer/polymer such that it can be released in a
controlled fashion when placed within the body. Preferably, the
copolymer/polymer matrix comprises a bio-degradable polymer.
[0220] As used herein, "release" of an agent refers to delivery of
an agent in a form that is bioavailable. For instance, the term
"release" includes degradation of a copolymer/polymer in which the
agent is incorporated in the copolymer/polymer backbone, or
appended to the copolymer/polymer backbone, to release free agent.
The term also includes degradation of a copolymer/polymer that
entraps molecules of the agent in the matrix of the
copolymer/polymer, thereby allowing the free agent to make direct
contact with the surrounding tissue or bone. The term "release"
also encompasses administration of an agent in a form that is
immediately bioavailable (i.e., not a sustained release
formulation).
[0221] As used herein, the terms "treat" and "treatment" can refer
to therapeutic treatment or to prophylactic or preventative
treatment, wherein the object is to prevent or decrease an
undesired physiological change or disorder, for example, such as
undesired physiological changes or disorders associated with wounds
(e.g., fibrous adhesions, inflammation, pain, etc.).
[0222] As used herein the phrase "fibrous adhesions" refers to
bands of fibrous tissue that join two surfaces in the body that are
not normally connected. They generally form after injury to an area
that results in increased inflammation. Surgery, trauma,
infections, radiation, and ischemia can all lead to adhesion
formation, with surgery being the most common cause. Fibrinous
bands, which are precursors to fibrous adhesions, are also
encompassed by this phrase.
[0223] As described herein, the phrase "adhesion barrier" refers to
a material that can be used to reduce deleterious internal scarring
(e.g., adhesions) following injury (e.g., surgery) by separating
adjacent surfaces of tissues and organs during healing. In certain
embodiments, the adhesion barrier is a film, fabric or gel. In
certain embodiments, the adhesion barrier is a barrier listed in
Table 2. In certain embodiments, the adhesion barrier is Seprafilm.
In certain embodiments, the adhesion barrier is Intercoat.
[0224] As used herein, the term "wound" refers to an injury to a
part or tissue of the body, especially one caused by physical
trauma and characterized by tearing, cutting, piercing, or breaking
of the tissue.
[0225] In certain embodiments, a polymer as described herein may be
administered "at a wound site" or by "contacting" a polymer with
the wound. As used herein, these phrases/terms may mean locally
administering the polymer so that it is in direct contact with the
wound; or locally administering the polymer to a location proximal
to the wound, so that the polymer can produce the desired or stated
therapeutic effect (e.g. prevention of fibrous adhesions, provision
of localized analgesia, etc.), at the site.
Formulations
[0226] The copolymers/polymers, microspheres and electrospun
nanofibers described herein can be formulated as pharmaceutical
compositions and administered to a mammalian host, such as a human
patient in a variety of forms adapted to the chosen route of
administration, i.e., parenterally, by intravenous, intramuscular,
topical or subcutaneous routes.
[0227] The present copolymers/polymers or microspheres may be
administered intravenously or intraperitoneally by infusion or
injection.
[0228] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the present copolymers/polymers or
microspheres that are adapted for the extemporaneous preparation of
sterile injectable or infusible solutions or dispersions.
[0229] For topical administration, the present copolymers/polymers
may be applied in pure form, i.e., when they are liquids. However,
it will generally be desirable to administer them to the skin as
compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0230] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the present
copolymers/polymers or microspheres can be dissolved or dispersed
at effective levels, optionally with the aid of non-toxic
surfactants. Adjuvants such as fragrances and additional
antimicrobial agents can be added to optimize the properties for a
given use. The resultant liquid compositions can be applied from
absorbent pads, used to impregnate bandages and other dressings, or
sprayed onto the affected area using pump-type or aerosol
sprayers.
[0231] Useful dosages of the present copolymers/polymers,
microspheres and electrospun nanofibers can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art; for example, see
U.S. Pat. No. 4,938,949.
[0232] The amount of the present copolymers/polymers, microspheres
or electrospun nanofibers required for use in treatment will vary
with the route of administration, the nature of the condition being
treated and the age and condition of the patient and will be
ultimately at the discretion of the attendant physician or
clinician. However, in one embodiment a suitable dose may be in the
range of from about 0.05 to about 100 mg/kg, e.g., from about 1 to
about 75 mg/kg of body weight per day, such as 3 to about 50 mg per
kilogram body weight of the recipient per day, preferably in the
range of 6 to 90 mg/kg/day, most preferably in the range of 15 to
60 mg/kg/day.
[0233] In one embodiment, the invention provides a composition
comprising the present copolymers/polymers formulated in such a
unit dosage form. The desired dose may conveniently be presented in
a single dose or as divided doses administered at appropriate
intervals, for example, as two, three, four or more sub-doses per
day. The sub-dose itself may be further divided, e.g., into a
number of discrete loosely spaced administrations.
[0234] The invention will now be illustrated by the following
non-limiting Examples.
Example 1
[0235] Fibrous adhesions are a common result of surgery that can
lead to chronic pain, infertility, and intestinal obstruction.
Available devices have been designed to act as barriers to adhesion
formation, but are not extensively used. Although they have been
shown to reduce adhesion formation and severity, the devices do not
significantly reduce complications resulting from adhesions. As
described herein is the enhancement of commercially available
devices by using salicylic acid (SA)-based poly(anhydride-esters)
(PAEs) that hydrolytically degrade to release salicylic acid and
physically admixed antioxidants at a controlled rate to potentially
reduce adhesion formation both physically and chemically.
[0236] Mechanical, surface, and thermal analyses, as well as in
vitro drug release assays and cytotoxicity studies will be used to
optimize potential devices. Optimized devices will be evaluated in
vivo for adhesion reduction efficacy and compared to the original,
commercially available devices. Efficacy will be determined by the
extent of adhesion formation in a rat model.
Section 1.
[0237] Fibrous adhesions are a serious complication that can arise
from trauma to the body as they can lead to chronic pain,
infertility, and intestinal obstruction. Adhesions are bands of
fibrous tissue that join two surfaces in the body which are not
normally connected. They generally form after injury to an area
that results in increased inflammation. Surgery, trauma,
infections, radiation, and ischemia can all lead to adhesion
formation, with surgery being the most common cause.
[0238] There are many different organs in the abdominal area that
can be affected by adhesions; therefore, the need to prevent
adhesion formation here is important. Table 1 lists the anatomical
sites and organs that were most commonly found to have adhesions
associated with them, and the percentage of abdominal laparoscopy
patients exhibiting adhesions on those organs after surgery.
TABLE-US-00001 TABLE 1 Adhesion Site Frequency to Anatomical Sites
of Abdominal Laparoscopy Patients Adhesion site % Adhered Trocar
Scar 71 Omentum 68 Small Bowel 67 Abdominal Wall 45 Colon 41 Liver
34 Female Reproductive Organs 23 Stomach 20 Spleen 9
[0239] The physiological pathway that leads to abdominal adhesion
formation has been well studied. After surgery, fibrinogen from the
blood leaks into the peritoneal cavity and forms a fibrin matrix.
This matrix forms into transient fibrinous bands that are either
broken down by fibrinolysis or used as a scaffold for fibroblasts
to create permanent fibrous adhesions. The occurrence of
fibrinolysis is dependent upon the levels of different cytokines
and enzymes (FIG. 1). Mesothelial cells of the injured peritoneum
release cytokines to recruit immune cells and fibroblasts.
Polymorphonuclear neutrophils are the first cells to appear.
Macrophages are recruited to the site around day 2 after surgery.
Mesothelial cells start to proliferate and begin forming new
peritoneum on day 3. The peritoneum regrows from mesothelial cells
floating freely in the peritoneal fluid and is therefore repaired
from many areas at once rather than needing to have cells migrate
into the damaged area from the undamaged edges. It is because of
this that the peritoneum is repaired in 7-10 days regardless of
whether the injury to the area is moderate or severe.
[0240] Fibrous adhesions have an enormous impact on the healthcare
system. It has been estimated that 95% of abdominal and pelvic
surgeries, including gynecologic, result in adhesions. Adhesion
related problems account for 6% of all hospital readmissions and 1%
of all hospitalizations in the United States. A comprehensive study
in Scotland found that 22% of patients who had abdominal surgery
were readmitted within a year, and 34.6% are readmitted within 10
years for an average of 2.1 times. In 1994, $1.3 billion was spent
on hospital costs for 446,000 abdominal adhesiolysis procedures, a
surgery to dissect fibrous adhesions. While adhesiolysis can help
alleviate some pain and complications associated with adhesions,
the effect is often temporary as the adhesions tend to grow back
after the procedure. The statistics on the number of secondary
surgeries needed due to adhesions are even more alarming when one
takes into account that the presence of adhesions makes such
secondary surgeries even more difficult and dangerous. Adhesions
increase surgery time, hospital stay, complications, blood loss,
morbidity, and mortality; they are the main reason for an otherwise
minimally invasive planned laparoscopy procedure to be switched to
an invasive laparotomy procedure. Adhesions are responsible for the
majority of trocar-related injuries. They also greatly increase the
chance of inadvertent enterotomy, cutting of the intestines, with
19% of reoperations of the abdomen resulting in inadvertent
enterotomies. This results in more postoperative complications,
more urgent laparotomies, higher admission rate to the ICU, and
longer hospital stays; if left undetected an inadvertent enterotomy
can lead to death. One of the most problematic consequences of
adhesion formation is small bowel obstruction (SBO). Studies
estimate that 74% of SBOs are caused by adhesions. Simple
obstructions result in 3-5% patient mortality; the mortality rate
rises to 30% if the bowel becomes strangulated, necrotic, or
perforated. If left untreated, SBOs will lead to fatal peritonitis.
In total, 2100-2400 patients die from intestinal adhesions with
bowel obstructions annually in the U.S. While there has been some
improvement in devices to reduce adhesion formation, no device has
been proven to significantly reduce the incidence of SBOs caused by
adhesions.
[0241] In addition to improved surgical techniques, both
pharmaceuticals and physical barriers have been explored as means
to prevent adhesion formation (Tingstedt et al., Eur Surg Res 39,
259-268 (2007); Ward, et al., Journal of Surgical Research, 165(1),
91-111 (2009)). Drugs tested for the prevention of adhesion growth
are those that affect the clotting cascade, the inflammatory
process, cell proliferation, extracellular matrix production, or
oxidative stress. Systemic administration of such drugs at
therapeutic levels can cause undesired side effects and delay
healing after surgery. There have been some attempts to inject the
drugs into the peritoneal cavity; however, most of these have shown
little to no efficacy in laboratory testing primarily due to the
tendency for drugs placed in the peritoneal cavity to be quickly
absorbed by the mesothelium and subsequently distributed throughout
the body.
[0242] Various solids, gels, and fluids have been used as physical
barriers. The main purpose of every barrier is to separate surfaces
that adhesions could potentially form between. The FDA has approved
only 5 barrier devices for human use. None of the devices have been
shown efficacious enough at reducing adhesion formation to warrant
their ubiquitous use. Based on the number of surgeries in which
adhesion barriers would be warranted and the number of barriers
sold, barriers are used in at best a little over 5% of abdominal
surgeries. The efficacy of these devices must be higher if they are
to be used more often.
[0243] There is a need for a more efficacious device for the
prevention of fibrous adhesions. Accordingly, as described herein
is a biodegradable device that can provide a physical barrier as
well as deliver sustained release of anti-inflammatory and
antioxidant drugs directly to the desired area as a pharmaceutical
treatment. This controlled sustained release of drugs may be
accomplished through the use of salicylic acid (SA)-based
poly(anhydride esters) (PAE).
Section 2.
[0244] 2.1. The Determination of the Composition, Formulation, and
Amount of SA-Based PAE that Gives the Most Optimal Drug Release
Profile for Adhesion Prevention and the Determination of how Best
to Incorporate the PAEs into Adhesion Barriers Currently on the
Market.
[0245] The current resorbable adhesion barrier devices on the
market have shown only moderate efficacy in patients. Two of these
devices, Seprafilm and Intercoat, are used in experiments described
below; however, other adhesion barriers could also be used.
Seprafilm (Genzyme Corp.), composed of hyaluronic acid and
carboxymethylcellulose, can only be used in open surgeries as it is
manufactured as a film that is placed on the most traumatized area
within the abdomen. Intercoat (Johnson & Johnson), composed of
polyethylene glycol (PEG) and carboxymethylcellulose, is an
injectable gel that can be used in either open or laparoscopic
surgeries.
[0246] The use of a SA-based PAE, or polyAspirin (PA), will allow
for sustained release of salicylic acid at the site of implantation
as the anhydride and ester bonds are hydrolytically labile and the
polymer will degrade to release salicylic acid and biocompatible
diacids (Prudencio, et al., Macromolecules 38, 6895-6901 (2005);
Erdmann, et al., Biomaterials 21, 1941-1946 (2000);
Whitaker-Brothers, et al., Journal of Biomedical Materials
Research. Part A. 76, 470-479 (2006)). Salicylic acid, the
prototypical non-steroidal anti-inflammatory drug (NSAID), is a
desirable agent for this application as it not only has
anti-inflammatory properties, but also acts as an anticoagulant and
an analgesic.
[0247] A method to optimally integrate PA with these current
devices will need to be determined. The type of device will dictate
the formulation of the PA. Ensuring that the form of the PA has the
necessary physical properties for each device is important.
Initially, PA microspheres are proposed for admixture with
Intercoat and PA containing electrospun membranes are proposed for
layering with Seprafilm.
[0248] The degradation rates of the polymers are critical to the
efficacy of these devices. Enough SA (e.g., corresponding to about
40-100 mg/day dosage) needs to be released from the device within
the first week of implantation to affect the growth of fibrous
adhesions (Muzii, L. et al., Human Reproduction 13, 1486-1489
(1998); Buerke, et al., American Heart Journal 130, 465-472
(1995)). However, if too much SA (e.g., corresponding to about 1700
mg/day) is released it can result no adhesion reduction and
interfere with the wound healing process responsible for closing
any incisions caused by surgery (Muzii, L. et al., Human
Reproduction 13, 1486-1489 (1998); Golan, et al., Human
Reproduction 10, 1797-1800 (1995)). One of the FDA guidelines for
resorbable adhesion barriers is that they not reduce tissue-holding
strength after sutures are removed. To ensure a beneficial amount
of SA is being released at all times, the optimal chemical
composition and amount of PA will need to be determined such that
the rate of SA release maintains a steady concentration that
results in the effective range as described above. Admixtures of SA
and SA-based diacids in the PA will also be considered as a means
of manipulating the SA release profile. The optimal composition and
amount of the PA needed will be determined separately for both the
film and the gel as the shape of the PA in the device and the
composition of the hydrogel will both affect the degradation of the
polymer and the release of the SA.
[0249] 2.2. The Incorporation of an Antioxidant into the SA-Based
PAE and Determination of the Release Profile of the Antioxidant and
its Effect on Polymer Degradation.
[0250] There have been numerous studies to evaluate the efficacy of
different drugs on adhesion prevention, however few have been shown
to be effective. To create a synergistic effect to better reduce
the incidence of adhesion formation, two drugs, an
anti-inflammatory and an antioxidant, are incorporated into the
barriers as described herein.
[0251] Two antioxidants have been widely researched for their
ability to prevent adhesions: Vitamin E and melatonin. Both have
been shown to significantly reduce abdominal adhesion formation in
rats when injected intraperitoneally (IP) (de la Portilla, F. et
al. Diseases of the Colon & Rectum 42, 2157-2161 (2004); Imai,
et al., European Journal of Obstetrics & Gynecology and
Reproductive Biology 149, 131-135 (2010)).
[0252] 2.3. The Determination of the Effects of Common
Sterilization Techniques on the Devices and Comparison of the
Efficacy of the Combination Devices Against their Equivalent
Non-polyAspirin Device In Vivo.
[0253] All medical devices need to be sterilized before
implantation. Common sterilization techniques can alter the polymer
in undesirable ways. It is proposed that once the final device
compositions are determined, the devices be subjected to two common
sterilization methods, namely electron beam and gamma ray radiation
sterilization (at various dosing levels), to determine if any
critical characteristics such as the degradation rate or mechanical
properties of the devices are altered by the sterilization
processes. If it is discovered that the degradation of the device
is affected, the composition of the polymer will be changed to
ensure that the desired release profile is obtained from the device
after sterilization.
[0254] To achieve market approval for a medical device in the
United States, it is necessary to show that the new device is both
safe and efficacious. The new device must be at least as
efficacious as other similar products on the market. The optimized
devices will need to be tested in vivo to determine if the addition
of polyAspirin has improved the efficacy of the original devices.
Such testing will be initiated in a rat model. The surgery model
consists of a ventral incision and intentional trauma to the
peritoneum to induce a wound healing response to promote adhesion
formation at the trauma site (Rajab, et al. Journal of Surgical
Research 161, 246-249 (2010)). The rats will be divided into groups
that will be treated with Seprafilm, Intercoat, Seprafilm+PA,
Intercoat+PA, Seprafilm+PA+antioxidant, Intercoat+PA+antioxidant,
or no barrier. The rats will be sacrificed at 14 days after
surgery. An investigator blinded to the treatment group will grade
the extent of adhesions.
Section 3.
[0255] 3.1. Polyanhydrides
[0256] Polyanhydrides are a class of hydrolytically degradable
polymers that have been widely used in several biomedical
applications. Polyanhydrides are primarily surface eroding, a
characteristic that makes them desirable for drug delivery
applications as this leads to a near zero order release of
molecules encapsulated within the polymer matrix
(Whitaker-Brothers, et al., Journal of Biomedical Materials
Research. Part A. 76, 470-479 (2006)).
[0257] 3.1.1. Salicylic Acid-Based Poly(Anhydride Esters)
[0258] A series of SA-based PAEs (polyAspirins) that are composed
of salicylic acid and various biocompatible diacid linker molecules
have been developed (Prudencio, et al., Macromolecules 38,
6895-6901 (2005); Schmeltzer, et al., Polymer Bulletin 49, 441-448
(2003); Schmeltzer, et al., Journal of Biomaterials Science:
Polymer Edition 19, 1295-1306 (2008); Carbone, et al.,
Macromolecular Rapid Communications 30, 1021 (2009)). These
polymers exhibit hydrolytic surface erosion degradation to release
free salicylic acid and biocompatible linker molecules (Scheme 1)
(Erdmann, et al., Biomaterials 21, 1941-1946 (2000)). The linker
can be used to alter the degradation rate of the polymer. SA-based
polymers with more hydrophobic linker molecules exhibit decreased
degradation rates (Prudencio, et al., Macromolecules 38, 6895-6901
(2005)).
##STR00015##
[0259] The advantage of incorporating a drug into the polymer
backbone, as opposed to merely physically mixing the drug into a
polymer that does not degrade into a bioactive molecule is the high
drug loading achieved with this method. The inherent drug loading
of PA can be up to 75% depending on the linker molecule, with the
ability to have higher drug loading via admixed free drugs. Polymer
systems with physically incorporated drugs cannot attain such a
high drug loading without affecting the polymer degradation and
drug release rates. The thermal and mechanical properties of PA
allows it to be manipulated into various shapes such as, disks,
films, microspheres, and fibers. The release profile of salicylic
acid from PA is observed to have a lag period of little to no drug
release followed by zero order release. The length of the lag
period is determined by the composition of the polymer (Prudencio
et al., Macromolecules 38, 6895-6901 (2005); Erdmann, et al.,
Biomaterials 21, 1941-1946 (2000); Yeagy, et al., Journal of
Microencapsulation 23, 643-653 (2006)).
[0260] 3.1.3. Admixtures of Free Drugs into PolyAspirin
[0261] Free drug molecules can be admixed into PA such that the
free drug is released along with the SA from the polymer backbone.
The release of free antimicrobials admixed into adipic-PA disks has
been studied in vitro by placing the disks into phosphate buffered
saline (PBS) pH 7.4 in an incubator shaker at 37.degree. C. and 65
rpm. PBS was collected and replaced at various time points and
analyzed by high-pressure liquid chromatography (HPLC) to determine
the concentration of both salicylic acid and the antimicrobial. It
was observed that the rate of antimicrobial release was dependent
upon the hydrophobicity of the antimicrobial; hydrophobic
antimicrobials were released at a slower rate than more hydrophilic
antimicrobials. The hydrophobicity of the antimicrobial did not
significantly affect the release of salicylic acid from the disks
(Johnson, et al., Journal of Biomedical Materials Research. Part A.
91, 671-678 (2009)).
[0262] SA and SA-based diacids were admixed into both fast and slow
degrading PA to observe the effect they would have on the lag
period in the release curve of the polymers. PA with adipic acid
and diethylmalonic acid as the linkers were chosen as the fast and
slow degrading polymers, respectively. SA, SA-based diacids with
linkers corresponding to their respective polymer, and a 1:1
mixture of SA and diacid were admixed into the polymers at 1, 5,
and 10 weight percentages. Approximately 160 mg of polymer
admixtures were placed in a 13 mm diameter mold and pressed for 10
minutes at 10,000 psi. This resulted in disks .about.1 mm thick.
All samples were made in triplicate. The disks were placed in glass
vials with 10 ml PBS pH 7.4 incubated at 37.degree. C. PBS was
collected and replaced at set time points and analyzed by UV
absorbance at 303 nm. SA concentration was quantified by comparing
absorbance values to a calibration curve of absorbance values for
SA solutions of known concentrations.
[0263] The cumulative SA release profiles of the samples are
presented in FIG. 2. The free SA and diacids removed the lag period
of SA release observed in both of the polymer systems. The SA
admixtures resulted in a burst release, where a relatively large
amount of SA was released in the early time points. The size of the
burst effect correlated with the weight percent of admixed SA in
the system. The diacid admixtures increased early SA release from
the systems, but did so with a less prominent burst profile. As
expected, the 1:1 mixtures of SA and diacid had an intermediate
effect between the two described above. The admixtures did not
increase the degradation rate of the polymers.
[0264] The ability to negate the early lag time of SA release from
PA is important for the proposed adhesion barriers, as fibrin
deposition occurs immediately following surgery and fibrinolysis
should occur within 3 days of surgery to mimic a proper healing
response that does not lead to adhesion formation.
[0265] 3.1.4. Cytotoxicity Studies
[0266] In vitro studies have been performed to assess the
cytotoxicity of PAs. Briefly, L929 fibroblast cells were seeded at
approximately 2000 cells/well in 3.times.96 well culture dishes. A
fourth 96 well plate is plated with triplicates of wells plated at
different cell seeding densities. Triplicates of wells in each dish
contained normal cell culture media, media with 0.10 mg/ml and 0.01
mg/ml of polymer dissolved in dimethyl sulfoxide (DMSO), and
controls of media with DMSO without polymer. After 24 hours, the 96
well dish with the different original cell seeding densities is
assessed using an MTS assay, which reacts with NADPH and NADH
produced in living cells, to indirectly measure the number of
living cells in a culture (Saed, et al., Fertility and Sterility
79, 164-168 (2003)). The MTS assay of the first dish is used to
create a calibration curve that will later be used to determine the
number of living cells present in the other plates. At 48, 72, and
96 hours, the other plates are assessed by MTS assay. Light
microscopy is used to observe any changes in cell morphology due to
PA. Previous studies have shown PA to be biocompatible at up to 0.1
mg/ml concentrations (Griffin, et al., Journal of Biomedical
Materials Research. Part A. 97(3), 230-242 (2011)).
[0267] 3.2. Electrospun Polymer Fibers
[0268] Electrospinning is a method to create nano to micro-sized
polymeric fibers (Demir, et al., Polymer 43, 3303-3309 (2002)). The
process involves creating an electric field between a charged
needle and a grounded collection surface. The polymer is dissolved
in a solvent and slowly ejected out of the needle where
electrostatic forces overcome surface tension to cause a stream of
polymer to move from the needle to the collection surface to form a
mat of fibers (Griffin, et al., Journal of Biomedical Materials
Research. Part A. 97(3), 230-242 (2011)).
[0269] Electrospun membranes have been tested previously for their
ability to prevent adhesions. Zong, et al created electrospun
poly(lactic-co-glycolic acid) (PLGA) membranes and studied their
ability to prevent abdominal adhesions in rats. PLGA membranes
alone decreased adhesion formation, but not significantly.
Membranes of PLGA and a poly(lactic acid) (PLA)-PEG copolymer did
show a significant decrease in adhesions (Zong, X. et al. Annals of
Surgery 240, 910-915 (2004)). The use of PEG and PLA as electrospun
membranes was further demonstrated by Yang, et al. They created
PEG:PLA block copolymers of differing ratios (Yang, et al., Journal
of Biomedical Materials Research. Part A. 82, 680-688 (2007)). In
vitro studies demonstrated that membranes with higher PEG:PLA
ratios exhibited decreased cell adhesion to the membrane (Yang, et
al., Acta Biomaterialia 5, 2467-2474 (2009)). In vivo studies
conducted with these membranes resulted in a significant decrease
of adhesion formation when a PLA membrane was implanted as opposed
to untreated controls but that a PEG:PLA membrane reduced adhesion
significantly more than the PLA membrane.
[0270] PAs, blended with higher molecular weight PLGA or poly(vinyl
pyrrolidone), have been used to create electrospun membranes (see,
e.g., FIG. 3) (Griffin, et al., Journal of Biomedical Materials
Research. Part A. 97(3), 230-242 (2011)). The PA-containing
membranes are thin and tear easily. This is why it is proposed that
the electrospun membranes be adhered to Seprafilm, to ensure that
the membrane remains intact during and after implantation.
[0271] 3.3. Microsphere Fabrication
[0272] Polymeric microspheres are advantageous over many other
forms of polymers for drug delivery as they are small enough to be
injected. PA has previously been formulated into microspheres with
size ranges of 2-20 .mu.m (FIG. 4) (Yeagy, et al., Journal of
Microencapsulation 23, 643-653 (2006)). This was accomplished by
using an oil-in-water single emulsion solvent evaporation technique
(Freitas, et al., Journal of Controlled Release 102, 313-332
(2005)). Briefly, PA is dissolved in dichloromethane (DCM). This
solution was then added to a 1% w/v aqueous solution of poly(vinyl
alcohol) (PVA) and homogenized for 2 minutes with a handheld
homogenizer. The solution was then allowed to stir for 15 minutes
to allow the DCM to evaporate. Microspheres were recovered by
centrifugation. PVA was then removed by a centrifugation wash.
Samples were frozen and lyophilized.
[0273] 3.4. Adhesion Barriers Currently on the Market
[0274] The 5 adhesion barrier products approved by the FDA for use
in the US are listed in Table 2. Many other devices have been
approved for use in countries other than the US but have not been
approved by the FDA (diZerga, et al., Reproductive BioMedicine
Online 17, 303-306 (2008)). One such product is Intercoat
(carboxymethylcellulose and polyethylene glycol, Ethicon), an
injectable gel that has been approved for use in Europe.
TABLE-US-00002 TABLE 2 FDA Approved Adhesion Prevention Products
Product Composition Company Description Preclude
polytetrafluoroethylene WL Gore Solid nonresorbable barrier
Seprafilm hyaluronic acid and Genzyme solid resorbable
carboxymethylcellulose Corp. barrier Interceed oxidized regenerated
Gynecare solid resorbable cellulose barrier REPEL-CV polylactic
acid and SyntheMed, solid resorbable polyethylene glycol Inc.
barrier Adept 4% icodextrin solution Baxter injectable solution
Biosurgery
[0275] 3.4.1. Seprafilm
[0276] With a 58% share of the abdominal adhesion barrier market,
Seprafilm is the industry standard. A multi-center study
demonstrated that Seprafilm use resulted in 51% of patients being
adhesion free after second-look laparoscopies as compared to 6% of
patients in the untreated group. The density of adhesions was also
lower in patients as compared to the control group. However a
systematic review of the literature found that Seprafilm decreased
adhesions but had no significant effect on SBO incidence. It did
however lead to more abdominal abscesses and leakage of gastric or
intestinal fluid at sites of anastomoses. Other systematic reviews
found that Seprafilm had no significant effect on readmission
rates, pregnancy rates, or pelvic pain in treated patients.
[0277] Many surgeons note that Seprafilm is brittle and sticky,
making it difficult to apply in patients; these characteristics
also exclude Seprafilm for use in laparoscopic surgeries.
[0278] 3.4.2. Intercoat
[0279] Intercoat has been approved for use in Europe since 2002 but
was not approved by the FDA for efficacy reasons (Fransen, Annals
of Surgical Innovation and Research 2 (2008)). A small clinical
study was conducted to determine the efficacy of Intercoat on
preventing adnexal adhesion formation, adhesions involving the
ovaries and fallopian tubes, in women undergoing gynecologic
surgeries, with 26 of the 28 patients undergoing adhesiolysis as a
part of their surgery. Formation of new adhesions was determined by
second look laparoscopies 6-10 weeks later. Adnexal adhesion score,
as defined by the American Fertility Society (ASF), was unchanged
in treated patients but was increased from 8.0 to 11.6 in untreated
patients. For adnexa with severe adhesions (ASF score .ltoreq.6)
before the surgery, treated group adhesion scores were reduced by
an average of 1.0 while the control group scores increased by an
average of 4.6. Overall, 34.5% of treated and 66.7% of untreated
adnexa had increased scores. A similar study on a larger group of
patients resulted in an ASF score reduction from 11.9 to 9.1 in the
treated group as opposed to an increase from 8.8 to 15.8 in the
untreated group. In this study only 7% of treated adnexa
demonstrated increased ASF score as opposed to 44% of untreated
adnexa. A 396 patient study of Intercoat use in spinal surgeries
demonstrated a reduction in the need for reoperations when compared
to when compared to what is experienced for this surgery, however,
as there were no controls for this study, the significance of the
findings cannot be determined.
[0280] A direct comparison of Seprafilm and Intercoat, as well as
Adept, was conducted by Rajab et al. in a rat model. Efficacy was
determined by adhesion area as a percent of the cauterized lesion
created during surgery. All three products significantly reduced
mean adhesion covered area as compared to control. Both Seprafilm
and Intercoat resulted in 20% adhesion-free animals in their
groups, as opposed to 0% in the Adept and control groups. The mean
area of adhesion incidence for Seprafilm (46%) was better than that
of Intercoat (55%), but not significantly so (Rajab, et al.,
Journal of Surgical Research 161, 246-249 (2010)).
[0281] 3.4.3. Cost-Benefit Analysis
[0282] Adhesion barriers are only used in a small percentage of the
estimated 9.9 million surgeries that they could be helpful for. One
study found that only 10% of patients undergoing abdomino-pelvic
surgeries were advised about the risk of adhesion and only 6% were
advised about prevention methods. The minimal usage of adhesion
barriers is primarily due to the fact that there are no large
randomized controlled trials that demonstrate the efficacy of
current adhesion barriers to significantly decrease the rate of
hospital readmissions due to adhesion related disorders. Without
proof that the adhesion barriers can save healthcare costs in the
future, their usage will remain minimal; it must be shown that
barriers can significantly reduce not just adhesion formation, but
also the complications of adhesions that lead to hospital
readmissions. A study from the United Kingdom estimated that a
product costing .English Pound.50 (.about.$80) per patient that
resulted in a 16% reduction of readmissions would payback the cost
of its investment in 3 years. A product costing .English Pound.200
(.about.$315) would need to have a reduction rate of 64.1% in order
to pay for itself in 3 years. With an average cost of about $200
per unit, and with some patients requiring more than one unit, the
devices currently available do not meet the criteria of being cost
effective for use in all patients undergoing abdominal surgery.
[0283] 3.5. Use of Drugs for the Prevention of Fibrous
Adhesions
[0284] Many different drugs have been investigated for their
potential to prevent adhesion formation. Some of the most common
classes of drugs studied are anti-inflammatories, antioxidants,
anticoagulants, fibrinolytics, and proteolytics. The
anti-inflammatory and antioxidant drugs serve to decrease the wound
healing response, thereby preventing the recruitment and
proliferation of cells that would form the fibrous adhesions. The
anticoagulants prevent fibrinous bands from forming whereas
fibinolytics and proteolytics break down the fibrinous bands. The
absence of these fibrinous bands would remove the scaffolding that
fibroblasts attach to when forming fibrous adhesions. The most
significant obstacle to the use of drugs for adhesion prevention is
the ability to target drugs to the specific area. This is a
critical problem as most of the drugs above, if given systemically
at high enough doses to be effective, would inhibit healing of
wounds received from surgery in addition to any other adverse side
effects that systemic delivery of those drugs would normally
cause.
[0285] 3.5.1. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)
[0286] NSAIDs are a class of drugs that are known to have
anti-inflammatory, analgesic (pain reducing), and antipyretic
(fever reducing) properties. The also inhibit fibroblast
proliferation and formation of granulation tissue. The most common
NSAID used is acetylsalicylic acid (aspirin). NSAIDs inhibit
cyclooxygenase (COX) enzymes, thereby preventing the synthesis of
prostoglandins, prostacyclins, and thromboxane. There are 2 COX
enzymes. COX-1 is constitutively expressed in most tissues at
constant levels. COX-2 is expressed in reaction to stimuli. It is
the inhibition of COX-2 that leads to the therapeutic effects of
NSAIDs. When fibroblasts harvested from IP adhesions and normal
peritoneum tissue were studied, it was observed that COX-2 was
expressed in the adhesion fibroblasts but not in the normal
peritoneal fibroblasts (Saed, et al., Fertility and Sterility 79,
1404-1408 (2003)). Adhesion fibroblasts were also found to have
lower levels of tissue plasminogen activator (tPA) and higher
levels of plasminogen activator inhibitor-1 (PAI-1). tPA and PAI-1
are important molecules in the fibrinolysis pathway; tPA is an
anti-coagulant and an activator of fibrinolysis, PAI-1 inhibits
tPA. Aspirin increases tPA levels and decreases PAI-1 (Buczko, et
al., Thrombosis Research 110, 331-334 (2003); Hammouda, et al.,
Thrombosis Research 42, 73-82 (1986)). Many studies have come to
competing conclusions as to the effect that aspirin has on
fibrinolysis. However, the effect aspirin has on fibrinolysis seems
to be dependent upon both dose and other signaling molecules in the
plasma. Low doses of aspirin have been shown to increase
fibrinolysis, while higher doses have an inhibitory effect on the
pathway.
[0287] Fibrinolysis is an important pathway in the prevention of
adhesions as it breaks down the fibrinous bands before they can
become permanent. The evidence that low doses of sustained aspirin
are preferable has been borne out in animal models. In a rat model,
animals were given either 0.35, 0.7 or 1.4 mg of aspirin every 6
hours for 48 or 96 hours after surgery. The rats that were given
the lowest dose for the longest time (0.35 mg, 96 hours) had the
fewest adhesions. The rats given higher doses for any amount of
time had the highest incidence of adhesions, however, the
difference between these groups was not significant and no group
was significantly different from the untreated control (Golan, et
al., Human Reproduction 10, 1797-1800 (1995)). In a rabbit model,
rabbits were given either 1.7 mg/kg/day or 28.0 mg/kg/day for 5
days after surgery. The low dose group had a significantly lower
adhesion rate at 44% than the high dose group at 77%, the control
had 100%. It was observed that the lower doses of aspirin
preferentially inhibited thrombaoxane rather than prostacyclin and
that this is what accounts for the increased efficacy of adhesion
(Muzii, L. et al. Human Reproduction 13, 1486-1489 (1998)).
[0288] Another NSAID, aceclofenac, was studied for its ability to
prevent adhesions. 5 mg/kg/day was injected intramuscularly into
rabbits for 7 days. A significant difference was observed in the
fibrous area between the control group and the treatment group. It
was also observed that the treatment group had an 8% decrease in
fibroblasts at the injury site (Sandoval, et al., Eur Spine J 17,
451-455 (2008)).
[0289] 3.5.2. Antioxidants
[0290] Oxidative stress leads to increased adhesion formation.
Vitamin E and melatonin are two antioxidants that have been
extensively investigated for their ability to mitigate this effect
and inhibit adhesion formation. In addition to reducing oxidative
stress, vitamin E also inhibits COX-2 and platelet aggregation. In
fact, vitamin E has been observed to potentiate the anticoagulant
effect of aspirin in vitro. This may be a useful characteristic in
the proposed devices as it may help the SA prevent the formation of
fibrinous bands. Vitamin E also inhibits TGF-.beta., which has an
anti-fibroblastic effect, and decreases collagen production which
may help prevent adhesions. In animal studies, vitamin E has been
shown to reduce adhesion formation by 80% when administered through
IP injection, but not when administered orally or intramuscularly
(de la Portilla et al., Diseases of the Colon & Rectum 42,
2157-2161 (2004)). In a rat model IP injections of vitamin E were
as effective as Seprafilm (Corrales, et al., Acta Cir rgica
Brasileira 23, 36-41 (2008)).
[0291] Melatonin is a more effective free radical scavenger than
vitamin E. It also has anti-inflammatory and anti-fibroblastic
properties as well. IP injection of melatonin has been demonstrated
to significantly reduce adhesion formation in rat and dog models
(Saeidi, et al., Interactive Cardiovascular and Thoracic Surgery 9,
26-28 (2009); Ara, et al. Life Sciences 77, 1341-1350 (2005)).
Further proof that melatonin has an effect on adhesion formation
has been demonstrated in animal models with pinealectomies,
surgeries to remove the pineal gland which normally produces
melatonin to regulate circadian rhythms. Adhesion formation in rats
with pineal glands removed had significantly more adhesions than
controls; when rats without pineal glands were given IP melatonin
adhesion formation was similar to the control. Rats with intact
pineal glands and injections of melatonin had significantly less
adhesions than the control (Ersoz, et al. Journal of
Gastroenterology and Hepatology 24, 1763-1767 (2009)).
[0292] 3.6. Sterilization Techniques
[0293] Two common sterilization techniques, electron beam
irradiation and gamma ray irradiation, were used to determine the
effect these techniques had on the adipic-PA and on poly(oCPX). The
polymers were sent to Johnson & Johnson's Sterile Process
Technology plant where they were subjected to 25 and 50 kGy of the
two types of radiation. These doses represent the typical and
typical maximum processing doses, respectively, generally used for
sterilization. Processed samples were tested for changes in
molecular weight (MW), glass transition temperature (T.sub.g),
infrared spectrum (IR), cytotoxicity, and degradation rates. No
significant differences were found in any of these characteristics
between irradiated samples and traveler samples (identical samples
that are subjected to all of the same conditions-shipping, storage,
etc., as the irradiated samples).
Section 4.
[0294] The development of resorbable adhesion barrier devices that
are more efficacious than those currently available on the market
by combining controlled release of drugs with a physical barrier
are described herein.
[0295] 4.1. The Determination of the Composition, Formulation, and
Amount of SA-Based FAE that Gives the Most Optimal Drug Release
Profile for Adhesion Prevention and the Determination of how Best
to Incorporate the PAEs into Adhesion Barriers Currently on the
Market.
[0296] 4.1.1. Preparation of PA Microspheres
[0297] Microspheres of adipic-PA will be prepared using an
oil-in-water single emulsion solvent evaporation technique as
described above. Adipic-PA has been shown to degrade relatively
quickly (approximately a week), which is a desired property for
this application. The microspheres will be characterized by SEM to
confirm a spherical shape and determine the microsphere size
distribution. The microspheres will also be characterized by
differential scanning calorimetry (DSC) to determine if their
T.sub.g is above physiological temperature (37.degree. C.). If the
T.sub.g is below 37.degree. C. the microspheres may not retain
their shape when they are at that temperature, thus potentially
affecting degradation rates.
[0298] 4.1.2. Admixture of Microspheres into Intercoat and In Vitro
Drug Release
[0299] The microspheres will be admixed into samples of Intercoat
at 0.1, 0.2, 0.3, 0.4 mg microspheres/ml Intercoat. These samples
will then be used for in vitro SA release studies by placing a
known mass of the sample into glass vials with 10 mL PBS at pH 7.4
rotating in an incubator set to 37.degree. C. and 80 rpm. The PBS
will be collected at specific time points, replaced with new PBS,
and placed back into the incubator. The collected PBS will be
analyzed on a UV/vis spectrophotometer set to measure the
absorbance at 303 nm. The absorbance value for each sample will be
compared to a calibration curve to determine the amount of
salicylic acid released into that sample. The release rates from
the above concentrations of microspheres will be used to determine
the optimal concentration to obtain a constant release of 42.5
.mu.g SA/ml Intercoat/day. This rate corresponds to the targeted
100 mg/day dose in an average 60 kg human given one unit of
Intercoat (40 ml) (Ersoz, N. et al., Journal of Gastroenterology
and Hepatology 24, 1763-1767 (2009)).
[0300] The optimal polymer and microsphere concentration will
degrade to give a release rate of 42.5 .mu.g SA/ml Intercoat/day
for about least 10 days. If incorporation into a hydrogel changes
the degradation rate of the adipic-PA such that the normal
degradation is drastically changed from what has been seen
previously, another polymer with a more hydrophobic (if the
degradation rate is too fast) or hydrophilic (if the degradation
rate is too slow) linker will be synthesized and formulated into
microspheres, and the in vitro studies repeated to obtain a more
desirable release curve.
[0301] 4.1.3. Electrospinning PA-Containing Membranes
[0302] SA-based PAEs will be admixed with high molecular weight
(.about.100 kDa) PEG (Sigma-Aldrich) and be electrospun into mats.
PEG has been demonstrated to be better than PLA at preventing
adhesions. If PA cannot be electrospun with PEG, PLGA will be used
as has been done before (Griffin, et al., Journal of Biomedical
Materials Research. Part A. 97(3), 230-242 (2011)).
[0303] The T.sub.g of the membranes will be analyzed by DSC. SEM
will be used to determine the fiber diameter distribution. The
mechanical properties of the membrane will be assessed by obtaining
the stress strain curve of the membrane. Measurements will be taken
for both dry and fully hydrated membranes. An optimal membrane
would be less brittle than Seprafilm at each state. The swelling
ratio of the membrane will also be assessed using Eq. 1 after being
hydrated in PBS for 24 hours.
Q = w s - w d w d Equation 1 ##EQU00001##
Q=swelling ratio w.sub.s=weight of swollen membrane w.sub.d=weight
of dry membrane
[0304] 4.1.4. Adhering PolyAspirin-Containing Electrospun Membranes
to Seprafilm
[0305] These membranes will then be adhered to samples of
Seprafilm. The most effective way to adhere these membranes to
Seprafilm will be determined based on the mechanical properties of
the combined device when evaluated under both the dry and the
hydrated conditions. Furthermore, adherence levels will be assessed
at both hydration states. The first methods attempted will be to
use small amounts of water, to make the Seprafilm sticky, or DCM,
to slightly dissolve the top layer of the membrane enough to make
it adhere to the Seprafilm.
[0306] Electrospun membranes of PA and another polymer were chosen
over spray-coated or solvent-cast films of PA alone due to the less
desirable mechanical properties of the latter-mentioned final
products. PA-containing electrospun membranes are much less brittle
and are more elastic than solid PA films. These are necessary
properties if the polymer is to remain attached to Seprafilm, as it
will expand when placed in an aqueous environment. The increased
surface area of a fibrous membrane, as compared to a solid film, is
also desirable as a method to increase SA release rates. The weight
percent of PA in the electrospun membrane will need to be
controlled such that the swelling of the membrane will be
comparable to Seprafilm in order to prevent disassociation of the
two layers when placed in an aqueous environment.
[0307] 4.1.5. In Vitro SA Release Studies from PA/Seprafilm
Device
[0308] In vitro SA release studies will be performed on the
PA/Seprafilm device similarly to those previously described for the
Intercoat and microsphere admixture. The composition of the
membrane will be adjusted as needed to obtain optimal mechanical
and degradation properties. The thickness of membrane layers
adhered to Seprafilm will be adjusted to obtain the desired 100 mg
SAJday/Seprafilm unit dose.
[0309] 4.1.6. Adjusting the Release of Salicylic Acid
[0310] PAs generally release SA in a linear fashion after a short
lag period. For the proposed devices, SA release will be desired
soon after implantation for the effect it can have on coagulation
and inflammation. For this, different weight percentages (to be
determined by the SA release curve of the polymer alone) of SA
and/or SA-based diacids will be admixed into the PA to give the
desired release curve.
[0311] 4.1.7. Cytotoxicity Studies
[0312] Both devices will be assessed for their in vitro
cytotoxicity toward L929 fibroblasts using methods similar to those
described previously in section 3.1.4. On day 1, the cells will be
plated 2000 cells/well in multiple 96 well plates with normal cell
culture media. A plate with a range of cell seeding densities will
also be plated for calibration of the MTS assay. Samples of the
devices will also be placed in separate cell culture media stocks
on day 1. At 24 hours, the media in the cell culture wells will be
replaced with media from the vials containing the device samples or
normal media in the case of the controls, and the MTS assay will be
used on the calibration plate. At 48 hours, one cell culture plate
will be assessed at that time using an MTS assay and trypan blue
exclusion assay for cell viability. The media in the cell culture
wells will again be replaced with fresh media from control and
sample vials. At 72 and 96 hours, the other sets of cells will be
assessed using the MTS and trypan blue assays. The cell media will
be replaced at 72 hours. Light microscopy images of cells will be
taken at each 24 hour time point to observe any changes in cell
morphology.
[0313] 4.1.8. Anticipated Results
[0314] It is anticipated that the incorporation of microspheres
into Intercoat may slightly decrease the degradation rate of the
microspheres as compared to microspheres free in PBS as the
hydrogel may protect the polymer surface from the shear forces of
moving liquid that can facilitate more rapid hydrolysis.
[0315] The electrospun membranes are expected to form without
problem as PA has successfully been electrospun with PLGA, and PEG
has been successfully electrospun as well (Griffin, et al., Journal
of Biomedical Materials Research. Part A. 97(3), 230-242 (2011);
Zong, X. et al., Annals of Surgery 240, 910-915 (2004); Yang, et
al., Journal of Biomedical Materials Research. Part A. 82, 680-688
(2007); Yang, et al., Acta Biomaterialia 5, 2467-2474 (2009)). The
membranes are expected to be less brittle than Seprafilm as the
PA/PLGA films are quite flexible, whereas Seprafilm is not. It is
also expected that the membranes will degrade faster than is
usually observed for PA as PEG is very hydrophilic and will allow
more water into the polymer matrix than would PA alone. The
increased surface area of the membrane fibers should also lead to a
faster degradation rate than a PA disk.
[0316] The admixture of SA and or SA-based diacids is expected to
negate any lag period in the drug release curves. This is a
desirable characteristic of the devices, as the anticoagulant
effect of the SA is most needed directly after implantation to
prevent the formation of the fibrinous bands that are precursors to
fibrous adhesions.
[0317] The cytotoxicity studies are expected to show little to no
toxicity from the devices as has been observed with other PA
devices.
[0318] If the homogeneous incorporation of microspheres into
Intercoat significantly slows the release of SA from the device,
the microspheres may be applied to the outer surface of the
Intercoat sample rather than distributed within it.
[0319] If proper adhesion of the two layers becomes an issue with
procedures described above, a PA/PEG admixture could be
spray-coated directly onto the Seprafilm.
[0320] 4.2. The Admixture of an Antioxidant into the SA-Based PAE
and Determination of the Release Profile of the Antioxidant and its
Effect on Polymer Degradation.
[0321] 4.2.1. Admixing Vitamin E into the PolyAspirin
[0322] Both vitamin E and melatonin have demonstrated the ability
to reduce adhesions in animal models. As there have been no studies
comparing the efficacy of the two against each other, it is
proposed that vitamin E be the first choice for the antioxidant
used as it is generally regarded as safe (GRAS) by the FDA. It also
acts synergistically with SA to reduce clotting which could be very
helpful in vivo by preventing fibrin deposition that can lead to
adhesions (Celestini, A. et al. Haematologica 87, 420-426
(2002).
[0323] Vitamin E will be admixed into the PA structures described
above. In vitro studies will be conducted, as described earlier, to
observe the release of vitamin E and its effect on the degradation
profile of the PA. HPLC will be used to measure the amounts of
drugs released. The HPLC method will entail passing the samples
through a C18 column with a mobile phase of 75:25 PBS(pH
2.5):acetonitrile at 1 ml/min with the detector set to measure at
both 303 and at the maximum absorbance wavelength of Vitamin E.
Peak areas will be compared to a calibration chart made from
measuring the peaks of standard solutions.
[0324] Previous studies testing the effect of vitamin E on adhesion
formation have been performed by dosing animals with approximately
20 mg vitamin E/kg immediately after induction of surgical lesions
(de la Portilla, F. et al. Diseases of the Colon & Rectum 42,
2157-2161 (2004); Corrales, et al., Acta Cit rgica Brasileira 23,
36-41 (2008)). The weight percent of admixed vitamin E will be
chosen so as to release 20 mg/kg within 24 hours and/or to ensure
no more than 25 mg/kg/day is released.
[0325] If the vitamin E significantly changes the degradation rate
of the PA, a different PA will be synthesized for this admixture to
achieve a polymer that releases SA at the same rate as the
previously developed devices.
[0326] 4.2.2. Anticipated Results
[0327] It is anticipated that the hydrophobic vitamin E may
decrease the degradation rate of the polymers, although this is
unlikely, as previous admixtures have not significantly changed PA
degradation rates. The above HPLC method has been used for
separation of SA but the mobile phase is mostly aqueous.
Accordingly, a gradient method from 100% PBS to 100% acetonitrile
at 1 ml/min is proposed may be an alternative method.
[0328] 4.3. The Determination of the Effects of Common
Sterilization Techniques on the Devices and Comparison of the
Efficacy of the Combination Devices Against their Equivalent
Non-polyAspirin Device In Vivo.
[0329] 4.3.1. Radiation Stability Tests
[0330] Optimized devices will be sent to Johnson & Johnson's
Sterile Process Technology plant where they will be exposed to 25
or 50 kGy of electron beam or gamma radiation. The molecular
weights, .sup.1HNMR and IR spectra, thermal properties, degradation
profiles, and cytotoxicity assays of the irradiated samples will be
compared to traveler samples.
[0331] The Seprafilm sample will also need to be observed for any
change in the ability of the two layers to adhere to each
other.
[0332] 4.3.2. In Vivo Studies in Rats
[0333] The different samples will be implanted into rats. The rats
will be broken into seven groups: 1) No barrier or drugs; 2)
Intercoat; 3) Seprafilm; 4) Intercoat+PA; 5) Seprafilm+PA; 6)
Intercoat+PA+Vitamin E; and 7) Seprafilm+PA+Vitamin E.
[0334] The surgery will consist of a ventral incision being made.
The parietal peritoneum will be electrocauterized and have five
stitches placed at the traumatized area (FIG. 5). The designated
barrier for that rat will be placed into the peritoneum and the
incision will be sutured closed. The rats will be sacrificed 14
days after surgery. An investigator who is blinded to the animal
group will measure the fraction of the lesion area covered in
adhesions. The healing of the incision line will be closely
monitored during the 2 weeks to observe any differences in healing
rate between different groups. The mean and standard deviation of
the fraction of lesion area covered by adhesions will be determined
for each group. Significance of differences between different
groups will be determined by one-way ANOVA with p.ltoreq.0.05.
[0335] 4.4.3. Anticipated Results
[0336] Previous radiation stability tests on adipic-PA indicated
that it is stable after exposure to typical sterilization doses of
electron beam and gamma radiation. The effect of radiation on the
other materials in these devices could affect the PA
characteristics, especially the degradation profile.
[0337] Devices with vitamin E may reduce adhesion more than the PA
devices without vitamin E, which will in turn reduce more adhesions
than the original devices. The untreated control is expected to
have severe, extensive adhesions. The synergistic effect that SA
and vitamin E may have on clotting could prevent closure of the
incision wound. A trial animal will be tested with a vitamin E
device to test for this before the rest of the group is operated on
to test this possibility. If the device does seriously impede wound
healing, one of two options will be chosen: reduce the amount of SA
and vitamin E in the device or replace the vitamin E with
melatonin.
Section 5. Summary
[0338] Described herein are methods to improve two adhesion barrier
devices by incorporating biodegradable polymers into the devices to
allow for targeted, controlled release of drugs, specifically
salicylic acid and vitamin E. The adhesion barriers currently
available on the market are not efficacious enough to warrant their
ubiquitous use. The devices described herein may lead to a better
cost-benefit ratio than current barriers, thereby leading to more
ubiquitous use in patients, resulting in fewer adhesion related
complications.
Example 2
Bioactive-Based Polyanhydrides for Controlled Drug Release in
Surgical Wounds
[0339] As described herein are experiments directed towards the
production of devices for wound healing applications where the
device is composed, in whole or in part, of a salicylic acid
(SA)-based poly(anhydride-ester) (SAPAE). SAPAEs have been
formulated by various methods that would allow them to be applied
to a wound site in various formulations, such as, e.g., a polymer
powder/microspheres in a liquid excipient (e.g., mineral oil),
electrospun mats, and polymers that are above their glass
transition temperature (T.sub.g) at room temperature such that they
can be easily applied to a surface.
[0340] The presence of these formulations at a wound site would
result in polymer degradation and subsequent release of SA. This
local release of SA over an extended period of time can help to
mitigate local pain, inflammation, and other wound healing
complications such as fibrous adhesions. The physical presence of
the polymer device at the wound site is another way in which these
devices can prevent fibrous adhesions.
[0341] There are many devices on the market that are designed to
decrease adhesion formation, most of these devices are solid films,
gels, or liquids that act solely as physical barriers to prevent
adhesion formation between the two surfaces they are separating.
These devices do no prevent adhesion formation at non-adjacent
sites. They also do not mitigate pain or inflammation. None of the
current devices combine physical barriers with controlled release
of drug. In contrast, the wound healing devices described herein
are unique in that they allow for a completely biodegradable device
that can be implanted at a wound site, such as a surgical incision,
to mitigate pain, inflammation, adhesion formation and other
problems during wound healing, while also providing a physical
barrier that can further help to prevent fibrous adhesion
formation.
Bioactive-Based Polyanhydrides
[0342] Bioactive-based polyanhydrides, specifically salicylic
acid-based poly(anhydride-esters), have been previously generated
(Schmeltzer, et al., Biomacromolecules, 6 (1) 359-367 (2005);
Prudencio, et al., Macromolecules, 38, 6895-6901 (2005); Carbone,
et al., Macromol Rapid Comm, 30, 1021-1026 (2009)). Salicylic acid,
a bioactive metabolite of aspirin, is a nonsteroidal
anti-inflammatory drug. It has analgesic and anti-inflammatory
properties as well as mild antimicrobial activity. The localized,
controlled release of salicylic acid at the surgery site can help
to alleviate post-operative pain and swelling, as well as mitigate
the inflammatory response that can lead to post-operative
complications such as fibrous adhesions.
[0343] Drug Loading and Control of Release
[0344] In these polymers, salicylic acid is chemically incorporated
within the backbone of the polymer and connected via biocompatible
linker molecules. When these polymers are placed into an aqueous
environment, the anhydride and ester bonds are hydrolytically
cleaved to release the drug (Scheme 3). This chemical incorporation
of the drug molecules prevents the burst release of drug typically
seen with polymeric drug delivery devices. It also allows very high
drug loading (>70%).
##STR00016##
[0345] The rate of drug release can be controlled by changing the
linker molecules used. Oxygen-containing linkers (such as
diglycolic acid) result in polymers that degrade in a matter of
days, linear aliphatic linkers (such as adipic acid) result in
polymers that degrade over weeks, and branched aliphatic linkers
(such as diethylmalonic acid) result in polymers that degrade over
months (FIG. 6). Copolymers can be prepared containing more than
one linker such that the release rate can be finely-tuned to
achieve a wide range of desired release rates/durations.
[0346] Incorporation of Other Drug Molecules
[0347] Polymers containing other NSAIDs, antioxidants, antibiotics,
and analgesics have also been developed. In addition to chemically
incorporating drugs into the polymer, it is also possible to
physically admix additional drug into the polymer matrix (similar
to most polymeric drug delivery applications) to achieve a
synergistic effect from the concurrent delivery of multiple drugs
at the implantation site (Johnson, et al., J Biomed Mater Res, Part
A, 91(3):671-8 (2009)).
[0348] Formulations
[0349] These polymers may be formulated for a wide range of
applications. For example, the polymers have been formulated into
gels and microspheres/powders (FIG. 7A) (Yeagy, et al., J.
Microencapsulation, 23 (6) 643-653 (2006). The microspheres/powders
can be dispersed within an excipient (e.g., liquid) to result in a
cream, ointment, or spray depending on the need, while still
maintaining controlled release of drug (FIG. 7C). Additionally,
these polymers may be formulated into flexible mats (e.g., electron
spun polymer mats) or films (e.g., electrospun films) (FIG. 7B)
(Griffin, et al., J Biomed Mater Res A, 97A (3) 230-242 (2011)).
For example, SAPAEs have been blended with
poly(lactide-co-glycolide) and PEG and formulated into microspheres
and electrospun films.
[0350] Stability
[0351] Storage stability studies indicate that if maintained at
5.degree. C. in a moisture-free environment, the polymers should be
stable for >12 months (deRonde, et al., Polymer Degradation and
Stability, 95, 1778-1782 (2010)). It has been demonstrated that the
polymers are readily sterilized by both electron beam and gamma
irradiation (up to 50 kGy) without adverse effects on the polymer
properties and drug release rates (Rosario-Melendez, et al.,
Polymer Degradation and Stability, 96, 1625-1630 (2011)).
[0352] In Vivo Results
[0353] These bioactive-based polymers have been implanted into both
animals (Erdmann, Biomaterials 21 (24) 2507-2512 (2000); Harten, et
al., J Biomed Mater Res A 72A (4) 354-362 (2005)) and humans
without evidence of irritation or inflammation.
[0354] In summary, it is possible to produce polyanhydride systems
that can finely control the release of drugs with modified release
rates and in vivo retention times. These polymers may be used for
the localized release of NSAIDs, which can provide the desired
analgesia and anti-inflammatory effects, or other biologically
active agents. Furthermore, these polymers are fully biodegradable
and do not result in irritation at the treatment site. They can be
formulated in multiple ways that would enable easy application at
the surgical site before suturing.
Copolymers: SAPAE Monomers and PEG Oligomers
[0355] Described below is the synthesis of copolymers comprised of
SAPAE monomers and PEG oligomers, which have the desired properties
that would allow them to be applied to a wound (Scheme 4). While an
SA-based PAE is discussed below, other bioactive-based
polyanhydrides (e.g., other nonsteroidal anti-inflammatory drugs,
anti-oxidants, antimicrobials, etc.) that would aid in the wound
healing process may also be used. Additionally, the SAPAEs may also
be blended with other polymers to create a film or gel that could
be used for wound healing purposes.
##STR00017##
[0356] These copolymers, which are comprised of SAPAE monomers and
PEG oligomers, are copolymerized at various ratios. For example,
ratios of adipic linked diacid:PEG have been generated at ratios of
4:1, 3:1, 2:1, 1:1, and 1:2. Mechanical properties of the
copolymers were evaluated. Copolymers with ratios of 2:1, 1:1, and
1:2 had mechanical properties considered beneficial for wound
healing applications, including for the prevention of fibrous
adhesion. Diethylmalonic diacid:PEG copolymers with ratios of 4:1
and 3:1 have also been generated. These copolymers have been
characterized by nuclear magnetic resonance spectroscopy, thermal
analysis, and gel permeation chromatography. They have also been
evaluated for their drug release profiles in phosphate buffered
saline. These polymers are synthesized by met condensation
polymerization, similar to other SAPAEs (Schmeltzer, et al.,
Biomacromolecules, 6 (1) 359-367 (2005); Prudencio, et al.,
Macromolecules, 38, 6895-6901 (2005); Carbone, et al., Macromol
Rapid Comm, 30, 1021-1026 (2009)). Briefly, the diacid and
carboxylated PEG oligomers are combined by desired weight ratio and
acetylated with acetic anhydride, excess solvent is removed by
evaporation, then the remaining monomers are heated at 180.degree.
C. under vacuum with constant stirring to achieve the
copolymer.
[0357] Generally, the higher the amount of salicylic acid based
monomers, the higher the glass transition temperature (T.sub.g).
Typical glass transition temperatures for four ratios of adipic
linked diacid:PEG are shown below in Table 3. These low T.sub.gs
allow them to be easily manipulated at room temperature.
TABLE-US-00003 TABLE 3 Adipic linked Diacid:PEG Ratio Glass
Transition Temperature (T.sub.g) 3:1 10.degree. C. 2:1 -5.degree.
C. 1:1 -25.degree. C. 1:2 -38.degree. C.
The polymers with the diethylmalonic linker had higher T.sub.gs
(23.degree. C. for 3:1) than similar adipic polymers (10.degree. C.
for 3:1).
[0358] The number average molecular weights of the polymers tends
to be 5-10 kDa but it is highly variable.
[0359] In vitro drug release studies show that the greater the PEG
percentage of the polymer, the faster it degrades. 50 mg samples of
the 1:2 adipic degrade in .about.4 days, while the 1:1 and 2:1 take
over a week.
[0360] Formulations
[0361] The copolymer is a viscous polymer that acts like a gel
(FIG. 8). These copolymers can have T.sub.g values below room
temperature such that the polymer can be easily applied to a wound
site, such as via a syringe or be extruded out of a tube (FIG. 8).
This aspect of the copolymers allows them to be easily applied to
various surfaces where they adhere well.
[0362] The copolymers, medical devices and compositions as
described herein can be implanted at a wound site, resulting in
localized analgesia and the mitigation of inflammation and fibrous
adhesion formation near the wound. This placement would result in
improved wound healing, as well as easing patient discomfort after
surgery with fewer side effects than systemic analgesia. Current
fibrous adhesion barrier devices have poor efficacy as they only
act as a physical barrier between adjacent surfaces. The present
copolymers, medical devices and compositions as described herein
would act as a physical barrier against adhesion formation while
also releasing SA that may help prevent adhesion formation in
distant regions, as oral acetylsalicylic acid was found to prevent
adhesion formation (Muzii L et al., Human reproduction, 13 (6):
1486-1489, 1998). SA would also provide the additional benefits of
local analgesia and mitigation of inflammation.
[0363] The invention also includes the subject matter of Example 3,
which is described in the following consecutively numbered
pages.
Example 3
Salicylic Acid-Based Poly(Anhydride-Esters) for the Prevention of
Fibrous Adhesions
[0364] Fibrous adhesions are bands of fibrous tissue that develop
due to increased inflammation after surgery or other trauma.
Depending on where they form, adhesions can lead to serious
complications such as chronic pain, infertility, and intestinal
obstruction. Current methods to prevent adhesion formation focus on
placing physical barriers at likely adhesion sites; however, they
do not prevent adhesion formation at distal sites nor do they
significantly decrease the incidence of adhesion related
complications. A salicylic acid (SA)- and poly(ethylene
glycol)-based poly(anhydride-ester) copolymer (SAPAE) has been
synthesized that degrades to release SA, an anti-inflammatory drug,
in a localized, controlled manner (FIG. 9A). This SAPAE can act as
both a physical barrier to adhesion formation as well as a
pharmaceutical treatment to better prevent adhesion formation.
Materials and Methods
[0365] Acetylated 1,6-bis(o-carboxyphenoxy)hexanoate (Prudencio,
Macromolecules, 2005, 38, 6895-6901) and poly(ethylene
glycol)bis(carboxymethyl ether) were melt polymerized in a 2:1
weight ratio to form the SAPAE as a random copolymer. In vitro
release studies were performed on 50 mg samples of polymer in
phosphate buffered saline (pH 7.4) in an incubator shaker
(37.degree. C., 60 rpm). Degradation media was collected at various
time points and analyzed for SA concentration. To determine the
cytotoxicity of the copolymer, L929 cells were cultured in media
with 0.1 mg/mL copolymer and 1% DMSO (with a DMSO control). Cell
viability was determined by MTS at 24, 48, and 72 hours. Primary
human macrophages were incubated with the copolymer (0.1 mg/mL, 1%
DMSO) and 100 nWmL lipopolysaccharide (LPS), a DMSO/LPS positive
control, and a DMSO (no LPS) negative control to monitor
TNF-.alpha. secretion (determined by ELISA assay) as a measure of
inflammation. Macrophage viability was also determined by MTS.
Results and Discussion
[0366] The synthesized polymer has a low glass transition
temperature (-5.degree. C.) that allows extrusion from a syringe at
room temperature for easy application in a surgical setting. The in
vitro drug release profile was observed to be close to zero-order
indicating a stable SA release rate over the first 10 days (FIG.
9B)--an important finding, as the first 7-10 days after surgery are
critical to adhesion formation. At the end of 10 days, polymer
still remained. Cell studies with both fibroblasts and macrophages
determined that the polymer is not cytotoxic at 0.1 mg/mL (FIG.
9C). The SAPAE at this concentration significantly (p<0.001)
decreases TNF-.alpha. secretion in LPS activated macrophages (FIG.
9D) indicating that the polymers are capable of reducing
inflammation that could lead to fibrous adhesions.
Conclusions
[0367] The aforementioned SAPAE degrades to release SA in a
controlled manner throughout the critical adhesion formation
period. It was also found to be non-cytotoxic at levels at
concentrations that have a significant anti-inflammatory effect in
vitro. In vivo efficacy studies of this SAPAE at reducing the
extent and severity of adhesion formation in a rat peritoneal
adhesion model will be performed.
Example 4
Flowable Salicylic Acid-Based Poly(Anhydride-Esters) for Injectable
Barrier Applications Introduction
[0368] Fibrous adhesions are bands of fibrous tissue that join two
surfaces in the body that are not normally connected. They
generally form after injury to an area that results in increased
inflammation. Surgery, trauma, infections, radiation, and ischemia
can all lead to adhesion formation, with surgery being the most
common cause. Adhesions are a serious problem that can lead to many
complications, including chronic pain, infertility, and intestinal
obstruction. Fibrous adhesions have an enormous impact on the
healthcare system. It has been estimated that 95% of abdominal and
pelvic surgeries, including gynecologic, result in adhesions.
Adhesion related problems account for 6% of hospital readmissions
and 1% of all hospitalizations in the United States. Over 400,000
abdominal adhesiolysis procedures, a surgery to dissect fibrous
adhesions, are performed annually. While adhesiolysis can help
alleviate some pain and complications associated with adhesions,
the effect is often temporary as the adhesions tend to grow back
after the procedure.
[0369] Adhesion-related complications often lead to additional
surgeries, which is particularly alarming when one takes into
account that the presence of adhesions makes such secondary
surgeries even more difficult and dangerous, increasing surgery
time, hospital stay, complications, blood loss, morbidity, and
mortality. While there has been some effort to develop devices to
reduce adhesion formation, no device has been proven to
significantly reduce the incidence of adhesion-related
complications (Al-Jaroudi et al., Adhesion Prevention in
Gynecologic Surgery. Obstetrical and Gynecological Survey. 2004;
59(5):360-7).
[0370] The physiological pathway that leads to abdominal adhesion
formation has been well studied. After surgery, fibrinogen from
blood in the peritoneal cavity form a fibrin matrix. This matrix
forms into transient fibrinous bands that degrade by fibrinolysis
or become a scaffold for fibroblasts to create permanent fibrous
adhesions. The occurrence of fibrinolysis is dependent upon the
levels of different cytokines and enzymes, with the first 7-10 days
after surgery being the most critical for adhesion formation.
[0371] Both physical and pharmaceutical methods have been
investigated to prevent adhesion formation (Tingstedt et al., Eur
Surg Res. 2007; 39:259-68; Ward et al., Journal of Surgical
Research. 2011; 165(1):91-111; Alpay et al., Seminars in
reproductive medicine. 2008; 26(4):313-21). Various solids, gels,
and fluids have been explored as physical barriers, the main
purpose of which is to separate surfaces where adhesions could
potentially form. The FDA has approved only 5 barrier devices for
human use. However, FDA approved devices are not efficacious enough
at reducing adhesion related complications to warrant their
ubiquitous use (Wiseman et al., Seminal Reproductive Medicine.
2008; 26:356-68; diZerga et al., Reproductive biomedicine online.
2008; 17(3):303-6; Wilson M S. Colorectal disease: the official
journal of the Association of Coloproctology of Great Britain and
Ireland. 2007; 9 Suppl 2:60-5. Epub 2007/10/27; Wilson et al.,
Colorectal disease: the official journal of the Association of
Coloproctology of Great Britain and Ireland. 2002;
4(5):355-60).
[0372] Drugs tested for adhesion prevention include primarily those
that affect the clotting cascade, the inflammatory process, cell
proliferation, extracellular matrix production, or oxidative
stress. Systemic administration of such drugs at therapeutic levels
can cause undesired side effects and delay healing after surgery.
Some attempts have been investigated to inject drugs into the
peritoneal cavity; however, most of these studies have shown low
efficacy in laboratory testing primarily because the mesothelial
membrane lining the peritoneal cavity quickly absorbs drugs and
subsequently distributes them throughout the body.
[0373] Salicylic acid-based poly(anhydride-esters) (SAPAEs) that
hydrolytically degrade to release salicylic acid (SA) and
biocompatible linker molecules have been developed (Erdmann et al.,
Biomaterials. 2000; 21:1941-6). SA, a non-steroidal
anti-inflammatory drug (NSAID), has been found to inhibit
cyclooxygenase-2 (COX-2) activity which is expressed in adhesion
fibroblasts but not in normal peritoneal fibroblasts (Saed et al.,
Fertility and sterility. 2003; 79(6):1404-8). SA is a desirable
agent for adhesion prevention as it not only has anti-inflammatory
properties, but also acts as an analgesic, potentially reducing
post-surgical pain. SAPAEs exhibit high drug loading capacities (up
to 75%) and are able to be manipulated into various geometries
depending on the application needs (Erdmann L, et al.,
Biomaterials. 2000; 21:1941-6; Schmeltzer et al., Polymer Bulletin.
2003; 49:441-8; Prudencio et al., Macromolecules. 2005;
38:6895-901; Schmeltzer et al., Journal of Biomaterials Science:
Polymer Edition. 2008; 19(10):1295-306; Carbone A L, et al.,
Macromolecular Rapid Communications. 2009; 30(12):1021). As
described herein, an SAPAE adhesion prevention material will allow
for sustained release of salicylic acid at the site of implantation
while also maintaining a temporary physical presence to block
adhesion formation.
[0374] The research described herein describes the development and
characterization of SAPAE:poly(ethylene glycol) (PEG) copolymers
with desirable mechanical and drug release properties for an
adhesion prevention device. The copolymers exhibited mechanical
properties similar to or better than current injectable barrier
devices on the market. In vitro drug release showed PEG content
controls SA release rates and cell studies confirmed
cytocompatibility and anti-inflammatory activity.
Materials and Methods
[0375] Materials
[0376] All chemicals and reagents, including poly(ethylene glycol)
(PEG) 20,000 Da, were purchased from Sigma-Aldrich (Milwaukee,
Wis.) and used as received.
[0377] .sup.1H NMR and FTIR Spectroscopies
[0378] .sup.1H spectra were recorded on a Varian 500 MHz
spectrometer using deuterated dimethyl sulfoxide (DMSO-d6) as the
solvent and internal reference. FTIR spectra were obtained using a
Thermo Nicolet/Avatar 360 spectrometer. Samples were dissolved in
dichloromethane and solvent-cast on NaCl plates. Each spectrum was
an average of 32 scans.
[0379] Molecular Weight
[0380] Gel permeation chromatography (GPC) was used to determine
polymer number-averaged molecular weight (M.sub.n) and
polydispersity index (PDI) using a Perkin-Elmer liquid
chromatography system consisting of a Series 200 refractive index
detector, a Series 200 LC pump, and an ISS 200 autosampler. Sample
automation and data processing were performed using a Dell OptiPlex
GX110 computer running Perkin-Elmer TurboChrom 4 software with a
Perkin-Elmer Nelson 900 Series Interface and 600 Series Link.
Polymer samples dissolved in dichloromethane (DCM, 10 mg/mL) were
filtered through 0.45 .mu.m poly(tetrafluoroethylene) syringe
filters. Samples were resolved on a Jordi divinylbenzene mixed-bed
GPC column (7.8.times.300 mm, Alltech Associates, Deerfield, Ill.),
with a DCM mobile phase and a flow rate of 1.0 mL/min. Molecular
weights were calibrated relative to broad polystyrene standards
(Polymer Source Inc., Dorval, Canada).
[0381] Thermal Properties
[0382] Differential scanning calorimetry (DSC) measurements were
carried out on a TA Instrument Q200 to determine glass transition
(T.sub.g) and melting (T.sub.m) temperatures. Measurements on
samples (4-8 mg) heated under nitrogen atmosphere to 200.degree. C.
at a heating rate of 10.degree. C./min and cooled to -50.degree. C.
at a rate of 10.degree. C./min with a two-cycle minimum were
performed. TA Instruments Universal Analysis 2000 software, version
4.5A, was used to analyze the data. T.sub.gs were calculated as
half Cp extrapolated.
[0383] Polymer Synthesis
[0384] SA-based diacid was synthesized according to previously
described methods (FIG. 10) (Prudencio et al., Macromolecules.
2005; 38(16):6895-901). Briefly, SA (2 equivalents (eq)) was
dissolved in tetrahydrofuran (THF) and pyridine (4 eq). Adipoyl
chloride (1 eq) was dissolved in THF and added drop-wise forming a
white suspension. The reaction mixture was stirred overnight,
quenched over water and acidified to pH 2 using concentrated
hydrochloric acid. The precipitate was filtered, washed with water
(3.times.250 mL), and dried in vacuo to yield diacid.
[0385] For the SAPAE homopolymer used in this study (referred to
hereafter as SAA, FIG. 10), diacid was activated in an excess of
acetic anhydride at room temperature, concentrated, and polymerized
via melt-condensation polymerization at 180.degree. C. for 5 h at
100 rpm in vacuo to yield a tan foam. M.sub.n=9,000 Da, PDI=1.2.
T.sub.g=45.degree. C.
[0386] For the SAPAE copolymers (referred to hereafter as SAA:PEG,
FIG. 10), diacid and poly(ethylene glycol)bis(carboxymethyl) ether
(M.sub.n 600, Sigma-Aldrich) were combined in weight ratios of 1:2,
1:1, and 2:1 and activated in an excess of acetic anhydride at room
temperature, concentrated, and polymerized via melt-condensation
polymerization at 180.degree. C. for 3 h at 100 rpm in vacuo to
yield a brown viscous liquid. Yield: 2.00 g (67%), brown viscous
liquid. .sup.1H NMR (500 MHz, DMSO-d.sub.6, .delta.) for SAA:PAE
copolymer: 8.21 (br, ArH), 7.93 (br, ArH), 7.77 (br, ArH), 7.39
(br, ArH), 4.41 (s, CH.sub.2), 4.02 (s, CH.sub.2), 3.46 (s,
CH.sub.2), 2.51 (br, CH.sub.2), 1.65 (br, CH.sub.2), peak
integration varied with SAA:PEG ratio. IR (solvent-cast DCM): 1775
cm.sup.-1 (C.dbd.O, anhydride), 1745 cm.sup.-1 (C.dbd.O,
ester).
[0387] Solvent-Casting SAA/PEG Blended Films
[0388] SAA (250 mg) and PEG 20,000 (250 mg) were dissolved in 1 mL
dichloromethane and cast into a Teflon drying dish (3 cm diameter).
The dish was left to evaporate overnight in a hood before being
placed into a vacuum desiccator for 24 hr at room temperature to
remove any remaining solvent.
[0389] Rheology
[0390] A Rheometrics SR-2000 parallel plate rheometer with the
temperature set to 25 or 37.degree. C. (TA Instruments, New Castle,
Del.) was used for rheological measurements. The top plate was
lowered to 0.5 mm. Oscillatory shear studies were performed ramping
the frequency from 0.1 to 10 rad/s at 2% shear strain. The SAA:PEG
copolymers shear viscosity was evaluated by ramping shear rates
from 0.1 to 1 rad/s for the 2:1 ratio, 0.1 to 100 rad/s for the 1:1
ratio, and 1 to 500 rad/s for the 1:2 ratio. Samples were analyzed
in triplicate.
[0391] Storage Stability
[0392] SAA:PEG copolymers (.about.0.5 g) were placed in 50 mL
centrifuge tubes at -20.degree. C., 4.degree. C., or 25.degree. C.
Tubes were flushed with dry nitrogen before storage. Copolymer
samples (1:2 ratio) were studied both with and without desiccant
(Drierite, W A Hammond Drierite Co. Ltd., Xenia, Ohio). A Kimwipe
(Kimberly-Clark, Irving, Tex.) taped to the tube cap suspended the
desiccant away from the polymer. All other ratios were tested
without desiccant only. M.sub.n and T.sub.g were analyzed for all
samples each week for 3 weeks. Samples were studied in singlet due
to the amount of time required for sample analysis.
[0393] In Vitro Drug Release
[0394] Polymers (50.+-.1 mg) were placed in aluminum pans (6.3 mm
diameter) to contain polymer spreading. Polymer-filled pans were
placed in 20 mL Wheaton glass scintillation vials containing 10 mL
phosphate buffered saline (PBS) at pH 7.4. Samples were incubated
at 37.degree. C. with agitation at 60 rpm in a controlled
environment incubator shaker (New Brunswick Scientific Co., Excella
E25, Edison, N.J.). All media was collected and replaced with fresh
PBS (10 mL) at pre-designated time points for 14 days. Spent media
was analyzed by UV spectrophotometry using a Perkin Elmer Lambda
XLS spectrophotometer (Waltham, Mass.) to specifically monitor SA
release. Measurements were obtained at .lamda.=303 nm, the maximum
absorbance of SA that does not overlap with other polymer
degradation products. Data were calculated against a calibration
curve of absorbance values from standard solutions of known SA
concentrations in PBS. Polymer remaining after 14 days was degraded
using basic water (pH>12) and SA was quantified to allow
normalization of percent release. Samples were studied in
triplicate.
[0395] In Vitro Cytotoxicity and Proliferation Assay
[0396] Polymer cytocompatibility was performed by culturing NCTC
clone 929 (strain L) mouse areolar fibroblast cells (ATCC,
Manassas, Va.) in media containing the dissolved polymers. These
L929 fibroblast cells are a standard cell type for
cytocompatibility testing as recommended by ASTM. Cell culture
media consisted of Dulbecco's Modified Eagle's Medium (DMEM,
Sigma-Aldrich, St. Louis, Mo.), 10% v/v fetal bovine serum (Atlanta
Biologicals, Lawrenceville, Ga.), 1% L-glutamate (Sigma) and 1%
penicillin/streptomycin. The polymers were dissolved in dimethyl
sulfoxide (DMSO) at 100, 50, 10, 5, and 1 mg/mL. These solutions
were then diluted with cell culture media to achieve concentrations
of 1, 0.5, 0.1, 0.05, and 0.01 mg/mL and 1% DMSO. A control with 1%
DMSO in media without polymer was prepared. Three 96-well plates
were seeded at an initial concentration of 2,000 cells per well
with each experimental group plated in triplicate. For the L929
fibroblasts, cell viability was determined by using a CellTiter
96.RTM.AQueous One Solution Cell Proliferation Assay (MTS, Promega,
Madison, Wis.) at 24, 48, and 72 hours. After 2 hr incubation with
MTS, the absorbance was recorded with a microplate reader at
.lamda.=490 nm. One-way ANOVAs followed by Bonferroni's all-pairs
comparison were used to determine significance (significantly
different if p<0.05).
[0397] TNF-.alpha. Secretion Assay
[0398] Human blood-derived monocytes (Blood Center of New Jersey,
East Orange, N.J.) were used to determine the polymer efficacy on
decreasing inflammatory cytokine secretion. The cell isolation and
purification protocol used was previously described by Kim et al.
(Kim et al., Experimental Hematology. 2009; 37(12):1445-53).
Briefly, peripheral blood mononuclear cells were collected from
blood of healthy donors by density gradient separation using
Ficoll-PLUS (GE Healthcare, Piscataway, N.J.). Red blood cells were
lysed by incubation in ammonium-potassium-chloride lysing buffer
for 5 min, washed with PBS and counted. Monocytes were cultured on
175 cm.sup.2 flasks (BD, Franklin Lakes, N.J.) at a concentration
of 8.times.10.sup.6 cells/mL in Roswell Park Memorial Institute
(RPMI) 1640 media (GIBCO BRL, Rockville, Md.). RPMI media was
supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL), 100
units/mL penicillin (GIBCO BRL), 100 .mu.g/mL streptomycin (GIBCO
BRL) and 400 mM L-glutamine (GIBCO BRL). Monocytes were allowed to
adhere for 2 h and then washed 3 times with PBS to remove
non-adherent cells. Monocytes were cultured for 7 days at
37.degree. C. and 5% CO.sub.2 in RPMI supplemented with 5 ng/mL
granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D
Systems, Minneapolis, Minn.) to generate macrophages.
[0399] After 7 days of culture, macrophages were washed once with
PBS and then detached with trypsin-EDTA (GIBCO) for 30 minutes at
room temperature. Cells were re-suspended in culture medium (RPMI),
counted, re-plated at 8.times.10.sup.3 cells/well in a 96 well
plate, and allowed to attach overnight. The following day, the
media was replaced with the various sample groups: polymer
containing media (0.2 mg/mL polymer, 10 ng/mL lipopolysaccharide
(LPS), 1% DMSO), a positive control (10 ng/mL LPS, 1% DMSO), and a
negative control (no LPS, 1% DMSO). All cell studies were performed
in triplicate. LPS (10 ng/mL) induced macrophage TNF-.alpha.
secretion. After 48 h, media was collected and TNF-.alpha.
secretion was determined with an enzyme-linked immunosorbent assay
kit against human TNF-.alpha. (BioLegend, San Diego, Calif.). A
CellTiter 96.RTM.AQueous One Solution Cell Proliferation Assay
(Promega, Madison, Wis.) was used to ensure that differences in
TNF-.alpha. secretion were not due to differences in cell
viability. A one-way ANOVA followed by Bonferroni's all-pairs
comparison was used to determine significance (significantly
different if p<0.05).
Results and Discussion
[0400] SAPAE:PEG Blended Films
[0401] SAPAE homopolymers are hard and glassy at physiological
temperatures, making them unfeasible as adhesion barrier devices on
their own. The first method attempted to create an adhesion barrier
material used SAA, the most well characterized SAPAE, blended with
PEG. PEG was chosen as it has favorable mechanical characteristics,
has been used in other barrier devices, and can inhibit protein
adsorption to surfaces, thereby decreasing the likelihood of cell
adhesion to the barrier as it degrades (Tziampazis et al.,
Biomaterials. 2000; 21(5):511-20). The polymers were concurrently
dissolved and solvent-cast to create a film. The resulting film
crumbled when removed from the Teflon dish it had been cast in.
Unlike typical solvent cast films of SAA, the film surface was not
smooth; the variegated surface of the blended films indicated
macroscopic phase separation.
[0402] DSC spectra of SAA, PEG, and the SAA/PEG film were
determined. If the polymer blend were completely miscible, the
thermal transitions for the blend would have intermediary values
between the homopolymer values transitions (Brostow et al.,
Materials Letters. 2008; 62(17-18):3152-5). The thermal transition
for the blend appears to be more additive than intermediary as the
SAA T.sub.g drop and PEG T.sub.m are still visible despite their
overlap. The DSC curves and film topography indicate immiscibility
and phase separation.
[0403] SAPAE:PEG Copolymers
[0404] Due to the poor mechanical properties and phase separation,
which could result in uneven degradation and drug release, other
methods of incorporating PEG with SAA were pursued. Specifically,
carboxylic acid-terminated PEG chains (Sigma-Aldrich) were
purchased for copolymerization with SAA monomers to allow the
copolymers to be formed using standard melt polymerization
techniques (FIG. 10).
[0405] Copolymer Characterization
[0406] The resulting copolymers formed brown viscous liquids at
room temperature (FIG. 8). These liquids were very sticky, a good
quality for an adhesion barrier as this property will help the
material adhere to the administered site and remain in place
throughout the healing process. .sup.1H NMR (FIG. 11) and FTIR
spectroscopies were used to confirm the products. .sup.1H NMR peak
integrations confirmed that theoretical and experimental SAA:PEG
ratios were similar. FTIR confirmed the presence of anhydride and
ester bonds in the various polymers. Typical M.sub.n, PDI, and
T.sub.g values for the copolymer ratios studied are summarized in
Table 4. A significant decrease in the copolymer T.sub.g as
compared to the homopolymer (45.degree. C.) was observed.
TABLE-US-00004 TABLE 4 Typical M.sub.n, PDI, and T.sub.g for
SAA:PEG Polymers. SAA:PEG Ratio M.sub.n (Da) PDI T.sub.g (.degree.
C.) 2:1 23,700 44.6 -5 1:1 16,500 22.2 -25 1:2 39,200 31.7 -35 1:0
9,000 1.2 45 (SAA homopolymer)
[0407] Rheology
[0408] With T.sub.gs below 0.degree. C., the polymers behaved as
viscous liquids at room temperature, as opposed to the glassy SADEM
homopolymers. To assess the copolymer mechanical properties,
rheological studies were performed. Initial oscillatory
measurements demonstrated a phase angle of approximately
90.degree.. This data indicates that the polymers primarily undergo
viscous deformation with negligible elastic deformation. The
results of subsequent linear shear viscosity ramping measurements
are given in Table 5. Shear viscosities decreased by an
order-of-magnitude as PEG content increased. Increasing the sample
temperatures from 25.degree. C. to 37.degree. C. resulted in a
decrease in shear viscosity by about half an order-of-magnitude.
These patterns suggest an ability to tailor copolymer rheological
properties by changing PEG content.
TABLE-US-00005 TABLE 5 Shear Viscosities of SAA:PEG Copolymers
Shear Shear Shear Thinning Shear Thinning SAA:PEG Viscosity at
25.degree. C. Observed Viscosity at 37.degree. C. Observed Ratio
(mPa s) (25.degree. C., rad/s) (mPa s) (37.degree. C., rad/s) 2:1
6.5 .times. 10.sup.7 .+-. N/A 8.5 .times. 10.sup.6 .+-. N/A 1.8
.times. 10.sup.7 3.7 .times. 10.sup.6 1:1 7.3 .times. 10.sup.6 .+-.
2 1.6 .times. 10.sup.6 .+-. 10 0.8 .times. 10.sup.6 0.4 .times.
10.sup.5 1:2 2.2 .times. 10.sup.5 .+-. 100 6.9 .times. 10.sup.4
.+-. 200 0.1 .times. 10.sup.5 0.1 .times. 10.sup.4
[0409] These shear viscosity values compare well with
Intercoat.RTM. (Ethicon, Somerville, N.J.), an injectable adhesion
barrier currently on the market. Intercoat.RTM. is a
carboxymethylcellulose and PEG blend with a viscosity of about
2.1.times.10.sup.5 mPas (diZerega et al., Journal of Biomedical
Materials Research Part B: Applied Biomaterials. 2007;
81B(1):239-50), similar to the 1:2 SAA:PEG copolymer described
here. Additionally, evidence suggests that as the viscosity of a
barrier increases, so does the efficacy. This result indicates that
the SAA:PEG copolymers may have mechanical properties suitable for
the prevention of adhesions. However, a balance must be made
between the ability of a material to remain in place in vivo and
the ease of surgical application. While both the 1:1 and 1:2
copolymers can be extruded from a syringe, the 2:1 copolymer can
only be extruded with extreme effort. The ease of application is an
important consideration for surgical use and suggests that the
optimal SAA:PEG ratio is below 2:1 for injectable applications.
[0410] Storage Stability
[0411] Copolymer storage stability is an issue as their degradation
over time affects both physicochemical properties and drug release
rates. Copolymers were stored in the freezer, refrigerator, and at
ambient temperatures. The molecular weight and glass transition
temperatures were monitored weekly for 3 weeks to assess the rate
of degradation of samples under the various conditions (FIG. 12).
As only one data set was taken, variability is observed, resulting
in increases in M.sub.n and T.sub.g at some time points. GPC column
issues resulting in fluctuating baselines could also have affected
M.sub.n measurements. However, there is a general trend observed
between groups. Colder environments slowed the rate of polymer
degradation. This fact can be seen in the dramatic differences in 0
and 3 week M.sub.n and T.sub.g values for polymers at 25.degree. C.
as compared to the slight differences between 0 and 3 weeks for
samples maintained at -20.degree. C. As PEG is hygroscopic, it is
expected to increase polymer degradation rates, thus, the copolymer
with the greatest amount of PEG (1:2 copolymer) was stored both
with and without desiccant to reduce degradation rates. The
desiccant effect is most obvious on the molecular weight, with only
slight differences observed in the T.sub.g. Due to these results,
all copolymers were subsequently stored in the freezer in a
secondary container with desiccant.
[0412] In Vitro Drug Release
[0413] SAPAEs are hydrolytically degradable and the incorporation
of hydrophilic PEG moieties was expected to significantly effect
drug release rates. FIG. 13 shows the SA release profiles from the
SAA:PEG copolymers. The 1:2 copolymer completely degraded in less
than a week, with 77% of incorporated SA released within the first
24 hours. The 1:1 and 2:1 copolymers exhibit lesser release within
the first day (37% and 15%, respectively). The initial release
correlated with the PEG content where increasing PEG led to greater
SA release. In comparison, the SAA homopolymer exhibits a 2-day lag
period (Ouimet et al., Journal of Bioactive and Compatible
Polymers. 2012; 27(6):540-9). For the 1:1 and 2:1 copolymers, after
the initial release, the release rate stabilized to give a more
linear profile. Average release after day 1 was 3.5% from the 1:1
samples and 2.8% from the 2:1 samples. On day 14, basic water was
used to completely degrade remaining polymer to determine total SA
content. SA content from remaining polymer was used to normalize
cumulative release data. After 14 days, the 1:1 and 2:1 copolymers
released 85% and 52% SA, respectively.
[0414] It has been reported that the critical period for adhesion
prevention is the first 7-10 days after injury (Moran, Colorectal
disease: the official journal of the Association of Coloproctology
of Great Britain and Ireland. 2007; 9 Suppl 2:39-44; van der Wal J
B, et al., Colorectal disease: the official journal of the
Association of Coloproctology of Great Britain and Ireland. 2007; 9
Suppl 2:9-13). While the 1:2 copolymer does not provide drug
release or physical barrier properties over this time period, many
factors should be considered. For example, the rate of degradation
in vitro and in vivo may vary dramatically. Additionally, the
amount of polymer used and how it is placed in the body will have
an effect on polymer duration in vivo. The increased shear stress
in vivo would also most likely result in increased degradation
rates. Alternatively, if the initial inflammatory response is
correctly modulated (inflammatory cytokine concentrations in the
peritoneal cavity peak within the first 24 hours after abdominal
surgery) (Sammour T, World Journal of Surgery. 2010; 34(4):704-20),
drug release and physical presence may not be as important at later
times and the polymer may not need to remain in the body for 10
days.
[0415] Cytocompatability
[0416] Copolymer (0.01 to 1 mg/mL) cytocompatibility was evaluated
over 3 days (FIG. 14). All polymers were cytotoxic when dissolved
at 1 mg/mL, compared to the 1% DMSO control. The polymers with
higher drug loading (1:1 and 2:1) were also toxic at 0.5 mg/mL
Below these levels, no significant toxicity over the three days was
observed. It should be noted, however, that in these studies, the
polymers were dissolved in solution, which increases polymer
degradation rate compared to expected in vivo degradation.
[0417] These levels of cytotoxicity are an important consideration
for in vivo use. Rapid polymer degradation could lead to locally
toxic effects if too much polymer is used or if the polymer is
placed in an area of the body that could not absorb the polymer
degradation products quickly. This rapid degradation should not
pose a problem in the peritoneal cavity, where most problematic
fibrous adhesions occur, as the peritoneal cavity absorbs fluids
rapidly.
[0418] Anti-Inflammatory Activity
[0419] Many inflammatory cytokines, such as TNF-.alpha., can lead
to adhesion cell phenotype differentiation. Macrophages were
exposed to 10 ng/mL LPS to elicit an immune response resulting in
TNF-.alpha. secretion. ELISA was used to monitor the copolymer
effect on macrophage TNF-.alpha. production. The copolymers
exhibited TNF-.alpha. knockdown in a dose-dependent manner
correlating with the amount of SA loading in the polymer (FIG. 15).
At 0.2 mg/mL, 2:1 and 1:1 copolymers significantly decreased
TNF-.alpha. expression while the 1:2 copolymer reduced expression
but not statistically significantly. Cytotoxicity assays were
performed to confirm that TNF-.alpha. knockdown was not due to cell
death (data not shown).
Conclusion
[0420] Fibrous adhesions are a prevalent medical issue. Currently
employed physical barrier devices and pharmaceutical regimens are
not efficacious at preventing adhesion related complications. The
SAA:PEG copolymers described herein combine these two approaches.
The polymers could be used as an injectable barrier substance to
physically prevent adhesion formation between tissue surfaces. They
could also provide controlled, sustained SA release which may be
able to prevent fibroblast differentiation into the adhesion
phenotype. Studies to assess adhesion prevention efficacy in vivo
may be performed subsequently (e.g., in vivo murine studies) by the
skilled artisan using known techniques.
[0421] The invention described herein also comprises compositions,
devices, methods of use and methods of treatment which are
disclosed herein, e.g., in Examples 1-4.
[0422] All documents cited herein are incorporated by reference.
While certain embodiments of invention are described, and many
details have been set forth for purposes of illustration, certain
of the details can be varied without departing from the basic
principles of the invention.
[0423] The use of the terms "a" and "an" and "the" and similar
terms in the context of describing embodiments of invention are to
be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. In addition to the order detailed herein, the
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate embodiments of invention and does not necessarily impose
a limitation on the scope of the invention unless otherwise
specifically recited in the claims. No language in the
specification should be construed as indicating that any
non-claimed element is essential to the practice of the
invention.
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