U.S. patent application number 14/478105 was filed with the patent office on 2015-07-23 for electrochemically degradable polymers.
The applicant listed for this patent is Trustees of Tufts College D/B/A Tufts University, Trustees of Tufts College D/B/A Tufts University. Invention is credited to Pericles Calias, Marc d'Alarcao.
Application Number | 20150202292 14/478105 |
Document ID | / |
Family ID | 36203625 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150202292 |
Kind Code |
A1 |
d'Alarcao; Marc ; et
al. |
July 23, 2015 |
ELECTROCHEMICALLY DEGRADABLE POLYMERS
Abstract
The present invention discloses polymeric materials that
incorporate a modified quinone moiety, either to cross-link the
polymer or as a monomeric unit of the polymer These polymeric
materials can be efficiently degraded through electrochemical
reduction of the quinone leading to rapid hydrolysis of the pendant
chemical groups and degradation of the polymer. Quinone-containing
compositions and methods of producing electrochemically degradable
polymers are disclosed. The methods and compositions of the present
invention can be used in a wide variety of applications, including,
but not limited to, drug delivery, tissue regeneration, biomedical
implants, and electronic systems.
Inventors: |
d'Alarcao; Marc; (Malden,
MA) ; Calias; Pericles; (Melrose, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trustees of Tufts College D/B/A Tufts University |
MEDFORD |
MA |
US |
|
|
Family ID: |
36203625 |
Appl. No.: |
14/478105 |
Filed: |
September 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11665234 |
Apr 1, 2008 |
8834901 |
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PCT/US2005/037248 |
Oct 14, 2005 |
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14478105 |
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60618654 |
Oct 14, 2004 |
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Current U.S.
Class: |
514/772.4 ;
514/772.3; 514/781 |
Current CPC
Class: |
C08F 8/50 20130101; A61K
47/34 20130101; A61K 41/00 20130101; A61K 47/32 20130101; A61K
9/0009 20130101; A61K 47/38 20130101 |
International
Class: |
A61K 41/00 20060101
A61K041/00; A61K 47/32 20060101 A61K047/32; A61K 47/34 20060101
A61K047/34; A61K 47/38 20060101 A61K047/38 |
Claims
1-3. (canceled)
4. A method of controlled release of pharmaceutical agents within a
subject comprising: implanting an electrically-controlled polymer
comprising at least one moiety of the formula: ##STR00009## wherein
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are
selected from the group comprising hydrogen, alkyl, aryl, alcohol,
ether, thiol, thioether, amine, cyano, halo, nitro, ketone,
aldehyde, ester, amide, thioester, carbonate, carbamate, and urea,
X and Y can be the same or different and are selected from the
group comprising vinyl sulfone, epoxide, alkyl halide, alkene,
amine, alcohol, acid halide, acid anhydride, sulfate, phosphate,
isocyanate, isothiocyanate, and thiol alkyl groups, wherein at
least one of X and Y is capable of degradation upon reduction of
the quinone; and electrically inducing chemical degradation of the
polymer thereby releasing pharmaceutical agents.
5. The method of claim 4, wherein the polymer further comprises at
least one monomer selected from the group comprising styrene,
acrylates, methacrylates, 1,3-butadiene, isoprene, 2-vinylpyridine,
ethylene oxide, acrylonitrile, methyl vinyl ketone,
alpha-cyanoacrylate vinylidene cyanide, propylene, butene,
isobutylene, phosphorus acid, phosphonous acid, phosphinous acid,
phosphoric acid, phosphonic acid, phosphinic acid, methylene bis
(phosphonic acid), poly(vinylphosponic acid), aziridine, spermine,
cadaverine, and putrecine.
6. The method of claim 4, wherein the step of electrically inducing
chemical degradation further includes applying voltage of at least
0.05 V.
7. The method of claim 4, wherein the step of electrically inducing
chemical degradation further includes utilizing a current producing
device that is controlled by the subject.
8. The method of claim 4, wherein the step of electrically inducing
chemical degradation further includes a step of sensing at least
one internal parameter within the subject.
9. The method of claim 4, wherein the step of electrically inducing
chemical degradation further includes a step of sensing at least
one external parameter.
10-13. (canceled)
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The technical field of this invention is degradable polymers
and, in particular, polymers that can be degraded in a controlled
manner.
BACKGROUND OF THE INVENTION
[0002] There is increased interest in the synthesis of new
degradable polymers that can be attributed, at least in part, to
the growing use of synthetic polymers in medical applications.
Degradable polymers are presently used in matrices for delivery of
bioactive substances, as scaffolding in tissue engineering, in
suture materials, for fracture fixation, in dental applications, as
sealants, as well as in other applications. Ideally, these
synthetic polymers should be capable of degradation and the
degradation products should be compatible with the human body.
[0003] Drug delivery systems can also benefit from the use of
degradable polymers, especially when they are designed so that they
are incapable of releasing their agent or agents until they are
placed in an appropriate biological environment. Depending upon the
polymer, the environmental change can involve pH, temperature, or
ionic strength, and the system can shrink, swell, or decompose upon
a change in any of these environmental factors. Biodegradable
polymers, for example, degrade within the body as a result of
natural biological processes, eliminating the need to remove a drug
delivery system after release of the active agent has been
completed.
[0004] Most biodegradable polymers are designed to degrade as a
result of hydrolysis of the polymer chains into biologically
acceptable, and progressively smaller, compounds. In some cases,
such as systems that employ polylactides, polyglycolides, or their
copolymers, the polymers will eventually break down to lactic acid
and glycolic acid, enter the Kreb's cycle, and be further broken
down into carbon dioxide and water and excreted through normal
processes. In some degradable polymer systems, the release rate can
be tailored for the application. For example, in systems that use
polyanhydrides or polyorthoesters, the degradation occurs primarily
at the surface of the polymer, resulting in a release rate that is
proportional to the surface area of the drug delivery system.
[0005] However, these biodegradable polymers do not allow for
controlled degradation of the polymer. For example, the
biodegradability of polyester polymers depends on the ability of
the ester linkage in the polymer backbone to hydrolyze or decompose
in the presence of water. Such polymers often do not allow for
predictable control over the rate of degradation once the polymer
is placed inside an aqueous environment. Moreover, such polymer
systems do not typically permit one to vary the release rate
following administration or implantation.
[0006] Thus, there is a need in the art for new compositions and
methods of synthesizing polymers that are capable of degrading in a
controlled manner, e.g., in response to changes in the local
environment or external stimuli.
SUMMARY OF THE INVENTION
[0007] The present invention discloses polymeric materials that
incorporate a modified quinone moiety, either to cross-link the
polymer or as a monomeric unit of the polymer. These polymeric
materials can be efficiently degraded through electrochemical
reduction of the quinone leading to rapid release of the pendant
chemical groups and degradation of the polymer. Quinone-containing
compositions and methods of producing electrochemically degradable
polymers are disclosed. The methods and compositions of the present
invention can be used in a wide variety of applications, including,
but not limited to, drug delivery, tissue regeneration, biomedical
implants, and electronic systems.
[0008] The invention is based, in part, upon the incorporation of a
modified quinone polymer moiety, either to cross-link the polymer
or as a monomer in the preparation of the polymer. The terms
"moiety," "quinone moiety," and "polymer moiety," as used herein,
are intended to encompass both polymer cross-linkers and monomeric
components of polymers. Electrochemical reduction of the quinone
within the polymer leads to rapid hydrolysis of the pendant
chemical groups and thereby results in degradation of the polymer
and alteration of its properties.
[0009] The invention makes use of modified quinone moieties that
can be incorporated into a polymer such that the resultant
polymeric materials can be controllably degraded via
electrochemical reduction. The electrochemically degradable
polymers can have the core structure shown below:
##STR00001##
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 can
be any organic functional groups including, but not limited to,
hydrogen, alkyl, aryl, alcohol, ether, thiol, thioether, amine,
cyano, halo, nitro, ketone, aldehyde, ester, amide, thioester,
carbonate, carbamate, and urea. Any chemical moiety used as a
reactive group in polymer cross-linking or as a reactive group in
polymerization could be appended to the quinone structure at X
and/or Y of structure (1). For example, the pendant groups X and Y
can be any functional groups subject to degradation upon reduction
of the quinone including, but not limited to, groups containing
vinyl sulfone, epoxide, alkyl halide, alkene, amine, alcohol, acid
halide, acid anhydride, sulfate, phosphate, isocyanate,
isothiocyanate, and thiol. The pendant groups X and Y can be
derived by substitution of any of the following elements: oxygen
(O), sulfur (S), selenium (Se), nitrogen (N), phosphorous (P),
and/or arsenic (As). The two groups X and Y can be identical or
different. The resulting quinone could be used, as a cross-linker
and/or a monomer, in the synthesis of electrochemically degradable
polymers. The polymeric material of the present invention can be
controllably degraded through electrochemical reduction. The
degradation can be done by subjecting the polymer to an electric
potential, a chemical reductant, or other agents that are capable
of inducing chemical degradation. In one embodiment, the
electrochemical reduction is induced by exposure to a change of
electric potential between about 0.05 to about 1.0 V relative to
Ag/AgCl reference electrode or between about 0.5 to about 1.0 V
relative to Ag/AgCl reference electrode. Since the Ag/AgCl
(silver/silver chloride) reference electrode is stable and easily
prepared, it is often used as the reference electrode of choice.
However, any technique for measuring electric potential can be
used.
[0010] In addition, the degradation of the electrically-degradable
polymers of the present invention can be modulated by varying the
quinone cross-linker at R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, and R.sub.6. Varying R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, and R.sub.6, especially R.sub.3 and R.sub.4, affects the
reduction potential of the quinone thereby affording an important
means for controlling the rate, extent, and/or conditions of
polymer degradation. For example, electron-donating groups such as
methoxyl or dimethylamino in positions R.sub.3 and/or R.sub.4 can
make the quinone less easily reduced and therefore can retard the
degradation of the polymer. By contrast, electron-withdrawing
groups, such as halogen or cyano in positions R.sub.3 and/or
R.sub.4, can make the quinone more easily reduced and therefore can
accelerate the degradation of the polymer.
[0011] In one embodiment, the invention provides an
electrically-degradable polymer moiety comprising a quinone
compound of the formula (1) wherein the polymer moiety is capable
of degrading upon exposure to a change in electric potential. The
quinone compound can be used to cross-link one or more monomers
selected from the group comprising styrene, acrylates,
methacrylates, 1,3-butadiene, isoprene, 2-vinylpyridine, ethylene
oxide, acrylonitrile, methyl vinyl ketone, alpha-cyanoacrylate
vinylidene cyanide, propyelene, butene, isobutylene, phosphorus
acid, phosphonous acid, phosphinous acid, phosphoric acid,
phosphonic acid, phosphinic acid, methylene bis (phosphonic acid),
poly(vinylphosponic acid), aziridine, spermine, cadaverine, and
putrecine.
[0012] The invention also provides a method of controlled release
of pharmaceutical agents within a subject comprising implanting an
electrically-controlled polymer derived from monomers and
cross-linked using quinone cross-linkers having the core structure
described above and electrically inducing chemical degradation of
the polymer thereby releasing pharmaceutical agents.
[0013] In another aspect, the invention provides a drug delivery
system comprising an electrically-degradable polymer comprising at
least one quinone moiety, one or more pharmaceutical agents bound
to the electrically-degradable polymer; and a current producing
device electrically coupled to the polymer. In this system, the
polymer is capable of undergoing electrochemical reduction
resulting in the hydrolysis of the cross-linkers and controlled
release of one or more pharmaceutical agents.
[0014] The electric current producing device can either provide a
constant current or variable current, e.g., one which varies in
response to changes in at least one internal parameter within the
subject or in response to one or more external parameters. In
another embodiment, the electrochemically-degradable polymers can
be used in tissue regeneration as temporary scaffolds for the
regeneration of various tissues. In addition, the
electrochemically-degradable polymers can be used as temporary
implants including vascular grafts, sutures, catheters, ligaments,
bone fixation devices (bone plates, screws, and staples), and
dental implants. Moreover, the electrically-degradable polymeric
systems of the present invention permit switching from a first
state to a second in response to a change in electric potential.
Therefore, these systems can have application in
microelectromechanical (MEM) devices, telecommunication devices and
lithography.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a schematic illustration of an
electrochemically-degradable polymer of the present invention using
styrene as the monomer;
[0016] FIG. 2 is a schematic illustration of an
electrochemically-degradable polymer of the present invention using
carboxymethylcellulose (CMC) as the cross-linked polymer;
[0017] FIG. 3 is a schematic illustration of an
electrochemically-degradable copolymer of the present invention
using 1,2-diaminohexane and quinone monomers; and
[0018] FIG. 4 is a three dimensional structure of the quinone acid
5.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The practice of the present invention employs, unless
otherwise indicated, conventional methods of organic and polymeric
chemistry within the skill of the art. Such techniques are
explained fully in the literature.
[0020] The terminology used herein is for describing particular
embodiments and is not intended to be limiting. Unless defined
otherwise, all scientific and technical terms are to be understood
as having the same meaning as commonly used in the art to which
they pertain. For the purposes of the present invention, the
following terms are defined below:
[0021] The term "alkyl" as used herein refers to an aliphatic
hydrocarbon group, which may be straight or branched-chain, having
about 1 to about 20 carbon atoms in the chain. Preferred alkyl
groups have 1 to about 12 carbon atoms in the chain. Branched means
that one or more lower alkyl groups such as methyl, ethyl or propyl
are attached to a linear alkyl chain. "Lower alkyl" means 1 to
about 4 carbon atoms in the chain, which may be straight or
branched. The alkyl may be substituted with one or more "alkyl
group substituents" which may be the same or different, and include
halo, cycloalkyl, hydroxy, alkoxy, amino, carbamoyl, acylamino,
aroylamino, carboxy, alkoxycarbonyl, aralkyloxycarbonyl, or
heteroaralkyloxycarbonyl. Representative alkyl groups include
methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl,
ethyl, n-propyl, propyl, n-butyl, 1-butyl, n-pentyl, 3-pentyl,
methoxyethyl, carboxymethyl, methoxycarbonylethyl,
benzyloxycarbonylmethyl, and pyridylmethyloxycarbonylmethyl.
[0022] The term "alkylene" as used herein refers to a straight or
branched bivalent hydrocarbon chain of 1 to about 6 carbon atoms.
The alkylene may be substituted with one or more "alkylene group
substituents" which may be the same or different, and include halo,
cycloalkyl, hydroxy, alkoxy, carbamoyl, carboxy, cyano, aryl,
heteroaryl or oxo. Preferred alkylene groups are the lower alkylene
groups having 1 to about 4 carbon atoms. Representative alkylene
groups include methylene, ethylene, and the like.
[0023] The term "amino" used herein refers to a group of formula
Z.sup.1Z.sup.2N-- wherein Z.sup.1 and Z.sup.2 are independently
hydrogen; acyl; or alkyl, or Z.sup.1 and Z.sup.2 taken together
with the N through which Z.sup.1 and Z.sup.2 are linked to form a 4
to 7 membered azaheterocyclyl. Representative amino groups include
amino (H.sub.2N--), methylamino, dimethylamino, diethylamino, and
the like.
[0024] The term "aryl" used herein refers to an aromatic monocyclic
or multicyclic ring system of 3 to about 14 carbon atoms,
preferably of 6 to about 10 carbon atoms. The aryl may be
substituted with one or more "ring system substituents" which may
be the same or different, and are as defined herein. Representative
aryl groups include phenyl, naphthyl, furyl, thienyl, pyridyl,
indolyl, quinolinyl or isoquinolinyl.
I. Polymeric Structures
[0025] The physical characteristics of the resulting polymer can be
controlled by varying the type of substituted monomers that are
cross-linked with the quinone cross-linker. The physical
characteristics are important in determining the consistency of the
polymer and what types of processing steps the polymer can
withstand and, thus will determine which applications particular
polymers will be most suited.
[0026] In one aspect of the invention, modified quinone moieties
can be incorporated into a polymer such that the resultant
polymeric materials can be controllably degraded via
electrochemical reduction. The electrochemically degradable
polymers can have the core structure shown below:
##STR00002##
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, and R.sub.6 can
be any organic functional groups including, but not limited to,
hydrogen, alkyl, aryl, alcohol, ether, thiol, thioether, amine,
cyano, halo, nitro, ketone, aldehyde, ester, amide, thioester,
carbonate, carbamate, and urea. Any chemical moiety used as a
reactive group in polymer cross-linking or as a reactive group in
polymerization could be appended to the quinone structure at X
and/or Y of structure 1. For example, the pendant groups X and Y
can be any functional groups subject to degradation upon reduction
of the quinone including, but not limited to, groups containing
vinyl sulfone, epoxide, alkyl halide, alkene, amine, alcohol, acid
halide, acid anhydride, sulfate, phosphate, isocyanate,
isothiocyanate, and thiol. The pendant groups X and Y can be
derived by substitution of any of the following elements: oxygen
(O), sulfur (S), selenium (Se), nitrogen (N), phosphorous (P),
and/or Arsenic (As). The two groups X and Y can be identical or
different. The resulting quinone could be used, as a cross-linker
and/or a monomer, in the synthesis of electrochemically degradable
polymers.
[0027] Electrically-degradable polymers cross-linked with a
modified quinone of the present invention can be made from
polymerization, condensation, or other reaction of any combination
of monomers selected from the group consisting of styrene,
acrylates, methacrylates, 1,3-butadiene, isoprene, 2-vinylpyridine,
ethylene oxide, acrylonitrile, methyl vinyl ketone,
alpha-cyanoacrylate vinylidene cyanide, propyelene, butene,
isobutylene, phosphorus acid, phosphonous acid, phosphinous acid,
phosphoric acid, phosphonic acid, phosphinic acid, methylene bis
(phosphonic acid), poly(vinylphosponic acid), aziridine, spermine,
cadaverine, and putrecine.
[0028] In one embodiment, electrically-degradable polymers
cross-linked with a modified quinone according to the methods of
the present invention can include polymers consisting of modified
carbohydrates including, but not limited to, derivatives of
cellulose, sucrose, chitosan, alginate, hyaluronic acid, guar gum,
and gelatin.
[0029] In yet another embodiment, electrically-degradable polymers
cross-linked with a modified quinone according to the methods of
the present invention can include polymers formed from the
polymerization, condensation or modification of amino acids
including, but not limited to, lysine, arginine, phenol, tyrosine,
and cysteine or modified versions thereof.
[0030] The resulting polymers of the present invention comprise one
or more linkages selected from the group consisting of ester,
ether, amine, amide, urethane, ketone, anhydride, carbonate,
phosphodiester, silicone, disulfide, urea, and phenolic.
[0031] The choice of linkage is based upon the desired use of the
resultant polymer. For example, the presence of an ester linkage
provides the necessary functionality to permit degradability,
particularly biodegradability, since the ester linkage undergoes
hydrolysis under mildly basic conditions, such as those found in
vivo. Other linkages, such as amides, require severe conditions in
order to decompose. The amide linkage requires more stringent
conditions and is not easily hydrolyzed even under strongly acidic
or basic conditions. Therefore, in vivo, the only available route
for cleavage of an amide bond is enzymatic, and that cleavage is
often specific to the amino acid sequence. The highly crystalline
nature of polyamides, e.g., nylon, further slows degradation by
preventing or blocking access to the amide bond by water molecules
and enzymes.
II. Examples of Quinone Structures
[0032] In one embodiment of the present invention, styrene can be
used as the monomer. An exemplary modified quinone cross-linker is
depicted as 1a
(R.sub.1.dbd.R.sub.2.dbd.R.sub.3.dbd.R.sub.4.dbd.R.sub.5.dbd.R.sub.6.dbd.-
CH.sub.3, X.dbd.Y=p-vinylaniline) shown in FIG. 1. In this example,
quinone 1a is used as a cross-linking agent in the synthesis of a
polystyrene-based polymer in place of the common cross-linking
agent, divinylbenzene. The product, polystyrene polymer 2a,
comprises the quinone moiety in the cross-links between strands.
Upon reduction, which can be either electrically, chemically, or by
some other method, hydroquinone-cross-linked polymer 3a is formed.
Polymer 3a can spontaneously undergo cleavage of the amide linkages
in the cross-links, leading to degraded polystyrene without
cross-links and thereby changing the material properties of the
polymer substantially.
[0033] In another embodiment of the present invention,
carboxymethylcellulose (CMC) can be crosslinked with a modified
quinone of the present invention. An example of an exemplary
modified quinone structure, 1b,
(R.sub.1.dbd.R.sub.2.dbd.R.sub.3.dbd.R.sub.4.dbd.R.sub.5.dbd.R.sub.6.dbd.-
CH.sub.3, X.dbd.Y=p-oxiridinoaniline) is shown in FIG. 2. In this
example, the modified quinone 1b is used as a cross-linking agent
for carboxymethylcellulose (CMC) in place of the common
cross-linking agent, epichlorohydrin. The product is CMC hydrogel
2b that contains the quinone moiety in the cross-links between
strands. Upon reduction, either electrically, chemically, or by
some other method, hydroquinone-crosslinked hydrogel 3b is formed.
Polymer 3b can spontaneously undergo cleavage of the amide linkages
in the cross-links, thus leading to a CMC derivative without
cross-links and thereby changing the material properties of the
hydrogel substantially.
[0034] In yet another embodiment of the present invention, a
copolymer can be formed using a quinone and 1,6-diaminohexane as
monomers. An example of a quinone useful for this embodiment is
depicted as the quinone structure is 1c
(R.sub.1.dbd.R.sub.2.dbd.R.sub.3.dbd.R.sub.4.dbd.R.sub.5.dbd.R.sub.6.dbd.-
CH.sub.3, X.dbd.Y.dbd.Cl) shown in FIG. 3. In this example, quinone
1c is used as one of the monomers in a condensation polymer with
1,6-diaminohexane. The product, the condensation quinone
acide-diamine copolymer 2c, contains the quinone moiety in
alternating units with the diaminohexane. Upon reduction,
hydroquinone-containing polymer 3c can be formed. Polymer 3c can
spontaneously undergo cleavage of the amide linkages holding the
polymer together, leading to completely degraded polymer.
[0035] Any chemical moiety used as a reactive group in polymer
cross-linking or as a reactive group in polymerization could be
appended to the quinone core structure 1 at X and/or Y. The
resulting quinone can be used in the electrochemically degradable
synthesis of polymers. The choice of modified quinone crosslinker
and monomers depends on the desired use of the resultant
polymer.
[0036] In some embodiments, the electrochemically-degradable
polymers of the present invention can be applied to or blended with
another biocompatible polymeric material, including biodegradable
or non-biodegradable polymeric materials. Combining the
electrochemically-degradable polymers with other polymeric material
allows for further control of the degradation rate.
III. Uses for Electrically Degradable Polymers
[0037] The methods and constructs of the present invention can be
used in drug delivery, tissue regeneration, biomedical implants,
electronic systems, microchip design, and/or chemical/biological
warfare.
[0038] The size and shape of the electrochemically degradable
polymers can be selected based upon the desired use. For example,
the polymers can be formulated into pellets, films, microspheres,
polymerizing gels, hydrogels, wafers, coatings, etc. In some
embodiments, the electrochemically degradable polymers of the
present invention can be formulated into microparticles, which can
be used, for example, in oral delivery systems and in
subcutaneously injected delivery systems. For example,
microparticles of poly(lactide-co-glycolide) (PLGA) can be prepared
in a fairly uniform manner to provide essentially nonporous
microspheres. Upon electrochemical stimulation, the polymers can be
induced to degrade resulting in polymer fragments. In some
embodiments, the polymer fragments can be adsorbed by the body.
Drug Delivery
[0039] Methods for sustained or controlled drug release can utilize
a drug dispersed in an electrically degradable polymer matrix,
which can be implanted, administered orally or injected. Exemplary
polymers to be used in such applications include poly(lactic acid)
and poly(lactic acid-co-glycolic acid) crosslinked with a modified
quinone of the present invention. These polymers undergo slow
hydrolysis in vivo, releasing the entrapped drug. The polymer
degradation products are the parent acids, which are absorbed by
the body.
[0040] Polymer/drug matrix particles to be administered via
injection must have a size range typically on the order of 200
microns or less. The size and morphology of polymer/drug matrix
particles depends upon the fabrication method employed, and the
formation of small polymer/drug matrix particles in which the drug
is a protein is currently limited to a few techniques. For example,
polymer/protein matrix particles comprising poly(lactic acid) and
either trypsin or insulin, can be prepared by both an oil/water
emulsion method and a neat mixing method at elevated temperature
(Tabata et al., J. Cont. Release 23: 55-64 (1993)). The
polymer/protein matrices thus formed can be subsequently ground
into granules.
[0041] In another embodiment, the electrochemically degradable
polymers can be used in drug delivery system comprising an
electrically degradable polymer and a current producing or electric
charge generating device. The electrically degradable polymer can,
for example, be used to house one or more pharmaceutical agents.
Alternatively, the electrochemically degradable polymer could be
used as a covering to drug reservoirs in implantable devices. The
current producing device can be electrically coupled to the
degradable polymer, such that the polymer is capable of undergoing
electrochemical reduction resulting in the hydrolysis of the
quinone cross-linkers and controlled release of one or more
pharmaceutical agents.
[0042] The electric current producing device can either provide a
constant current or variable current, e.g., one which varies in
response to changes in at least one internal parameter within the
subject or in response to one or more external parameters.
[0043] Non-limiting examples of the internal parameter include
diagnostic markers (such as cancer markers including
carcinoembryonic antigen (CEA), prostate-specific antigen (PSA),
alpha-fetoprotein (AFP), beta-human chorionic gonadotropin
(.beta.-HCG), carbohydrate antigen 125 (CA-125), carbohydrate
antigen 15-3 (CA 15-3), carbohydrate antigen 19-9 (CA 19-9),
Beta.sub.2 (.beta..sub.2)-microglobulin, lactate dehydrogenase),
cholesterol, blood pressure, temperature, energy expenditure,
activity level, heart rate, blood acidity, blood alcohol, ammonia,
ascorbic acid, bicarbonate, bilirubin, blood volume, calcium,
carbon dioxide pressure, carbon monoxide, CD4 cell count,
ceruloplasmin, chloride, complete blood cell count (CBC), copper,
creatine, kinase (CK or CPK), creatine kinase isoenzymes,
creatinine, cytokines, electrolytes (calcium, chloride, magnesium,
potassium, sodium), erythrocyte sedimentation rate (ESR or
Sed-Rate), glucose, hematocrit, hemoglobin, iron, iron-binding
capacity, lactate (lactic acid), lactic dehydrogenase, lead,
lipase, zinc, lipids, cholesterol, triglycerides, liver function
tests (i.e., bilirubin (total), phosphatase (alkaline), protein
(total and albumin), transaminases (alanine and aspartate),
prothrombin (PTT)), magnesium mean corpuscular hemoglobin (MCH),
mean corpuscular hemoglobin concentration (MCHC), mean corpuscular
volume (MCV), osmolality, oxygen pressure, oxygen saturation
(arterial), phosphatase, phosphatase, phosphorus, platelet count,
potassium, prostate-specific antigen (PSA), total blood proteins,
albumin, globulin, prothrombin (PTT), pyruvic acid, red blood cell
count (RBC), sodium, thyroid-stimulating hormone (TSH),
transaminase, alanine (ALT), aspartate (AST), urea nitrogen (BUN),
BUN/creatinine ratio, uric acid, vitamin A, white blood cell count
(WBC), etc. Changes in one or more internal parameter can be
continuously monitored. An automatic turn-on protocol can be
triggered once the change in one or more internal parameter reaches
a preset limit. The current producing device can also be capable of
being controlled externally, by, for example, the subject and/or
doctor. For example, the drug delivery system can be used as an
on-demand delivery of analgesics wherein NSAIDS or other pain
medication can be controllably released when the subject activates
the current producing device upon sensing pain.
[0044] Non-limiting examples of the external parameter that can be
monitored by the current producing device include
biochemical/biological agents (i.e., aerosolized or lyophilized
agents like Bacillus anthracia, Yersia pestis, Francisella
tularensis, brucellosis, tularemia, and Venezuelan Equine
Encephalitis ("VEE"), Bacillus globigii, Clostridium perfringens,
Clostridium botulinum, ricin, SEB (Staphococcal Enterotoxin B)),
chemical agents such as cyanide gas and mustard gas and those
including organo-phosphate compounds such as those known as GA, GB,
GD, GF, and VX, viruses responsible for diseases such as smallpox,
chicken pox, german measles, herpes, hepatitis, AIDS, rabies,
polio, and influenza (See, for example, U.S. Pat. No. 6,777,228 and
U.S. Pat. No. 6,472,155 for systems for monitoring biological
agents, which are hereby incorporated by reference). For example,
the drug delivery system can be used as an on-demand delivery of
antidotes for biochemical/biological warfare wherein antidotes
(i.e., penicillin for bubonic plague) could be controllably
released when the external parameter exceeds a predetermined
threshold.
[0045] The electrically degradable polymer can be used either
inside or outside the body in proximity to the area to be treated.
The system has applications in transdermal, subcutaneous and
intravenous use. For example, this system can be used as a
transdermal drug delivery system for transdermal delivery of
medical or veterinary pharmaceutical agents.
[0046] In some embodiments, the electrically degradable polymer can
be use in a drug delivery system having uses in treatment or
monitoring of conditions such as, for example, pain, arrhythmia,
cancer, diabetes, angiogenesis, restenosis, edema, infection,
infectious diseases, sepsis, post operative adhesions, cell
signaling, immunologic responses, tissue/implant rejection,
neurodegenerative diseases, and hormone imbalances.
Tissue Engineering
[0047] In another embodiment, the electrochemically-degradable
polymers can be used in tissue regeneration as a temporary scaffold
for the regeneration of various tissues including, but not limited
to, cartilage, epithelium, cardiac, skeletal, vascular, and may
also be used as a temporary nerve guide. The
electronically-degradable polymers can be used to seed any
combination of cell types including, but not limited to,
endothelial cells, parenchymal cells such as hepatocytes, stem
cells, Islet cells, and other organ cells, muscle cells, cells
forming bone and cartilage such as osteoblasts and chondrocytes and
nerve cells, from mammalian tissue or lower animals and
genetically-engineered cells. A combination of polymers can be
formed prior to cell growth and attachment prior to in vivo or ex
vivo use. The electrochemically-degradable polymers can blended
with another polymeric material, applied as a coating on the
surface of another material, or be used to form the material
itself.
[0048] Porous polymer scaffolds comprising electrically degradable
polymers can be shaped into articles for tissue engineering and
tissue guided regeneration and repair applications, including
reconstructive surgery. Scaffold applications include the
regeneration of tissues such as nervous, musculoskeletal,
cartilaginous, tendenous, hepatic, pancreatic, ocular,
integumentary, arteriovenous, urinary or any other tissue forming
solid or hollow organs. Scaffolds can be used as materials for
vascular grafts, ligament reconstruction, adhesion prevention and
organ regeneration. In one embodiment, the polymer scaffold
provides physical support and an adhesive substrate for isolated
cells during in vitro culturing and subsequent in vivo
implantation. in the human body. An alternate use of electrically
degradable polymer scaffolds is to implant the scaffold directly
into the body without prior culturing of cells onto the scaffold in
vivo. Once implanted, cells from the surrounding living tissue
attach to the scaffold and migrate into it, forming functional
tissue within the interior of the scaffold. Regardless of whether
the scaffold is populated with cells before or after implantation,
the scaffold is designed so that as the need for physical support
of the cells and tissue diminishes over time, the scaffold can
degrade upon electrical stimulation. The controllable degradation
of the electrically degradable scaffold can be catalyzed via
reduction of the modified quinone cross-linkers of the polymer
scaffold. For example, once the doctor determines that engineered
tissue has regenerated, a voltage can be supplied, either inside or
outside the body, causing degradation of the scaffold.
[0049] Materials which can be used for tissue engineering
(implantable matrices) include sutures, tubes, sheets, adhesion
prevention devices (typically films, polymeric coatings applied as
liquids which are polymerized in situ, or other physical barriers),
and wound healing products (which vary according to the wound to be
healed from films and coating to support structures). Both normal
and genetically engineered nerve cells optionally can be seeded on
the implants, to help replace lost function.
[0050] As described by Langer et al., J. Ped. Surg. 23(1), 3-9
(1988), WO88/03785 and EPA 88900726.6 by Massachusetts Institute of
Technology, a matrix for implantation to form new tissue should be
a pliable, non-toxic, porous template for vascular ingrowth. The
pores should allow vascular ingrowth and the seeding of cells
without damage to the cells or patient. These are generally
interconnected pores in the range of between approximately 100 and
300 microns. The matrix should be shaped to maximize surface area,
to allow adequate diffusion of nutrients and growth factors to the
cells. In the preferred embodiment, the matrix is cross-linked with
a modified quinone and formed of an electrically-degradable
bioabsorbable, or biodegradable, synthetic polymer such as a
polyanhydride, polyorthoester, or polyhydroxy acid such as
polylactic acid, polyglycolic acid, and copolymers or blends
thereof. Non-degradable materials can also be used to form the
matrix. Examples of suitable materials include ethylene vinyl
acetate, derivatives of polyvinyl alcohol, teflon, nylon,
polymethacrylate and silicon polymers. The preferred non-degradable
materials are ethylene vinyl acetate meshes and polyvinyl alcohol
sponges. Commercially available materials may be used. Polymers for
use in the matrix can be characterized for polymer molecular weight
by gel permeation chromatography (GPC), glass transition
temperature by differential scanning calorimetry (DSC), thermal
stability by thermal gravimetric analysis (TGA), bond structure by
infrared (IR) spectroscopy, toxicology by initial screening tests
involving Ames assays and in vitro teratogenicity assays, and by
implantation studies in animals for immunogenicity, inflammation,
release and degradation studies.
[0051] The electrically-degradable polymers may be implanted in
vivo into a patient in need of therapy to repair or replace damaged
cells or tissue, such as nervous system tissue. Scaffolds for
tissue engineering can be coated with, or made of,
electrically-degradable polymers to enhance regeneration, growth or
function of implanted cells or cells which migrate into, attach and
proliferate within the implanted matrices. Materials which can be
used for implantation include sutures, tubes, sheets, adhesion
prevention devices (typically films, polymeric coatings applied as
liquids which are polymerized in situ, or other physical barriers),
and wound healing products (which vary according to the wound to be
healed from films and coating to support structures). To enhance
the effectiveness of the treatment, compositions which further
promote nervous tissue healing, such as proteins, antibodies, nerve
growth factors, hormones, and attachment molecules, can be applied
together with the polymer, and optionally can be covalently
attached to the polymer or a polymeric support material. Those
skilled in the art can readily determine exactly how to use these
materials and the conditions required without undue
experimentation.
[0052] Molecules such as attachment molecules or bioactive
molecules such as growth factors can be provided on the
electrically-degradable polymers, and may be optionally covalently
or non-covalently attached to the polymers. Attachment molecules
are defined as any natural or synthetic molecule which is
specifically bound by cell surface receptors. These include natural
and synthetic molecules having one or more binding sites. Examples
of natural molecules are extracellular matrix factors such as
fibronectin and laminin. Examples of synthetic molecules are
peptides containing the binding sites of fibronectin. In some
embodiments, attachment of the cells to the polymer is enhanced by
coating the polymers with compounds such as basement membrane
components, gelatin, gum arabic, collagens types I, II, III, IV,
and V, fibronectin, laminin, glycosaminoglycans, mixtures thereof,
and other materials known to those skilled in the art of cell
culture. Extracellular matrix molecules (ECM) include compounds
such as laminin, fibronectin, thrombospondin, entactin,
proteoglycans, glycosaminoglycans and collagen types I through XII.
Other natural attachment molecules include simple carbohydrates,
complex carbohydrates, asialoglycoproteins, lectins, growth
factors, low density lipoproteins, heparin, poly-lysine, thrombin,
vitronectin, and fibrinogen. Synthetic molecules include peptides
made using conventional methods to incorporate one or more binding
sites such as R G D from fibronectin, L I G R K K T from
fibronectin and Y I G S R from laminin.
[0053] Methods for attaching biological molecules to polymeric
substrates available in the art may be used. Methods for applying
attachment molecules to substrates include: attachment of molecules
to substrate by applying attachment molecules in a solution such as
PBS or a high pH, carbonate buffer and adsorption of the molecules
to the substrate surface; ionic binding of attachment molecules to
substrate; covalent binding of molecules to the substrate surface
by chemical reactions using reagents such as glutaraldehyde or
carbodiimide; and drying of attachment molecules on the substrate
surface.
Biomedical Implants
[0054] In another embodiment, the electrochemically-degradable
polymers of the present invention can be used in biomedical
implants. For example, the electrochemically-degradable polymers
can be used as temporary implants including vascular grafts,
sutures, catheters, ligaments, bone fixation devices (i.e., bone
plates, screws, and staples), and dental implants.
[0055] Additional biomedical applications for electrically
degradable polymers include use with fracture fixation, for
example, as absorbable orthopedic fixation devices. In particular,
such electrically degradable polymers permit treatment of bone
fractures through fixation, providing good tissue/material
compatibility, and facile molding (into potentially complex shapes)
for easy placement. Controlled degradation of the electrically
degradable polymers permits optimum bone function upon healing. The
materials can reestablish the mechanical integrity of the bone and
subsequently degrade to allow new bone formation to bear load and
remodel. These electrically degradable polymers maintain mechanical
integrity while undergoing a gradual degradation and loss in size
permitting bone ingrowth. In contrast to the traditional use of
steel fixation devices, the electrically degradable polymer-based
device is advantageous in those situations where the device is not
needed permanently or would require removal at a later point in
time. Also, metallic orthopedic devices shield stress during
healing and can lead to bone atrophy.
[0056] Non-limiting examples of other polymeric materials that can
be blended or coated with the electrically-degradably polymers
include biocompatible materials which are not biodegradable, such
as poly(styrene), poly(esters), polyurethanes, polyureas,
poly(ethylene vinyl acetate), poly(propylene), poly(methaerylate),
poly(ethylene), poly(ethylene oxide), glass, polysilicates,
poly(carbonates), teflon, fluorocarbons, nylon, and silicon rubber.
Other useful materials include biocompatible, biodegradable
materials such as poly(anhydrides), poly(hydroxy acids) such as
poly(glycolic acid) and poly(lactic acid),
poly(lactide-co-glycolide), poly(orthoesters),
poly(propylfumerate), proteins and polymerized proteins such as
collagen, and polysaccharides and polymerized polysaccharides, such
as glycosaminoglycans, heparin and combinations thereof.
Electronic Systems
[0057] The electrically-degradable polymeric systems of the present
invention permit reasonably rapid switching from a first state to a
second in response to a change in electric potential. By way of
examples, these systems can have application in
microelectromechanical (MEM) devices, telecommunication devices and
lithography. For example, it can be employed as switches in
photonic applications, such as a crossbar switch router for a fiber
optic communications network, as actuable valves in microfluidicic
systems, MEMs, and other electronic systems, such as to switch
optical data packets. In addition, the electrically-degradable
polymers can be used as a masking element in microchip design.
[0058] The electrically-degradable polymers can be used to provide
an integral switching mechanism within a high density interconnect
(HDI) circuit environment. Previous MEM based switches and
actuators required the insertion of individual MEM parts into the
HDI circuit and the subsequent routing of signals to the MEM
structure, particularly when a large number of switches were
required or high isolation of the switched signals was desired. The
use of an integral MEM switch within an HDI structure will allow
switches to be positioned in desired locations with a minimum of
signal diversion and routing. In addition, it will not be necessary
to handle and insert the fragile MEM actuators into cavities in the
HDI circuit and suffer the yield loss of this insertion process.
The use electrically-degradable polymers to fabricate integral
switching mechanisms within HDI architecture will ultimately result
in a lower cost system.
[0059] In one embodiment, a MEM based switch structure or actuator
can be fabricated using traditional HDI processing steps. The
switch structure can be operated by selectively passing current
through the electrically-degradable polymer layers thereby causing
them to heat above the transition temperature and causing a
deformation of the heated layer.
[0060] In addition, polymers of the present invention can be
designed such that they can be environmentally friendly. The
creation of polymers in today's society and the exponential use in
all areas of society has also created environmental concerns over
whether such polymers will be able to degrade over time or will end
up in landfills forever. Electrically-degradable polymers can be
used to reduce the stress on the environment caused be the
increasing use of polymeric materials.
EXAMPLES
[0061] The following examples illustrate practice of the invention.
These examples are for illustrative purposes only and are not
intended in any way to limit the scope of the invention
claimed.
[0062] The present invention relates to polymeric materials capable
of being degraded when exposed to an electric current. In
particular, electrochemical reduction of the modified quinone
moiety, which can be used to cross-link the polymer, can cleave the
polymer resulting in efficient degradation. The following examples
illustrate the synthesis of one exemplary polymer, polystyrene,
using the methods of the present invention.
[0063] The general synthesis described in the examples comprise the
three reaction steps illustrated below.
##STR00003##
Example 1
Synthesis of Bislactone 4
##STR00004##
[0065] This example demonstrates an exemplary method of
synthesizing bislactone 4. Methane sulfonic acid (1 mL) can be
heated to 70.degree. C. in an oil bath with stirring. To this,
p-xylohydroquinone (91 mg, 0.658 mmol) and methyl
.beta.,.beta.-dimethylacrylate (195 .mu.L, 179 mg, 1.49 mmol) can
be added and the reaction is allowed to proceed for 15 h at
70.degree. C. After cooling to 20.degree. C., the reaction mixture
can be diluted with ice water (15 mL) and extracted with 4.times.20
mL of ethyl ether. The organic phase can then be washed with
2.times.50 mL of sat NaHCO.sub.3, dried with MgSO.sub.4, and the
solvent can be removed in vacuo. A light orange solid (198 mg) will
result. Purification by flash column chromatography
(CH.sub.2Cl.sub.2) will yield 58.4 mg (0.180 mmol, 27%) of the
lactone as a white solid. R.sub.F 0.44 (CH.sub.2Cl.sub.2); mp
280-282.degree. C.; .sup.1H NMR (CDCl.sub.3) .delta. 2.59 (s, 4H,
CH.sub.2), 2.42 (s, 6H, CH.sub.3), 1.48 (s, 12H, CH.sub.3). (Anal.
Calcd for C.sub.18H.sub.22O.sub.4:C, 71.50; H, 7.33. Found: C,
71.22; H, 7.33.)
Example 2
Synthesis of Quinone Acid
(R.sub.1.dbd.R.sub.2.dbd.R.sub.3.dbd.R.sub.4.dbd.R.sub.5.dbd.R.sub.6.dbd.-
CH.sub.3, X.dbd.Y.dbd.OH)
##STR00005##
[0067] This example demonstrates an exemplary method of
synthesizing quinone acid
(R.sub.1.dbd.R.sub.2.dbd.R.sub.3.dbd.R.sub.4.dbd.R.sub.5.dbd.R.sub.6.dbd.-
CH.sub.3, X.dbd.Y.dbd.OH). The lactone 4 (150 mg) was dissolved in
15 mL of THF and 1M aqueous LiOH (15 mL) was added. The resulting
turbid solution was stirred vigorously in an uncapped vessel at
20.degree. C. for 4 h, after which TLC (silica, 5%
EtOH/CH.sub.2Cl.sub.2) indicated complete reaction. The reaction
mixture adjusted to pH 3 by addition of 6M aqueous HCl, and the
mixture was extracted with EtOAc (3.times.50 mL). The extracts were
washed with H.sub.2O (50 mL), brine (50 mL), dried (MgSO.sub.4) and
the solvent was evaporated to produce 159 mg (95%) of essentially
pure quinone acid 5 as a yellow solid. The crude acid was
recrystallized from hexane-ethanol to produce an analytically pure
sample as bright yellow crystals: mp 164-7.degree. C. .sup.1H NMR
(CDCl.sub.3) .delta. 2.79 (s, 4H, CH.sub.2), 2.08 (s, 6H), 1.42 (s,
12H). ESI MS (neg. ion mode) m/z 335 (M-H). UV .lamda..sub.max 257,
343 nm. Anal. Calcd for C.sub.18H.sub.24O.sub.6/NH.sub.3:C, 61.17;
H, 7.70, N, 3.96, O, 27.16. The structure of the compound was
confirmed by x-ray crystallography and shown in FIG. 4.
Example 3
Synthesis of Quinone Cross Linkers
A. Synthesis of Quinone Esters
##STR00006##
[0069] This example demonstrates an exemplary method of
synthesizing a quinone ester. With time, the quinone acid can
convert to a mixture of at least three compounds. This conversion
is likely due to intramolecular Michael addition of the acid moiety
to the quinone or to the carbonyl, much like what was observed by
Cohen in a related compound (R. T. Borchardt and L. A. Cohen J. Am.
Chem. Soc. 1973, 95, 8308). By analogy to Cohen's work, the product
mixture will equilibrate and the equilibrating mixture can be
converted into any desired ester, simply by stirring with the
corresponding alcohol in the presence of an acid catalyst. Thus,
any of the quinone esters for use as degradable cross-linking
agents according to the methods of the present invention can be
available by this or a related synthesis.
[0070] For example, the quinone acid chloride (product structure;
R.dbd.Cl) can be generated by treatment of the equilibrating
mixture with oxalyl chloride. The acid chloride can easily be
converted into needed amides by treatment with the corresponding
amines. Alternatively, amides can be prepared by reaction of the
equilibrating mixture with the corresponding amines in the presence
of N,N'-dicyclohexyl-carbodiimide (DCC) or other condensing agents,
such as diisoproplycarbodiimide (DIC). In the DCC coupling method,
the carboxylic acid initially will form a reactive intermediate
with the carbodiimide, an O-acylisourea (Sheehan et al., J. Am.
Chem. Soc., 1955, 77, 1067-1068). Depending on the exact reaction
conditions, the adduct can be converted into a symmetrical
anhydride, in the presence of excess carboxylic acid, or into an
active ester in the presence of a hydroxy component. In either
case, insoluble dicyclohexylurea (DCU) is formed as co-product and
the anhydride or active ester can be isolated.
[0071] The diisopropyl analog of DCC, N,N'-diisopropylcarbodiimide
(DIC) is preferable in some embodiments since the corresponding
urea derivative is more soluble in organic solvents such as DCM and
DMF. For carbodiimide-mediated couplings, 1-hydroxybenzotriazole
(HOBt) (Koonig, W. and Geiger, R. Chem. Ber. 1970, 103, 2034-2040)
can be added, generating an O-acyl-1-hydroxybenzotriazole, which is
a very powerful acylating reagent. Alternatively,
3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (Dhbt-OH) can be
used.
[0072] In addition to DCC and DIC, several other in situ acylating
reagents can be used, including, but not limited to,
2-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorofluorophosphate (HBTU),
2-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate, TBTU, (Dourtoglou, V. and Gross, B. Synthesis
1984, 573-574); BOP (benzotriazolyl N-oxytrisdimethaminophosphonium
hexafluorofluorophosphate), and PyBOP (benzotriazolyl
N-oxytrispyrrolidinophosphonium hexafluorofluorophosphate) (Coste,
J. et al. Tetrahedron Lett. 1990, 31, 205-208), which all requiring
the presence of an activating base.
B. Synthesis of Sulfones
##STR00007##
##STR00008##
[0074] This example demonstrates an exemplary method of
synthesizing a sulfone cross-linker. The quinone acid 5 (100 mg)
was dissolved in a solution of the amine (67 mg, 2.2 eq) in dry DMF
(5.0 mL) and DCC (135 mg, 2.2 eq) was added. The reaction was
stirred at room temperature for 6 h, then evaporated to dryness.
The residue was purified by chromatography (EtOAc-hexane, 1:5)
providing the sulfide amide 6.
[0075] The sulfide amide 6 (10 mg) was added to a solution of 30%
aqueous hydrogen peroxide (0.5 mL) in acetic acid (1.0 mL) at
0.degree. C. The reaction mixture was stirred at 0.degree. C. for
12 h, then allowed to warm to room temperature. The mixture was
evaporated to dryness and then dissolved in EtOH. The ethanol
solution of the sulfone 7 could be used directly in polymer
cross-linking reactions. Preferably, the sulfone 7 should be
prepared immediately prior to use.
[0076] While the present invention has been described in terms of
specific methods and compositions, it is understood that variations
and modifications will occur to those skilled in the art upon
consideration of the present invention.
[0077] Those skilled in the art will appreciate, or be able to
ascertain using no more than routine experimentation, further
features and advantages of the invention based on the
above-described embodiments. Accordingly, the invention is not to
be limited by what has been particularly shown and described,
except as indicated by the appended claims. All publications and
references are herein expressly incorporated by reference in their
entirety.
* * * * *