Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NITRIC OXIDE-PRODUCING HYDROGEL MATERIALS
Field of the Invention
The present invention relates to photopolymerizable hydrogel
materials that produce physiologically relevant amounts of nitric oxide (NO)
for prolonged periods of time.
This application claims priority to U.S.S.N. 60/152,054 filed
September 2, 1999.
Background of the Invention
Endothelial cells, normally present as a monolayer in the intimal
layer of the arterial wall, are believed to play an important role in the
regulation of smooth muscle cell proliferation in vivo. Endothelial cells are
seriously disrupted by most forms of vascular injury, including that caused
by percutaneous transluminal coronary angioplasty and similar procedures.
Approximately 35-50% of patients treated by percutaneous transluminal
coronary angioplasty experience clinically significant renarrowing of the
artery, or restenosis, within six months of the initial treatment. Restenosis
is
due, at least in part, to migration and proliferation of smooth muscle cells
in
the arterial wall along with increases in secretion of matrix proteins to form
2 0 an obstructive neointimal layer within the arterial wall. Similar issues
limit
the performance of vascular grafts. The processes that regulate arterial
wound healing following vascular injury, such as that caused by angioplasty,
are as yet poorly understood, but are believed to involve a complex cascade
of blood and vessel wall-derived factors.
2 5 Numerous factors that stimulate intimal thickening and restenosis
have been identified through administration of exogenous proteins, genetic
alteration of cells, or through the blockade of certain signals using
antibodies
or other specific growth factor inhibitors. These smooth muscle cell
mitogens and chemoattractants derive from both the blood or thrombus
3 o formation and from the vessel wall itself. Endothelial cells produce a
number of substances known to down-regulate smooth muscle cell
proliferation, including heparin sulfate, prostacyclin (PG12), and NO.
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NO is an endothelium-derived target molecule useful for the
prevention of restenosis because, in addition to limiting the proliferation of
smooth muscle cells (Garg et al., 1989), NO reduces platelet aggregation (de
Graaf et al., 1992; Radomski et al., 1987), increases endothelial cell
proliferation (Ziche et al., 1993), and attenuates leukocyte adhesion (Lefer
et
al., 1993), all of which are highly desirable for the reduction of intimal
thickening and restenosis (Reviewed by Loscalzo, 1996). Because of the
complexity of the restenotic process, approaches that act upon multiple
targets are the most likely to be successful.
The mechanisms whereby NO affects these multiple responses are
not fully understood as yet, but it is known that NO activates soluble
guanylate cyclase by binding to its heme moiety, thereby elevating the levels
of cyclic guanosine monophosphate (cGMP), an intracellular second
messenger with multiple cellular effects (Moro et al., 1996). The effects of
NO can often be mimicked by the administration of cGMP or more stable
derivatives of cGMP (Garg et al., 1989). In addition, NO has been found to
inhibit ribonucleotide reductase, an enzyme that converts ribonucleotides
into deoxy ribonucleotides, thus significantly impacting DNA synthesis
(Lepoivre et al., 1991; Kwon et al., 1991 ), as well as several enzymes
2 0 involved in cellular respiration (Stuehr et al., 1989).
A number of molecules that produce NO under physiological
conditions (NO donors) have been identified and evaluated both in vitro and
in vivo. NO donor molecules exert biological effects minvcking those of NO
and include S-nitrosothiols (Diodati et al, 1993; Lefer et al., 1993; DeMeyer
2 5 et al., 1995), organic nitrates (Ignarro et al., 1981 ), and complexes of
NO
with nucleophiles (Diodati et al., 1993; Diodati et al., 1993; Maragos et al.,
1993). Most of these have been low molecular weight molecules that are
administered systemically and have short half lives under physiologic
conditions, thus exerting effects upon numerous tissue types with a brief
3 0 period of activity. In addition, L-arginine is often thought of as a NO
donor,
as L-arginine is a substrate for NO synthase, and thus administration of L-
arginine increases endogenous NO production and elicits responses similar
to those caused by NO donors in most cases (Cooke et al., 1992).
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The development of NO-releasing polymers containing
NO/nucleophile complexes has been reported by Smith et al., (1996). These
materials were capable of releasing NO for as long as 5 weeks in vitro and
were able to limit smooth muscle cell proliferation in culture and to reduce
platelet adherence to vascular graft materials in an arterio-venous shunt
model. These materials show promise for numerous clinical applications
where localized NO production would be desired, such as anti-thrombotic
coating materials for catheters, but probably will not be useful for the
direct
treatment of tissues in vivo as these materials suffer from a number of
1 o disadvantages. These polymers may be produced as films, powders, or
microspheres, but they cannot be formed in situ in direct contact with cells
and tissues, thus making it difficult to strictly localize NO treatment to a
tissue and potentially causing issues with the retention of the polymer at the
site of application. The formulation issues will also make local
administration during laparoscopic or catheter-based procedures difficult or
impossible. Additionally, biocompatibility of the base polymer is a serious
issue for implantable, NO-releasing polymers, especially those intended for
long-term use, as inflammatory and thrombotic responses may develop after
the cessation of NO release.
2 0 It would be more efficient if these compounds could be administered
solely to the site in need of treatment, and in some cases, reduce or
eliminate
side effects due to systemic administration of the agents, particularly over
prolonged time periods.
It is therefore an object of the present invention to provide reagents
2 5 for controlled release of NO and/or compounds modulating NO levels at a
particular site, following local or topical application.
It is a further object of the present invention to provide methods for
treatment of conditions involving inflammatory responses by providing
hydrogel materials releasing compound modulating NO levels at the site of
3 0 application.
Summary of the Invention
Biocompatible polymeric materials releasing or producing NO, most
preferably photopolymerizable biodegradable hydrogels capable of releasing
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physiological amounts of NO for prolonged periods of time, are applied to
sites on or in a patient in need of treatment thereof for disorders such as
restenosis, thrombosis, asthma, wound healing, arthritis, penile erectile
dysfunction or other conditions where NO plays a significant role. The
polymeric materials can also be formed into films, coatings, or
microparticles. The polymers are typically formed of macromers, which
preferably include biodegradable regions, and have bound thereto groups that
are released in situ to elevate or otherwise modulate NO levels at the site
where treatment is needed. The macromers can form a homo or hetero-
dispersion or solution, which is polymerized to form a polymeric material,
that in the latter case can be a semi-interpenetrating network or
interpenetrating network. Compounds to be released can be physically
entrapped, covalently or ionically bound to macromer, or actually form a part
of the polymeric material. Hydrogels can be formed by ionic and/or covalent
crosslinking. Other active agents, including therapeutic, prophylactic, or
diagnostic agents, can also be included within the polymeric material.
Brief Description of the Invention
Figure 1 is a schematic of the synthesis of S-nitrosocysteine
hydrogels {Acryloyl-PEG-CYSNO).
2 0 Figure 2 is a schematic of the synthesis of acryloyl-PEG-Lysines NO-
nucleophile complex hydrogels.
Figure 3 is a schematic of the synthesis of acryloyl-PEG-DETA-NO-
nucleophile complex hydrogels.
Figure 4 is a graph showing the temporal release (%NO released over
2 5 time in days) of NO from acryloyl-PEG-Lyss-NO hydrogels at pH 7.4
(circles) and pH 3 (squares).
Figure 5 is a graph showing the temporal release (%NO released over
time in days) of NO from acryloyl-PEG-DETA-NO hydrogels at pH 7.4
(circles) and pH 2 (squares).
3 0 Figure 6 is a graph showing the temporal release (%NO released over
time in hours) of NO from PEG-CYSNO hydrogels at pH 7.4 (circles) and
pH 2 (squares).
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Figure 7 is a graph showing the temporal release (pmol NO released
per gram of polymer over time in hours) of NO from PVA-NO-bFGF
hydrogels at pH 7.4, 37°C.
Figures 8A and 8B are graphs showing that acryloyl-PEG-Lysine-NO
hydrogels inhibit the proliferation of smooth muscle cells. Figure 8A, % of
control cell number, hydrogel formulation. Figure 8B, % of control cell
number, soluble polymer.
Figures 9A and 9B are graphs showing the inhibition of SMC
proliferation by NO released from acryloyl-PEG-DETA-NO hydrogels
(Figure 9A) and soluble polymer (Figure 9B), as a percentage of the control.
Figures l0A and lOB are graphs showing inhibition of SMC
proliferation by NO released from acryloyl-PEG-CYSNO hydrogels (Figure
l0A) and soluble polymer (Figure 108), as a percentage of controls.
Figure 11 is a graph comparing the degree of inhibition of smooth
muscle cell growth by NO released from hydrogels: acryloyl-PEG-Lys-NO,
acryloyl-PEG-DETA-NO, and acryloyl-PEG-CYSNO, compared to control
hydrogel with NO. The %inhibition of smooth muscle cell growth is
determined by comparing the cell growth for each NO-releasing hydrogel to
a control PEG-diacrylate hydrogel.
2 o Figure 12a is a graph showing the temporal release of NO,
micromolar NO released/gram of gel over time in hoursfrom PVA-NO-bFGF
hydrogels at pH 7.4, 37°C. Figure I2b is a graph showing the temporal
release (% of theoretical bFGF released per gram of gel over time in hours)
from PVA-Cys-NO-bFGF hydrogels at pH 7.4, 37°C.
2 5 Detailed Description of the Invention
I. Polymeric Materials for Release of NO
The polymeric materials are biocompatible and release or produce
NO. In various preferred embodiments, the polymers are also biodegradable,
form hydrogels, polymerize in situ and are tissue adherant. These properties
3 0 are conferred by the selection of the macromer components as well as
addition of various groups to the components.
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The term "polymerizable" means that the regions have the capacity to
form additional covalent bonds resulting in macromer interlinking, for
example, carbon-carbon double bonds of acrylate-type molecules. Such
polymerization is characteristically initiated by free-radical formation
resulting from photon absorption of certain dyes and chemical compounds to
ultimately produce free-radicals, although it can be obtained using other
methods and reagents known to those skilled in the art.
A. Polymeric Materials
The polymeric materials must be biocompatible, i.e., not eliciting a
significant or unacceptable toxic or immunogenic response following
administration to or implantation into an individual.
A number of polymeric materials are known which are
biocompatible, including both natural and synthetic polymers. Examples
include proteins (of the same origin as the recipient), polysaccharides such
as
chondroitin sulfate and hyaluronic acid, polyurethanes, polyesters,
polyamides, and acrylates. Polymers can be degradable or non-degradable.
Most polymeric materials will be selected based on a combination of
properties conferred by the various components, which may include a water
soluble regions such as PEG or PVA, biodegradable regions such as regions
2 0 that degrade hydrolytically, and groups that can be used to polymerize the
macromers in situ.
Water-Soluble and/or Tissue Adhesive Regions
There are a variety of water soluble materials that can be
incorporated into the polymers. The term "at least substantially water
soluble" is indicative that the solubility should be at least about 5 g/100 ml
of
aqueous solution. In preferred embodiments, the core water soluble region
can consist of polyethylene glycol), polyethylene oxide), polyvinyl
acetate), polyvinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline),
polyethylene oxide)-co-poly(propyleneoxide) block copolymers,
3 0 polysaccharides or carbohydrates such as hyaluronic acid, dextran, heparan
sulfate, chondroitin sulfate, heparin, or alginate, or proteins such as
gelatin,
collagen, albumin, or ovalbumin.
Hydrophilic (i.e., water soluble) regions will generally be tissue
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adhesive. Both hydrophobic and hydrophilic polymer including large
number of exposed carboxylic groups will be tissue or bioadhesive. Ligands
such as RGD peptides and lectins which bind to carbohydrate molecules on
cells can also be bound to the polymer to increase tissue adhesiveness.
Degradable Regions
Polyesters (Holland et aL, 1986 Controlled Release, 4:155-180) of a-
hydroxy acids (viz., lactic acid, glycolic acid), are the most widely used
biodegradable materials for applications ranging from closure devices
(sutures and staples) to drug delivery systems (U.S. Patent No. 4,741,337 to
Smith et al.; Spilizewski et al., 1985 J. Control. Rel. 2:197-203). In
addition
to the poly(hydroxy acids), several other polymers are known to biodegrade,
including polyanhydrides and polyorthoesters, which take advantage of
labile backbone linkages, as reported by Domb et al., 1989 Macromolecules,
22:3200; Heller et al., 1990 Biodegradable Polymers as Drug Delivery
Systems, Chasin, M. and Langer, R., Eds., Dekker, New York, 121-161.
Polyaminoacids have also been synthesized since it is desirable to have
polymers that degrade into naturally occurring materials, as reported by
Miyake et al., 1974, for in vivo use.
The time required for a polymer to degrade can be tailored by
2 0 selecting appropriate monomers. Differences in crystallinity also alter
degradation rates. Due to the relatively hydrophobic nature of these
polymers, actual mass loss only begins when the oligomeric fragments are
small enough to be water soluble. Hence, initial polymer molecular weight
influences the degradation rate.
2 5 The biodegradable region is preferably hydrolyzable under in vivo
conditions. Hydrolyzable groups may be polymers and oligomers of
glycolide, lactide, s-caprolactone, other a-hydroxy acids, and other
biologically degradable polymers that yield materials that are non-toxic or
present as normal metabolites in the body. Preferred poly(a-hydroxy acids
3 0 are poly(glycolic acid), poly(DL-lactic acid) and poly(L-lactic acid).
Other
useful materials include poly(amino acids), poly(anhydrides),
poly(orthoesters), and poly(phosphoesters). Polylactones such as poly(e-
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caprolactone), poly(s-caprolactone), poly(b-valerolactone) and poly(gamma-
butyrolactone), for example, are also useful.
Biodegradable regions can also be constructed from polymers or
monomers using linkages susceptible to biodegradation by enzymes, such as
ester, peptide, anhydride, orthoester, and phosphoester bonds. Degradable
materials of biological origin are well known, for example, crosslinked
gelatin. Hyaluronic acid has been crosslinked and used as a degradable
swelling polymer for biomedical applications (U.S. Patent No. 4,987,744 to
delta Valle et al., U.S. Patent 4,957,744 to Delta Valle et al. (1991) Polym.
l0 Mater. Sci. Eng., 62:731-735]).
Biodegradable Hydrogels
A number of polymers have been described which include both water
soluble regions and biodegradable regions. Sawhney et al., (1990) J. Biomed.
Mater. Res. 24:1397-141 l, copolymerized lactide, glycolide and E-
caprolactone with PEG to increase its hydrophilicity and degradation rate.
U.S. Patent No. 4,716,203 to Casey et al. ( 1987) synthesized a PGA-PEG-
PGA block copolymer, with PEG content ranging from 5-25% by mass.
U.S. Patent No. 4,716,203 to Casey et al. (1987) also reports synthesis of
PGA-PEG diblock copolymers, again with PEG ranging from 5-25%. U.S.
2 o Patent No. 4,526,938 to Churchill et al. (1985) described noncrosslinked
materials with MW in excess of 5,000, based on similar compositions with
PEG; although these materials are not water soluble. Cohn et al. (1988) J.
Biomed. Mater. Res. 22:993-1009 described PLA-PEG copolymers that
swell in water up to 60%; these polymers also are not soluble in water, and
2 5 are not crosslinked. The features that are common to these materials is
that
they use both water-soluble polymers and degradable polymers, and that they
are insoluble in water, collectively swelling up to about 60%.
U.S. Patent No. 5,410,016 issued on April 25, 1995 to Hubbell, et al.,
describes materials which are based on polyethylene glycol (PEG), because of
3 0 its high biocompatible and thromboresistant nature, with short polylacdde
extensions to impart biodegradation and acrylate termini to allow rapid
photopolymerization without observable heat production. These materials are
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readily modified to produce hydrogels which release or produce NO.
The polymerizable regions are separated by at least one degradable
region to facilitate uniform degradation in vivo. There are several variations
of these polymers. For example, the polymerizable regions can be attached
directly to degradable extensions or indirectly via water soluble
nondegradable sections so long as the polymerizable regions are separated by
a degradable section. For example, if the macromer composition contains a
simple water soluble region coupled to a degradable region, one
polymerizable region may be attached to the water soluble region and the
l0 other attached to the degradable extension or region. In another
embodiment, the water soluble region forms the central core of the macromer
composition and has at least two degradable regions attached to the core. At
least two polymerizable regions are attached to the degradable regions so
that, upon degradation, the polymerizable regions, particularly in the
polymerized gel form, are separated. Conversely, if the central core of the
macromer composition is formed by a degradable region, at least two water
soluble regions can be attached to the core and polymerizable regions
attached to each water soluble region. The net result will be the same after
gel formation and exposure to in vivo degradation conditions.
2 0 In another embodiment, the macromer composition has a water
soluble backbone region and a degradable region affixed to the macromer
backbone. At least two polymerizable regions are attached to the degradable
regions, so that they are separated upon degradation, resulting in gel product
dissolution. In a further embodiment, the macromer backbone is formed of a
2 5 nondegradable backbone having water soluble regions as branches or grafts
attached to the degradable backbone. Two or more polymerizable regions
are attached to the water soluble branches or grafts. In another variation,
the
backbone may be star shaped, which may include a water soluble region, a
biodegradable region or a water soluble region which is also biodegradable.
3 0 In this general embodiment, the star region contains either water soluble
or
biodegradable branches or grafts with polymerizable regions attached
thereto. Again, the polymerizable regions must be separated at some point
by a degradable region.
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Polymerizable groups.
The polymerizable regions are preferably polymerizable by
photoinitiation by free radical generation, most preferably in the visible or
long wavelength ultraviolet radiation. The preferred polymerizable regions
are acrylates, diacrylates, oligoacrylates, dimethacrylates,
oligomethoacrylates, or other biologically acceptable photopolymerizable
groups. A preferred tertiary amine is triethanol amine.
Useful photoinitiators are those which can be used to initiate by free
radical generation polymerization of the macromers without cytotoxicity and
within a short time frame, minutes at most and most preferably seconds.
Preferred dyes as initiators of choice for LWUV initiation are ethyl eosin,
2,2-dimethoxy-2-phenyl acetophenone, other acetophenone derivatives, and
camphorquinone. In all cases, crosslinking and polymerization are initiated
among copolymers by a light-activated free-radical polymerization initiator
such as 2,2-dimethoxy-2-phenylacetophenone or a combination of ethyl
eosin (10'x-10-2 milliM) and triethanolamine (0.001 to 0.1 M), for example.
The choice of the photoinitiator is largely dependent on the
photopolymerizable regions. For example, when the macromer includes at
least one carbon-carbon double bond, light absorption by the dye causes the
2 0 dye to assume a triplet state, the triplet state subsequently reacting
with the
amine to form a free radical which initiates polymerization. Preferred dyes
for use with these materials include eosin dye and initiators such as 2,2-
dimethyl-2-phenylacetophenone, 2-methoxy-2-phenylacetophenone, and
camphorquinone. Using such initiators, copolymers may be polymerized in
2 5 situ by long wavelength ultraviolet light or by laser light of about 514
nm,
for example.
Initiation of polymerization is accomplished by irradiation with light
at a wavelength of between about 200-700 nm, most preferably in the long
wavelength ultraviolet range or visible range, 320 nm or higher, most
3 0 preferably about 514 nm or 365 nm.
There are several photooxidizable and photoreducible dyes that may
be used to initiate polymerization. These include acridine dyes, for example,
acriblarine; thiazine dyes, for example, thionine; xanthine dyes, for example,
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rose bengal; and phenazine dyes, for example, methylene blue. These are
used with cocatalysts such as amines, for example, triethanolamine; sulphur
compounds, for example, RS02R~; heterocycles, for example, imidazole;
enolates; organometallics; and other compounds, such as N-phenyl glycine.
Other initiators include camphorquinones and acetophenone derivatives.
Thermal polymerization initiator systems may also be used. Such
systems that are unstable at 37°C and would initiate free radical
polymerization at physiological temperatures include, for example,
potassium persulfate, with or without tetramethyl ethylenediamine;
1 o benzoylperoxide, with or without triethanolamine; and ammonium persulfate
with sodium bisulfate.
Other initiation chemistries may be used besides photoinitiation.
These include, for example, water and amine initiation schemes with
isocyanate or isothiocyanate containing macromers used as the
polymerizable regions.
Preferred Embodiments
In the preferred embodiment, the polymeric materials are a
biodegradable, polymerizable and at least substantially water soluble
macromer composition. The first macromer includes at least one water
2 0 soluble region, at least one NO carrying region and at least one free
radical-
polymerizable region. The second macromer includes at least one water
soluble region and at least two free radical polymerizable regions. The
regions can, in some embodiments, be both water soluble and biodegradable.
The macromer composition is polymerized by exposure of the polymerizable
2 5 regions to free radicals generated, for example, by photosensitive
chemicals
and dyes.
Examples of these macromers are PVA or PEG-oligoglycolyl-
acrylates. The choice of appropriate end caps permits rapid polymerization
and gelation. Acrylates are preferred because they can be polymerized using
3 0 several initiating systems, e.g., an eosin dye, by brief exposure to
ultraviolet
or visible light. A poly(ethyleneglycol) or PEG central structural unit (core)
is preferred on the basis of its high hydrophilicity and water solubility,
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accompanied by excellent biocompatibility. A short oligo or poly(a-
hydroxy acid), such as polyglycolic acid, is selected as a preferred chain
extension because it rapidly degrades by hydrolysis of the ester linkage into
glycolic acid, a harmless metabolite. Although highly crystalline
polyglycolic acid is insoluble in water and most common organic solvents,
the entire macromer composition is water-soluble and can be rapidly gelled
into a biodegradable network while in contact with aqueous tissue fluids.
Such networks can be used to entrap and homogeneously disperse water-
soluble drugs and enzymes and to deliver them at a controlled rate. Further,
they may be used to entrap particulate suspensions of water-insoluble drugs.
Other preferred chain extensions are polylactic acid, polycaprolactone,
polyorthoesters, and polyanhydrides. Polypeptides may also be used. Such
"polymeric" blocks should be understood to include timeric, trimeric, and
oligomeric blocks.
PVA contains many pendant hydroxyl groups. These hydroxyl
groups are easily reacted to form side chains such as various crosslinking
agents and nitric oxide donors. PVA is water soluble and has excellent
biocompatiblity. Modification of PVA to attach methacrylate groups via a
diacetal bond with the pendant hydroxyl groups and addition of an
2 0 appropriate photoinitiator enables the PVA to be photopolymerized to form
hydrogels under long wavelength UV light. In another preferred
embodiment, the hydrogel is formed from modified polyvinyl alcohol (PVA)
macromers, such as those described in U.S. Patent Nos. 5,508,317,5,665,840,
5,849,841, 5,932,674, 6,011,077, 5,939,489, and 5,807,927. The macromers
disclosed in U.S. Patent No. 5,508,317, for example, are PVA prepolymers
modified with pendant crosslinkable groups, such as acrylamide groups
containing crosslinkable olefinically unsaturated groups. These macromers
can be polymerized by photopolymerization or redox free radical
polymerization, for example. The starting polymers are, in particular,
3 0 derivatives of polyvinyl alcohol or copolymers of vinyl alcohol that
contain,
for example, a 1,3-diol skeleton. The crosslinkable group or the further
modifier can be bonded to the starting polymer skeleton in various ways, for
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example through a certain percentage of the 1,3-diol units being modified to
give a 1,3-dioxane, which contains a crosslinkable radical, or a further
modifier in the 2-position. Another possibility is for a certain percentage of
hydroxyl groups in the starting polymer to be esterified by means of an
unsaturated organic acid, these ester-bonded radicals containing a
crosslinkable group. The hydrophobicity of these macromers can be
increased by substituting some of the pendant hydroxyl groups with more
hydrophobic substituents. The properties of the macromers, such as
hydrophobicity, can also be modified by incorporating a comonomer in the
macromer backbone. The macromers can also be formed having pendant
groups crosslinkable by other means.
B. NO groups or Modulating Compounds
A number of molecules that produce NO under physiological
conditions (NO donors) have been identified and evaluated both in vitro and
in vivo, including S-nitrosothiols, organic nitrates, and complexes of NO
with nucleophiles. L-arginine is a NO donor, since L-arginine is a substrate
for NO synthase, and thus administration of L-arginine increases endogenous
NO production and elicits responses similar to those caused by NO donors in
most cases. Other NO donors include molsidomine, CAS754, SPM-5185,
and SIN-1. Other compounds capable of producing and/or donating NO may
also be used. These include organic nitrates, nitrosylating, compounds,
nitrosoesters, and L-arginine.
The molecules which produce NO, or release or generate NO, are
preferably attached to regions containing nucleophiles and/or thiols such as
2 5 S-nitrosothiols capable of forming a complex with NO.
C. Prophylactic, Therapeutic and Diagnostic Agents
The polymeric materials can also be used for drug delivery,
preferably localized release of prophylactic, therapeutic or diagnostic agents
at the site where the materials are needed, although the polymeric materials
3 0 can be loaded with agent to be released systemically. These agents include
proteins or peptides, polysaccharides, nucleic acid molecules, and simple
organic molecules, both natural and synthetic. Representative materials
include antibiotics, antivirals, and antifungal drugs, anti-inflammatories
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(steroidal or non-steroidal), hormones, growth factors, cytohines, neuroactive
agents, vasoconstrictors and other molecules involved in the cardiovascular
responses, enzymes, antineoplastic agents, local anesthetics, antiangiogenic
agents, antibodies, drugs affecting reproductive organs, and oligonucleotides
such as antisense oligonucleotides. Diagnostic materials may be radioactive,
bound to or cleave a chrornogenic substrate, or detectable by ultrasound, x-
ray, mri, or other standard imaging means.
These agents can be mixed with macromer prior to polymerization,
applied into or onto the polymer, or bound to the macromer prior to or at the
time of polymerization, either covalently or ionically, so that the agent is
released by degradation (enzymatic or hydrolytic) or diffusion at the site
where the polymer is applied.
II. Methods of Use
A. Coatings; Films; Microparticles
Although described primarily with respect to in vivo treatment, it is
apparent that the polymeric materials described herein can be used in cell
culture, on cell culture substrates, or as coatings on medical implants or
devices such as stems or catheters, or formed using standard techniques into
microparticles or other types of formulations which may be used in or
2 0 administered to a patient.
B. Therapeutic Applications
Polymeric materials capable of releasing physiological amounts of
NO for prolonged periods of time can be applied to sites on or in a patient in
need of treatment thereof. Representative disorders or conditions that can be
2 5 treated with NO include restenosis, thrombosis, asthma, wound healing,
arthritis, and penile or female erectile dysfunction. The material is
typically
applied as a macromer solution and polymerized in situ, although
polymerization can be initiated prior to application.
Wound Xealing
3 0 The formulations are particularly useful for treatment of wounds,
such as ulcers and burns. ... all types of wounds, including burns, surgical
wounds, and open leg and foot wounds. There are generally three types of
open leg wounds, termed ulcers: venous stasis ulcers, generally seen in
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sedentary elderly people when blood flow to the leg becomes sluggish;
decubitus ulcers, also termed pressure sores or bed sores, which occurs most
often in people who are bedridden and are unable to frequently change
position; and diabetic foot ulcers, caused by poor blood circulation to the
feet. Due to the aging of the population, there will likely be a greater
demand for effective and user friendly wound treatments in the near future.
The term "wound" as used herein refers to all types of tissue injuries,
including those inflicted by surgery and trauma, including burns, as well as
injuries from chronic or acute medical conditions, such as atherosclerosis or
diabetes.
Treatment of Restenosis
A preferred application is a method of reducing the effects of
restenosis on post-surgical patients. The method includes coating the surface
within an artery with an aqueous solution of light-sensitive free radical
polymerizable initiator and a number of macromers. The coated artery is
subjected to a Xenon arc laser inducing polymerization of the macromers.
As the newly polymerized macromer composition is formed, the
physiological conditions within the artery will induce the release of NO. This
release will be strictly localized for prolonged periods of time.
2 0 Prevention of Surgical Adhesions.
A preferred application is a method of reducing formation of
adhesions after a surgical procedure in a patient. In one embodiment the
method includes coating damaged tissue surfaces in a patient with an
aqueous solution of a light-sensitive free-radical polymerization initiator
and
2 5 a macromer solution as described above. The coated tissue surfaces are
exposed to light sufficient to polymerize the macromer. The light-sensitive
free-radical polymerization initiator may be a single compound (e.g., 2,2-
dimethoxy-2-phenyl acetophenone) or a combination of a dye and a
cocatalyst (e.g., ethyl eosin and triethanol amine).
3 0 Tissue Adhesives.
Another use of the polymers is in a method for adhering tissue
surfaces in a patient. In one embodiment the macromer is mixed with a
photoinitiator or photoinitiator/cocatalyst mixture to form an aqueous
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mixture and the mixture is applied to a tissue surface to which tissue
adhesion is desired. The tissue surface is contacted with the tissue with
which adhesion is desired, forming a tissue junction. The tissue junction is
then irradiated until the macromers are polymerized.
Tissue Coatings.
In a particularly preferred application of these macromers, an
ultrathin coating is applied to the surface of a tissue, most preferably the
lumen of a tissue such as a blood vessel. One use of such a coating is in the
treatment or prevention of restenosis, abrupt reclosure, or vasospasm after
vascular intervention. An initiator is applied to the surface of the tissue,
allowed to react, adsorb or bond to tissue, the unbound initiator is removed
by dilution or rinsing, and the macromer solution is applied and polymerized.
This method is capable of creating uniform polymeric coating of between
one and 500 microns in thickness, most preferably about twenty microns,
which does not evoke thrombosis or localized inflammation.
Tissue Supports.
The polymeric materials can also be used to create tissue supports by
forming shaped articles within the body to serve a mechanical function.
Such supports include, for example, sealants for bleeding organs, sealants for
2 0 bane defects and space-fillers for vascular aneurisms. Further, such
supports
can include strictures to hold organs, vessels or tubes in a particular
position
for a controlled period of time.
Controlled drug delivery.
As noted above, the polymeric materials can be use as carriers for
2 5 biologically active materials such as therapeutic, prophylactic or
diagnostic
agents, including hormones, enzymes, antibiotics, antineoplastic agents, and
cell suspensions. The polymeric material may be used to temporarily
preserve functional properties of an agent to be released, as well as provide
prolonged, controlled release of the agent into local tissues or systemic
3 0 circulation.
In a variation of the method for controlled drug delivery in which
agent is mixed with the macromer solution then polymerized in situ, the
macromers are polymerized with the biologically active materials to form
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microspheres or nanoparticles containing the biologically active material.
The macromer, photoinitiator, and agent to be encapsulated are mixed in an
aqueous mixture. Particles of the mixture are formed using standard
techniques, for example, by mixing in oil to form an emulsion, forming
droplets in oil using a nozzle, or forming droplets in air using a nozzle. The
suspension or droplets are irradiated with a light suitable for
photopolymerization of the macromer.
These materials are particularly useful for controlled drug delivery of
hydrophilic materials, since the water soluble regions of the polymer enable
access of water to the materials entrapped within the polymer. Moreover, it
is possible to polymerize the macromer composition containing the material
to be entrapped without exposing the material to organic solvents. Release
may occur by diffusion of the material from the polymer prior to degradation
and/or by diffusion of the material from the polymer as it degrades,
depending upon the characteristic pore sizes within the polymer, which is
controlled by the molecular weight between crosslinks and the crosslink
density. Deactivation of the entrapped material is reduced due to the
immobilizing and protective effect of the gel and catastrophic burst effects
associated with other controlled-release systems are avoided. When the
2 0 entrapped material is an enzyme, the enzyme can be exposed to substrate
while the enzyme is entrapped, provided the gel proportions are chosen to
allow the substrate to permeate the gel. Degradation of the polymer
facilitates eventual controlled release of free macromolecules in vivo by
gradual hydrolysis of the terminal ester linkages.
2 5 III. Examples
As demonstrated by examples 1-3, three classes of NO-producing,
PEG-based polymers have been synthesized and their NO release rate
constants determined in vitro under physiological conditions. The biological
response to appropriate materials has been evaluated in vitro using cultured
3 0 smooth muscle cells and endothelial cells and in vivo using a rat carotid
artery injury model that resembles restenosis in man. The materials include
BAB block copolymers of polyethylene glycol (A) with polycysteine (B) that
are subsequently reacted with NaN02 to form S-nitrosothiols, BAB block
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copolymers of polyethylene glycol ("PEG") (A) and diethylenetriamine
("DETA") (B) that are subsequently reacted with NO gas to form
nucleophile/NO complexes, and BAB block copolymers of polyethylene
glycol {A) and polylysine (B) that are subsequently reacted with NO gas to
form nucleophile/NO complexes. All polymers are further terminated with
reactive acrylate groups to allow rapid photopolymerization in situ.
Such materials would be expected to have good biocompatibility,
provided that a water soluble, biocompatible polymer such as PEG
comprises the bulk of the material and has a sufficiently high molecular
weight, and to slowly biodegrade due to the presence of two ester bonds and
two amide bonds in each polymer chain. These three materials were selected
as they are expected to have vastly different release kinetics: nucleophile/NO
complexes have been shown to release NO for up to 5 weeks (Smith el al.,
1996), while the half life of S-nitrosocysteine is 0.023 hours (Mathews et
al.,
1993). The amount of NO produced by these copolymers may be tailored by
altering the ratio of polyethylene glycol (PEG) to cysteine or lysine.
An advantage of these macromer compositions are that they can be
polymerized rapidly in an aqueous surrounding. Precisely conforming, semi-
permeable, biodegradable films or membranes can thus be formed on tissue
2 0 in situ to serve as biodegradable barners, as carriers for living cells or
other
biologically active materials, and as surgical adhesives. The polymer shows
excellent biocompatibility, as seen by a minimal fibrous overgrowth on
implanted samples. Hydrogels for the models were gelled in situ from
water-soluble precursors by brief exposure to long wavelength ultraviolet
(LWUV) light, resulting in formation of an interpenetrating network of the
hydrogel with the protein and glycosaminoglycan components of the tissue.
As demonstrated by examples 4 and 5, three types of PVA hydrogels
were made and demonstrated release of NO and incorporated drug (bFGF):
PVA-NH2-NO hydrogels; PVA-Cys-NO hydrogels; PVA-NO-bFGF
3 0 hydrogels. The results are similar to those for the PEG based hydrogels.
Example 1: Synthesis of PEG-Cys Macromers
As shown in Figure 1, an acryloyl-PEG-CYSNO polymer was
formed by first reacting polyethylene glycol N-hydroxysuccinimide
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monoacrylate (ACRL-PEG-NHS, MW 3400, commercially available from
Shearwater Polymers, Huntington, AL) with L-cysteine at an 1:2 molar ratio
in 50 mM sodium bicarbonate buffer (pH 8.5) far 2 hours; the product was
then dialyzed in a cellulose ester membrane (Molecular weight cutoff 500,
Spectrum Labs, Laguna Hills, CA) in diH20, and lyophilized. Analysis of
the acryloyl-PEG-Cys copolymer was performed using gel permeation
chromatography {GPC) with an evaporative light scattering detector and a
UV detector at 260 nm (Polymer Laboratories, Amherst, MA). Successfi~l
synthesis of acryloyl-PEG-Cys was determined by a shift in the position of
the peak from the evaporative light scattering detector. The copolymer was
then reacted with an equimolar amount of NaN02 at pH 2 and 37°C for 20
minutes to form S-nitrosocysteine. Conversion of thiol groups to S-
nitrosothiols was measured using the Ellman's assay (Hermanson, 1995).
After adjusting the pH of the solution to 7.4, the acryloyl-PEG-CYSNO
polymer was incorporated into photopolymerizable hydrogels by mixing
with PEG-diacrylate (MW 3400) at a 1:10 molar ratio in aqueous solution
with 1500 ppm 2,2-dimethoxy-2-phenyl acetophenone as a long wavelength
ultraviolet initiator. 0.15% N-vinyipyrrolidone was present in this mixture
as it was used as a solvent for the photoinitiator. Exposure to UV light (365
2 0 nm, 10 mW/cm2) was used to crosslink the polymer, resulting in conversion
to a hydrogel (Sawhney et al., 1993). Production of NO by the hydrogels
was quantified using the Griess assay.
Example 2: Synthesis of PEG-Lys Macromers.
As shown in Figure 2, for acryloyl-PEG-Lyss-NO hydrogels, a
copolymer of ACRL-PEG-NHS (MW 3400, Shearwater Polymers) and
poly-L-lysine {DP=5) was synthesized by reacting at an equimolar ratio in
50 mM sodium bicarbonate (pH 8.5). The resultant copolymer was
analyzed via GPC, then dissolved in water and reacted with NO gas in an
evacuated vessel, thus forming NO-nucieophile complexes with the amine
3 0 groups on the lysine side groups. The extent of conversion of amine groups
to NO-nucleophile complexes was measured using the ninhydrin assay, and
crosslinked hydrogels were formed as described above in Example 1.
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Example 3: Synthesis of DETA-NO-nucleophile complex hydrogels.
Diethylenetriamine (DETA, Aldrich, Milwaukee, WI) was reacted
with ACRL-PEG-NHS (MW 3400, Shearwater Polymers) in 50 mM sodium
bicarbonate buffer (pH 8.5) at an equimolar ratio, lyophilized, and analyzed
via GPC as described above. The copolymer was then dissolved in water
and exposed to NO gas to form NO-nucleophile complexes as described for
PEG-Lyss-NO and assayed for amine content using the ninhydrin assay. The
PEG-DETA-NO was lyophilized and then photopolymerized as described
above to form hydrogels, as shown in Figure 3.
l0 Example 4: Synthesis of PVA-NH2-NO hydrogels
Polyvinyl alcohol) (Hoechst, Mowiol 4-88) was dissolved in diH20
and warmed to 95°C in a round bottom Mask under continuous stirring.
After
one hour, the solution was cooled to room temperature, and a crosslinkable
acetal group, methacrylamidoacetaldehyde dimethyl acetal (NAAADA) was
added. The amine acetal, gamma-aminobutyraldehyde diethyl acetal, was
also added, and the mixture was acidified using glacial acetic acid and 37%
hydrochloric acid. The mixture was allowed to stir at room temperature for
nine hours, after which the pH was adjusted to pH 3.6 using triethylamine.
In order to purify the polymer, the solution was then diafiltered through a
2 o MW 3000 cellulose membrane against diH20 at 6.5 times the volume of
polymer solution. The polymer concentration was adjusted to 22% w/v
using diafiltration, and the pH was adjusted to 7.4 with 1N NaOH. The
amine concentration of the polymer was determined using the ninhydrin
assay.
In order to form the NO donor bound to the PVA-NH2, the
neutralized amine-modified polymer was placed in a round bottom flask with
stopcock. The flask was evacuated and filled with nitric oxide gas until the
desired conversion of amines to NO nucleophile complexes was achieved.
Photocrosslinked hydrogels were formed from the PVA-NH2-NO by adding
3 0 0.1 % Irgacure 2959 (Ciba-Geigy) photoinitiator (based on total solution
volume) and then exposing to UV light (2 mW/cmz, 365 nm) for 30 seconds.
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Addition of the photoinitiator brings the final polymer concentration to 20%
w/v.
Example 5: Synthesis of PVA-Cys-NO hydrogels
PVA-NH2 was synthesized as described above. The amine terminus
of cysteine was acetylated using acetic anhydride, and the carboxyl end of
the cysteine was coupled to the PVA-NH2 using water-soluble EDAC
chemistry. The resulting PVA-Cys was then purified using diafiltration and
brought to a concentration of 22% w/v. PVA-Cys-NO was formed by
adding sodium nitrite at an equimolar amount to cysteine residues, adjusting
the pH to 2, and incubating at 37°C for 15 minutes. The extent of
reaction of
cysteine to Cys-NO was assayed using both the Ellman's and Griess
reactions. The photoinitiator, 2,2-methyl-2-phenylacetophenone was
dissolved in N-vinylpyrrolidone at a concentration of 600 mg/ml and added
to the polymer solution (0.1 % based on total solution volume). The polymer
was then crosslinked under UV light for 30 seconds and placed in HEPES
buffered saline, pH 7.4, 37°C.
Example b: Release of bFGF from PVA-NO-bFGF hydrogels.
For PVA-NO-bFGF hydrogels, the above procedure was used to
make the PVA-NO polymer. Immediately prior to exposure to UV light, 25
2 0 ~,g/ml bFGF was added to the polymer solution and mixed well. Gels were
crosslinked as described earlier and stored in HEPES buffered saline, pH 7.4,
37°C. Release of bFGF was quantified using the BCA assay (Pierce
Chemicals), and NO release was assayed using the Griess reaction.
Example 7: NO-release Rates from acryloyl-PEG-Lyss-NO hydrogels
2 5 Following preparation and photopolymerization of the NO-releasing
materials as described above, the hydrogels were weighed and stored in
HEPES buffered saline, pH 7.4, at 37°C. Aliquots of the buffer
were
removed at each time point and replaced with fresh buffer. The samples
from each time point were then analyzed for nitrite content using a
3 0 colorimetric assay based on the Griess reaction.
NO release kinetics of hydrogels stored in buffer at various pH levels
were also investigated in order to explore possible storage conditions for the
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hydrogels. At acidic pH levels, release of NO from the hydrogels was
significantly inhibited.
NO release from acryloyl-PEG-Lyss-NO hydrogels is shown in
Figure 4.
NO release from acryloyl -PEG-DETA NO hydrogels is shown in
Figure S.
NO release from acryloyl-PEG-CYSNO hydrogels is shown in Figure
6.
Example 8: NO-release Rates from PVA-NO-bFGF hydrogels
The release of NO release from PVA-NO-bFGF hydrogels was
determined in the same manner as Example 7 and is shown in Figure 7.
Figures 12a and 12b. respectively, show the temporal release of NO
and a growth factor, bFGF, over time from PVA-NO-bFGF hydrogels.
Release of NO continues for well over 12 hours, while the growth factor is
completely released within the first 5 hours.
Example 9: Effects of NO-releasing Macromers on Cultured Smooth
Muscle Cells: Proliferation and Viability
In order to assess the potential of a material for the reduction of
smooth muscle cell proliferation after vascular injury, cultured smooth
2 0 muscle cells were grown in the presence of NO-releasing materials, and the
effects of those materials on the cells evaluated. Smooth muscle cells
isolated from Wistar-Kyoto rats (passage 11-1S, provided by T. Scott-
Burden) were cultured in Minimum Essential Medium supplemented with
10% FBS, 2 mM L-glutamine, S00 units penicillin, and 100 mg/L
streptomycin, at 37°C in a S% C02 environment. The cells were seeded
into
24-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) at a
density of 10,000 cells/cm2. NO donors in either soluble or hydrogel form
were added to the media in the wells one day after seeding. At 4 days
culture, cell numbers were determined by preparing single cell suspensions
3 0 with trypsin and counting three samples from each group using a Coulter
counter (Multisizer #0646, Coulter Electronics, Hialeah, FL).
The effects of NO donors in solution on the proliferation of SMCs
were first investigated by performing a NO dose response curve, whereupon
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cells were cultured with a range of NO donor concentrations ( 1 pM - 10
mM) in order to identify appropriate dosages for hydrogel studies. NO-
nucleophile complexes (Lys-NO and DETA-NO) were formed by reacting
either L-lysine or DETA with NO gas in water for 24 hours. Soluble Cys-
NO was synthesized by reacting an equimolar amount of L-cysteine with
NaN02 at pH 2 and 37°C for 20 minutes. All NO donor solutions were
adjusted to pH 7.4 prior to addition to cell cultures.
Smooth muscle cell proliferation in the presence of NO-producing
and control hydrogeis was then investigated using the optimal NO dose
determined above. Hydrogels containing acryloyl-PEG-Lys-NO, acryloyl-
PEG-DETA-NO, and acryloyl-PEG-CYSNO were formed as described
above, except that the gel solutions were sterile filtered through 0.2 ~tm
syringe filters (Gelman Sciences, Ann Arbor, MI) prior to adding 2,2-
dimethoxy-2-phenyl acetophenone. PEG-diacrylate hydrogels containing no
NO donors were used as a control. The hydrogels were photopolymerized in
cell culture inserts (8 pm pore size, Becton Dickinson, Franklin Lakes, NJ)
and placed in the media over the cultured cells.
. All three hydrogel NO donors significantly inhibited SMC growth
(p < 0.0001 ). The number of smooth muscle cells remained near that of the
seeding density, which ranged from 10-15% of the final control cell number
for all experiments.
Inhibition of SMC proliferation by acryloyl-PEG-Lyss-NO hydrogels
is shown in Figure 8A, compared to the macromer solution control shown in
Figure 8B. Both significantly inhibited SMC proliferation.
2 5 Inhibition of SMC proliferation by acryloyl-PEG-DETA-NO-
nucleophile complex hydrogels is shown in Figure 9A, compared to the
macromer solution control shown in Figure 9B. Both significantly inhibited
SMC proliferation.
Inhibition of SMC proliferation by acryloyl-PEG-CYSNO hydrogels
3 0 is shown in Figure 10A, compared to the macromer solution control shown
in Figure l OB. Both significantly inhibited SMC proliferation.
Inhibition of SMC proliferation by acryloyl-PEG-CYSNO hydrogels,
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acryloyl-PEG-DETA-NO hydrogels, and acryloyl-PEG-Lys-NO hydrogels is
compared to the control hydrogel in Figure 11. All of the NO hydrogels
significantly inhibited SMC growth.
Example 5: Effects of NO-releasing Macromers on Platelet Adhesion
in vitro
The effect of NO release on platelet adhesion was investigated to
assess the potential of these materials for prevention of thrombosis. Blood
was obtained from a healthy volunteer by venipuncture and anticoagulated
with 10 U/ml heparin. Platelets and white blood cells were fluorescently
labeled with mepacrine at a concentration of 10 pM. A solution of 2.5
mg/ml collagen I in 3% glacial acetic acid in diH20 was prepared and
applied to glass slides for 45 minutes in a humidified environment at room
temperature. Acryloyl-PEG-CYSNO and PEG-diacrylate hydrogels were
prepared as described above and incubated with the labeled whole blood at
37°C for 30 minutes. The hydrogels were removed and the blood was then
incubated with the collagen-coated glass slides (two per group) for 20
minutes at 37°C and then rinsed with HBS. Platelet counts per field of
view
at 40x were counted under a fluorescent microscope (Zeiss Axiovert 135,
Thornwood, NY) in four randomly chosen areas per slide.
2 0 Photos of platelets which had been exposed to control PEG-diacrylate
or acryloyl-PEG-CYSNO hydrogels demonstrate that exposure to the NO-
releasing hydrogels inhibits platelet adhesion to thrombogenic surfaces.
Glass slides coated with collagen were used as a thrombogenic surface to
which platelets would normally adhere. When the blood was incubated with
control PEG-diacrylate hydrogels, 69.25 f 4.46 (mean ~ SD) adherent
platelets were observed per field of view. This number was reduced to 7.65
f 6.16 platelets pre field of view when blood was pre-exposed to the
acryloyl-PEG-CYSNO hydrogels (p < 0.0001).
24
