Note: Descriptions are shown in the official language in which they were submitted.
WO 01/70199 CA 02403818 2002-09-20 PCT/USO1/08806
MATRICES CONTAINING NITRIC OXIDE DONORS
AND REDUCING AGENTS AND THEIR USE
BACKGROUND OF THE INVENTION
This invention relates to matrices that release nitric oxide. In particular,
the invention
relates to matrices containing a compound that releases nitric oxide (NO) and,
optionally, a
reducing agent that promotes NO release from the matrix. The invention also
relates to uses of
such matrices.
There is a widespread need for techniques that improve surface properties of
blood-
contacting surfaces, e.g. to prevent platelet aggregation and neutrophil
adhesion, and to prevent
infection, which can result in deleterious effects. By modifying blood-contact
properties of
such surfaces, one can reduce or eliminate the need for systemic anti-
coagulation therapy,
extend the life expectancy of long-term implanted blood-contacting devices
such as vascular
grafts, and improve the performance of shorter-term interventional devices,
such as urinary and
vascular catheters.
Invasive therapy such as vascular catheterization can be complicated by local
infection
and induced sepsis, which usually causes the failure of the therapy and is
often life-threatening.
About 6% ~ 10% catheters used for long-term venous access become infected
(Bernard RW, et
al., "Subclavian vein catheterization: a prospective study. II. Infectious
complications," Ann
Surg 173:191, 1971; Uldall PR, Joy C, Merchant N., "Further experience with a
double-lumen
subclavian cannula for hemodialysis, Trans Am Soc Artif Intern Organs 28:71,
1982).
The catheter can allow microorganisms to gain access directly into the
patient's
vascular system. Biomaterials may alter host humoral and cellular immune
response. The
relatively hydrophobic property of the biomaterial makes it easy for bacteria
to adhere to its
surface. Endoscopic catheters and instruments suffer similar problems. Efforts
have been made
to reduce catheter infection, such as modifying the biomaterial surface to
diminish bacterial
adhesion, and binding antibiotics to the surface of biomaterials. However,
none of these has
been successfully used in clinical practice, and administering antibiotics
systemically is
unsatisfactory. Catheter-induced infection still remains a problem to be
solved.
As early as 1927, Warburg's study suggested that nitric oxide could reversibly
and
irreversibly inhibit the respiratory enzyme of yeast cells, that reversible
inhibition could
restrain bacteria growth, and that irreversible inhibition may kill bacteria.
(Warburg, O., 1927,
"The inhibition of carbon monoxide and of nitric oxide on respiration and
fermentation,"
Biochem Z. 189:354-380). There has been little progress on this front.
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Another common complication from the use of inserted devices or devices used
for
extracorporeal flow of bodily fluids is platelet aggregation and
thrombogenesis. There are
several known techniques which have been dried to reduce thrombogenicity of
medical devices
by surface modification or coating. Several types of heparin coatings
(covalent and ionic) have
been produced. The performance of these coatings has been disappointing and
none have been
accepted for routine clinical practice. Phosphorylcholine coatings, marketed
by
Biocompatibles, Ltd., and described in U.S. 5,658,561, are at a very early
stage of development
and have not been well demonstrated.
Another technique to prevent thrombogenesis is release of NO from polymer
films
containing nitroso-containing compounds. Espadas-Torre, C., et al.,
"Thromboresistant
chemical sensors using combined nitric oxide release/ion sensing polymeric
films," J. Am.
Chem. Soc., 1997, 119:2321-2322. Nitric oxide-containing compounds may be
characterized
into several groups. (1) N-nitroso compounds are stable and do not readily
release NO absent
hydrolysis. In addition, N-nitroso compounds present risks of carcinogenicity.
(2) A variety of
S-nitrosothiols are known to generate NO in vivo. (3) C-nitroso compounds tend
to be stable
and release NO at body temperature, as in Rosen et al., U.S. 5,665,077. (4)
Nitrosyl-containing
organometallic compounds are described in Rosen et al., U.S. 5,797,887.
According to the
latter patent, decomposition of a nitrosyl-containing organometallic compound,
such as
nitroprusside, into NO is restricted by a polymer coating with a small
porosity that inhibits the
diffusion of blood-borne reductants to the NO-releasing compound; yet this
small porosity
allows NO to diffuse through the polymer into the surrounding fluid. There is
a need for
matrices demonstrating enhanced release of NO.
Green, U.S. 5,944,444, describes release of NO from biodegradable polymer
matrices
containing nitrites in an acid environment. The picomolar concentrations of NO
released are
undesirably low, and are not sustained over time. The requirement of a low pH
environment is
inconsistent with use at physiological pH as in blood and other tissues.
Green et al., US patent 5,814,666, describes N-nitroso compounds (NONOates)
that
release NO with antimicrobial effect upon hydrolysis when injected or
ingested. Use of
NONOates is incompatible with generating NO by reduction.
Polymer matrices containing porosigens taught in the prior art, e.g., Eury, et
al., U.S.
5,605,696, designed to facilitate the release of the therapeutic drug from the
polymer coating
into the vasculature, are unsatisfactory for enhancing nitric oxide release
from nitric oxide
donors.
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Nitroprusside (as in, for example, sodium nitroprusside or SNP) has drawbacks
when
administered systematically as a NO donor, including short biological half
time and systemic
effects. There is a need for techniques that would prolong SNP biological
effects and limit
SNP effects to a local area.
Folts et al., WO 95/07691, describes using S-nitroso and other NO adducts
mixed with
bovine serum albumin on blood-contacting surfaces to inhibit platelet
deposition. Such
compositions are not biostable and allow the NO adduct to leach into the
blood.
SUMMARY OF THE INVENTION
The invention relates to compositions that release NO and uses thereof. The
self
contained system of the invention may be used as a drug delivery device or a
coating on a
medical device that contacts blood or other body fluids to bring about
biological effects.
Desired biological effects include preventing aggregation of platelets and
inhibiting
proliferation of tissue within or near the device (which could decrease
functioning of the
device), and antimicrobial effects. Further, the favorable effects of the NO
release include
reducing damage caused by the device itself, and providing a broadened
therapeutic benefit.
The invention provides a composition comprising a biostable preferably
hydrophobic
matrix, a reducible NO donor, and an intrinsic reductant reactably associated
together with the
reducible NO donor within the matrix, that may release an effective amount of
NO from the
matrix when wetted in a target medium for a sustained period, independently of
the presence or
absence of extrinsic reducing agents, and inhibits the release of the NO
donor. This invention
does not require an acidic pH to release NO from the donor, as is the case in
Green, U.S.
5,944,444. The target liquid is preferably at physiological pH. Preferred
target media include
biological fluids, particularly blood.
The matrix may comprise a polymer. Nitric oxide donors may be nitrosyl-
containing
organometallic compounds, or S-nitroso compounds. Preferably, the NO donor is
a reducible
NO donor such as sodium nitroprusside or S-nitrosoglutathione and may be
present in an
amount between about 0.1 % and about SO% and preferably from about 1 % to
about 10%.
Reductants that may be suitable for use in the composition of the invention
include ascorbic
acid, cysteine, glutathione, penicillamine, N-acetylcysteine, iodine,
hydroquinone,
mercaptosuccinic acid, thiosalicylic acid, methylthiosalicylic acid,
dithiothreitol,
dithioerythritol, 2-mercaptoethanol, and FeCl2. Other reductants presently
known or hereafter
discovered may be used it they are compatible with the NO donor. The reductant
is preferably
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present in a concentration from about 0.1% to about 25% and preferably between
about 1% and
about 10%.
In another aspect, the invention is a medical device having as a blood-
contacting
surface a composition comprising a hydrophobic matrix, a reducible NO donor,
and an intrinsic
reluctant reactably associated together with the reducible NO donor within the
matrix, that
may release an effective amount of NO from the matrix when wetted in a target
medium for a
sustained period, independently of the presence or absence of extrinsic
reducing agents. The
composition of the blood contacting surface of the medical device may be one
in which the NO
donor is nitroprusside or S-nitrosoglutathione, the matrix comprises silicone,
and the reluctant
is ascorbic acid, cysteine, glutathione, penicillamine, N-acetylcysteine,
glutathione,
mercaptosuccinic acid, thiosalicylic acid, methylthiosalicylic acid,
dithiothreitol,
dithioerythritol, 2-mercaptoethanol or FeCl2.
In yet another aspect, the invention is a composition comprising means for
releasing
NO in the presence of a reducing agent, and means for incorporating the NO
releasing
compound with a reducing agent together in a hydrophobic matrix.
In an additional aspect, the invention is a method for improving the
performance of a
device in a target medium by providing the device with a surface comprising a
hydrophobic
matrix comprising a compound that releases NO in the presence of a reluctant,
and associated
therewith a reluctant, the matrix being capable of releasing NO into the
target medium in an
amount effective to produce a desired effect. The desired effect may be to:
inhibit cell
proliferation, retard growth of cancer cells, act as a second messenger in
stimulating host
immune response toward bacteria, viruses, fungi, parasites and other microbes
and cancer cells,
promote gastrointestinal motility, stimulate penile erection, relax the uterus
during pregnancy,
dilate blood vessels, inhibit platelet adhesion, aggregation, and activation,
inhibit neutrophil
adhesion, and regulate smooth muscle tone. Inhibition of target cell growth is
particularly
preferred.
In still a further aspect, the invention is a method comprising providing a
first
compound that releases NO when reduced, providing a second compound that
reduces the first
compound, the first and second compounds being associated together within a
hydrophobic
matrix, contacting the hydrophobic matrix with a target medium, allowing the
second
compound to reduce the first compound so as to produce NO, and selectively
allowing the NO
to be released from the matrix into the target medium.
In still another aspect, the solid matrix is at the surface of a device, and
the step of
providing the solid matrix may comprise coating the surface of a device with
the solid matrix.
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The contacting step may comprise inserting the solid matrix into the target
medium, or if the
solid matrix coats an internal surface of a container such as a vessel or
tubing, the contacting
step preferably comprises placing the biological medium into the container.
The matrix may
optionally be withdrawn from the biological medium. The device may be an
interventional
medical device such as a urinary tract catheter or blood catheter.
The method is effective where the biological medium has a non-acid pH, such
that NO
is released at a non-acid pH, or a physiological pH (typically neutral or
above, although lower
in some tissues). Nitric oxide production from the NO donor is not pH
dependent.
The solid hydrophobic matrix preferably consists essentially of a matrix
forming solid
and the nitric oxide donor, or the matrix may comprise a reductant reactably
associated with
the nitric oxide donor. The solid matrix is preferably formed by a hydrophobic
polymer, which
may be one or more selected from the group consisting of silicone,
polyvinylchloride,
polystyrene, PMMA, polyolefins, and polytetrafluorocarbons. In one embodiment,
toxic
byproducts are produced with the nitric oxide from the nitric oxide donor and
the solid matrix
inhibits release of the toxic byproducts.
The nitric oxide donor is preferably nitroprusside. The NO donor may be
S-nitrosoglutathione. One or more donors may be used depending on the
circumstances.
A biological medium is a preferred target medium. The biological medium is
preferably a biological fluid such as blood or urine or interestitial fluid.
It may be a non-fluid
tissue such as skin, cells, or a urethral lining.
The target cells are preferably one or more selected from the group consisting
of
bacteria, fungi, virally infected cells, parasitic microorganisms, and cancer
cells. The method
is preferably effective such that the growth rate inhibition is at least about
25%, preferably
about SO%, or greater than about 90%. In most preferred embodiments, the
method kills target
cells. More particularly, the method may extend the length of time for 50% of
saturation to
occur (TSO) in a growth medium by 25%, 50%, double, or longer. The method may
reduce the
count of cells that grow on a surface such as an interventional catheter
within a given period by
25%, SO%, or 90%. Most preferably, the method completely prevents growth of
cells on such
surfaces.
In other aspects of the invention, a method comprises: providing a device
coated with a
solid hydrophobic matrix comprising a NO donor retained within the matrix; and
contacting
the coated device with a target medium containing target cells; the NO donor
reacting within
the matrix to produce NO, and the NO, but not the NO donor, being released
from the solid
hydrophobic matrix and thereby inhibiting growth of target cells in the
vicinity of the device.
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A method of using a medical device in a biological medium according to the
invention
comprises: a step for achieving contact between the medical device and the
target medium; a
step for producing NO non-hydrolytically from a NO donor within a solid matrix
at the surface
of the device; and a step for releasing NO from the device in the target
medium over a
sustained period without releasing the NO donor, in an amount effective to
inhibit growth of
target cells in the target medium.
According to an embodiment of the invention, a target medium contacting
surface is
provided that releases NO, thereby having improved properties such as that it
is less
susceptible to thrombosis and infection, and thus has reduced occlusion and
lower likelihood of
failure. Compositions that include Nitrosyl-containing organometallic
compounds or S-
nitrosothiols that release NO upon reaction with a reductant may be reactably
associated with a
reductant in a matrix, preferably a hydrophobic polymer, present as a coating
or at a device
surface. In these systems, the nitrosyl-containing organometallic compound is
preferably
nitroprusside, the S-nitrosothiol is preferably S-nitrosoglutathione, the
hydrophobic polymer
matrix preferably comprises silicone, and the reductant is preferably ascorbic
acid or
glutathione. The coating inhibits the diffusion into the polymer matrix of
blood-borne
reductants, but is nonetheless able to release NO without exposure to light or
hydrolysis.
A further embodiment of the invention envisions providing a tissue contacting
surface
that releases NO, thereby having improved properties such that it is less
susceptible to
infection, and has lower likelihood of failure by for example, inhibiting cell
proliferation such
as myointimal hyperplasia. Such a coating is able to release NO without
hydrolysis. Nitric
oxide may be generated by reduction, thermolysis, nucleophilic decomposition,
electrophilic
decomposition, catalysis and combinations thereof. Reduction is a preferred
pathway for
generating nitric oxide; thus, preferred nitric oxide releasing compositions
include a reductant.
The claimed invention relies on a specific kind of NO donor: a therapeutic
agent
precursor that produces NO in therapeutic amounts, such as SNP or S-
nitrosoglutathione
(GSNO). Preferred compositions include a reductant such as ascorbate, retained
together with
the NO donor in the matrix. Decomposition of SNP or GSNO by ascorbic acid
within the
matrix produces a by-product, NO. It is NO, not SNP or GSNO, which diffuses
from within
the polymer into the blood stream or other bodily fluids.
Advantages of this invention include:
1) Toxic byproducts of NO donor decomposition, such as cyanide in the case of
nitroprusside, may be trapped in the coating, preventing or reducing toxic
response to these
byproducts.
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2) Release of effective amounts of NO according to the invention occurs within
a
controlled solid matrix, and does not involve releasing the NO donor into the
biological
medium to generate NO there, under poorly controllable conditions.
Additional advantages of compositions according to the invention that contain
a
reducing agent in the matrix include:
3) NO release does not depend on exterior reducing agents, light or
hydrolysis. It can
provide a controlled release of NO by varying the concentration of the
reductant in the polymer
that is applied onto the surface of implanted devices and catheters.
4) The inventive methods of using coatings and devices permit more accurate
design
and control of NO release than was previously possible. The release is
independent of the
individual patient's metabolic conditions. There is preferably no need for
light, hydrolysis or
additional coating components to bring about NO release.
The invention differs from the prior art in the use of nitrosyl-containing
organometallic
compounds, S-nitroso compounds, and C-nitroso compounds as nitric oxide-
releasing
antimicrobial agents, in a device coating with a biostable matrix that
includes and retains such
compounds, where the device exhibits cytotoxic or cytostatic effects.
This invention provides advantages that were not previously appreciated,
including the
possibility of exactly controlling the NO release pattern without regard to
individual patient
blood characteristics or hydrolytic pathways for generating NO and the
possibility of reducing
systemic use of antibiotics in conjunction with invasive medical procedures.
This invention satisfies a long felt-need for insertable medical devices that
do not
promote infection, and can instead reduce microbial growth and promote other
desirable
properties. This invention is contrary to the teachings of the prior art such
as Green, US patent
5,814,666 which disfavored nitrosyl-containing organometallic compounds such
as sodium
nitroprusside because they require activation to release NO.
In some compositional aspects, this invention differs from the prior art in
modifications
that were not previously known or suggested. The compositions in the prior art
lack reductants
along with NO donors, release NO donors into the target medium or release NO
donors
through hydrolytic pathways.
Compositions of the invention satisfy a long felt need for a composition that
releases
NO in a controlled pattern. This invention is contrary to the teachings of the
prior art in that it
associates nitroso-containing compounds with reductants in the polymer to
release NO,
whereas the prior art taught inhibiting the ability of reductants to diffuse
into the polymer.
WO 01/70199 - CA 02403818 2002-09-20 PCT/USO1/08806
S Further objectives and advantages will become apparent from a consideration
of the
description, drawings, and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is better understood by reading the following detailed
description with
reference to the accompanying figures and tables, in which like reference
numerals refer to like
elements throughout, and in which
Figure 1 shows NO release from SNP/Si coating containing 1 % L-ascorbic acid
(LAA)
in the dark.
Figure 2 shows NO release from SNP/Si coating containing 10% L-ascorbic acid
(LAA) in the dark.
Figure 3 shows NO release from GSNO/Si coating containing 3% L-ascorbic acid
(LAA) in the dark.
Figure 4 shows the inhibitory effects of SNP/Si coating on S. aureus growth.
Figure 5 shows the inhibitory effects of SNP/Si coating on S. aureus growth
starting
from a lower bacterial concentration.
Figure 6 shows the inhibitory effects of SNP/Si coating on E. coli growth.
Figure 7 shows the inhibitory effects of SNP/Si coating on E. coli growth
starting from
a lower bacterial concentration.
Figure 8 shows that SNP and GSNO with a reducing agent inhibits growth of
bacteria.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In describing preferred embodiments of the present invention illustrated in
the
drawings, specific terminology is employed for the sake of clarity. However,
the invention is
not intended to be limited to the specific terminology so selected. It is to
be understood that
each specific element includes all technical equivalents which operate in a
similar manner to
accomplish a similar purpose. Each reference cited here is incorporated by
reference as if each
were individually incorporated by reference.
A device according to the invention may be a medical, veterinary, or
laboratory device
having a surface that contacts a biological medium in use. These include blood
vessel and
urinary tract implants such as catheters, stems, intracorporeal or
extracorporeal blood circuits,
endoscopy equipment, insertable laparascopic devices, implants of bone,
polymer, metal, or
composites, artificial joints, membranes, tubing, grafts, and other devices
inserted into
biological media. The materials from which these devices may he made include
plastic,
_g_
WO 01/70199 CA 02403818 2002-09-20 PCT/USO1/08806
stainless steel, nitinol, dacron, polytetrafluoroethylene, and countless other
materials known to
practitioners.
A "NO donor" refers to a compound that releases NO on decomposition. A
"reducible
NO donor" refers to a nitrosyl-containing compound that releases NO in the
presence of a
reducing agent under the mild conditions encountered within a biostable
hydrophobic polymer
matrix. In general, NO donors include reducible NO donors and others.
A target cell is any cell or cell population that is targeted for growth
inhibition or
killing. Examples include bacteria, fungi, viruses, parasitic microorganisms,
cancer cells, and
cells that are foreign or undesirable in a patient animal such as a human or
animal. Growth
inhibition means that the method results in a growth rate slower than that
which would be
present in the absence of the inventive method. The extent of inhibition may
be small or
complete, and the method may involve killing cells (reversing the growth of
the population).
The target medium is one that does not prevent the NO donor from reacting
within the
matrix to produce NO and release it into the medium. Nitric oxide is generally
considered
hydrophobic. Typically the target medium is a biological medium, such as an
aqueous liquid
like blood, urine, interstitial fluid, or cell growth medium in vitro. The
liquid is preferably at
physiologic pH or is pH neutral, i.e. having a pH greater than about 5, and
most preferably has
a pH of about 7 or slightly above, such as blood. The medium may also be
tissue such as skin,
internal tracts, or interstitial tissue.
Nitrosyl-containing organometallic compounds, such as sodium nitroprusside,
are
readily susceptible to reduction, and are preferred. S-nitroso compounds, such
as S-
nitrosoglutathione, may be paired with a suitable reducing agent in a matrix
according to the
invention, and are preferred as well. Preferably, the release of NO from the
NO donor is not
pH dependent. The practitioner will be able to use such nitrosyl-containing
organometallic or
S-nitroso compounds, selecting those that generate NO in the presence of a
reducing agent and
a hydrophobic matrix, without toxic byproducts.
The reaction which generates NO from a NO donor is preferably non-hydrolytic
because there is no water present or limited amounts present in the solid
phase of the biostable
matrix. For reducible NO donors, NO is generated and released in effective
amounts by
reduction, although other mechanisms may also operate to a limited extent,
such as photolysis,
thermolysis, hydrolyis, or other mechanisms. This is in contrast to use of
nitrites and
NONOates, and other compounds that generate NO primarily by hydrolysis.
Reductive
degradation of reducible NO donors in the presence of reductants according to
the invention
does not preclude generating NO to some extent by other mechanisms.
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Reducing agents according to the invention include ascorbic acid and others
that are
effective to reduce the reducible NO donor in the polymer matrix. The
reductant must be
selected to be compatible with the reducible NO donor. Examples of other
reducing agents
include cysteine, penicillamine, N-acetylcysteine, glutathione,
mercaptosuccinic acid,
thiosalicylic acid, methylthiosalicylic acid, dithiothreitol,
dithioerythritol, 2-mercaptoethanol,
and FeClz.
A biostable matrix according to the invention is preferably hydrophobic, that
is, one
that absorbs a limited amount of water, preferably less than 10-20%, although
other, less
hydrophobic polymers absorbing 50% or 100% of their weight in water, or
higher, may also be
suitable according to the invention. Any biostable matrix is useable as long
as it retains the
nitric oxide donor, reductant, if present, and other reactants and by-
products, while releasing
nitric oxide, and prevents unwanted or uncontrolled reactions resulting from
water penetration.
The matrix may be hydrated before contacting the biological medium. Polymer
matrices are
preferred for their simplicity, although ceramic or other types of alloys
could accomplish the
same function. Silicone is a preferred polymer. Other hydrophobic polymer
examples include
but are not limited to: PVC, polystyrene, polymethylmethacrylate (PMMA),
polyolefins,
polyfluorocarbons, etc. When reducible NO donors are used, the hydrophobic
matrix must
entrap and retain the reducible NO donor and reductant together in a reactive
relationship so
they are not released in a significant amount, but must permit the NO to be
released. For
example, a polyurethane matrix releases ascorbic acid and is therefore
incompatible with the
inventive compositions absent modification according to the invention.
The matrix is biostable in that it is not appreciably biodegradable or
bioabsorbable.
The matrix inhibits release of the reductant, the NO donor, toxic and other
reactants and
byproducts during an effective period of use from several minutes to several
months,
preferably at least about 12 hours, and more preferably at least about one
day.
The matrix is biostable, meaning that it does not degrade in the target medium
particularly when the target medium is a biological medium. Of course, the
stability relates to
the medium and some media and uses require a more durable matrix. If the
matrix is not
sufficiently stable it will either physically wear off or dough off, or
dissolve, or degrade
chemically in the medium, yielding uncertain dosage and uncontrolled release
of NO donor and
by-products. The matrix is selected so that it can retain the NO donor and
reductant for an
effective product life, allow them to react to produce NO, and allow the NO to
be released
from the matrix. Thus, the invention employs a self contained solid phase NO
releasing system
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that is not dependent on the nature of the target medium or reactions that may
occur in it, to
produce desirable biological effects.
The invention permits effective concentrations of NO to be released into a
physiological environment over a sustained period. The amount of components
released from
the matrix into a medium depends on their concentration, the rate of release,
and time. It is
important that there is no deleterious effect from the release of any
component from the matrix,
either on the medium itself, or in terms of interfering with desirable effects
of NO. The matrix
inhibits the release of the NO donor and preferably there is no release of
other components
such as the optional reductant, NO donor, or byproducts other than NO that
would cause a
discernable deleterious effect or interference with the NO.
Preferably, the amount of NO released is greater than about 10 nmoles.
Sustained
release in this context means that the concentration does not drop below a
threshold of
effectiveness and/or remains within a certain proportion of the initial
concentration for a
suitable period. For example, in some applications it is desirable that the
concentration not
drop by more than one order of magnitude, e.g., 1 nmole, over a two week
period. In other
applications the period of sustained release may need to be shorter (e.g.
minutes) or longer (e.g.
months). In yet other applications, the effective range may be broader.
In its compositional aspects, the invention provides a new NO releasing
mechanism.
The NO donor, preferably nitroprusside or S-nitrosoglutathione, reacts with
the intrinsic
reducing agents, and generates NO at a more rapid rate than that described in
Rosen, U.S.
5,797,877. Nitric oxide is released, and nitroprusside, for instance and
reducing agents, as well
as the byproducts of nitroprusside decomposition, are trapped in the polymer
matrix. This NO
releasing mechanism is confirmed by the following experimental results
detailed in the
examples:
1. Pores created by washing out lactose did not improve NO release from SNP in
a
silicone coating.
2. A SNP/silicone coating plus L-ascorbic acid (either 1 % or 10%) did release
NO
in the dark.
3. A GSNO/silicone coating plus L-ascorbic acid (LAA, 3%) did release NO in
the
dark, and release of NO was considerably greater than GSNO in the absence of
L-ascorbic acid.
Thus, the reducible NO donors, SNP and GSNO, when incorporated into a silicone
coating with reducing agents release NO at a rate greater than SNP or GSNO
alone. They are
also cytostatic and/or cytotoxic.
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S The antimicrobial method aspect of the invention is intended not to produce
toxicity to
healthy cells of the target animal or patient in in vivo applications. The
effective amount of NO
to be released depends on the target cells, the target medium, and the desired
degree of
inhibition or killing, and the sensitivity of the host tissue, as can readily
be determined by a
person of ordinary skill. Specifically excluded from the meaning of inhibition
of target cell
growth in this context is inhibition of platelet aggregation as known in U.S.
Patent No.
5,797,887, which is not a proliferation cell growth phenomenon. Thus, the
inventive method
relates to inhibition of non-platelet target cell growth. In this application,
inhibition of platelet
aggregation and anti-restenosis effects are referred to specifically but not
as inhibition of target
cell growth.
1 S The invention is better understood upon consideration of the following non-
limiting
examples illustrating preferred embodiments of the invention. Periods skilled
in the art may
identify other embodiments which are within the scope of the invention upon
consideration of
the examples.
EXAMPLES
Materials
Silicone
RTV-12A O1G, from GE, Batch# HB156
RTV-12C OlP, from GE, Batch# HD213
L-Ascorbic Acid, from Sigma, Lot# 48H1038
L-Cysteine, from Sigma, Lot # 107H09382
Glutathione, from Sigma, Lot# 48H3502
Sodium nitroprusside (SNP), from Sigma, Lot# 96H3502
S-nitroso-L-glutathione, Lot #125H4124
Sulfanilamide, from Sigma, Lot# 77H0150
N-(1-Naphthyl)ethylenediamine, from Aldrich, Lot# 01715LW
24 well untreated tissue culture plate from Becton Dickinson Labware Lot#
17348
Phosphate buffered saline (PBS) from Sigma, Lot 88H6073 (NaCI 120 mM, KCl 2.7
mM, and phosphate buffer 10 mM, pH 7.4 at 25°C)
Griess Reagents
RT1: dissolve 5 g Sulfanilamide in 500 ml 5% H3P04
RT2: dissolve 0.5 g N-(1-Naphtyl) ethylenediamine in 500 ml distilled water.
Mix RT1 and RT2 in the ratio 1:1 before use.
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Methods
Method 1 Plate coating
RTV-12A and RTV-12C were mixed in a ratio of 20:1 (v/v) and 0.2 ml of the
silicone
mixture was added to wells of a 24 well plate. Other additives, such as SNP,
GSNO, reducing
agents, or lactose were added in different experiments. The coating procedure
was done at
room temperature and in reduced light.
Method 2 Nitrite assay
Accumulation of nitrite was determined colorimetrically by mixing 0.5 mL each
of
culture medium and freshly prepared Griess reagent [0.1% N-(1-
naphthyl)ethylenediamine in
water and 1 % sulfanilamide in 5% phosphoric acid, mixed 1:1 ] (Green, et al.,
Anal. Biochem
126, 131-138, 1982.). Concentrations of nitrite were estimated by comparing
absorbance at
550 nanometers against standard solutions of sodium nitrite prepared in the
same medium.
Nitrite indicates presence of nitric oxide and/or nitroprusside.
EXAMPLE 1
SNP, a NO donor according to the invention, is retained within a solid
silicone matrix,
even if it is rendered porous by including lactose as a porosigen in the
matrix and then washing
out the lactose. Lactose (1% and 10%, w/v) was added to SNP and silicone
mixtures that were
added to wells of a 24 well plate. PBS was added to each of the coated wells.
The plate was
wrapped with foil and placed in the dark. A sample was collected every 24
hours for nitrite
assay, and the buffer was replaced with fresh PBS. No significant nitrite
concentrations were
detected in the samples over a ten-day test period. The results demonstrate
that even with
voids left from washed out lactose, a silicone matrix did not release SNP into
the medium.
EXAMPLE 2
The reducing agent L-ascorbic acid improves NO generation from a hydrophobic
matrix
containing the NO donor, SNP. L-ascorbic acid was added to a SNP/Si coated
surface. In the
same experimental conditions as mentioned above, that is, in the dark, SNP/Si
plus L-ascorbic
acid coatings released NO in a dose-dependent manner (Figures 1 and 2). Nitric
oxide
production reached a peak at 7-8 days with 1 % and 10% L-ascorbic acid. Peak
concentrations
were 32 ~M and 150 ~M, respectively.
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The effectiveness of L-ascorbic acid in increasing NO release is in contrast
to the lack
of effect of lactose, as shown above. These data suggest that porosigen
effects did not
contribute to NO produced in SNP/Si plus L-ascorbic acid coatings.
Further, there is evidence to show that SNP/Si plus L-ascorbic acid coatings
release NO
rather than SNP itself. First, SNP without reductant is not released as shown
above.
Second, if SNP itself were being released, a first order decline should be
observed day
by day as the NO donor concentration in the matrix diminishes. To the
contrary, in this
experiment, NO release into the fresh buffer increases with time, which is
inconsistent with
leaching of SNP from the matrix. Rather, there is a second order effect
perhaps as NO
accumulates in the matrix, although the mechanism is unclear.
EXAMPLE 3
The reducing agent L-ascorbic acid improves NO generation from a hydrophobic
matrix containing the nitric oxide donor, GSNO. L-ascorbic acid was added to a
GSNO/Silicone coated surface. In the same experimental conditions as mentioned
above, that
is, in the dark, GSNO/Silicone produced only 2 ~M of NO after 1 day. In
contrast,
GSNO/Silicone plus L-ascorbic acid coated surface released 10 ~M NO after 1
day (Figure 3).
EXAMPLES 4-5
Materials and Methods
Tryptic Soy Agar (4% w/v) and Tryptic Soy Broth (30% w/v), Becton Dickinson,
containing digested casein, soy powder, and dextrose
VWR Sterile Petri Dish (Polystyrene), 100 X 15 mm
Flask Coating: Silicones RTV 12A and RTV 12 C were mixed in a ratio 20:1
(v/v).
SNP powder was mixed with RTV mixture; 10 ml RTV mixture or 10 ml SNP/RTV
mixture
was put into each flask and cured 24 hours in dark. All procedures were
performed in reduced
light and room temperature.
Nitric oxide release from SNP/Si coating: The coated flask was filled with
PBS, or
TSB 15 ml. The flasks were placed in a shaking incubator, shaking speed 200
RPM @ 37°C.
Samples were collected for nitrite assay. A curve of accumulation of nitrite
was generated.
Bacterial growth curve: 15 ml TSB was placed in each flask. Equal amount of
bacteria
was added to each flask. The flasks were placed in a shaking incubator,
shaking 200 RPM @
37°C. Samples were collected for O.D. measurement. An accumulation
curve were generated.
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Bacterial growth on agar: 4 grams TSA was dissolved in distilled water, and
autoclaved
at 121°C for 15 minutes. When the agar cooled to ~SO°C, 15 ml
agar was placed into each tube,
and equal amounts of bacteria were added to each. Then the agar and bacteria
mixture was cast
on culture dishes. The dishes were placed into an incubator @ 37°C. The
clone number was
counted at 24 hours.
EXAMPLE 4
SNP/silicone coatings inhibit bacteria growth. Flasks were coated with
silicone
containing 1%, 5%, and 10% SNP (w/v). A flask coated with only silicone was
used as control
(see method 1). Light absorbency was measured (@ 600 nm) to evaluate bacteria
growth.
Figures 4 and 5 present the results of experiments with S. aureus. Figures 6
and 7 show
the results of experiments with E. coli. A very high titer of bacteria, about
400,000 cells, was
transferred to each flask (Figures 4, 6). Compared with control, SNP/Si
coating inhibits the
growth of S. aureus and E. coli in a dose-dependent manner. At even 100 times
higher starting
concentration of bacteria, a dose-dependent effect was still noted, but the
effect was less
dramatic than shown in Figures 4 and 6 due to saturation. These experiments
were repeated
with about 1000 bacteria introduced at the beginning (Figures 5, 7). Here, the
presence of SNP
at S% produced dramatic inhibition of bacterial growth. These results show
that 1) SNP/Si
coating inhibits bacteria growth, both S. aureus and E. coli (gram-positive
and gram-negative,
respectively); 2) the inhibition is SNP concentration-dependent; and 3) the
inhibition effect is
related to bacteria number -- higher concentrations of SNP, and presumably of
NO, are needed
to inhibit very high bacterial number.
It was noted that there was NO release from SNP/silicone in TSB at SNP
concentrations as low as 1 %. In contrast, in PBS, NO was released at 10% SNP.
This
establishes that different concentrations of NO donor may be required to
achieve effective
concentrations in different biological systems.
EXAMPLE 5
Nitric oxide release from SNP inhibits bacterial growth on agar. Agar
containing
different concentrations of SNP was used to test the effects of NO release
from SNP on
bacteria growth. Both S. aureus and E. coli were tested. After 24 hours
culture, bacteria
number were counted. No bacteria were found in the dishes containing 5% and
10% SNP.
Bacterial numbers in dishes of control and 1 % SNP were counted. With both S.
aureus and E.
coli, the experiment showed that 1 % SNP inhibits both strains of bacteria,
significantly, and
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S% and 10% SNP kill S. aureus and E. coli completely; no bacterial growth was
observed.
These results support the existence of a dose-dependent relationship between
release of NO
from a nitrosyl-containing organometallic compound and cell growth inhibition.
The results
also support the use of matrices that are less hydrophobic than silicone.
EXAMPLE 6
Segments of polyurethane catheter for extracorporeal blood dialysis (available
from
Bard Access) were coated by dipping in a solution of silicone in
tetrahydrofuran and with or
without the other components, and allowed to dry. The dipping process was
repeated three
times. The coatings tested were: 1 ) silicone, as control; 2) silicone plus 1
% (w/v) L-ascorbic
acid (AA) as control; 3) silicone plus 5% (w/v) SNP and 1 % AA; and 4)
silicone plus 1 % S-
nitrosoglutathione (GSNO) (Sigma) and 1 % AA. The coated catheter segments
were placed in
15 ml plastic test tubes containing 10 ml Tryptic Soy Broth. An equal amount
of E. coli was
added to each tube. The tubes were put in a shaking incubator. The speed was
set at 200 RPM,
and temperature 37°C. Samples were collected for O.D. measurement every
hour. Cumulative
growth curves were plotted.
The experimental results are shown in Figure 4. The controls (silicone and
ascorbic
acid) showed classical growth over a 12-hour period. In contrast, the test
samples were
effective in eliminating growth of bacteria during the time period of the
study. Similar results
would be expected for S. aureus and other microbes. Also, the enhanced release
of NO from
the coated catheter surfaces would have other desirable biological effects
such as preventing
platelet aggregation.
The embodiments illustrated and discussed in this specification are intended
only to
teach those skilled in the art the best way known to the inventors to make and
use the
invention. Nothing in this specification should be considered as limiting the
scope of the
present invention. The above-described embodiments of the invention may be
modified or
varied, and elements added or omitted, without departing from the invention,
as appreciated by
those skilled in the art in light of the above teachings. It is therefore to
be understood that,
within the scope of the claims and their equivalents, the invention may be
practiced otherwise
than as specifically described.
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