U.S. patent application number 09/752223 was filed with the patent office on 2001-09-06 for novel polymers for delivering nitric oxide in vivo.
Invention is credited to Stack, Richard S., Stamler, Jonathan S., Toone, Eric J..
Application Number | 20010020083 09/752223 |
Document ID | / |
Family ID | 22294044 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010020083 |
Kind Code |
A1 |
Stamler, Jonathan S. ; et
al. |
September 6, 2001 |
Novel Polymers for Delivering nitric oxide in vivo
Abstract
Disclosed are novel polymers derivatized with at least one -SNO
group per 1200 atomic mass unit of the polymer. In one embodiment,
the S-nitrosylated polymer has stabilized --S--nitrosyl groups. In
another embodiment the S-nitrosylated polymer prepared by
polymerizing a compound represented by the following structural
formula: 1 R is an organic radical. Each X' is an independently
chosen aliphatic group or substituted aliphatic group. Preferably,
each X' is the same and is a C2-C6 alkylene group, more
preferably--CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. p and m are independently a
positive integer such that p+m is greater than two. The polymers of
the present invention can be used to coat medical devices to
deliver nitric oxide in vivo to treatment sites.
Inventors: |
Stamler, Jonathan S.;
(Chapel Hill, NC) ; Toone, Eric J.; (Durham,
NC) ; Stack, Richard S.; (Chapel Hill, NC) |
Correspondence
Address: |
David E. Brook, Esq.
HAMILTON, BROOK, SMITH & REYNOLDS, P. C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Family ID: |
22294044 |
Appl. No.: |
09/752223 |
Filed: |
December 29, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09752223 |
Dec 29, 2000 |
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09103225 |
Jun 23, 1998 |
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6232434 |
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09103225 |
Jun 23, 1998 |
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08691862 |
Aug 2, 1996 |
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5770645 |
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Current U.S.
Class: |
528/373 ;
424/402; 528/422; 528/425; 604/1 |
Current CPC
Class: |
A61L 2300/416 20130101;
A61L 2300/114 20130101; A61K 31/785 20130101; A61L 33/068 20130101;
B82Y 5/00 20130101; A61L 31/10 20130101; C08L 5/16 20130101; A61L
2300/42 20130101; A61L 31/10 20130101; A61L 29/10 20130101; C08G
75/00 20130101; A61K 47/6951 20170801; C08B 37/0012 20130101; C09D
105/16 20130101; A61L 29/16 20130101; A61L 31/16 20130101; A61L
33/08 20130101; A61K 47/61 20170801; Y10T 428/1393 20150115; C08L
5/16 20130101 |
Class at
Publication: |
528/373 ;
528/422; 528/425; 424/402; 604/1 |
International
Class: |
C08G 075/00 |
Claims
What is claimed is:
1. A polymer derivatized with at least one --SNO group per 1200
atomic mass units of the polymer.
2. The polymer of claim 1 comprising at least one --SNO group per
600 atomic mass units of the polymer.
3. An article capable of releasing NO wherein the article is coated
with a polymer comprising at least one --SNO group per 1200 amu of
the polymer.
4. The article of claim 3 wherein the article is a medical device
for implantation in a subject or a tube or catheter for contacting
the bodily fluid of a subject.
5. A method of delivering nitric oxide to a treatment site in a
subject or to a bodily fluid comprising the steps of: a) providing
a medical device coated with a polymer having at least one --SNO
group per 1200 amu of the polymer; and b) implanting the medical
device at the treatment site or contacting the bodily fluid with
the medical device.
6. A method of preparing an article capable of releasing NO
comprising coating an article with at least one --SNO group per
1200 amu of the polymer.
7. The method of claim 6 wherein the article is a medical device
for delivering nitric oxide to a treatment site in a subject or a
tube or catheter for contacting a bodily fluid of a subject.
8. The polymer of claim 1 wherein the polymer is non-peptidyl.
9. The polymer of claim 2 wherein the polymer is non-peptidyl.
10. A method of replacing a loss of NO groups from an
S-nitrosylated polymer, said method comprising the step of
contacting the S-nitrosylated polymer with an effective amount of a
gaseous nitrosylating agent.
11. The method of claim 10 wherein the gaseous nitrosylating agent
is nitric oxide or nitrosyl chloride.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/103,225, filed Jun. 23, 1998, which is a
continuation-in-part of U.S. application Ser. No. 08/691,862 (now
U.S. Pat. No. 5,770,645), filed Aug. 2, 1996. The entire teachings
of these applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Many modern medical procedures require that synthetic
medical devices remain in an individual undergoing treatment. For
example, coronary and peripheral procedures involve the insertion
of diagnostic catheters, guide wires, guide catheters, PTCA balloon
catheters (for percutaneous transluminal coronary angioplasty) and
stents in blood vessels. In-dwelling sheaths (venous and arterial),
intraaortic balloon pump catheters, tubes in heart lung machines,
GORE-TEX surgical prosthetic conduits and in-dwelling urethral
catheters are other examples. There are, however, complications
which can arise from these medical procedures. For example, the
insertion of synthetic materials into lumen can cause scaring and
restenosis, which can result in occlusion or blockage of the lumen.
Synthetic materials in the blood vessels can also cause platelet
aggregation, resulting in some instances, in potentially
life-threatening thrombus formation.
[0003] Nitric oxide (referred to herein as "NO") inhibits the
aggregation of platelets. NO also reduces smooth muscle
proliferation, which is known to reduce restenosis. Consequently,
NO can be used to prevent and/or treat the complications such as
restenosis and thrombus formation when delivered to treatment sites
inside an individual that have come in contact with synthetic
medical devices. In addition, NO is anti-inflammatory, which would
be of value for in-dwelling urethral or TPN catheters.
[0004] There are, however, many shortcomings associated with
present methods of delivering NO to treatment sites. NO itself is
too reactive to be used without some means of stabilizing the
molecule until it reaches the treatment site. NO can be delivered
to treatment sites in an individual by means of polymers and small
molecules which release NO. However, these polymers and small
molecules typically release NO rapidly. As a result, they have
short shelf lives and rapidly lose their ability to deliver NO
under physiological conditions. For example, the lifetime of
S--nitroso--D, L-penicillamine and S-nitrosocysteine in
physiological solution is no more than about an hour. As a result
of the rapid rate of NO release by these compositions, it is
difficult to deliver sufficient quantities of NO to a treatment
site for extended periods of time or to control the amount of NO
delivered.
[0005] Polymers containing groups capable of delivering NO, for
example polymers containing diazeniumdiolate groups (NONOate
groups), have been used to coat medical devices. However,
decomposition products of NONOates under oxygenated conditions can
include nitrosamines (Ragsdale et al., Inorg. Chem. 4:420 (1965),
some of which may be carcinogenic. In addition, NONOates generally
release NO, which is rapidly consumed by hemoglobin and can be
toxic in individuals with arteriosclerosis. Further, the elasticity
of known NO-delivering polymers is generally inadequate, making it
difficult to coat medical devices with the polymer and deliver NO
with the coated device under physiological conditions. Protein
based polymers have a high solubility in blood, which results in
short lifetimes. Finally, many NO-delivering polymers cannot be
sterilized without loss of NO from the polymer and amounts of NO
delivered are limiting.
[0006] There is, therefore, a need for new compositions capable of
delivering NO to treatment sites in a manner which overcomes the
aforementioned shortcomings.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to novel polymers with
--SNO groups, also referred to as S-nitrosyl groups. Polymers with
--SNO groups are referred to as "S-nitrosylated polymers". The
polymers of the present invention generally have at least one --SNO
group per 1200 atomic mass units (amu) of the polymer, preferably
per 600 amu of the polymer.
[0008] In one embodiment, the S-nitrosylated polymer has stabilized
--S-nitrosyl groups. The polymer generally comprises at least one
stabilized S-nitrosyl group per 1200 amu, and often one stabilized
S-nitrosyl group per 600 amu. An S-nitrosyl group can be stabilized
by a free thiol or a free alcohol from the same molecule. Each
stabilized--SNO group is stabilized by a different free alcohol or
thiol group. Thus, a polymer with a stabilized S-nitrosyl group
generally has at least one free alcohol and/or thiol group per 1200
amu of polymer, preferably per 600 amu of polymer. Another
embodiment of the present invention is an S-nitrosylated polymer
prepared by polymerizing a compound represented by Structural
Formula (I): 2
[0009] R is an organic radical.
[0010] Each X' is an independently chosen aliphatic group or
substituted aliphatic group. Preferably, each X' is the same and is
a C2-C6 alkylene group, more preferably--CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--.
[0011] p and m are independently a positive integer such that p+m
is greater than two. Preferably, p+m is less than or equal to about
8.
[0012] Another embodiment of the present invention is a method of
preparing an S-nitrosylated polymer with stabilized --S-nitrosyl
groups. The method comprises polymerizing a compound represented by
Structural Formula (I).
[0013] In another embodiment of the present invention, the
S-nitrosylated polymer is an S-nitrosylated polythiolated
polysaccharide. Preferably, the polythiolated polysaccharide is a
polythiolated cyclodextrin.
[0014] Another embodiment of the present invention is an
S-nitrosylated polymer prepared by reacting a polythiolated polymer
with a nitrosylating agent under conditions suitable for
nitrosylating thiol groups. Preferably, the polythiolated polymer
is a polythiolated polysaccharide.
[0015] Another embodiment of the present invention is a method of
preparing an S-nitrosylated polymer. The method comprises reacting
a polymer having a multiplicity of pendant thiol groups, i.e., a
polythiolated polymer, with a nitrosylating agent under conditions
suitable for nitrosylating free thiol groups. In a preferred
embodiment, the polythiolated polymer is a polythiolated
polysaccharide.
[0016] Another embodiment of the present invention is an article
which is capable of releasing NO. The article is coated with at
least one of the polymers of the present invention. The article can
be any device for which a useful result can be achieved by NO
release, including a medical device suitable for implantation at a
treatment site in a subject (individual or animal). The medical
device can then deliver nitric oxide to the treatment site in the
subject. In another example, the article is a tube or catheter for
contacting a bodily fluid of a subject.
[0017] Another embodiment of the present invention is a method of
delivering nitric oxide to a treatment site in an individual or
animal. The method comprises providing a medical device coated with
an S-nitrosylated polymer of the present invention. The medical
device is then implanted into the individual or animal at the
treatment site. Nitric oxide can be delivered to a bodily fluid,
for example blood, by contacting the bodily fluid with a tube or
catheter coated with one or more of the polymers of the present
invention.
[0018] Yet another embodiment of the present invention is a method
of preparing an article capable of releasing NO, e.g., a medical
device for delivering nitric oxide to a treatment site in an
individual or animal or a tube or catheter for contacting a bodily
fluid. The method comprises coating the article with an
S-nitrosylated polymer of the present invention.
[0019] Polymers with stabilized S-nitrosyl groups and polymers
obtained by polymerizing compounds represented by Structural
Formula (I) can cause vasodilation in bioassays (Example 16). These
polymers have also been found to deliver NO for extended periods of
time lasting at least several weeks (Example 15). Thus, they are
expected to be useful as coatings on medical devices for
implantation in subjects, thereby delivering NO at treatment
sites.
[0020] Medical devices coated with S-nitrosylated polythiolated
polysaccharides are effective in reducing platelet deposition and
restenosis when implanted into animal models. Specifically, stents
coated with an S-nitrosylated .beta.-cyclodextrin or an
S-nitrosylated .beta.-cyclodextrin complexed with
S-nitroso-N-acetyl-D, L-penicillamine or S-nitroso-penicillamine
resulted in decreased platelet deposition when inserted into the
coronary or cortoid arteries of dogs compared with stents which
lacked the polymer coating (Example 12). It has also been found
that S-nitrosylated .beta.-cyclodextrin and S-nitrosylated
.beta.-cyclodextrin complexed with S-nitroso-N-acetyl-D,
L-penicillamine cause vasodilation in bioassays (Examples 8 and
10). Furthermore, the disclosed S-nitrosylated polysaccharides have
been found to deliver NO-related activity for extended periods of
time and to exhibit increased shelf stability compared with
compounds presently used to deliver NO in vivo.
[0021] A further advantage of the S-nitrosylated polysaccharides is
that they lack the brittleness of other NO-delivering compositions
and have sufficient elasticity to coat and adhere under
physiological conditions to medical devices such as stents.
BREIF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a graph illustrating the number of platelets
deposited per square centimeter on stents coated with
S-nitrosylated P-cyclodextrin and on uncoated control stents which
had been implanted in the arteries of dogs.
[0023] FIG. 2 is a graph illustrating the number of --S-NO groups
per cyclodextrin on the product resulting from the reaction of
per-6-thio-p-cyclodextrin with one (1X), two (2X), three (3X), six
(6X) and ten (10X) equivalents of acidic nitrite.
[0024] FIG. 3 is the visible/ultraviolet spectrum of a reaction
mixture comprising .beta.-cyclodextrin and a 50 fold excess of
acidic nitrite, taken at intervals of (1) 5 minutes, (2) fifteen
minutes, (3) thirty minutes, (4) forty-five minutes, (5) sixty
minutes, (6) seventy five minutes and (7) ninety minutes.
DETAILED DESCRIPTION OF THE INVENTION
[0025] As used herein "polymer" has the meaning commonly afforded
the term. Examples include homopolymers (i.e., polymers obtained by
polymerizing one type of monomer), co-polymers (i.e., polymers
obtained by polymerizing two or more different types of monomers),
including block copolymers and graft copolymers, dendritic
polymers, crosslinked polymers and the like. Suitable polymers
include synthetic and natural polymers (e.g. polysaccharides) as
well as polymers prepared by condensation, addition and ring
opening polymerizations. Also included are rubbers, fibers and
plastics. Polymers can be hydrophilic, amphiphilic or hydrophobic.
The polymers of the present invention are typically non-peptide
polymers.
[0026] A polymer with "stabilized S-nitrosyl groups" comprises,
along with the S-nitrosyl groups, one free thiol group or free
alcohol group for each stabilized S-nitrosyl group and has a
half-life for NO release which is significantly greater than for
the corresponding compound or polymer without free thiol or alcohol
groups (e.g., about two times greater, preferably about ten times
greater). Although Applicants do not wish to be bound by any
particular mechanism, it is believed that an S-nitrosyl group can
be stabilized by the interaction between a free thiol or a free
alcohol group and the --S-nitrosyl group. A stabilizing interaction
can formed, for example, when a free thiol or alcohol is located
within three covalent bonds of (alpha to) an S-nitrosyl group. In
another example, a stabilizing interaction can be formed when a
free thiol or alcohol can be brought within about one to one and a
half bond lengths of an S-nitrosyl group by energetically
accessible conformational rotations of covalent bonds within the
molecule.
[0027] A polymer with stabilized S-nitrosyl groups generally has a
half life for NO release greater than about two hundred hours and
often greater than about one thousand hours. Most known compounds
which release NO have half-lives for NO release that is less than
about twelve hours.
[0028] The term "organic radical", as it is used herein, refers to
a moiety which comprises primarily hydrogen and carbon, but can
also include small amounts of other non-metallic elements such as
sulfur, nitrogen, oxygen and halogens. R, when taken together with
the remainder of the molecule represented by Structural Formula
(I), is a small organic molecule and typically has a molecular
weight less than about 2000 amu, more typically less than 1000 amu.
Thus, the compound represented by Structural Formula (I) is not a
protein, polypeptide or polysaccharide.
[0029] An organic radical can also comprise functional groups which
do not significantly decrease the stability of --S-nitrosyl groups.
Suitable functional groups include those which: 1) are
substantially inert with respect to --S-nitrosyl groups, i.e.,
groups which do not substantially increase the rate, for example,
double the rate, of NO release from NO-releasing molecules; and 2)
do not substantially interfere with the nitrosylation of free thiol
groups, i.e. do not substantially decrease the yield (e.g., a 50%
decrease in yield) of the nitrosylation or cause the formation of
significant amounts of by-products. Examples of suitable functional
groups include alcohols, thiols, amides, thioamides, carboxylic
acids, aldehydes, ketones, halogens, double bonds, triples bonds
and aryl groups (e.g, phenyl, naphthyl, furanyl, thienyl and the
like).
[0030] Aliphatic groups include straight chained, branched or
cyclic C.sub.1-C.sub.8 hydrocarbons which are completely saturated
or which contain one or more units of unsaturation.
[0031] Suitable substituents for an aliphatic group are those
which: 1) are substantially inert with respect to --S-nitrosyl
groups, i.e., groups which do not substantially increase the rate,
for example, double the rate of NO release from NO-releasing
molecules; and 2) do not substantially interfere with the
nitrosylation of free thiol groups, i.e. do not substantially
decrease the yield of the nitrosylation (e.g., a 50% decrease in
yield) or cause the formation of significant amounts of
by-products. Examples of suitable substituents include halogens, C1
-C5 straight or branched chain alkyl groups, alcohols, carboxylic
acids, amides, thioamides, and the like.
[0032] Compounds represented by Structural Formula (I)
spontaneously polymerize over a period of several days to about two
weeks at room temperature to form a rubberlike NO-releasing
polymer. The polymerization is generally carried out neat, but can
also carried out in solution at concentrations greater than about
0.1 M, for example, in a suitable solvent such as diethyl ether.
These polymers release NO for at least several weeks (Example 15).
They have been shown to relax aortic smooth muscle in vitro
(Example 16). Thus, they show great promise as coatings for medical
devices to deliver NO to treatment sites in vivo.
[0033] The preparation of compounds represented by Structural
Formula (I) is described in co-pending U.S. patent application
"STABLE NO-DELIVERING COMPOUNDS" (Attorney Docket No. DUK97-03),
filed on Jun. 23, 1998, the entire teachings of which are hereby
incorporated herein by reference. Briefly, the compound is formed
by nitrosylating an esterified polyol represented by Structural
Formula (II): 3
[0034] R is an organic radical, as described above.
[0035] n is an integer greater than two, preferably an integer from
three to about ten. More preferably, n is an integer from three to
about eight.
[0036] Each X is independently a thiol-bearing aliphatic group or a
substituted thiol bearing aliphatic group. Preferably, each X is
the same thiol-bearing aliphatic group. Examples of suitable
thiol-bearing aliphatic groups include --CH.sub.2SH,
--CH.sub.2CH.sub.2SH, -- CH.sub.2CH.sub.2CH.sub.2SH and
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2SH. Suitable substituents for an
aliphatic group are provided above.
[0037] The nitrosylation of the esterified polyol is carried out by
reacting the esterified polyol with a nitrosylating agent at room
temperature. Suitable nitrosylating agents are described below.
Preferably about 0.5 to about 0.7 equivalents of nitrosylating
agent per free thiol and free alcohol are used.
S-nitroso-N-acetyl-D, L-penicillamine (SNAP) is a preferred
nitrosylating agent for preparing compounds represented by
Structural Formula (I). The nitrosylating agent is preferably added
to the esterified polyol. The nitrosylation can be carried out neat
or in solvents such as dimethyl sulfoxide, dimethyl formamide or
acetonitrile at concentrations greater than about 0.01 M.
[0038] Polymers prepared by polymerizing compounds represented by
Structural Formula (I) comprise monomer units represented by
Structural Formula (III): 4
[0039] R is an organic radical, as described above.
[0040] Each X is independently a substituted or unsubstituted
aliphatic group. Preferably, every X is the same. Examples of X
include alkylene groups such as --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2-- or --CH.sub.2CH.sub.2CH.sub.2CH--.
[0041] In Structural Formula (III), p is zero or a positive
interger and m is a positive integer. Preferably, p+m is less than
or equal to eight.
[0042] Preferably, the monomer is represented by Structural Formula
(IV): 5
[0043] R' is an organic radical such that
--X--CO--O--R'--O--CO--X-- is R. X, m and p are as described for
Structural Formula (III).
[0044] S-nitrosylated polymers comprising monomers units
represented by Structural Formulas (III) or (IV) can be
crosslinked, as described above. For example, after an --SNO group
in a monomer unit represented by Structural Formulas (III) or (IV)
releases NO, the sulfur atom is available to form a disulfide bond
with a thiol group in another S-nitrosylated polymer molecule. In
one example, the crosslinking monomer unit is represented by
Structural Formula (V): 6 7
[0045] R" is an organic radical, as described above.
[0046] m' is an integer greater than 1 and less than or equal to
m+1.
[0047] p' is an integer greater than or equal to one and less than
or equal to p+1.
[0048] p, m and X are as described for Structural Formula
(III).
[0049] S-Nitrosylated polymers can also be prepared from polymers
having a multiplicity of pendant thiol groups, referred to herein
as "polythiolated polymers", by reacting with a nitrosylating agent
under conditions suitable for nitrosylating free thiol groups.
[0050] Suitable nitrosylating agents are disclosed in Feelisch and
Stamler, "Donors of Nitrogen Oxides", Methods in Nitric Oxide
Research edited by Feelisch and Stamler, (John Wiley & Sons)
(1996), the teachings of which are hereby incorporated into this
application. Suitable nitrosylating agents include acidic nitrite,
nitrosyl chloride, compounds comprising an S-nitroso group
S-nitroso-N-acetyl-D, L-penicillamine (SNAP), S-nitrosoglutathione
(SNOG), N-acetyl-S-nitrosopenicillaminyl-S-n- itrosopenicillamine,
S-nitrosocysteine, S-nitrosothioglycerol, S-nitrosodithiothreitol
and S-nitrosomercaptoethanol), an organic nitrite (e.g. ethyl
nitrite, isobutyl nitrite, and amyl nitrite) peroxynitrites,
nitrosonium salts (e.g. nitrosyl hydrogen sulfate), oxadiazoles
(e.g. 4-phenyl-3-furoxancarbonitrile) and the like.
[0051] Nitrosylation of a polythiolated polymer with acidic nitrite
can be, for example, carried out in an aqueous solution with a
nitrite salt, e.g. NaNO.sub.2, KNO.sub.2, LiNO.sub.2 and the like,
in the presence of an acid, e.g. HCl, acetic acid, H.sub.3PO.sub.4
and the like, at a temperature from about -20.degree. C. to about
50.degree. C., preferably at ambient temperature. Generally, from
about 0.8 to about 2.0, preferably about 0.9 to about 1.1
equivalents of nitrosylating agent are used per thiol being
nitrosylated. Sufficient acid is added to convert all of the
nitrite salt to nitrous acid or an NO.sup.+ equivalent. Specific
conditions for nitrosylating a polythiolated polymer with acidic
nitrite are provided in Example 3.
[0052] Nitrosylation of a polythiolated polymer with NOCI can be
carried out, for example, in an aprotic polar solvent such as
dimethylformamide or dimethylsulfoxide at a temperature from about
-20.degree. C. to about 50.degree. C., preferably at ambient
temperature. NOCl is bubbled through the solution to nitrosylate
the free thiol groups. Specific conditions for nitrosylating a
polythiolated polymer with NOCl are provided in Example 4.
[0053] Polythiolated polymers can be formed from polymers having a
multiplicity of pendant nucleophilic groups, such as alcohols or
amines. The pendant nucleophilic groups can be converted to pendant
thiol groups by methods known in the art and disclosed in Gaddell
and Defaye, Angew. Chem. Int. Ed. Engl. 30:78 (1991) and Rojas et
al., J. Am. Chem. Soc. 117:336 (1995), the teachings of which are
hereby incorporated into this application by reference.
[0054] In one example, the S-nitrosylated polymer is an
S-nitrosylated polysaccharide. Examples of suitable S-nitrosylated
polysaccharides include S-nitrosylated alginic acid, k-carrageenan,
starch, cellulose, fucoidin, cyclodextrins such as
.alpha.-cyclodextrin, .beta.-cyclodextrin and .gamma.-cyclodextrin.
Other suitable examples are disclosed in Bioactive Carbohydrates,
Kennedy and White eds., (John Wiley Sons), Chapter 8, pages
142-182, (1983) the teachings of which are incorporated herein by
reference. Polysaccharides have pendant primary and secondary
alcohol groups. Consequently, S-nitrosylated polysaccharides can be
prepared from polythiolated polysaccharides by the methods
described hereinabove. Preferred polysaccharides include
cyclodextrins, for example .alpha.-cyclodextrin,
.beta.-cyclodextrin and .gamma.-cyclodextrin. The polysaccharide is
first converted to a polythiolated polysaccharide, for example, by
the methods disclosed in Gaddell and Defaye and Roj as et al. In
these methods primary alcohols are thiolated preferentially over
secondary alcohols. Preferably, a sufficient excess of thiolating
reagent is used to form perthiolated polysaccharides.
Polysaccharides are "perthiolated" when all of primary alcohols
have been converted to thiol groups. Specific conditions for
perthiolating .beta.-cyclodextrin are given in Examples 1 and 2.
Polythiolated and perthiolated polysaccharides can be nitrosylated
in the presence of a suitable nitrosylating agents such as acidic
nitrite (Example 3) or nitrosyl chloride (Example 4), as described
above.
[0055] In one aspect, an excess of acidic nitrite is used with
respect to free thiol groups when preparing an S-nitrosylated
polysaccharide, for example an S-nitrosylated cyclodextrin. An
excess of acidic nitrite results in a polysaccharide with pendant
--S--NO and --O--NO groups. The extent of O-nitrosylation is
determined by how much of an excess of acidic nitrite is used. For
example, nitrosylation of per-6-thio-.beta.-cyclodextrin with a 50
fold excess of acidic nitrite results in a product comprising about
ten moles of NO for each cyclodextrin (Example 14), or about 1 mole
of NO per 140 amu. Nitrosylation of per-6-thio-.beta.-cyclodextrin
with a 100 fold excess of acidic nitrite results in a product
comprising about 21 moles of NO for each cyclodextrin (Example 14),
or about 1 mole of NO per 70 amu. Specific conditions for the
preparation of .beta.-cyclodextrin with pendant --O--NO and --S--NO
groups are described in Example 14.
[0056] In another aspect, a polythiolated polysaccharide can be
prepared by reacting the alcohol groups, preferably the primary
alcohol groups, on the polysaccharide with a reagent which adds a
moiety containing a free thiol or protected thiol to the alcohol.
In one example the polysaccharide is reacted with a bis
isocyanatoalkyldisulfide followed by reduction to functionalize the
alcohol as shown in Structural Formula (VI): 8
[0057] Conditions for carrying out this reaction are found in
Cellulose and its Derivatives, Fukamota, Yamada and Tonami, eds.
(John Wiley & Sons), Chapter 40, (1985) the teachings of which
are incorporated herein by reference. One example of a
polythiolated polysaccharide which can be obtained by this route is
shown in Structural Formula (VII): 9
[0058] It is to be understood that agents capable of nitrosylating
a free thiol, in some instances, also oxidize free thiols to form
disulfide bonds. Thus, treating a polythiolated polymer (e.g.
polythiolated polysaccharides such as polythiolated cyclodextrins)
with a nitrosylating agent, e.g. acidified nitrite, nitrosyl
chloride, S-nitrosothiols can, in some instances, result in the
formation of a crosslinked S-nitrosylated polymer matrix. A
"polymer matrix" is a molecule comprising a multiplicity of
individual polymers connected or "crosslinked" by intermolecular
bonds. Thus, in some instances the nitrosylating agent nitrosylates
some of the thiols and, in addition, crosslinks the individual
polymers by causing the formation of intermolecular disulfide
bonds. Such polymer matrices are encompassed by the term
"S-nitrosylated polymer" and are included within the scope of the
present invention.
[0059] The quantity of --S--NO groups present in the composition
can be determined by the method of Saville disclosed in
"Preparation and Detection of S-Nitrosothiols," Methods in Nitric
Oxide Research, edited by Feelisch and Stamler, (John Wiley &
Sons) pages 521-541, (1996). To calculate the amount of NO per
molecular weight of polymer, the polymer concentration, e.g.
carbohydrate concentration, is also determined. Carbohydrate
concentration can be determined by the method disclosed in Dubois
et al., Anal. Chem. 28:350 (1956). When an excess of the
nitrosylating agent is used and when the nitrosylating agent is of
a sufficient size, it can be incorporated, or "entwined," within
the polymeric matrix by the intermolecular disulfide bonds which
crosslink the individual polymer molecules, thereby forming a
complex between the polymer and the nitrosylating agent.
[0060] S-nitrosylated polysaccharides, in particular S-nitrosylated
cyclized polysaccharides such as S-nitrosylated cyclodextrins, can
form a complex with a suitable nitrosylating agent when more than
one equivalent of nitrosylating agent with respect to free thiols
in the polythiolated polysaccharide is used during the
nitrosylation reaction, as described above. Generally, between
about 1.1 to about 5.0 equivalents of nitrosylating agent are used
to form a complex, preferably between about 1.1 to about 2.0
equivalents.
[0061] Nitrosylating agents which can complex with an
S-nitrosylated cyclic polysaccharide include those with the size
and hydrophobicity necessary to form an inclusion complex with the
cyclic polysaccharide. An "inclusion complex" is a complex between
a cyclic polysaccharide such as a cyclodextrin and a small molecule
such that the small molecule is situated within the cavity of the
cyclic polysaccharide. The sizes of the cavities of cyclic
polysaccharides such as cyclodextrins, and methods of choosing
appropriate molecules for the preparation of inclusion complexes
are well known in the art and can be found, for example, in Szejtli
Cyclodextrins In Pharmaceutical, Kluwer Academic Publishers, pages
186-307, (1988) the entire relevant teachings of which are
incorporated herein by reference.
[0062] Nitrosylating agents which can complex with an
S-nitrosylated cyclic polysaccharide also include nitrosylating
agents with a sufficient size such that the nitrosylating agent can
become incorporated into the structure of the polymer matrix of an
S-nitrosylated polysaccharide. As discussed earlier, in certain
instances nitrosylation of polythiolated polymers can also result
in the crosslinking of individual polymer molecules by the
formation of intermolecular disulfide bonds to give a polymer
matrix. Suitable nitrosylating agents are those of an appropriated
size such that the nitrosylating agent can be incorporated into
this matrix. It is to be understood that the size requirements are
determined by the structure of each individual polythiolated
polymer, and that suitable nitrosylating agents can be routinely
determined by the skilled artisan according to the particular
S-nitrosylated polymer being prepared.
[0063] Nitrosylating agents which can form a complex with
S-nitrosylated cyclodextrins include compounds with an S-nitroso
group (S-nitroso-N-acetyl-D, L-penicillamine (SNAP),
S-nitrosoglutathione (SNOG),
N-acetyl-S-nitrosopen-icillaminyl-S-nitrosopenicillamine,
S-nitrosocysteine, S-nitrosothioglycerol, S-nitrosodithiothreitol,
and S-nitrosomercaptoethanol), an organic nitrite (e.g. ethyl
nitrite, isobutyl nitrite, and amyl nitrite), oxadiazoles (e.g.
4-phenyl-3-furoxancarbonitrile), peroxynitrites, nitrosonium salts
and nitroprusside and other metal nitrosyl complexes (See Feelisch
and Stamler, "Donors of Nitrogen Oxides," Methods in Nitric Oxide
Research edited by Feelisch and Stamler, (John Wiley & Sons)
(1996). As discussed in greater detail below, NO delivery times and
delivery capacity of S-nitrosylated cyclodextrins are increased by
the incorporation of nitrosylating agents. The extent and degree to
which delivery times and capacity are increased is dependent on the
nitrosylating agent.
[0064] Specific conditions for forming a complex between an
S-nitrosylated cyclodextrin and a nitrosylating agent are provided
in Examples 5 and 6. Conditions described in these examples result
in nitrosylation of at least some of the free thiols in the
polysaccharide. Because an excess of nitrosylating agent is used
with respect to the quantity of free thiols in the polysaccharide
is used, the resulting composition contains unreacted nitrosylating
agent. Evidence that the S-nitrosylated polysaccharide forms a
complex with the nitrosylating agent comes from the discovery,
reported herein, that the rate of NO release from the reaction
product of per-(6-deoxy-6-thio).beta.-thiocyclodextrin and
S-nitroso-N-acetylpenicillamine is extended compared with
S-nitroso-N-acetylpenicillamine alone (Example 10).
[0065] Although Applicants do not wish to be bound by any
particular mechanism, it is believed that incorporation of a
nitrosylating agent into the S-nitrosylated cyclic polysaccharide
allows both the polysaccharide and the nitrosylating agent to
deliver NO at a treatment site. It is also believed that the
interaction between the cyclic polysaccharide and the nitrosylating
agent results in stabilization of the --S--NO functional group in
the nitrosylating agent. It is further believed that the presence
of a nitrosylating agent in the composition serves to feed, i.e.
replenish, the nitrosyl groups in the S-nitrosylated
polysaccharide, thereby serving to extend the lifetime during which
the polymer can serve as an NO donor. The degree to which the
lifetime of an S-nitrosylated cyclic polysaccharide can be extended
is determined by the stability of the S-nitrosyl group when the
nitrosylating agent is a thionitrite. The stability of --S--NO
groups is dependent on a number of factors; the ability of --S--NO
groups to chelate metals facilitates homolytic breakdown; tertiary
--S--NO groups are more stable than secondary --S--NO groups which
are more stable than primary groups; --S--NO groups which fit into
the hydrophobic pocket of cyclodextrins are more stable than those
which do not; the proximity of amines to the --S--NO group
decreases stability; and modification at the position .beta. to the
--S--NO group regulates stability.
[0066] In one embodiment, the present invention is a composition
comprising a polymer of the present invention and at least one
other ingredient which endow the polymer with desirable
characteristics. For example, plasticizers and elastomers can be
added to the composition to provide the polymer with greater
elasticity. Generally, suitable plasticizers and elastomers are
compounds which are: 1) biocompatible, i.e. which cause minimal
adverse reactions such as platelet and protein deposition in an
individual to which it is administered and 2) which are soluble in
the polymer capable of delivering NO and which can, in turn,
solubilize said polymer. Examples of suitable plasticizers include
polyalkylene glycols such as polyethylene glycols. Preferred
plasticizers are those which can also deliver NO, for example
nitrosothioglycerol.
[0067] Another embodiment of the present invention is a method of
delivering NO to a treatment site in a subject (individual or
animal) using the novel polymers and compositions of the present
inventions to deliver NO. A "treatment site" includes a site in the
body of an individual or animal in which a desirable therapeutic
effect can be achieved by contacting the site with NO. An
"individual" refers to a human. Suitable animals include veterinary
animals such as dogs, cats and the like and farm animals such as
horses, cows, pigs and the like.
[0068] Treatment sites are found, for example, at sites within the
body which develop restenosis, injury or thrombosis as a result of
trauma caused by contacting the site with a synthetic material or a
medical device. For example, restenosis can develop in blood
vessels which have undergone coronary procedures or peripheral
procedures with PTCA balloon catheters (e.g. percutaneous
transluminal angioplasty). Restenosis is the development of scar
tissue from about three to six months after the procedure and
results in narrowing of the blood vessel. NO reduces restenosis by
inhibiting platelet deposition and smooth muscle proliferation. NO
also inhibits thrombosis by inhibiting platelets and can limit
injury by serving as an anti-inflammatory agent.
[0069] A treatment site often develops at vascular sites which are
in contact with a synthetic material or a medical device. For
example, stents are often inserted into blood vessels to prevent
restenosis and re-narrowing of a blood vessel after a procedure
such as angioplasty. Platelet aggregation resulting in thrombus
formation is a complication which may result from the insertion of
stents. NO is an antiplatelet agent and can consequently be used to
lessen the risk of thrombus formation associated with the use of
these medical devices. Other examples of medical devices which
contact vascular sites and thereby increase the risk of thrombus
formation include sheaths for veins and arteries and GORE-TEX
surgical prosthetic.
[0070] Treatment sites can also develop at non-vascular sites, for
example at sites where a useful therapeutic effect can be achieved
by reducing an inflammatory response. Examples include the airway,
the gastrointestinal tract, bladder, uterine and corpus cavemosum.
Thus, the compositions, methods and devices of the present
invention can be used to treat respiratory disorders,
gastrointestinal disorders, urological dysfunction, impotence,
uterine dysfunction and premature labor. NO delivery at a treatment
site can also result in smooth muscle relaxation to facilitate
insertion of a medical device, for example in procedures such as
bronchoscopy, endoscopy, laparoscopy and cystoscopy. Delivery of NO
can also be used to prevent cerebral vasospasms post hemorrhage and
to treat bladder irritability, urethral strictures and biliary
spasms.
[0071] Treatment sites can also develop external to the body in
medical devices used to treat bodily fluids temporarily removed
from body for treatment, for example blood. Examples include
conduit tubes within heart lung machines, tubes of a dialysis
apparatus and catheters.
[0072] The method of delivering NO to a treatment site in an
individual or animal comprises implanting a medical device coated
with a polymer of the present invention at the treatment site. NO
can be delivered to bodily fluids, for example blood, by contacting
the bodily fluid with a medical device coated with a polymer of the
present invention. Examples of treatment sites in an individual or
animal, medical devices suitable for implementation at the
treatment sites and medical devices suitable for contacting bodily
fluids such as blood are described in the paragraphs
hereinabove.
[0073] "Implanting a medical device at a treatment site" refers to
bringing the medical device into actual physical contact with the
treatment site or, in the alternative, bringing the medical device
into close enough proximity to the treatment site so that NO
released from the medical device comes into physical contact with
the treatment site. A bodily fluid is contacted with a medical
device coated with a polymer of the present invention when, for
example, the bodily fluid is temporarily removed from the body for
treatment by the medical device, and the polymer coating is an
interface between the bodily fluid and the medical device. Examples
include the removal of blood for dialysis or by heart lung
machines.
[0074] In one embodiment of the present invention, an article, for
example a medical device, tube or catheter, is coated with a
polymer of the present invention. In one example, the article is
coated with an S-nitrosylated polysaccharide, preferably a cyclic
S-nitrosylated polysaccharide, and even more preferably an
S-nitrosylated cyclodextrin. A mixture is formed by combining a
solution comprising a polythiolated polysaccharide with an article
insoluble in the solution. The mixture is then combined with a
nitrosylating agent under conditions suitable for nitrosylating
free thiol groups, resulting in formation of an S-nitrosylated
polysaccharide. In an aqueous solution, the S-nitrosylated
polysaccharide precipitates from the solution and coats the
article. In polar aprotic solvents such as dimethylformamide (DMF)
or dimethylsulfoxide (DMSO), the article can be dipped into the
reaction mixture and then dried in vacuo or under a stream of an
inert gas such as nitrogen or argon, thereby coating the article.
Suitable nitrosylating agents include acidified nitrite,
S-nitrosothiols, organic nitrite, nitrosyl chloride, oxadiazoles,
nitroprusside and other metal nitrosyl complexes, peroxynitrites,
nitrosonium salts (e.g. nitrosyl hydrogensulfate) and the like.
[0075] In another example, the article is coated with a polymer
having stabilized S-nitrosyl groups or a polymer obtained by
polymerizing a compound represented by Structural Formula (I). The
polymer is dissolved in a suitable solvent. The article is then
dipped into the solution and then dried in vacuo or under a stream
of an inert gas such as nitrogen or argon, thereby coating the
article. Alternatively, the article is coated with a compound
represented by Structural Formula (I) which is then allowed to
polymerize.
[0076] The polymers of the present invention are not brittle, and
consequently remain adhered to the article, even under
physiological conditions. Thus, these polymers are particularly
suited for coating medical devices which are to be implanted in
patients for extended periods of time.
[0077] It is to be understood that other methods of applying
polymer coatings to articles, including methods known in the art,
can be used to coat articles with the polymers of the present
invention.
[0078] Another embodiment of the present invention is a method of
replacing a loss of NO groups from an S-nitrosylated polymer. As
discussed above, NO is lost from S-nitrosylated compounds over
time. In addition, sterilization of medical instruments containing
S-nitrosylated compounds also results in the loss of NO from
S-nitrosylated compounds. The loss of NO from S-nitrosylated
compounds reduces the capacity of the compound to deliver NO to a
treatment site. NO groups can be replaced by contacting the
S-nitrosylated polymer with an effective amount of a gaseous,
nitrosylating agent such as nitrosyl chloride or nitric oxide.
[0079] The invention is further illustrated by the following
examples, which are not intended to be limiting in any way.
EXEMPLIFICATION
EXAMPLE 1
Preparation of Per-(6-deoxy-6-iodo)-.beta.-iodocyclodextrin
[0080] .beta.-Cyclodextrin (20.0 g, 17.6 mmol, 123 mmol primary
hydroxyl) was added to a stirred solution of triphenylphosphine
(97.2 g, 371 mmol, 3 eq per primary hydroxyl) and iodine chips
(93.5 g, 371 mmol, 3 eq per primary hydroxyl) in dimethylformamide
(DMF) (400 mL); the mixture warmed on addition. The solution was
placed in an oil bath at 80.degree. C. for 20 hours, then permitted
to cool to room temperature DMF (350 mL) was removed under reduced
pressure to yield a thick, the dark syrup was roughly one-third the
volume of the original solution. To this syrup, cooled in an ice
bath, was added 160 mL of 3 M NaOMe; the pH was found to be 9 (pH
paper with a drop of water). After addition, the syrup was
permitted to warm to room temperature and stirred for an additional
1 hour. The syrup was then poured into MeOH (3600 mL) to give a
small amount of precipitate. Water (1000 mL) was added slowly to
the MeOH solution, yielding a milky white precipitate in the dark
brown solution. The precipitate was removed by filtration to give a
yellow solid that was washed several times with MeOH (1000 mL
total) to remove most of the color, giving a tan solid that was
Soxhlett-extracted for >12 hours and dried under high vacuum to
give 19.84 of an off-white solid (59%).
EXAMPLE 2
Preparation of Per-(6-deoxy-6-thio).beta.-thiocyclodextrin
[0081] Per-(6-deoxy-6-iodo)-.beta.-cyclodextrin (19.84 g, 10.4
mmol, 72.9 mmol primary iodide) was dissolved in DMF (210 mL) and
thiourea (6.3 g, 82.8 mmol, 1.13 eq) was added. The solution was
stirred at 70.degree. C. under nitrogen for 48 hours. DMF was
removed under reduced pressure to give an orange oil, which was
added to aqueous NaOH (5.4 g in 1000 mL, 135 mmol) to give a white
precipitate on stirring. The solution was heated to a gentle reflux
for 1 hour, which effected full solvation of the precipitate, then
cooled, which resulted in formation of a precipitate that was
removed by filtration and washed with water (this precipitate was
not used). The solution was acidified with 1 M KHSO.sub.4 to give a
fine white precipitate that was filtered and washed with water,
then air-dried overnight. The precipitate was suspended in water
(700 mL), then solvated by addition of 70 mL of aqueous 1 M NaOH,
then re-precipitated with 90 mL of aqueous 1 M KHS0.sub.4. The
precipitate was filtered, air-dried overnight, then dried under
high vacuum to give 6.0 g (46%) of an off-white solid, mp
289.degree. C. (dec).
EXAMPLE 3
Nitrosylation of Per-6-thio-.beta.-cyclodextrin with Acidic
Nitrite
[0082] Per-(6-deoxy-6-thio)-.beta.-cyclodextrin (500 mg, 0.401
mmol, 2.81 mmol primary thiol) was dissolved in 0.5 M aqueous NaOH
(10 mL) to give a faintly yellow solution. A mixture of 2.8 mL 1 M
aqueous NaNO.sub.2 (2.8 mmol, 1 equivalent per mole free thiol) and
2 M HCl (15 mL) was quickly added to give a brick-red precipitate.
The precipitate was pelleted by centrifuge, and the acidic
supernatant was removed by syringe. Deionized water was added and
the precipitate was agitated to full dispersion. The
centrifugation/supernatant removal process was repeated six times
(until the supernatant was neutral to pH paper), giving a deep red
pellet in a small amount of water.
EXAMPLE 4
Nitrosylation of per-6-thio-.beta.-cyclodextrin with Nitrosyl
Chloride in DMF
[0083] Per-(6-deoxy-6-thio)-.beta.-cyclodextrin (50 mg, 0.04 mmol,
0.28 mmol primary thiol) was dissolved in DMF (1 mL). Nitrosyl
chloride was bubbled through to give a deep brown solution. The
solvent can be removed in vacuo or under a stream of an inert gas
such as nitrogen or argon to afford the polymer product.
EXAMPLE 5
Nitrosylation of Per-6-thio-.beta.-cyclodextrin with
S-Nitroso-N-Acetylpenicillamine
[0084] Per-(6-deoxy-6-thio)-.beta.-cyclodextrin (32.3 mg, 0.0259
mmol, 0.181 mmol primary thiol) was dissolved in 1 mL 1 M NaOH.
D(+)-S-nitroso-N-acetylpenicillamine (57.0 mg, 1.4 eq per thiol)
was added to give a deep-red precipitate. The precipitate was
pelleted by centrifuge, and the acidic supernatant was removed by
syringe. Deionized water was added and the precipitate was agitated
to full dispersion. The centrifugation/supematant removal process
was repeated four times (until the supernatant was neutral to pH
paper), giving a deep red pellet in a small amount of water.
EXAMPLE 6
Nitrosylation of Per-6-thio-.beta.-cyclodextrin with
S-Nitroso-N-Acetylpenicillamine in Dimethylformamide
[0085] Per-(6-deoxy-6-thio)-.beta.-cyclodextrin (10 mg, 0.0080
mmol, 0.056 mmol primary thiol) was dissolved in 1 mL DMF.
D(+)-S-nitroso-N-acetylpen- icillamine (17.7 mg, 0.080 mmol, 1.4 eq
per thiol) was added to give a green solution. After standing for 2
hours, the solution had turned deep red. The solvent can be removed
in vacuo or under a stream of an inert gas such as nitrogen or
argon to afford the polymer product.
EXAMPLE 7
Method for Assaying Nitric Oxide Release
[0086] The capacity of a compound to cause relaxation of vascular
smooth muscle, measured by the degree and duration of vasodilation
resulting from exposure of a blood vessel to the compound, is a
measure of its ability to deliver NO in vivo. Methods reported in
Stamler et al., Proc. NatL. Acad. Sci. USA 89:444 (1992), Osborne
et al., J Clin. Invest. 83:465 (1989) and the chapter by Furchgott
in Methods in Nitric Oxide Research, edited by Feelisch and
Stamler, (John Wiley & Sons) (1996), were used to measure
vascular smooth muscle contraction. Because lower concentrations of
NO are required to inhibit platelet aggregation than vasodilation,
measurement of smooth muscle contraction provides a good indication
of whether a composition delivers sufficient NO to reduce platelet
aggregation.
[0087] New Zealand White female rabbits weighing 3-4 kg were
anesthetized with sodium pentobarbital (30 mg/kg). Descending
thoracic aorta were isolated, the vessels were cleaned of adherent
tissue, and the endothelium was removed by gentle rubbing with a
cotton-tipped applicator inserted into the lumen. The vessels were
cut into 5-mm rings and mounted on stirrups in 20 mL organ baths.
The rings were suspended under a resting force of 1 g in 7 ml of
oxygenated Kreb's buffer (pH 7.5) at 37.degree. C. and allowed to
equilibrate for one hour. Isometric contractions were measured on a
Model 7 oscillograph recorder connected to transducers (model TO3C,
Grass Instruments, Quincy, MA). Fresh Krebs solution was added to
the bath periodically during the equilibration period and after
each test response. Sustained contractions were induced with 7
.mu.M norepinephrine prior to the addition of the test
compound.
EXAMPLE 8
[0088] Delivery of Nitric Oxide by a Polmer Coated Stent
[0089] The ability of S-nitrosylated .beta.-cyclodextrin (referred
to as "free polymer") to cause continuous vasodilation was compared
with the NO-related activity of a stent coated with S-nitrosylated
.beta.-cyclodextrin. S-nitrosylated .beta.-cyclodextrin was
obtained by the method described in Example 3. Polymer-coated
stents were obtained by suspending a stent in the reaction mixture
prepared according to the procedure described in Example 3, thereby
allowing the precipitated S-nitrosylated .beta.-cyclodextrin to
coat the stent. Alternatively, polymer-coated stents were obtained
by dipping a stent into a reaction mixture prepared by the method
of Example 4. In either case, the polymer-coated stent was then
dried in vacuo or under a stream of a nitrogen. The delivery of NO
by the polymer coated stent and by the free polymer was assayed
according to the procedure described in Example 7.
[0090] The polymer coated stent resulted in continuous vasodilation
for more than one hour. Removal of the stent resulted in immediate
restoration of tone, indicative of continuous NO release.
[0091] A fresh polymer coated stent was added to the organ chamber.
The stent was then removed from the organ chamber and transferred
to a second organ chamber. Similar levels of smooth muscle
relaxation were observed to occur in each organ chamber, which is
indicative of continuous release of NO from the S-nitrosylated
.beta.-cyclodextrin.
EXAMPLE 9
Stability of Polymers Prepared by Nitrosylating
Per-6-Thio-.beta.-Cyclodex- trin with
S-Nitroso-N-Acetylpenicillamine
[0092] The S-nitrosylated polymer prepared by the method described
in Example 5 was placed on a metal base and dried in vacuo or under
a stream of nitrogen to give a brown solid. This solid had an
absorabance of about 15 in the visible range from about 540 to
about 600 nanometers. Concentrations of NO in the 1.0 mM range are
sufficient to give an absorbance of about 0.15 in this region of
the visible spectrum.
[0093] The polymer was then stored and protected from light for
three weeks. The absorbance in the region from about 540-600
nanometers was essentially unchanged, indicating retention of S--NO
by the polymer. In addition, the ability of the compound to cause
vasodilation, as measured by the assay described in Example 7, also
remained essentially unchanged over the three week period.
EXAMPLE 10
[0094] Incorporating S-Nitroso-N-Acetylpenicillamine Into
S-Nitrosylated Polymers Increases the Nitric Oxide Delivering
Capacity and Half-Life of the Polymers
[0095] S-Nitroso-penicillamine, S-nitrosylated .beta.-cyclodextrin
(prepared according to the procedure in Example 3) and
S-nitrosylated .beta.-cyclodextrin complexed with
S-nitroso-penicillamine (prepared according to the procedure in
Example 5) were assayed by the method described in Example 7 for
their ability to cause vasodilation. In addition, the half-lives
for these compositions in physiological solution were measured. The
half-life is time required for the composition to lose one half of
its bound NO. The amount of NO in the composition is determined by
the method of Saville, as described in Example 13.
[0096] S-nitrosylated .beta.-cyclodextrin complexed with
S-nitroso-penicillamine was found to deliver several orders of
magnitude more NO in physiological solution than
S-nitroso-penicillamine. In addition, S-nitroso-penicillamine was
able to deliver NO for no more than about one hour, while
S-nitrosylated .beta.-cyclodextrin complexed with
S-nitroso-penicillamine had a half-life of greater than forty
hours. This result indicates that incorporating
S-nitroso-penicillamine into the polymer matrix results in
stabilization of the S-nitroso-penicillamine --S--NO group.
[0097] Incorporation of S-nitroso-penicillamine into the polymer
matrix of S-nitrosylated .beta.-cyclodextrin resulted in an
extension of the time period during which nitric oxide can
released. The half-life of S-nitrosylated .beta.-cyclodextrin was
greater than about eighteen hours, while the half-life of
S-nitrosylated .beta.-cyclodextrin complexed with
S-nitroso-penicillamine was greater than about forty hours. This
result indicates that it is possible to extend the time period
during which S-nitrosylated polymers can release NO, based on the
type of NO donor that is incorporated into the polymer matrix. This
result also suggests that the NO donor is "empowering" the polymer
with NO activity, thus serving to extend the polymer lifetime.
EXAMPLE 1
Assay For Determining Antiplatelet Effects
[0098] Venous blood, anticoagulated with 3.4 mM sodium citrate was
obtained from volunteers who had not consumed acetylsalicylic acid
or any other platelet-active agent for at least 10 days.
Platelet-rich plasma was prepared by centrifugation at 1 50 xg for
10 minutes at 25.degree. C. Platelet counts were determined with a
Coulter Counter (model ZM).
[0099] Aggregation of platelet-rich plasma was monitored by a
standard nephelometric technique, in which 0.3-ml aliquots of
platelets were incubated at 37.degree. C. and stirred at 1000 rpm
in a PAP-4 aggregometer (Biodata, Hatsboro, Pa.).
[0100] S-Nitrosylated .beta.-cyclodextrin, prepared according to
the method described in Example 3, was incubated at concentrations
of 1 .mu.M, 10 .mu.M and 100 .mu.M in 400 .mu.L of platelet rich
plasma for 3 minutes. Aggregations were induced by adding 100 .mu.L
of 10 .mu.M ADP. Controls were run in the absence of polymer.
Aggregations were quantified by measuring the maximal rate and
extent of change of light transmittance and are expressed as
normalized value relative to control aggregations.
[0101] Dose-dependent inhibition of ADP-induced platelet
aggregation was observed over the range of 1 .mu.M to 100 .mu.M
S-nitrosylated .beta.-cyclodextrin. Inhibition of platelet
aggregation was observed, even at the lowest concentration.
EXAMPLE 12
[0102] Inhibition of Platelet Deposition in Dogs by S-Nitrosylated
.beta.-Cyclodextrin Coated Stent
[0103] Platelets play a central role in the development of acute
closure as well as late restenosis following angioplasty. Potent
inhibitors of the platelet glycoprotein II.sub.B/III.sub.A when
given systemically have been shown to be effective in reducing 30
day and 6 month clinical events following high risk angioplasty.
This benefit, however, has come at the expense of higher rates of
bleeding complications. By delivery of a potent glycoprotein
II.sub.B/III.sub.A inhibitor locally, the benefits of platelet
inhibition may be attained without the risk of systemic platelet
inhibition. The purpose of this study is to determine the local
platelet inhibitory effects of cyclodextrin-nitric oxide.
[0104] Methods
[0105] Seven mongrel dogs were studied. After diagnostic
angiography, stents were implanted into the LAD and LCX arteries.
The first 3 animals received plain 8 mm corrugated metal ring
stents and the remaining 4 were given SNO-cyclodextrin coated
stents. Coronary dimensions were obtained utilizing on-line QCA
measurements and stents were appropriately sized to achieve a
1.2-13:1 stent to artery ratio. Prior to stent implantation,
autologous platelets were labeled with Indium 111 oxime, reinfused
and allowed to recirculate for 1 hour. The assigned stents were
then deployed at 10-14 ATMs and quantitative coronary angiography
was repeated. Platelets were allowed to circulate an additional 24
hours then the study was terminated for platelet deposition
analysis.
[0106] Results
[0107] Platelet deposition on plain metal stents was greater than
on NO coated stents although the difference was not statistically
significant: 5.19.+-.5.78 vs. 4.03.+-.5.33 platelets
.times.10.sup.8/cm.sup.2p=0.5827. However, 4 of the 6 metal
controls had greater platelet deposition than any of the NO coated
stents. The mean for the metal controls was affected by 2 very low
values. These data suggest that the drug prevents above baseline
platelet deposition as was seen in 4 of the 6 metal stents without
NO coating. The number of platelets/square centimeter on each of
the control stents and on each of the coated stents are shown in
FIG. 1.
EXAMPLE 13
Determination of the Amount of S-Nitrosylation in S-Nitrosylated
Polysaccharides
[0108] Determination of Carbohydrate Concentration
[0109] The amount of carbohydrate present is determined by the
following disclosed in Dubois et al., Anal. Chem. 28:350 (1956).
Two milliliters of carbohydrate solution containing between 10 and
70.gamma. of carbohydrate are pipetted into a colorimetric tube,
and 0.05 ml of 80% phenol is added. Then 5 ml of concentrated
sulfuric acid is added rapidly, the stream of acid being directed
against the liquid surface rather than against the side of the test
tube in order to obtain good mixing. The tubes are allowed to stand
10 minutes, then they are shaken and placed for 10 to 20 minutes in
a water bath at 25.degree. C. to 30.degree. C. before readings are
taken. The color is stable for several hours and readings may be
made later if necessary. The absorbance of the characteristic
yellow-orange color is measured at 490 m.mu. of hexoses and 480
m.mu. for pentose and uronic acids. Blanks are prepared by
substituting distilled water for sugar solution. The amount of
sugar may then be determined by reference to a standard curve
previously constructed for the particular sugar under
examination.
[0110] All solutions are prepared in triplicate to minimize errors
resulting from accidential contamination with cellulose lint.
[0111] Determination of R--S--NO Concentration
[0112] The concentration of R--S--NO groups in a sample is based on
the method reported in Saville, Analyst 83:620 (1958). By this
method, R--S--NO groups are converted into an azo dye. The
concentration of this dye is determined by measuring the absorbance
at 540 nm (.epsilon..about.50,000 M.sup.-1 cm.sup.-1). The
procedure is as follows:
[0113] Reagents
[0114] Solution A: sulfanilamide 1% dissolved in 0.5 M HCl.
[0115] Solution B: same solution as used in A to which 0.2%
HgCl.sub.2 Solution C: 0.02% solution of
N-(1-naphthyl)-ethylenediamine dihydrochloride dissolved in 0.5 M
HC1.
[0116] Procedure
[0117] A given volume (50 .mu.1-1m) of the sample to be assayed is
added to an equivalent volume of solution A and solution B. The two
samples are set aside for 5 minutes to allow formation of the
diazonium salt, after which an equivalent volume of solution C is
added to each mixture. Color formation, indicative of the azo dye
product, is usually complete by 5 minutes. The sample absorbance is
then read spectrophotometrically at 540 nm. The RSNO is quantified
as the difference in absorbance between solution B and A. (i.e. B -
A). In the event that the background nitrite concentration is high
(i.e. increased background in A), the accuracy of the measurement
can be increased by the addition of an equivalent volume of 0.5%
ammonium sulfamate in acid (45 mM) 5 minutes prior to the addition
of sulfanilamide. The nitrous acid in solution reacts immediately
with excess ammonium sulfamate to form nitrogen gas and
sulfate.
[0118] Concentrations of thiol greater than 500 .mu.M in samples
may interfere with the assay if nitrite is also present at
micromolar concentration. Because nitrite will nitrosate
indiscriminantly under the acidic conditions employed, thiols will
effectively compete for reaction with sulfanilamide (present at 50
mM in this assay) as their concentration approaches the millimolar
range. This will lead to artifactual detection of RSNO. The problem
can be avoided by (1) keeping the ratio of thiol to sulfanilamide
< 0.01, (2) first alkylating thiols in the solution, or (3)
adding free thiols to standards to correct for the potential
artifact.
[0119] S-nitrosylated .beta.-cyclodextrin was prepared as described
in Example 3 using 1 mM perthiolated .beta.-cyclodextrin and 1) one
equivalent (1X); 2) two equivalents (2X); three equivalents (3X);
six equivalents (6X); and ten equivalents (10X) of acidic nitrite.
The carbohydrate concentration and the --S--NO concentration of
each resulting carbohydrate polymer was then determined, as
described above. The results are shown in FIG. 2. A six fold excess
of acidified nitrite results in about three --S--NO groups per
molecule of cyclodextrin, or about one --S--NO group per 470
molecular weight. The use of three and ten equivalents of acidified
nitrite results in a product with between about 2 and 2.5 --S--NO
groups per cyclodextrin.
EXAMPLE 14
Preparation of O- and S-Nitrosylated .beta.-Cyclodextrin
[0120] .beta.-Cyclodextrin with pendant --O--NO and --S--NO groups
was prepared according to the procedure described in Example 3
except that 50 and 100 equivalents of acidic nitrite were used.
[0121] The formation of --O--NO groups is accompanied by an
increase in absorbance in the 320-360 nm range of the
ultraviolet/visible spectrum. Because --S--NO groups also absorb in
this region of the ultraviolet/visible spectrum, confirmation of
O-nitrosylation is provided by the observation that the increase in
absorbance in the 320-360 nm region is accompanied by no further
increase in the --S--NO concentration. The concentration of --S--NO
is determined by the method of Saville, described in Example 13.
The amount of --O--NO present in the polymer can be determined by
the intensity of the absorbance in the 320-360 nm region and the
loss of NO from media. The quantity of --O--NO per molecular weight
can be calculated by first determining the carbohydrate
concentration, as described in Example 13 above.
[0122] FIG. 3 shows the ultraviolet/visible spectrum of the
.beta.-cyclodextrin in the presence of a 50 fold excess of acidic
nitrite, as described above. As can be seen, the absorbance in the
340-350 nm region increases over time, with a maximum being reached
after about 45 minutes. The combined concentration of --O--NO and
--S--NO groups was determined to be about 10 NO groups per
cyclodextrin when a 50 fold excess of acidic nitrite were used or
about one NO group per 140 amu. The combined concentration of
--O--NO and --S--NO groups was determined to be about 21 NO groups
per cyclodextrin when a 100 fold excess of acidified nitrite were
used or about one NO group per 67 amu.
EXAMPLE 15
General Procedure for the Preparation of Polymers With Stabilized
S-Nitrosylated Groups
[0123] All precursor thiols were obtained from Sigma-Aldrich
Chemical Co. and were used without further purification.
Tertiary-butyl nitrite (TBN, 96%) was purchased from Aldrich
Chemical Co. and was used without further purification.
[0124] Polythiol and TBN were mixed neat and allowed to stir at
room temperature. 0.5 equivalents of TBN were used for each
equivalent of free thiol present in the polythiol. The reaction
vessel was then sealed to exclude oxygen and wrapped in aluminum
foil to exclude light.
[0125] The following polythiols were reacted with TBN according to
the procedure described in the previous paragraph:
[0126] Polythiol 1-Trimethylolpropane Tris(3-Mercaptopropionate)
10
[0127] Polythio 2-Pentaerythritol
Tetrakis-(3-Mercaptopropionate)
[0128] Polythiol 3-1,2,6-Hexanetriol Trithioglycolate 11
[0129] Polythiol 4-Trimethylolpropane Tris(2-Mercaptoacetate)
[0130] In each case, the reaction mixture rapidly turned a deep red
after mixing the polythiol and TBN. The red color is indicative of
S-nitrosylation. After standing for about two weeks, each reaction
mixture appeared as a pink-gel like solid. The color persisted in
each case for at least three weeks, indicating that the polymers
retained the ability to release NO was retained during this period
of time. As shown in Example 16, polymers that were one week old
released sufficient NO to relax vascular smooth muscle.
EXAMPLE 16
Relaxation of Vascular Smooth Muscle by the Polymers With
Stabilized S-nitrosyl Groups
[0131] The capacity of the polymers prepared in Example 15 to relax
vascular smooth muscle was determined by the method described in
Example 7, modified to use descending thoracic aorta obtained from
Wistar rats. 20.5 mg of Polythiol 1, 7.1 mg of Polythiol 2, 25.5 mg
of Polythiol 3 and 6.6 mg of Polythiol 4 were tested independently.
All polymers had been prepared at least one week prior to testing.
In each case, relaxation of the smooth muscle occurred within one
minute of adding the test polymer.
[0132] Equivalents
[0133] Those skilled in the art will know, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
* * * * *