U.S. patent application number 10/565573 was filed with the patent office on 2009-05-28 for stabilization and ionic triggering of nitric oxide release.
Invention is credited to Daniel J. Smith.
Application Number | 20090136410 10/565573 |
Document ID | / |
Family ID | 34115373 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090136410 |
Kind Code |
A1 |
Smith; Daniel J. |
May 28, 2009 |
Stabilization and ionic triggering of nitric oxide release
Abstract
Provided is a method for producing nitric oxide that employs an
ion exchange resin. Also provided is a method for producing nitric
oxide that combines a salt with a gel or cream. A method is
provided for producing nitric oxide that combines a Ph adjuster
with a diazeniumdiolate-containing compound or composition.
Inventors: |
Smith; Daniel J.; (Stow,
OH) |
Correspondence
Address: |
ROETZEL AND ANDRESS
222 SOUTH MAIN STREET
AKRON
OH
44308
US
|
Family ID: |
34115373 |
Appl. No.: |
10/565573 |
Filed: |
July 26, 2004 |
PCT Filed: |
July 26, 2004 |
PCT NO: |
PCT/US04/23867 |
371 Date: |
February 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60490218 |
Jul 25, 2003 |
|
|
|
Current U.S.
Class: |
423/405 |
Current CPC
Class: |
B01J 39/04 20130101;
A61K 9/0014 20130101; A61K 9/145 20130101; C01B 21/24 20130101;
A61K 47/585 20170801; B01J 41/04 20130101; A61K 33/00 20130101 |
Class at
Publication: |
423/405 |
International
Class: |
C01B 21/24 20060101
C01B021/24 |
Claims
1. A method for producing nitric oxide comprising: producing nitric
oxide by using an ionic exchange resin.
2. The method of claim 1, wherein the ionic exchange resin is an
anionic exchange resin.
3. The method of claim 2, wherein the anionic exchange resin has a
counter ion selected from the group consisting of ascorbate,
nitrite, a weak-acid anion, lactate, and a
diazeniumdiolate-containing composition.
4. The method of claim 1, wherein the ionic exchange resin is a
cationic exchange resin.
5. The method of claim 4, wherein the cationic exchange resin has a
hydrogen-atom counter ion.
6. The method of claim 1, wherein the ionic exchange resin is in a
gel or cream.
7. A method for producing nitric oxide comprising the step: mixing
a salt with a cream, gel, or combination thereof to produce nitric
oxide.
8. The method of claim 7, wherein the salt is sodium chloride,
sodium phosphate, or sodium acetate.
9. The method of claim 7, wherein the cream or gel is an ion-free
hydrogel, an off-white-oil-in-water vanishing cream, or a
combination thereof.
10. The method of claim 7, wherein the cream or gel has an ionic
exchange resin therein.
11. The method of claim 10, wherein the ionic exchange resin is an
anionic exchange resin.
12. The method of claim 11, wherein the anionic exchange resin has
a counter ion selected from the group consisting of ascorbate,
nitrite, a weak acid anion, lactate, and a
diazeniumdiolate-containing composition.
13. The method of claim 10, wherein the ionic exchange resin is an
cationic exchange resin.
14. The method of claim 13, wherein the cationic exchange resin has
a hydrogen atom counter ion.
15. The method of claim 12, further comprising reacting a
hydrogen-atom cation with the ascorbate to produce ascorbic
acid.
16. The method of claim 12, further comprising reacting ascorbic
acid with the nitrite to form nitric oxide.
17. The method of claim 12, further comprising reacting a hydrogen
cation with the diazeniumdiolate-containing composition to produce
nitric oxide.
18. A method for producing nitric oxide comprising the step:
producing nitric oxide by adding a Ph adjuster to a nanofiber
having a diazeniumdiolate functional group.
19. The method of claim 18, wherein the nanofiber is a linear
polyethylenimine fiber.
20. The method of claim 18, wherein the nanofiber is an electrospun
nanofiber.
21. The method of claim 18, wherein the Ph adjuster is phosphate,
lactate, citrate, or a combination thereof.
22. A method for producing nitric oxide comprising the step:
producing nitric oxide by adding a Ph adjuster to a nanoparticle
having a diazeniumdiolate functional group.
23. The method of claim 22, wherein the nanoparticle is cellulose,
polystyrene, cm cellulose, or chitosan.
24. The method of claim 22, wherein the Ph adjuster is phosphate,
lactate, citrate, or a combination thereof.
25. The method of claim 22, wherein the nanoparticle is within or
attached to an electrospun nanofiber.
Description
[0001] This application claims priority of U.S. Provisional Patent
Application Ser. No. 60/490,218, which is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to a method for producing nitric
oxide. More specifically, this invention relates to using ionic
exchange resins in the production of nitric oxide. This invention
is also directed to using Ph adjusters in combination with
diazeniumdiolate-containing compounds (NONOates) to produce nitric
oxide. The invention is further directed to producing nitric oxide
by mixing a cream with a salt.
BACKGROUND OF THE INVENTION
Ionic Exchange Resins
[0003] Ionic exchange resins are known. Most modern ionic exchange
resins, or ionic exchangers, consist of a synthetic polymer
backbone or matrix to which is attached a functional group that
gives each ionic exchanger its specific properties. Ionic exchange
resins are produced in various physical forms depending on the end
use for the resin. Most commonly, they are used as small spherical
beads or granules, but they can be made into membranes, fibers,
tubes, cloth, or foams. By special manufacturing techniques, the
polymers, especially in the bead form, may be made with porous
structures instead of the conventional solid gel resin structure.
Such resins are called macroporous or macroreticular resins.
[0004] The functional groups that are distributed throughout the
resin structure contain fixed electric charges or ion-active
groups, each of which is associated with a mobile counter ion of
opposite charge. These mobile ions are capable of reacting with or
exchanging with other ions of like sign when they are in contact
with a solution containing such ions. It is important that ionic
exchange resins swell to a certain extent in aqueous or liquid
solution so that the solution can diffuse into the resin and come
into contact with the active sites.
[0005] When the fixed electrical charges within the resin matrix
are negative (when the fixed functional group is a sulfonic group,
for example), the mobile ions are cations and the resin is said to
be a cationic exchange resin. Conversely when the fixed groups are
positively charged, the mobile ions are anions and the resin is an
anionic exchanger.
[0006] The polymer matrices are usually cross-linked to make them
insoluble and to give them mechanical strength and stability. The
extent of cross-linking must be controlled so as to give good
mechanical properties to the resin while permitting enough water
absorption and swelling to ensure good ionic exchange activity.
[0007] Ionic exchange has been defined as the reversible
interchange of ions between a solid and a liquid phase in which
there is no permanent change in the structure of the solid. This
means that ionic exchangers are not consumed by ordinary usage, but
when they are exhausted, they can be regenerated or reconverted to
their original state and reused. Ionic exchange is regarded as a
unit process in chemical engineering and it has many applications.
One of the best known and largest applications is water softening,
in which calcium and magnesium ions, which cause water hardness,
are removed from the water and exchanged for sodium ions from the
resin. When the resin is exhausted, it is brought back to its
original state by treatment with a sodium chloride solution. By a
more complex process, water may be not only softened, but
completely deionized. Ionic exchange resins are widely used to
treat boiler feed water, process water, and to perform a large
number of separations and reactions in the manufacture of
chemicals, foods, pharmaceuticals, electronic devices, and many
other products.
[0008] Ionic exchange is a widespread phenomenon in nature,
occurring in living cells and in soils, for example. Ionic exchange
materials include silicates, phosphates, fluorides, humus, wool,
proteins, cellulose, alumina, glass, and many others. The first
industrial ionic exchangers were probably inorganic aluminum
silicates, used for softening water and treating sugar solutions.
Later on, it was discovered that sulfonated coal is a relatively
effective ionic exchange material, but such materials are fragile
and are useful only under restricted operating conditions. In the
United States nearly all ionic exchange applications use synthetic
polymer resins.
Nitric Oxide
[0009] At room temperature nitric oxide (NO) is a gas that can
participate in many chemical reactions. There are many known
biological and medical uses of NO. A nonlimiting list of some of
these uses include:
[0010] Blood Flow: NO relaxes the smooth muscle in the walls of the
arterioles. At the time of each systole, the endothelial cells that
line the blood vessels release a puff of NO. This diffuses into the
underlying smooth muscle cells causing them to relax and thus
permit a surge of blood to pass through easily.
[0011] Nitroglycerine, which is often prescribed to reduce the pain
of angina, does so by Generating nitric oxide, which relaxes the
walls of the coronary arteries and arterioles.
[0012] NO also inhibits the aggregation of platelets and thus keeps
inappropriate clotting from interfering with blood flow.
[0013] Kidney Function: Release of NO around the glomeruli of the
kidneys increases blood flow through them thus increasing the rate
of filtration and urine formation.
[0014] Penile Erection: The erection of the penis during sexual
excitation is mediated by NO released from nerve endings close to
the blood vessels of the penis. Relaxation of these vessels causes
blood to pool in the blood sinuses producing an erection.
[0015] The popular prescription drug sildenafil citrate inhibits
the breakdown of NO and thus enhances its effect.
[0016] Peristalsis: The wavelike motions of the gastrointestinal
tract are aided by the relaxing effect of NO on the smooth muscle
in its walls.
[0017] Because of the many well-known uses of NO, there is
therefore a need in the art for additional methods directed to NO
production and its delivery at target locations.
SUMMARY OF THE INVENTION
[0018] In general the present invention provides a method for
producing nitric oxide comprising producing nitric oxide by using
an ionic exchange resin.
[0019] The present invention also includes a method for producing
nitric oxide comprising mixing a salt with a cream, gel, or
combination thereof to produce nitric oxide.
[0020] The present invention also includes a method for producing
nitric oxide comprising producing nitric oxide by adding a Ph
adjuster to a nanofiber having a diazeniumdiolate functional
group.
[0021] The present invention further includes a method for
producing nitric oxide comprising producing nitric oxide by adding
a Ph adjuster to a nanoparticle having a diazeniumdiolate
functional group.
[0022] The present invention includes an embodiment that uses an
ion exchange resin, and that embodiment allows for ionic triggering
of the release or production of nitric oxide. An additional
advantage of the present invention is that it provides a gel or
cream delivery system for producing or delivering nitric oxide to a
target region or specific location. An additional advantage to
using a cream or gel delivery system is that the surface area of
the delivery vehicle provides an advantage over other prior art
methods for delivering nitric oxide.
Terminology
[0023] Nitric oxide is a well-known compound that is a colorless
gas at room temperature.
[0024] Ionic exchange resins are well-known. An ionic exchange
resin is typically a polymer with electrically-charged sites at
which one anion may replace another. Synthetic ionic exchange
resins are usually cast as porous beads with a considerable
external and porous surface area where ions can attach. Anionic
exchange resins and cationic exchange resins are two specific types
of ionic exchange resins. Anionic exchange resins are exchange
resins that have a plurality of positively-charged sites, and
cationic exchange resins are ionic exchange resins that have a
plurality of negatively-charged sites.
[0025] Counter ions are the ions that form ion pairs with the
charged sites on an ionic exchange resin. More specifically, when
an ionic exchange resin is a cationic exchange resin, the counter
ion is a cation that forms an ion pair with a negatively-charged
site on the exchange resin. When the ionic exchange resin is an
anionic exchange resin, the counter ion is an anion that forms an
ion pair with the positively-charged site on the anionic exchange
resin.
[0026] A salt is generally known as a compound that is formed when
the hydrogen atom of an acid is replaced by a metal or a metal
equivalent.
[0027] A Ph adjuster is a composition that either increases or
decreases the Ph of a reaction medium.
[0028] A nanofiber is a fiber having a characteristic dimension on
the nanoscale.
[0029] A nanoparticle is a particle having a characteristic
dimension on the nanoscale.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 provides structures of polyamine NONOates.
Specifically provided are ethyl putreanate diazeniumdiolate sodium
salt (EPN-Na), methyl putreanate diazeniumdiolate sodium salt
(MePN-Na), putreanine diazeniumdiolate sodium salt (PuN-Na), and
diethylenetriamine diazeniumdiolate (DETA-NO);
[0031] FIG. 2 is general synthesis of putreanine esters.
[0032] FIG. 3 is UV detection of polyamine NONOates in 0.1M of
NaOH.
[0033] FIG. 4 after at least 68 hours in deionized water at room
temperature, the subject NONOate beads showed no absorption between
245-260 nm. As shown in the plot, an anion is required to exchange
the NONOate.
[0034] FIG. 5 is titration of 4.3 mg of MePN-DOWEX with 1M solution
of NaCl.
[0035] FIG. 6 is a diagram of the loading of the diazeniumdiolates
with DOWEX-ionic exchange resin.
[0036] FIG. 7 is a table of the summary of physical properties of
putreanine based diazeniumdiolates.
[0037] FIG. 8 is a table of kinetics results of NONOate-DOWEX.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0038] This invention is generally directed to a method for
producing nitric oxide by using an ion exchange resin. The ionic
exchange resin is either a cationic exchange resin or an anionic
exchange resin. Using an ionic exchange resin allows a user to
ionically trigger the release of anionic or cationic reactants from
an ion exchange resin, whereby the anionic or cationic reactants
preferably proceed to participate in producing nitric oxide via a
chemical reaction.
[0039] This invention is further directed to producing NO by using
a Ph adjuster used in combination with a
diazeniumdiolate-containing composition.
[0040] This invention is further directed to mixing a salt with a
cream, gel, or combination thereof to produce nitric oxide.
[0041] As mentioned above, an embodiment of this invention
preferably employs an ionic exchange resin that forms an ion pair
with a counter ion, wherein the counter ion can be displaced by a
different and typically stronger cation or anion. There is no
limitation on useful ion exchange resins, and any ion exchange
resin can be employed in the present invention. The cation or anion
that displaces a counter ion on the ionic exchange resin can come
from any source. Preferably, the displacing anion or cation is
generated from a salt that has dissociated. Salts that are useful
in generating these displacing cations and anions can be selected
from the group consisting of sodium chloride, sodium phosphate, or
sodium acetate. There is, however, no limitation on the salts that
can be used in the present invention.
[0042] When an anionic exchange resin is employed, its counter ion
is preferably selected from the group consisting of ascorbate,
nitrate, a diazenium-diolate (a NONOate functional group)
containing compound, or a combination thereof. There is, however,
no limit on the anionic counter ions that can be used in this
invention.
[0043] In an embodiment of the present invention, the ionic
exchange resins are preferably positioned or reside in a cream,
gel, or combination thereof. Employable creams and gels include
ion-free hydrogels, off-white-oil-in-water vanishing cream, or
combinations thereof. The cream or gel preferably provides an inert
reaction medium wherein the ionic exchange resins can reside. The
cream or gel also preferably provides a medium or phase wherein a
salt can dissociate and thereby form a salt cation and a salt
anion. A salt cation or salt anion can displace a counter ion and
thereby facilitate nitric oxide production.
[0044] In a preferred embodiment, an ascorbate anion is a counter
ion on an anionic exchange resin. The ascorbate anion preferably
reacts with a hydrogen cation to produce ascorbic acid.
Additionally, a nitrite counter ion preferably reacts with the
ascorbic acid to produce nitric oxide.
[0045] In yet another embodiment, a diazeniumdiolate-containing
compound is a counter ion on an anionic exchange resin that
preferably reacts with a hydrogen cation to produce nitric
oxide.
[0046] In still another embodiment, the diazeniumdiolate-containing
compound is a polymer having a diazeniundiolate functional group.
More preferably, the polymer is a polyethylenimine nanofiber having
a diazeniumdioate functional group. More preferably, the nanofiber
is an electrospun nanofiber, and any electrospun nanofiber having a
diazeniumdiolate functional group can be employed. Preferably, a Ph
adjuster is added to a nanofiber having a diazeniumdioate
functional group in order to produce nitric oxide. The Ph adjuster
is preferably selected from phosphate, lactate, citrate, or
combinations thereof. There is no limitation on useful polymers or
Ph adjusters that can be employed.
[0047] In yet another embodiment, nitric oxide can be produced by
adding a Ph adjuster to a nanoparticle having a diazeniumdiolate
functional group. Preferably, the nanoparticle is made of
cellulose, polystyrene, cm cellulose, chitosan or a combination
thereof. In this embodiment, the Ph adjuster is selected from
phosphate, lactate, citrate, or combination thereof. There is no
limitation on useful polymers or Ph adjusters that can be
employed.
[0048] In still another embodiment, the nanoparticle having a
diazeniumdiolate functional group is within or attached to a
nanofiber. This can be achieved by incorporating the nanoparticle
into an electrospinnable solution following by electrospinning.
[0049] In another embodiment, a nitrite anion forms an ion pair
with an anionic exchange resin. For this particular embodiment, the
ion pair will herein be referred as the exchange-resin composition.
The exchange-resin composition is preferably in the presence of dry
ascorbic acid, such that a dry mix of the exchange-resin
composition and ascorbic acid is created. To this dry mix of the
exchange-resin composition and ascorbic acid, a gel having a salt
is added to the dry mix. The salt preferably dissociates or is
dissociated within the gel. The dissociated salt forms a salt
cation and a salt anion within the gel. The salt anion preferably
displaces the nitrite counter ion and facilitates the nitrite
counter ion to react with the ascorbic acid and thereby produces
nitric oxide.
[0050] In another embodiment, a diazeniumdiolate-containing
composition is a counter ion on an anionic exchange resin.
Preferably, this exchange resin and its diazeniumdiolate-containing
counter ion is suspended in a salt-free gel. The gel is then
applied to a target region on skin or tissue, and salt from the
skin or tissue preferably dissociates within the salt-free gel to
thereby create salt anions and salt cations. The salt anions
preferably proceed to exchange the counter ion, i.e., the
diazeniumdiolate-containing compound, away from the exchange resin.
Once the diazeniumdiolate compound is no longer a counter ion on
the exchange resin, the diazeniumdiolate functional group
preferably reacts to produce nitric oxide.
[0051] In yet another embodiment, an anionic exchange resin and a
cationic exchange resin are employed in combination. Preferably,
the anionic exchange resin has nitrite as a counter ion, and
cationic exchange resin has protons as a counter ion. These two
exchange-resin compositions, i.e., the anionic exchange resin in
combination with its nitrite counter ion and the cationic exchange
resin in combination with its proton or hydrogen counter ion are
either in a dry mix or suspended within an ion-free gel. Upon
addition of a salt to the gel, the salt dissociates and the salt
cation displaces the hydrogen counter ion. Additionally, these salt
ions displaces the nitrite counter ion. Once the anionic counter
ion and the cationic counter ion are displaced from their
respective exchange resins, the nitrite and hydrogen cation react
to produce nitric oxide.
[0052] And still another embodiment, three anionic exchange resins
are employed. A first ionic exchange resin is an anionic exchange
resin wherein nitrite is the counter ion. The second ionic exchange
resin is an anionic exchange resin wherein ascorbate in the counter
ion. The third ionic exchange resin is a cationic exchange resin
wherein hydrogen is the counter ion. Preferably, the first, second,
and third ionic exchange resin compositions, i.e., each of these
exchange resins in combinations with it's respective counter ion,
are mixed within an ion-free gel. To the gel, a salt is added. The
salt then dissociates and creates a salt cation and a salt anion.
The salt cation preferably displaces the hydrogen counter ion on
the third cationic exchange resin. Once displaced from its cationic
exchange resin, a hydrogen cation reacts with an ascorbate counter
ion to produce ascorbic acid. The ascorbic acid then proceeds to
react with nitrite on the first anionic exchange resin to thereby
yield nitric oxide.
EXPERIMENTAL
[0053] In order to demonstrate the practice of the present
invention, the following examples have been prepared and tested.
The examples should not, however, be viewed as limiting the scope
of the invention. The claims will serve to define the
invention.
Synthesis of Polyamines Derivatives: Putreanine Esters
[0054] Synthesis of Ethyl Putreanate (EP)
[0055] The synthesis was begun by dissolving 5 ml of ethyl acrylate
(46.1 mmol) in 150 ml distilled THF which was dispensed into an
addition funnel. This solution was added drop-wise over 8 hours
with constant stirring by a magnetic stirrer to a solution of three
molar excess (14 ml, 138 mmol) of 1,4-diaminobutane in 200 ml of
distilled THF. Once all of the ethyl acrylate was added, the
reaction was stirred at room temperature for an additional 16
hours. The reaction was performed under nitrogen atmosphere. After
this period of time the solvent (THF) was removed by
roto-evaporation to leave an oily product (light yellow color). The
1,4-diaminobutane was removed by bulb-to-bulb vacuum distillation
[17].
[0056] Hydrolysis of EP: Putreanine Sulfate Salt
[0057] This reaction was done by dissolving 3.18 g of EP in
deionized water followed by The addition of 1 ml of concentrated
sulfuric acid. The reaction mixture was allowed to reflux for one
hour at 50-60.degree. C. The solvent was removed by
roto-evaporation leaving an oily clear product. Hot ethanol was
added to the reaction flask. The crystals were formed after being
kept in the refrigerator overnight [18]. They were filtered and
washed with cold isopropyl alcohol and then dried under nitrogen
atmosphere.
Nitric Oxide Modification
[0058] Sodium Salt of Methyl Putreanate NONOate (MeN-Na)
[0059] The NO modified compound was prepared by dissolving ethyl
putreanate in 1.5 excess molar ratio of MeONa/MeOH, in a high
pressure glass bottle (ACE Glass) with a magnetic stir bar. The
stirred mixture was purged with nitrogen gas and then connected to
a NO gas tank. The mixture was then brought to 100 psi of NO and
left to react for 8 days under continuous stirring. After this time
the NO gas was released, and the mixture was purged and flushed
with nitrogen. The product was isolated by removal of solvent using
a roto evaporator. The creamy color product (solid) was dried under
the hood. The presence of the NONOate was confirmed by UV
absorbance at 245 nm.
[0060] Sodium Salt of Putreanine NONOate (PuN-Na)
[0061] The NO modified compound was prepared by dissolving
"putreanine sulfate salt" in 1.5 excess molar ratio of MeONa/MeOH
in a high pressure glass bottle (ACE Glass) with a magnetic stir
bar. The stirred mixture was purged with nitrogen gas and then
connected to NO gas tank. The mixture was then brought to 100 psi
of NO and left to react for 8 days under continuous stirring. After
this time the NO gas was released, and the mixture was purged and
flushed with nitrogen. The product was isolated by removal of
solvent using a roto evaporator. The sample was then washed with
ethyl ether. The white product (solid) was dried with air at room
temperature. The presence of the NONOate was confirmed by UV
absorbance at 240 nm.
[0062] Sodium Salt of Ethyl Putreanate NONOate (EPN-Na)
[0063] The NO modified compound was prepared by dissolving ethyl
putreanate in 1.5 excess molar ratio of EtONa/EtOH in high pressure
glass bottle (ACE Glass) with a magnetic stir bar. The stirred
mixture was purged with nitrogen gas and then connected to a NO gas
tank. The mixture was then brought to 100 psi of NO and left to
react for 8 days under continuous stirring. After this time the NO
gas was released, and the mixture was purged and flushed with
nitrogen. The product was isolated by removal of solvent using a
roto evaporator. The sample was washed then with ethyl ether
leaving a yellow product (solid). The presence of the NONOate was
confirmed by UV absorbance at 250 nm.
NO Donors Loading
[0064] Loading
[0065] DOWEX 1.times.400 ionic exchanger resin was treated with 1M
solution of sodium hydroxide for 24 hours, in order to modify the
chloride ions of the resin with hydroxyl groups. The sample of the
treated resin was washed with deionized water until neutral and
then dried. A flame test was used to confirm the removal of
chloride ions; a positive result for the presence of chloride is an
observable green color. NONOates were added to the resin in cold
water, and the sample was kept on ice and stirred overnight [19].
Then the sample was washed several times with deionized water and
dried under nitrogen.
[0066] Stability Tests of NONOate-DOWEX
[0067] A sample of NONOate-DOWEX was analyzed using a UV
spectrophotometer. A 1M solution of sodium chloride was used to
exchange the NONOate from the resin. To determine the stability of
the NONOate-DOWEX, the sample was kept in water for a few days,
taking the UV absorption in the region of 220-400 nm every 24
hours.
Nitric Oxide Release Kinetic Experiments
[0068] NO release profiles were performed by rapidly dissolving
approximately 10.0 mg of the NONOate in 25 ml of PBS solution at
physiological conditions (pH 7.4). The same stock solution of the
buffer was used for baseline determination. The kinetic program was
set up to keep a constant temperature of the sample (25.degree. C.
or 37.degree. C.) and also to take measurements at the maximum
absorptivity every 3 minutes for 20 hours. The rate of NO release
(k) was determined with the best fit curve to the first order
exponential decay [ln (abs.sub.inf/abs) vs. time] using Microsoft
Office Excel 2002. The half-life of each NONOate was determined
with the following equation:
t.sub.1/2=-ln (0.5)/k
The same procedure was followed for the nitric oxide release from
NONOate-DOWEX using 2.6 mg in 4 ml of PBS at pH 7.41; the only
difference was that the sample was centrifuged. The supernatant was
placed in a UV cell for the experiment. Each experiment was done in
triplicate, and the Q-test was applied.
Nitric Oxide Modification of Polyamine Derivatives
[0069] After 8 days of reaction with NO, the solvent was removed
from the reaction flask producing the sodium salt of the NONOate,
which UV spectroscopy showed the absorption in the range of 240-260
nm. The addition of the sodium salt of the solvent increases the
hydrophilicity of the species making them more hydrophilic; as a
consequence they dissolve easily in aqueous solutions. This
modification did not show a relevant effect on nitric oxide release
of the NONOate in non-salt form. The extinction coefficient and NO
release profiles are summarized in FIG. 7. The kinetics profiles of
these NO donors is in the range of 0.5-2.0 hours at 37.degree. C.,
which is comparable with 3-morpholinosydnonimine hydrochloride
(SIN-1), which was found to be 1-2 hours in PBS pH 7.4 at
37.degree. C. [21] . SNI-1 has been shown to be a potent
vasodilator in vivo and in vitro as well as inhibiting smooth
muscle cell mitogenesis and proliferation [22, 23].
Loading and Stability of NONOates in an Anionic Exchange Resin
[0070] DOWEX 1.times.4 400 ionic exchange resin is a cationic
resin, which consists of trimethyl benzyl ammonium chloride (TMBAC)
(Cl.sup.- Me.sub.3.sup.+-N--CH.sub.2-Ph). Chloride ion has a strong
affinity for this resin; a high concentration of sodium hydroxide
was required in order to exchange the chloride ions with hydroxyl
groups. The exchange of the chloride ions was confirmed by a
negative flame test; this was not observed with the treated resin.
This process facilitates the attachment of the NONOates and also
prevents decomposition of the NO donor.
[0071] The stability of the complex NONOate-DOWEX was evaluated by
leaving the sample in water at room temperature along with exposure
to light. For at least 68 hours, the sample appeared to be stable
under such conditions. The exchange of the NONOate was successful
with the addition of an anion, in this case chloride ion. Once
released, the NONOates show absorption at their maximum wavelength
(see FIG. 3). However, other anions such as iodide, carbonate,
bicarbonate, nitrate and phosphate were able to exchange the
NONOate from the resin. The concentration of the anion required to
exchange the NONOate varies from 50 mM to 144 mM (in the case of
sodium chloride (see FIG. 4), which is close to the concentration
of the saline solution (136 mM).
[0072] Kinetic profiles of the NONOates that were released from the
resin are summarized in FIG. 8. The behavior of the NO release from
the NONOates that had been attached to the resin was similar to the
"free" salt form of those NONOates. There are some increases for
DETAN and EPN and a decrease for MePN. The synthesized polyamines
kept the same range of 0.5 to 3 hours for the NO release, which was
observed on the free sodium salt diazeniumdiolates (see FIG.
7).
[0073] Various modifications and alterations that do not depart
from the scope and spirit of this invention will become apparent to
those skilled in the art. This invention is not to be duly limited
to the illustrative embodiments set forth herein.
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