U.S. patent number 5,789,447 [Application Number 08/527,314] was granted by the patent office on 1998-08-04 for nitric oxide releasing compounds as protective agents in ischemia reperfusion injury.
This patent grant is currently assigned to The Board of Supervisors of Louisiana State University and Agricultural and Mechanical College, N/A, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Daniel Neil Granger, Matthew B. Grisham, Ingeborg Hanbauer, Murali C. Krishna, James B. Mitchell, Angelo Russo, David A. Wink, Jr..
United States Patent |
5,789,447 |
Wink, Jr. , et al. |
August 4, 1998 |
Nitric oxide releasing compounds as protective agents in ischemia
reperfusion injury
Abstract
The present invention provides a method for treating oxygen free
radical induced tissue damage associated with ischemia reperfusion
injury, wherein nitric oxide is delivered to target cells/tissues
through the administration of a nitric oxide-containing compound
that spontaneously releases nitric oxide under physiological
conditions without requiring the presence of oxygen.
Inventors: |
Wink, Jr.; David A.
(Hagerstown, MD), Mitchell; James B. (Damascus, MD),
Russo; Angelo (Bethesda, MD), Krishna; Murali C.
(Derwood, MD), Hanbauer; Ingeborg (Chevy Chase, MD),
Grisham; Matthew B. (Shreveport, LA), Granger; Daniel
Neil (Shreveport, LA) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
N/A (Baton Rouge, LA)
The Board of Supervisors of Louisiana State University and
Agricultural and Mechanical College (N/A)
|
Family
ID: |
22518169 |
Appl.
No.: |
08/527,314 |
Filed: |
September 12, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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146610 |
Nov 2, 1993 |
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Current U.S.
Class: |
514/611;
514/642 |
Current CPC
Class: |
A61P
1/00 (20180101); A61P 9/00 (20180101); A61P
1/04 (20180101); A61P 11/00 (20180101); A61P
25/28 (20180101); A61K 31/13 (20130101); A61K
31/15 (20130101); A61P 9/12 (20180101); A61P
37/06 (20180101); A61P 7/00 (20180101); A61P
35/00 (20180101); A61P 3/08 (20180101); A61P
43/00 (20180101); A61P 9/10 (20180101); A61K
31/198 (20130101); A61P 9/08 (20180101); A61K
31/14 (20130101); A61P 29/00 (20180101) |
Current International
Class: |
A61K
31/13 (20060101); A61K 31/14 (20060101); A61K
31/185 (20060101); A61K 31/15 (20060101); A61K
31/198 (20060101); A61K 031/40 () |
Field of
Search: |
;514/642,611 |
References Cited
[Referenced By]
U.S. Patent Documents
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|
|
5250550 |
October 1993 |
Keefer et al. |
5714511 |
February 1998 |
Saavedra et al. |
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Other References
Carey et al., Circulatory Shock, 38, 209-216 (1992). .
Lefer et al., J. Cardiovasc. Pharmacol., 22 (Suppl. 7), S27-S33
(1993). .
Lefer et al., Circulation, 88 (5 (1)), 2337-2350 (1993). .
Masini et al., Biol. Nitric Oxide, Proc. 2nd Int. Meet. (Moncada,
ed.), 1, 190-192 (1992)..
|
Primary Examiner: Burn; Brian M.
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
This is a continuation of copending application Ser. No.
08/146,610, now abandoned, filed on Nov. 2, 1993.
Claims
What is claimed is:
1. A method of treating oxygen free radical induced tissue damage
associated with ischemia reperfusion injury in a mammal, which
method comprises administering to a mammal having ischemia
reperfusion injury a treatment amount of a nitric oxide-containing
compound that can spontaneously release nitric oxide under
physiological conditions in the absence of oxygen, said treatment
amount being sufficient to protect against oxygen free radical
induced tissue damage.
2. The method of claim 1, wherein said compound is a compound of
formula: ##STR8## wherein b and d are independently zero or one; x,
y, and z are independently 2-12; and R.sub.1 -R.sub.5 are
independently hydrogen, C.sub.3 -C.sub.8 cycloalkyl, C.sub.1
-C.sub.12 straight or branched chain alkyl, benzyl, benzoyl,
phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl or
2,2,2-trichloro-t-butoxycarbonyl.
3. The method of claim 1, wherein the ischemia reperfusion injury
is associated with a condition or disease selected from the group
consisting of transplantation, trauma, inflammation, stroke,
seizure, rheumatoid arthritis, atherosclerosis, cancer, dementia,
diabetes, hypertensive crisis, ulcers, lupus, sickle cell anemia,
ischemic bowel syndrome, pulmonary emboli, Ball's syndrome,
pancreatitis, heart attack, and aging.
4. The method of claim 1, wherein the compound is administered by
an injection method selected from the group consisting of
intravenous and local injections.
5. A method of preventing oxygen free radical induced tissue damage
associated with the onset of ischemia reperfusion injury in a
mammal, which method comprises administering to a mammal at
immediate risk for ischemia reperfusion injury a prophylactic
amount of a nitric oxide-containing compound that can spontaneously
release nitric oxide under physiological conditions in the absence
of oxygen, said prophylactic amount being sufficient to protect
against oxygen free radical induced tissue damage.
6. The method of claim 5, wherein the ischemia reperfusion injury
is associated with a condition or disease selected from the group
consisting of transplantation, trauma, inflammation, stroke,
seizure, rheumatoid arthritis, atherosclerosis, cancer, dementia,
diabetes, hypertensive crisis, ulcers, lupus, sickle cell anemia,
ischemic bowel syndrome, pulmonary emboli, Ball's syndrome,
pancreatitis, heart attack, and aging.
Description
TECHNICAL FIELD OF THE INVENTION
The present invention is directed to a method of using nitric
oxide-containing compounds that spontaneously release nitric oxide
under physiological conditions without requiring the presence of
oxygen to treat oxygen free radical mediated tissue damage
associated with ischemia reperfusion injury.
BACKGROUND OF THE INVENTION
Oxygen free radicals, such as superoxide and peroxide, have been
implicated in the genesis of many disease and degenerative states
(Ames et al., PNAS USA, 78, 6858-6862 (1981); Halliwell et al.,
FEBS, 307, 108-112 (1992); Halliwell et al., Arch. Biochem.
Biophys., 246, 501-514 (1986); Halliwell et al., Biochem. J., 219,
1-14 (1984); Minotti et al., J. Biol. Chem., 262, 1098-1104
(1987)). For example, oxygen-mediated biological damage has been a
mechanism involved in inflammation, ischemia reperfusion injury,
stroke, rheumatoid arthritis, atherosclerosis, cancer, and aging.
The chemistry of oxygen-mediated biological damage involves the
Haber-Weiss cycle, in which Fenton-type intermediates, i.e.,
combinations of peroxide and metal, are generated:
(Minotti et al. (1987), supra; Halliwell et al. (1992), supra).
Study of mammalian (Mitchell et al., Biochem., 29, 2802-2807
(1990)) and bacterial (Imlay et al., Science, 240, 640-642 (1988))
cell cultures has revealed that the primary cytotoxic agent is
peroxide (Mitchell et al., supra), the major source of which is an
extracellular superoxide and peroxide generating system involving
hypoxanthine (HX) and xanthine oxidase (XO).
Intravital microscopic studies of tissues exposed to ischemia and
reperfusion have revealed an acute inflammatory response that is
characterized by enhanced protein efflux and increased adherence
and emigration of leukocytes in postcapillary venules (Oliver et
al., Inflammation, 15, 331-346 (1991); Lehr et al., J. Clin.
Invest., 87, 2036-2041 (1991); Messmer et al., Adv. Exp. Med.
Biol., 242, 95 (1988); and Yasahara et al., Am. J. Physiol., 261,
H1626-H1629 (1991)). NO has been shown to block platelet
aggregation (Rubanyi et al., Biochem. Biophys. Res. Comm., 181,
1392-1397 (1991)) and reduce platelet adhesion to endothelial cell
monolayers (Radomski et al., Lancet, 2, 1057-1058 (1987)).
Leukocyte-endothelial cell adhesive interactions in postcapillary
venules has been inhibited by NO (Kubes et al., PNAS USA, 88,
4651-4655 (1991)) and inhibition of NO production increases
microvascular permeability in cat small intestine (Kubes et al.,
Am. J. Physiol., 262 (Heart Circ. Physio. 31), H611-H615 (1992a)).
NO also has been reported to modulate protein extravasation in rat
coronary (Filep et al., Br. J. Pharmacol., 108, 323-326 (1993)) and
intestinal (Hutcheson et al., Br. J. Pharmacol., 101, 815-820
(1990)) circulation treated with proinflammatory mediators. In
addition, diminished basal NO release after myocardial ischemia and
reperfusion has been shown to promote neutrophil adherence to cat
coronary endothelium. Whole organ studies have demonstrated an
accumulation of neutrophils in postischemic tissues (Romson et al.,
Circulation, 67, 1016-1023 (1983); Simpson et al., J. Clin.
Invest., 81, 624-629 (1988); Smith et al., Am. J. Physiol., 256,
H789-H793 (1989); Entman et al., FASEB J., 5, 2529-2537 (1991)),
attenuation of ischemia-reperfusion-induced vascular injury in
animals rendered neutropenic with neutrophil anti-serum (Simpson et
al., supra; Hernandez et al., Am. J. Physiol., 253, H699-H703
(1987); Carden et al., Circ. Res., 66, 1436-1444 (1990)), and a
reduction in reperfusion-induced vascular leakage by monoclonal
antibodies which prevent leukocyte adhesion (Hernandez et al.,
supra; Carden et al., supra; Adkins et al., J. Appl. Physiol., 69,
2012-2018 (1990)), all of which suggest a role for leukocytes as
mediators of the microvascular dysfunction elicited by ischemia
reperfusion. Such studies have also led to the recognition that
leukocyte-endothelial cell adhesion may be a rate-limiting step in
the pathogenesis of ischemia-reperfusion-induced tissue injury. In
general, ischemia and reperfusion of mucosa and microvasculature
result in increased mucosal permeability, leukocyte-endothelial
cell adhesion, increased vascular permeability, platelet
aggregation, and vascular thrombosis (Siegfried et al., Am. J.
Physiol., 263 (Heart Circ. Physiol. 32), H771-H777 (1992); Kubes,
Am. J. Physiol., 262 (Gastrointest. Liver Physiol. 25), G1138-G1142
(1992b); and Kubes et al., (1992a), supra). Endothelial dysfunction
and parenchymal tissue injury produced by ischemia reperfusion has
been reviewed along with pharmacological agents known to exert
protective effects (Lefer et al., Annu. Rev. Pharmacol. Toxicol 31,
71-90 (1993)).
NO in high concentration has been suggested as a cytotoxic agent in
ischemia reperfusion injury (Beckman, Nature, 345, 27-28 (1990))
and neurotoxicity (Dawson et al., PNAS USA, 88, 7797-7801 (1991)).
The NO has been proposed to react with superoxide or peroxide
generated by endogenous hypoxanthine/xanthine oxidase to form
peroxynitrite anion, OONO.sup.- (Beckman, supra), which has been
invoked as a mediator in ischemia reperfusion injury (Beckman,
supra) and lipid peroxidation (Radi et al., Arch. Biochem.
Biophys., 288, 481-487 (1991)) and as a primary cytotoxic agent
generated by macrophages (Beckman et al., PNAS USA, 87, 1620-1624
(1990)).
Based on evidence that nitric oxide synthase (NOS) inhibitors
increase tissue damage during in vivo ischemia reperfusion within
the cerebral cortex and that nitric oxide prevents damage during
ischemia reperfusion events in the brain and heart, it has been
suggested that NO can also function as a cytoprotective agent
(Johnson et al., Critical Care Medicine, 19, 244-252 (1991);
Morikawa et al., Am. J. Physiol., 263, H1632-H1635 (1992); Masini
et al., Agents and Actions, 33, 53-56 (1991); Siegfried et al., J.
Pharm. Expt. Ther., 260, 668-675 (1992); Gambassi et al.,
Pharmacol. Res., 25, 11-12 (1992); and Linz et al., J. Mol. Cell
Cardiol., 24, 909-919 (1992)). Primary neuronal cell cultures
exposed to concentrations of NO as high as 1 mM show no adverse
effects (Hanbauer et al., Neuroreports, 3, 409-412 (1992);
Kiedrowski et al., Mol. Pharmacol., 41, 779-784 (1992)).
Administration of a NO-generating compound, such as nitroglycerin
or nitroprusside, has been described to reduce NMDA
receptor-mediated neuronal damage (U.S. Pat. No. 5,234,956).
However, such compounds suffer from the disadvantages of metabolic
as opposed to spontaneous release of NO and slow release rates
(Lipton et al., Nature, 364, 626-630 (1993)). The nitrone DMPO has
been shown to reduce neuronal cell death more efficiently than the
nitric oxide synthase inhibitor L-N-nitroarginine (Lafon-Cazal et
al., Nature, 364, 535-537 (1993)). Additional studies have shown
that NO, though present, plays only a minimal role in the
pathological effects associated with ischemia reperfusion injury
(Woditsch et al., Am. J. Physiol., 263, H1390-H1396 (1992);
Jaescheke et al., Life Sciences, 50, 1797-1804 (1992)) or tumor
necrosis factor (TNF) mediated cytotoxicity (Fast et al., J.
Leukoc. Biol., 52, 255-261 (1992)). In fact, many of the biological
events in which NO has been proposed as a toxin occur concurrently
with the production of reactive oxygen species, e.g., immune
responses and ischemia reperfusion injury. Reactive oxygen
metabolites and granulocyte activation have been implicated in the
ischemia-reperfusion-induced microvascular injury (Granger, Am. J.
Physiol., 255, H1269-H1275 (1988)).
In view of these reports, nitric oxide and nitrovasodilators, such
as sodium nitroprusside and SIN-1, have been suggested to protect
against ischemia reperfusion injury (Aoki et al., Am. J. Physiol.,
258 (Gastrointest. Liver Physiol. 21), G275-G281 (1990); Kubes et
al., Gastroenterology, 104 (4 Suppl.), Abstract 728 (1993); Andrews
et al., Gastroenterology, 104 (4 Suppl.), Abstract A33 (1993);
Masini, supra; Masini et al., Int. Arch. Allergy Appl. Immunol.,
94, 257-258 (1991); and Johnson, supra). The protective role of
nitric oxide has been supported by a showing that nitric oxide
could quench Fenton-type oxidation (Kanner et al., Arch. Biochem.
Biophys., 289, 130-136 (1991)).
An attempt to provide nitric oxide in vivo involved the
administration of high concentrations of nitric oxide in the gas
phase. However, such a method damages lung tissue and results in
the destruction of the nitric oxide by various chemical reactions,
such as the diffusion-controlled oxidation of oxyhemoglobin in the
blood, before it reaches the target cells or tissues.
In view of the disadvantages inherent in methods of treating
oxygen-free radical mediated tissue damage associated with ischemia
reperfusion which utilize nitric oxide gas or nitric
oxide-containing compounds that do not spontaneously release NO in
the presence or absence of oxygen, it is an object of the present
invention to provide a method of treating oxygen free radical
mediated tissue damage which overcomes the disadvantages of other
methods. It is a related object of the present invention to provide
a method of delivering nitric oxide to cells at risk of being
injured or injured by ischemia reperfusion. It is another object of
the present invention to provide a method of delivering nitric
oxide to cells at risk of being injured or injured by ischemia
reperfusion by means of an agent, in particular a water-soluble
agent, that spontaneously releases NO under physiological
conditions in the presence or absence of oxygen. It is a further
object of the present invention to provide for such delivery in a
controlled and predictable manner. These and other objects and
advantages of the present invention, as well as additional
inventive features, will be apparent from the description of the
invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of treating oxygen free
radical mediated tissue damage associated with ischemia reperfusion
injury, including that associated with transplantation, trauma,
inflammation, stroke, seizure, rheumatoid arthritis,
atherosclerosis, cancer, dementia, diabetes, hypertensive crisis,
ulcers, lupus, sickle cell anemia, ischemic bowel syndrome,
pulmonary emboli, Ball's syndrome, pancreatitis, heart attack, and
aging. In the method, nitric oxide is delivered to target cells in
a controlled and predictable manner through the administration of a
nitric oxide-containing compound. The nitric oxide-containing
compound spontaneously releases nitric oxide under physiological
conditions in the presence or absence of oxygen.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graph of log of surviving fraction versus time (min)
of exposure of cells to hypoxanthine/xanthine oxidase in the
presence and absence of a nitric oxide-releasing compound.
FIG. 1B is a graph of log of surviving fraction versus time (min)
of exposure of cells to hydrogen peroxide in the presence and
absence of DEA/NO breakdown products.
FIG. 2A is a graph of log of surviving fraction versus hydrogen
peroxide concentration (.mu.M) for cells exposed to hydrogen
peroxide for 1 hr in the presence and absence of a nitric
oxide-releasing compound.
FIG. 2B is a graph of log of surviving fraction versus hydrogen
peroxide concentration (.mu.M) for cells in the presence and
absence of the breakdown products of a nitric oxide-releasing
compound.
FIG. 3 is a graph of .sup.3 H-dopamine concentration (pmol) versus
hydrogen peroxide concentration (.mu.M) for ventral mesencephalic
cells in the presence and absence of a nitric oxide-releasing
compound.
FIGS. 4A-C are bar graphs of NO donors and NO synthesis inhibitors
versus number of adherent leukocytes per 100 .mu.m, emigrated
leukocytes per field, and % albumin leakage, respectively, for 20
min ischemia and 30 min reperfusion with data shown for 10 min and
30 min reperfusion.
FIGS. 5A and B are graphs of % albumin leakage versus adherent
leukocytes per 100 .mu.m and emigrated leukocytes per field,
respectively.
FIG. 6 is a bar graph of compounds versus leukocyte-platelet
aggregates per 5 min.
FIG. 7 is a bar graph of compounds versus % degranulated mast
cells.
FIG. 8 is a bar graph of nitrite/nitrate concentration (.mu.M)
versus carotid artery and superior mesenteric vein (SMV) before and
after ischemia reperfusion (I/R).
FIG. 9 is a bar graph of .sup.51 Cr-EDTA clearance
(ml/min.times.100 g) versus time (min) for control, untreated,
SpNO-treated, and SIN-1-treated animals.
FIG. 10 is a graph of water absorption (ml/min.times.100 g) versus
time (min) for control, untreated, SpNO-treated, and SIN-1-treated
animals.
FIG. 11 is a bar graph of lymph flow (ml/min.times.100 g) versus
time (min) for control, untreated, SpNO-treated, and SIN-1-treated
animals.
FIG. 12 is a bar graph of lymph protein clearance (ml/min.times.100
g) versus time (min) for control, untreated, SpNO-treated, and
SIN-1-treated animals .
FIG. 13 is a bar graph of mm Hg versus time (min) for control,
untreated, SpNO-treated, and SIN-1-treated animals.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It has been discovered, surprisingly, that nitric oxide-containing
compounds that spontaneously release NO under physiological
conditions without requiring the presence of oxygen can be used to
treat oxygen free radical mediated tissue damage associated with
ischemia reperfusion injury. The present invention thus provides a
method of treating oxygen free radical mediated tissue damage
associated with ischemia reperfusion injury. In accordance with the
method of the present invention a nitric-oxide containing compound
is administered to a mammal at risk for or having ischemia
reperfusion injury in an amount sufficient to treat oxygen free
radical mediated tissue damage associated with ischemia reperfusion
injury. The nitric oxide-containing compounds used in the present
inventive method spontaneously release nitric oxide under
physiological conditions in the presence or absence of oxygen.
In keeping with the present invention, a series of amine
derivatives of dimeric nitric oxide (NONOates) are particularly
useful. The NONOates have been shown to release nitric oxide in a
predictable manner under physiological conditions (Maragos et al.,
J. Med. Chem., 34, 3242-3247 (1991)). The half-lives of the
NONOates can range from 1 minute to several days (Hrabie et al., J.
Org. Chem., 58, 1472-1476 (1993)) and, accordingly, offer
advantages over compounds, such as spermidine and spermine, by
having characteristically prolonged half-lives in solution. The
NONOates have been employed in various studies of cytostasis
(Maragos et al., Cancer Res., 53, 564-568 (1993)), cytotoxicity,
mutagenicity (Wink et al., Science, 254, 1001-1003 (1991)), nitric
oxide-mediated dopamine release in nerve cell cultures, and nitric
oxide-mediated inhibition of platelet aggregation (Keefer et al.,
in Biology of Nitric Oxide, 2, Enzymology, Biochemistry.
Immunology, Moncada et al., eds., Portland Press, Chapel Hill,
N.C., pages 153-156, (1992)). Vasorelaxation of aortic ring strips
was shown to correlate linearly to the concentration of nitric
oxide release from the NONOates (Maragos et al., supra). NONOates
also have been shown to be effective in the treatment of
cardiovascular disorders and hypertension (U.S. Pat. Nos.
4,954,526, 5,155,137, and 5,212,204 and WO 93/07114) and have been
suggested to be effective in cancer chemotherapy (Maragos et al.
(1993), supra). The potential utility of the NONOates in other
biomedical applications also has been suggested (Maragos et al.
(1991), supra; Keefer et al.(1992), supra).
Several types of NONoates are useful in the method of the present
invention. One type of NONOates useful for treating oxygen free
radical mediated tissue damage associated with ischemia reperfusion
injury in a mammal are NONOates of the formula:
wherein R.sub.1 and R.sub.2 are the same or different and are
selected from the group consisting of hydrogen, a C.sub.1 -C.sub.8
alkyl, a C.sub.6 -C.sub.10 aryl, a C.sub.4 -C.sub.10 heterocyclic
nitrogen-containing radical, a C.sub.6 -C.sub.10 aryl substituted
with a C.sub.1 -C.sub.3 alkyl, and a C.sub.3 -C.sub.10 cycloalkyl,
either or both of which R groups may be substituted by 1-3
substituents, which may be the same or different and are selected
from the group consisting of halo, hydroxy, C.sub.1 -C.sub.8
alkoxy, amino, amido, formyl, carboxy, and nitro, with the proviso
that both R.sub.1 and R.sub.2 cannot be hydrogen; and wherein X is
a pharmaceutically acceptable cation, a pharmaceutically acceptable
metal center, or a pharmaceutically acceptable organic group
selected from the group consisting of a C.sub.1 -C.sub.8 alkyl,
acyl, and amido; and wherein Y is 1 to 3 and is consistent with the
valence of X, sufficient to treat the oxygen free radical mediated
tissue damage.
The term "C.sub.1 -C.sub.8 alkyl" is used to refer to branched and
straight chain hydrocarbon radicals of 1-8 carbons, such as methyl,
ethyl, propyl, isopropyl, butyl, 2-butyl, tert-butyl, amyl,
isoamyl, hexyl, heptyl, octyl, and the like. The term "C.sub.6
-C.sub.10 aryl" is used to refer to aromatic cyclic hydrocarbon
radicals of 6-10 carbons, such as phenyl, naphthyl and the like,
and the term "C.sub.4 -C.sub.10 heterocyclic nitrogen-containing
radical" is used to refer to radicals such as pyrrolyl, pyridinyl,
quinolinyl, isoquinolinyl, and the like. Similarly, "C.sub.3
-C.sub.10 cycloalkyl" is used to refer to nonaromatic cyclic
hydrocarbon radicals of 3-10 carbons, such as cyclopropyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The
terms "halo" and "halogen" are intended to include fluorine,
chlorine, bromine, and iodine. Other terms should be given those
meanings normally ascribed to such terms by those of skill in the
art.
The term "pharmaceutically acceptable cation" as used herein means
any cation biologically compatible in a mammal and includes
alkylammonium cations, e.g., isopropyl ammonium cation and the
like; alkali metals, e.g., sodium, potassium, lithium, and the
like; and alkaline earth metals, e.g., calcium, barium, magnesium,
and the like. The only essential characteristic of the cation
chosen is that it not be biologically incompatible in a mammal.
The term "pharmaceutically acceptable metal center" as used herein
means a central metal ion, having a valence of 1 to 3 attached by
coordinate links to one or more nonmetal atoms of each of the Y
organic groups of the above formula. The term "central metal ion"
as used herein includes biologically acceptable metal ions selected
from alkali metals, such as sodium, potassium, lithium, and the
like; alkaline earth metals, such as calcium, magnesium, barium,
and the like; transition metals, including iron, copper, nickel,
zinc, and the like; Group III metals including aluminum and the
like, and lanthanide series metals. The only principal requirement
for the central metal ion chosen is biological compatibility in a
mammal.
The term "pharmaceutically acceptable organic group" as used herein
refers to those biologically acceptable organic groups that
covalently bond to the organic grouping of the compound of the
above formula to form ethers and other derivatives thereof.
Acceptable organic groups include lower alkyls, acyl, amido, and
the like.
Additional types of nitric oxide-releasing compounds useful in the
method of the present invention include the nitric oxide-releasing
NONOates of Formulas II, III and IV: ##STR1## wherein b and d are
independently zero or one; x, y, and z are independently 2-12;
R.sub.1 -R.sub.8 are independently hydrogen, C.sub.3 -C.sub.8
cycloalkyl, C.sub.1 -C.sub.12 straight or branched chain alkyl,
benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl,
t-butoxycarbonyl or 2,2,2-trichloro-t-butoxycarbonyl; R.sub.9 is
hydrogen or a C.sub.1 -C.sub.12 straight or branched chain alkyl; B
is ##STR2## f is 0-12, with the proviso that when B is the
substituted piperazine moiety ##STR3## then f is 2-12; and g is
2-6. The group --N.sub.2 O.sub.2.sup.- has the structure
##STR4##
Preferred among the compounds of Formulas II and III are those
compounds wherein R.sub.1 -R.sub.7 are independently hydrogen,
C.sub.3-8 cycloalkyl, C.sub.1-12 straight or branched chain alkyl,
benzyl, or acetyl. More preferred are those compounds wherein
R.sub.1 -R.sub.7 are independently hydrogen, methyl, ethyl, benzyl
or acetyl, and x, y and z are 2-4. Most preferred are those
compounds wherein R.sub.1 -R.sub.7 are independently hydrogen,
methyl, benzyl or acetyl, and x, y and z are 2-4.
Preferred among the compounds of Formula IV are those compounds
wherein R.sub.8 is C.sub.5-6 cycloalkyl, C.sub.1-4 straight or
branched chain alkyl, benzyl or acetyl. More preferred are those
compounds wherein R.sub.8 is methyl, ethyl, benzyl or acetyl, and
most preferred are those compounds wherein R.sub.8 is methyl or
acetyl.
In addition to the nitric oxide-releasing compounds of Formulas
I-IV, the following nitric oxide-releasing compounds of the Formula
V, VI, VII and VIII are useful in the present inventive method as
follows: ##STR5## wherein J is an organic or inorganic moiety,
preferably a moiety which is not linked to the nitrogen of the
N.sub.2 O.sub.2 group through a carbon atom, M.sup.+x is a
pharmaceutically acceptable cation, where x is the valence of the
cation, a is 1 or 2, and b and c are the smallest integers that
result in a neutral compound, preferably such that the compound is
not a salt of alanosine or dopastin, as described in U.S. Pat. No.
5,212,204; ##STR6## wherein R.sub.1 and R.sub.2 are independently
selected from the group consisting of a straight chain or branched
chain C.sub.1 -C.sub.12 alkyl group and a benzyl group, or else
R.sub.1 and R.sub.2 together with the nitrogen atom are bonded to
form a heterocyclic group, preferably a pyrrolidino, piperidino,
piperazino or morpholino group, M.sup.+x is a pharmaceutically
acceptable cation, and x is the valence of the cation, as described
in U.S. Pat. No. 5,039,705;
wherein M is a pharmaceutically acceptable metal, or, where x is at
least two, a mixture of two different pharmaceutically acceptable
metals, L is a ligand different from (R.sup.1 R.sup.2 N--N.sub.2
O.sub.2) and is bound to at least one metal, R.sup.1 and R.sup.2
are each organic moieties and may be the same or different, x is an
integer of from 1 to 10, x' is the formal oxidation state of the
metal M, and is an integer of from 1 to 6, y is an integer of from
1 to 18, and where y is at least 2, the ligands L may be the same
or different, z is an integer of from 1 to 20, and K is a
pharmaceutically acceptable counterion to render the compound
neutral to the extent necessary, as described in U.S. patent
application Ser. No. 07/858,885, filed Mar. 27, 1992; and ##STR7##
wherein R.sub.1 and R.sub.2 are independently chosen from
C.sub.1-12 straight chain alkyl, C.sub.1-12 alkoxy or acyloxy
substituted straight chain alkyl, C.sub.2-12 hydroxy or halo
substituted straight chain alkyl, C.sub.3-12 branched chain alkyl,
C.sub.3-12 hydroxy, halo, alkoxy, or acyloxy substituted branched
chain alkyl, C.sub.3-12 straight chain olefinic and C.sub.3-12
branched chain olefinic which are unsubstituted or substituted with
hydroxy, alkoxy, acyloxy, halo or benzyl, or R.sub.1 and R.sub.2
together with the nitrogen atom to which they are bonded form a
heterocyclic group, preferably a pyrrolidino, piperidino,
piperazino or morpholino group, and R.sub.3 is a group selected
from C.sub.1-12 straight chain and C.sub.3-12 branched chain alkyl
which are unsubstituted or substituted by hydroxy, halo, acyloxy or
alkoxy, C.sub.2-12 straight chain or C.sub.3-12 branched chain
olefinic which are unsubstituted or substituted by halo, alkoxy,
acyloxy or hydroxy, C.sub.1-12 unsubstituted or substituted acyl,
sulfonyl and carboxamido; or R.sub.3 is a group of the formula
--(CH.sub.2).sub.n -ON.dbd.N(O)NR.sub.1 R.sub.2, wherein n is an
integer of 2-8, and R.sub.1 and R.sub.2 are as defined above; with
the proviso that R.sub.1, R.sub.2 and R.sub.3 do not contain a halo
or a hydroxy substituent .alpha. to a heteroatom, as described in
U.S. patent application Ser. No. 07/950,637, filed Sep. 22,
1992.
In addition to the nitric oxide-releasing compounds described
above, other nitric oxide-containing compounds that spontaneously
release NO under physiological conditions and do not require the
presence of oxygen can be used in the present inventive method.
These compounds include S-nitroso adducts of the formula
O.dbd.N--S--R, wherein R is selected from the group consisting of a
C.sub.1 -C.sub.8 alkyl, a C.sub.6 -C.sub.10 aryl, a C.sub.4
-C.sub.10 heterocyclic nitrogen-containing radical, a C.sub.6
-C.sub.10 aryl substituted with a C.sub.1 -C.sub.3 alkyl, and a
C.sub.3 -C.sub.10 cycloalkyl, which R groups may be substituted by
1-3 substituents, which may be the same or different and are
selected from the group consisting of halo, hydroxy, C.sub.1
-C.sub.8 alkoxy, amino, amido, formyl, carboxy, and nitro.
Preferred S-nitroso adducts include S-nitroso adducts of peptides
and proteins, particularly S-nitroso-N-acetyl penicillamine (SNAP)
(Morley et al., J. Cardiovasc. Pharmacol., 21, 670-676 (1993);
Feelisch, J. Cardiovasc. Pharmacol., 17, S25-S33 (1991); Stamler et
al., PNAS (USA), 89, 7674-7677 (1992); and Stamler et al., PNAS
(USA), 89, 444-448 (1992)). Such adducts offer the advantages of
cell-targeting methods, through the use of adducts of S-nitroso
cell-specific antibodies and adducts of S-nitroso peptides that
mimic recognition sequences of receptor ligands.
The compounds used in the present inventive method are
characterized in that they are highly soluble in physiological
solutions and release NO spontaneously without the need for
enzymatic conversion. The release of NO, in particular the rate of
release, can be controlled by the choice of the nucleophile moiety,
is independent of the presence of oxygen, and is not accompanied by
overtly toxic byproducts.
The compounds used in the present inventive methods may be
synthesized according to methods that are well known in the art. It
is preferred that appropriate amines be obtained from suitable
commercial suppliers and reacted with nitric oxide under suitable
conditions to obtain the desired compound. Suitable commercial
suppliers include, among others, Aldrich Chemical Co., Milwaukee,
Wis.
Once a suitable amine has been synthesized or otherwise obtained
(e.g., from a commercial supplier), it may then be reacted with
nitric oxide to obtain a compound for use in the present invention.
For example, one of the methods of Drago et al., J. Am. Chem. Soc.,
83, 1819-1822 (1961), may be used to react a suitable primary amine
with nitric oxide. Certain diamines may be prepared in accordance
with Garrido et al., J. Org. Chem., 49, 2021-2023 (1984). Certain
triamines may be prepared in accordance with Bergeron, Accts. Chem.
Res., 19, 105-113 (1986). Bergeron, in J. Ore. Chem., 53, 3108-3111
(1988), also describes various methods that may be used to prepare
tetraamines. Carboni et al., Tet. Let., 29, 1279-1282 (1988),
discloses techniques that are relevant to the preparation of di-,
tri-, and tetraamines. Other methods that may be employed in
synthesis are described in U.S. Pat. Nos. 4,954,526 and
5,155,137.
Once a suitable amine has been prepared or commercially obtained,
it may then be reacted with nitric oxide to produce one of the
nitric oxide-containing compounds to be used in the present
inventive methods. Suitable methods are described in the '526 and
'137 patents, for example. If certain of the amines to be reacted
with nitric oxide contain additional nitrogen, oxygen, or other
heteroatoms, suitable blocking groups may be employed to prevent
the reaction of such atoms with nitric oxide. The blocked
heteroatoms may then be unblocked after the Drago reaction of the
amine with nitric oxide. Such blocking/deblocking agents and
methods of using them are known in the art.
Once the desired nitric oxide adduct has been prepared, a
pharmaceutically acceptable salt thereof can be prepared, if
desired. For example, the potassium salt of the compound can be
prepared by reacting the compound with potassium hydroxide in
ethanol or similar solution. Alternatively, sodium, calcium, and
magnesium salts, among others, can be prepared.
The nitric-oxide releasing compounds can be used in the method of
the present invention in many forms, including by way of
illustration as the compounds per se or in the form of their
pharmaceutically acceptable salts and derivatives. The compounds
can be used alone or in appropriate combination with one or more
other compounds/derivatives of nitric-oxide releasing compounds or
with other active compounds. It should be understood, however, that
the salt or derivative should not be one that renders the compound
unstable or insoluble in water or toxic at the doses
contemplated.
S-nitroso adducts of peptides and proteins form readily as
described in the art. See, for example, the two papers by Stamler
et al., supra.
The nitric-oxide releasing compounds can also be incorporated into
a polymeric matrix as described in U.S. patent application Ser. No.
07/935,565. Incorporation of the N.sub.2 O.sub.2.sup.- functional
group into a polymeric matrix provides a polymer-bound nitric
oxide/nucleophile adduct composition that can be applied with
specificity to a biological target site. Site-specific application
of a polymer-bound adduct enhances the selectivity of action of the
nitric oxide releasing N.sub.2 O.sub.2.sup.- functional group. If
N.sub.2 O.sub.2.sup.- functional groups attached to the polymer are
necessarily localized, then the effect of their nitric oxide
release will be concentrated in the tissues with which they are in
contact. If the polymer is soluble, selectivity of action can still
be arranged, for example, by attachment to or derivatization of an
antibody specific to the target tissue. Similarly, attachment of
N.sub.2 O.sub.2.sup.- groups to small peptides that mimic the
recognition sequences of ligands for important receptors provides
localized concentrated effect of nitric oxide release, as would
attachment to oligonucleotides capable of site-specific
interactions with target sequences in a nucleic acid.
Additionally, incorporation of the N.sub.2 O.sup.2.sup.- functional
group into a polymer matrix can reduce the propensity of the nitric
oxide/nucleophile adduct for the relatively rapid release of nitric
oxide. This prolongs the release of nitric oxide by the N.sub.2
O.sub.2.sup.- functional group, and allows for efficient dosing to
achieve a desired biological effect so the frequency of dosing can
be reduced.
While not being bound to any particular theory, it is believed that
longevity of nitric oxide release in the polymer-bound nitric
oxide/nucleophile adduct compositions of the present invention is
to be attributed both to the physical structure of the composition
and to electrostatic effects. Thus, it is believed that if the
polymer is an insoluble solid, N.sub.2 O.sub.2.sup.- groups near
the surface of the particle should be available for rapid release
while those that are more deeply imbedded are sterically shielded,
requiring more time and/or energy for the nitric oxide to work its
way into the medium. Unexpectedly, it has been found that
increasing positive charge in the vicinity of an N.sub.2
O.sub.2.sup.- functional group also tends to increase the half-life
of nitric oxide generation. The mechanism of this rate retardation
may be attributable simply to repulsive electrostatic interactions,
i.e., increasing the number of H.sup.+ -repelling positive charges
in the vicinity of the N.sub.2 O.sub.2.sup.- groups inhibits attack
of positively charged H.sup.+ ions on the N.sub.2 O.sub.2.sup.-
functional group and slows the rate of its H.sup.+ - catalyzed
decomposition. For example, by attaching amino groups to the
polymeric support that are capable of forming the nitric
oxide-releasing N.sub.2 O.sub.2.sup.- functional group on reaction
with nitric oxide, partially converted structures can be produced
on less-than-exhaustive treatment with nitric oxide that after
exposure to water contain a large number of positively charged
ammonium centers surrounding the N.sub.2 O.sub.2.sup.- group that
electrostatically inhibit the approach of H.sup.+ ions capable of
initiating nitric oxide loss from the nitric oxide releasing
N.sub.2 O.sub.2.sup.- functional group.
The nitric oxide-releasing N.sub.2 O.sub.2.sup.- functional groups
that are bound to the polymer generally are capable of releasing
nitric oxide in an aqueous environment spontaneously upon
contacting an aqueous environment, i.e., they do not require
activation through a redox reaction or electron transfer such as is
required for glyceryl trinitrate and sodium nitroprusside. Some of
the nitric oxide/nucleophile complexes useful in the context of the
present invention do require activation by particular means, but
only as necessary to free the nitric oxide releasing
X[N(O)NO].sup.- group in the vicinity of the particular cells of
interest. As an example, covalent attachment of a protecting group
to the anionic [N(O)NO].sup.- function provides a means of
postponing nitric oxide release until the molecule reaches an organ
capable of metabolically removing the protecting group. By choosing
a protecting group that is selectively cleaved by enzymes specific
to a cell or tissue of interest, for example, the action of the
nitric oxide/nucleophile complex can be targeted to maximize the
desired effect. While the polymer-bound nitric oxide releasing
compositions of the present invention are capable of releasing
nitric oxide in an aqueous solution, such a compound preferably
releases nitric oxide under physiological conditions.
The nitric oxide releasing N.sub.2 O.sub.2 functional group for
attachment to a polymer is preferably a nitric oxide/nucleophile
adduct, e.g., a complex of nitric oxide and a nucleophile, most
preferably a nitric oxide/nucleophile complex which contains the
anionic moiety X[N(O)NO].sup.-, where X is any suitable nucleophile
residue. The nucleophile residue is preferably that of Formula I,
such as a primary (e.g., X.dbd.(CH.sub.3).sub.2 CHNH, as in
(CH.sub.3).sub.2 CHNH[N(O)NO]Na) or secondary amine (e.g.,
X.dbd.(CH.sub.3 CH.sub.2).sub.2 N, as in (CH.sub.3 CH.sub.2).sub.2
N[N(O)NO]Na), or a polyamine (e.g., X=spermine, as in the
zwitterion H.sub.2 N(CH.sub.2).sub.3 NH.sub.2.sup.+
(CH.sub.2).sub.4 N[N(O)NO].sup.- (CH.sub.2).sub.3 NH.sub.2, or
X=3-(n-propylamino) propylamine, as in the zwitterion CH.sub.3
CH.sub.2 CH.sub.2 N[N(O)NO].sup.- CH.sub.2 CH.sub.2 CH.sub.2
NH.sub.3 .sup.+), or a derivative thereof. Such nitric
oxide/nucleophile complexes are stable solids and are capable of
delivering nitric oxide in a biologically usable form at a
predictable rate.
The nucleophile residue for polymer attachment is preferably not an
entity such as that of sulfite (e.g., X.dbd.SO.sub.3, as in
NH.sub.4 O.sub.3 S[N(O)NO]NH.sub.4) even though the complex is a
stable compound, since it is capable of releasing nitric oxide in
an aqueous environment only under harsh, nonphysiological
conditions.
Other suitable nitric oxide/nucleophile complexes for attachment to
a polymer include those having the formulas of Formulas II-VIII
above.
Any of a wide variety of polymers can be used to make polymer
NONOates. It is only necessary that the polymer selected is
biologically acceptable. Illustrative of polymers suitable for use
in the present invention are polyolefins, such as polystyrene,
polypropylene, polyethylene, polytetrafluorethylene, polyvinylidene
difluoride, polyvinylchloride, derivatized polyolefins such as
polyethylenimine, polyethers, polyesters, polyamides such as nylon,
polyurethanes, biopolymers such as peptides, proteins,
oligonucleotides, antibodies and nucleic acids, starburst
dendrimers, and the like.
The physical and structural characteristics of the polymers
suitable for use in the present invention are not narrowly
critical, but rather will depend on the end use application. It
will be appreciated by those skilled in the art that where the
polymer-bound nitric oxide/nucleophile adduct compositions of the
present invention are intended for topical, dermal, percutaneous,
or similar use, they need not be biodegradable. For some uses, such
as ingestion or the like, it may be desirable that the polymer of
the polymer-bound compositions slowly dissolves in a physiological
environment or that it is biodegradable.
The nitric oxide-releasing complexes having N.sub.2 O.sub.2.sup.-
functional groups, including the compounds described above, may be
bound to the polymer support in a number of different ways. For
example, the compounds described above may be bound to the polymer
by coprecipitation of such compounds with the polymer.
Coprecipitation involves, for example, solubilizing both the
polymer and the nitric oxide/nucleophile compound and evaporating
the solvent.
Alternatively, nitric oxide releasing N.sub.2 O.sub.2.sup.-
functional groups may be bound to the polymer by formation of a
nitric oxide/nucleophile complex of the types and having the
formulas of those described above, in situ on the polymer. The
N.sub.2 O.sub.2.sup.- functional group may be attached to an atom
in the backbone of the polymer, or it may be attached to a group
pendant to the polymer backbone, or it may simply be entrapped in
the polymer matrix. Where the N.sub.2 O.sub.2.sup.- functional
group is in the polymer backbone, the polymer includes in its
backbone sites which are capable of reacting with nitric oxide to
bind the nitric oxide for future release. For example, where the
polymer is polyethylenimine, the polymer includes nucleophilic
nitrogen atoms which react with nitric oxide to form the N.sub.2
O.sub.2.sup.- functional group at the nitrogen in the backbone.
Where the N.sub.2 O.sub.2.sup.- functional group is a group pendant
to the polymer backbone, the polymer contains, or is derivatized
with, a suitable nucleophilic residue capable of reacting with
nitric oxide to form the N.sub.2 O.sub.2.sup.- functionality.
Reaction of the polymer which contains a suitable nucleophilic
residue, or of the suitably derivatized polymer with nitric oxide
thus provides a polymer-bound nitric oxide-releasing N.sub.2
O.sub.2.sup.- functional group. To form the polymer-bound nitric
oxide releasing N.sub.2 O.sub.2.sup.- functional group, it is
generally preferred to impart a net charge to the polymer near the
site on the polymer where the N.sub.2 O.sub.2.sup.- functional
group is to be formed. The resulting polymer bound nitric
oxide-releasing compounds may then be administered as described
below or may be formed into an implant for implantation in or near
a site of ischemia reperfusion injury for example.
The present inventive methods can be utilized in vitro for
scientific and research purposes. However, the methods of the
present invention have particular usefulness in in vivo
applications, such as treating oxygen free radical mediated tissue
damage associated with ischemia reperfusion injury. "Treating"
means protecting against the onset of oxygen free radical mediated
tissue damage, where the method is used prior to the onset of
ischemia reperfusion, as well as protecting against further oxygen
free radical mediated tissue damage, where ischemia reperfusion
injury has already been initiated. The method is believed to
accomplish the objective of treating oxygen free radical mediated
tissue damage by intercepting reactive oxygen species or by forming
metal nitrosyl complexes, which prevent formation of reactive
oxygen species. The NO is also believed to increase blood flow in
damaged tissue, thereby increasing oxygenation. Accordingly, the
present inventive methods have both prophylactic and therapeutic
benefits. The present invention includes the administration to a
mammal, particularly a human, at risk for or having ischemia
reperfusion injury an amount of one or more of the nitric-oxide
releasing compounds previously described or pharmaceutically
acceptable salts or derivatives thereof or polymers, alone or in
combination with one or more other pharmaceutically active
compounds, in a pharmaceutically acceptable composition sufficient
to treat the oxygen free radical mediated tissue damage.
The nitric oxide-releasing compound or polymer preferably is
administered as soon as possible after it has been determined that
a mammal, particularly a human, is at immediate risk for ischemia
reperfusion injury or has just begun to realize ischemia
reperfusion injury. It is expected that, in most situations, the
nitric oxide-releasing compound will be administered within about
15 minutes to about 60 minutes of injury, i.e., before or after
injury as appropriate. When it is possible to predict the onset of
ischemia reperfusion, e.g., associated with transplantation, the
compound or polymer should be administered immediately upon
knowledge of need. It is expected that, in such situations, the
nitric-oxide-releasing compound will be administered within about
15 minutes before the onset of ischemia reperfusion. When ischemia
reperfusion injury has already begun, the compound or polymer
should be administered as soon as possible after the onset of
ischemia reperfusion. It is expected that, in such situations, the
nitric oxide-releasing compound will be administered within about
15 minutes to about 60 minutes after the onset of ischemia
reperfusion. The term to treat the oxygen free radical mediated
tissue damage will depend, in part, upon the particular nitric
oxide-releasing compound or polymer used, the amount administered,
the method of administration, and the cause and extent of oxygen
free radical mediated damage anticipated or realized.
The present inventive method is useful in treating oxygen free
radical mediated tissue damage associated with any condition or
disease state associated with or characterized by ischemia
reperfusion, wherein "ischemia," otherwise known as hypoemia,
refers to a region of localized tissue anemia due to the
obstruction of arterial blood flow to the region and wherein
"reperfusion" refers to the restoration of blood flow to the
ischemic region. Examples of such conditions and disease states
include transplantation, trauma, inflammation, stroke, seizure,
rheumatoid arthritis, atherosclerosis, cancer, dementia, diabetes,
hypertensive crisis, ulcers, lupus, sickle cell anemia, ischemic
bowel syndrome, pulmonary emboli, Ball's syndrome, pancreatitis,
heart attack, and aging, for example. Accordingly, use of the term
"ischemia reperfusion" is intended to encompass these and other
conditions involving ischemia reperfusion.
One skilled in the art will appreciate that suitable methods of
administering a nitric oxide-releasing compound useful in the
method of the present invention to a mammal are available. Although
more than one route can be used to administer a particular
compound, a particular route can provide a more immediate and more
effective reaction than another route. Accordingly, the described
methods are merely exemplary and are in no way limiting.
The dose administered to an animal, particularly a human, in
accordance with the present invention should be sufficient to
effect the desired response, i.e., treatment of oxygen free radical
mediated tissue damage, in the animal over a reasonable time frame.
One skilled in the art will recognize that dosage will depend upon
a variety of factors including the strength (i.e., nitric oxide
release capability) of the particular compound employed, the age,
species, condition or disease state, and body weight of the animal,
as well as the amount of cells or tissue about to be affected or
actually affected by ischemia reperfusion injury. The size of the
dose will also be determined by the route, timing and frequency of
administration as well as the existence, nature, and extent of any
adverse side-effects that might accompany the administration of a
particular compound and the desired physiological effect. It will
be appreciated by one of skill in the art that various conditions
or disease states, in particular chronic conditions or disease
states, may require prolonged treatment involving multiple
administrations.
Suitable doses and dosage regimens can be determined by
conventional range-finding techniques known to those of ordinary
skill in the art. Generally, treatment is initiated with smaller
dosages, which are less than the optimum dose of the compound.
Thereafter, the dosage is increased by small increments until the
optimum effect under the circumstances is reached. The present
inventive method will typically involve the administration of about
0.1 to about 100 mg of one or more of the compounds or polymers
described above per kg body weight.
The present invention also provides a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an amount of a
nitric-oxide containing compound sufficient to treat oxygen free
radical-mediated tissue damage. The carrier may be any of those
conventionally used and is limited only by chemico-physical
considerations, such as solubility and lack of reactivity with the
compound, and by the route of administration. It will be
appreciated by one of skill in the art that, in addition to the
following described pharmaceutical composition, the compounds of
the present inventive method may be formulated as inclusion
complexes, such as cyclodextrin inclusion complexes, or
liposomes.
Examples of pharmaceutically acceptable acid addition salts for use
in the present inventive pharmaceutical composition include those
derived from mineral acids, such as hydrochloric, hydrobromic,
phosphoric, metaphosphoric, nitric and sulfuric acids, and organic
acids, such as tartaric, acetic, citric, malic, lactic, fumaric,
benzoic, glycolic, gluconic, succinic, and arylsulphonic, for
example p-toluenesulphonic acids.
The pharmaceutically acceptable excipients described herein, for
example, vehicles, adjuvants, carriers or diluents, are well-known
to those who are skilled in the art and are readily available to
the public. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the active compounds
and one which has no detrimental side effects or toxicity under the
conditions of use.
The choice of excipient will be determined in part by the
particular compound, as well as by the particular method used to
administer the composition. Accordingly, there is a wide variety of
suitable formulations of the pharmaceutical composition of the
present invention. The following formulations for oral, aerosol,
parenteral, subcutaneous, intravenous, intramuscular,
interperitoneal, rectal, and vaginal administration are merely
exemplary and are in no way limiting.
Injectable formulations are among those formulations that are
preferred in accordance with the present inventive methods. The
requirements for effective pharmaceutical carriers for injectable
compositions are well known to those of ordinary skill in the art
(See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,
Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250,
(1982), and ASHP Handbook on Iniectable Drugs, Toissel, 4th ed.,
pages 622-630 (1986)). It is preferred that such injectable
compositions be administered intravenously or locally, i.e., at or
near the site of ischemia reperfusion injury.
Topical formulations are well-known to those of skill in the art
and are suitable in the context of the present invention for
application to skin and hair as radiation protection.
Formulations suitable for oral administration can consist of (a)
liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets, tablets, lozenges, and troches, each containing
a predetermined amount of the active ingredient, as solids or
granules; (c) powders; (d) suspensions in an appropriate liquid;
and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant, suspending
agent, or emulsifying agent. Capsule forms can be of the ordinary
hard- or soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers, such as lactose,
sucrose, calcium phosphate, and corn starch. Tablet forms can
include one or more of lactose, sucrose, mannitol, corn starch,
potato starch, alginic acid, microcrystalline cellulose, acacia,
gelatin, guar gum, colloidal silicon dioxide, croscarmellose
sodium, talc, magnesium stearate, calcium stearate, zinc stearate,
stearic acid, and other excipients, colorants, diluents, buffering
agents, disintegrating agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible excipients.
Lozenge forms can comprise the active ingredient in a flavor,
usually sucrose and acacia or tragacanth, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to the active ingredient, such excipients
as are known in the art.
The compounds or polymers of the present invention, alone or in
combination with other suitable components, can be made into
aerosol formulations to be administered via inhalation. These
aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. They also may be formulated as pharmaceuticals for
non-pressured preparations, such as in a nebulizer or an atomizer.
Such spray formulations may be used to spray mucosa.
Formulations suitable for parenteral administration include aqueous
and non-aqueous, isotonic sterile injection solutions, which can
contain anti-oxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The compound may be administered in
a physiologically acceptable diluent in a pharmaceutical carrier,
such as a sterile liquid or mixture of liquids, including water,
saline, aqueous dextrose and related sugar solutions, an alcohol,
such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such
as propylene glycol or polyethylene glycol, dimethylsulfoxide,
glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol,
ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a
fatty acid ester or glyceride, or an acetylated fatty acid
glyceride with or without the addition of a pharmaceutically
acceptable surfactant, such as a soap or a detergent, suspending
agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or
emulsifying agents and other pharmaceutical adjuvants.
Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral.
Suitable fatty acids for use in parenteral formulations include
oleic acid, stearic acid, and isostearic acid. Ethyl oleate and
isopropyl myristate are examples of suitable fatty acid esters.
Suitable soaps for use in parenteral formulations include fatty
alkali metal, ammonium, and triethanolamine salts, and suitable
detergents include (a) cationic detergents such as, for example,
dimethyl dialkyl ammonium halides, and alkyl pyridinium halides,
(b) anionic detergents such as, for example, alkyl, aryl, and
olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-.beta.-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
The parenteral formulations will typically contain from about 0.5
to about 25% by weight of the active ingredient in solution.
Preservatives and buffers may be used. In order to minimize or
eliminate irritation at the site of injection, such compositions
may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations will typically
range from about 5 to about 15% by weight. Suitable surfactants
include polyethylene sorbitan fatty acid esters, such as sorbitan
monooleate and the high molecular weight adducts of ethylene oxide
with a hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
Additionally, the compounds and polymers useful in the present
inventive methods may be made into suppositories by mixing with a
variety of bases, such as emulsifying bases or water-soluble bases.
Formulations suitable for vaginal administration may be presented
as pessaries, tampons, creams, gels, pastes, foams, or spray
formulas containing, in addition to the active ingredient, such
carriers as are known in the art to be appropriate.
The following examples further illustrate the present invention
and, of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
This example describes the treatment of peroxide-mediated
cytotoxicity in Chinese hamster V79 fibroblasts by nitric-oxide
containing compounds that spontaneously release nitric oxide under
physiological conditions without requiring the presence of
oxygen.
Chinese hamster V79 lung fibroblasts were cultured in F12 medium
supplemented with 10% fetal calf serum and antibiotics. Cell
survival was assessed by clonogenic assay with 85-95% plating
efficiency. Stock cultures of exponentially growing cells were
trypsinized, rinsed, and plated (7.times.10.sup.5 cells/dish) into
a number of 100 cm.sup.2 petri dishes and incubated 16 hr at
37.degree. C. prior to experimental protocols.
V79 cells exposed to hydrogen peroxide or HX/XO have been shown to
serve as a good model for the study of reactive oxygen species in
events such as ischemia reperfusion (Mitchell et al., supra, Gelvan
et al., PNAS USA, 88, 4680-4684 (1991)). It has been shown that
exposure of V79 cells to hydrogen peroxide results in
dose-dependent cytotoxicity (Mitchell et al., supra). HX/XO induced
cytotoxicity was not treated by the addition of superoxide
dismutase. In contrast, catalase addition treated the cytotoxicity,
indicating that hydrogen peroxide was the predominant toxin
(Mitchell et al., supra).
HX and XO were purchased from Boehringer Co. (Indianapolis, Ind.).
Nitrite, diethylamine, sulfinamide, diethylenetriaminepentaacetic
acid (DETAPAC) and N-(1-naphthyl)ethylenediaminedihydrogenchloride
(NEDD) were purchased from Aldrich (Milwaukee, Wis.).
Cytosine-.beta.-D-arabinofuranoside and ferricytochrome c were
purchased from Sigma (St. Louis, Mo.). DEA/NO and SPER/NO were
synthesized as previously described (Maragos et al., (1991),
supra).
Exponentially growing cultures of Chinese hamster V79 fibroblasts
were exposed to either HX/XO (final concentration of HX was 0.5 mM;
0.08 units/ml XO were used) for various amounts of time (FIG. 1A)
or to various concentrations of hydrogen peroxide for one hour
(FIG. 1B). DEA/NO, SPER/NO, nitrite, or diethylamine (final
concentration of 1 mM) were added to parallel cultures immediately
prior to addition of HX/XO or hydrogen peroxide. Additionally, 1 mM
DEA/NO was added to medium in the absence of cells, incubated at
37.degree. C. for 1 hr or 16 hrs, and then added just prior to the
addition of hydrogen peroxide to evaluate the effects of DEA/NO
which had released NO. After treatment, cell monolayers were rinsed
twice with phosphate-buffered saline (PBS), trypsinized, counted
and plated in triplicate for macroscopic colony formation. Each
dose determination was plated in triplicate and experiments were
repeated a minimum of two times. Plates were incubated for 7 days,
after which colonies were fixed with methanol/acetic acid (3:1),
stained with crystal violet, and counted. Colonies containing more
than 50 cells were scored.
The activity of XO was monitored in the absence and presence of 1
mM DEA/NO by two different assays. Superoxide-induced reduction of
ferricytochrome c to ferrocytochrome c was monitored
spectrophotometrically at 550 nm (Fridovich, Handbook of Method for
Oxygen Radical Research, pages 213-215 (1985)). The reaction was
carried out in a 1 ml volume in aerated phosphate buffer (pH 7.8,
50 mM) containing 50 .mu.M DETAPAC. HX was maintained at 2.5 mM and
ferricytochrome c at 20 .mu.M. The reactions were initiated with
the addition of XO (final concentration of 0.2 units/ml). The
activity of XO in the absence and presence of 1 mM DEA/NO was
directly monitored by measuring the production of uric acid
spectrophotometrically at 305 nm for 10 min under the same
conditions but in the absence of ferricytochrome c. All enzymatic
assays and chemical reactions were done at 37.degree. C.
Anaerobic solutions of 1 mM hydrogen peroxide in 10 mM phosphate
buffer, pH 7.4, were mixed with 1 mM NO. No rapid formation of
nitrate/nitrite (<1000 s) was observed at 210 nm using
stopped-flow techniques (Wink et al., Chem. Res. Toxicol., 6, 23-27
(1993)). In addition, the nitrosation of sulfinamide in an aerobic
solution (100 mM phosphate buffer, pH 7.4) by intermediates of the
NO/O.sub.2 reaction followed by subsequent diazotization with NEDD
to form the azo dye was not inhibited in the presence of 1 mM
hydrogen peroxide; thus, the consumption of NO by hydrogen peroxide
was not significant under these conditions. The rate of
decomposition of DEA/NO was unaffected by the presence of 1 mM
hydrogen peroxide; likewise, hydrogen peroxide was not consumed by
DEA/NO or intermediates of the DEA/NO decomposition reaction as
measured by the production of I.sub.3.sup.- (Hochanadel, J. Phys.
Chem., 56, 587-594 (1952)).
FIG. 1A is a graph of log of surviving fraction versus time (min),
wherein the lines represent the exposure of cells to
hypoxanthine/xanthine oxidase in the absence of a nitric-oxide
releasing compound (control, O), in the presence of 0.1 mM
(.tangle-solidup.) and 1 mM (.circle-solid.) DEA/NO, and in the
presence of 1 mM SPER/NO (.box-solid.). FIG. 1A shows that exposure
of V79 cells for various time intervals to HX/XO in the absence of
a nitric oxide-releasing compound resulted in cell killing.
Although 0.1 mM DEA/NO provided marginal protection against HX/XO
generated superoxide radicals, 1 mM DEA/NO significantly protected
against cell killing resulting from superoxides generated from
hypoxanthine/xanthine oxidase. Accordingly, these results clearly
show that hydrogen peroxide-mediated cytotoxicity can be prevented
by the presence of an NO-generating compound. SPER/NO (1 mM), which
releases NO 40 times slower than DEA/NO, also protected V79 cells
against HX/XO induced cytotoxicity but to a lesser extent than
DEA/NO. Given that the amount of NO released from SPER/NO is less
than that released from DEA/NO, this further suggests that NO
released from these complexes is responsible for the
protection.
Another possible toxic agent in mammalian cells is peroxynitrite
anion (OONO.sup.-), which would be expected to form in the presence
of O.sub.2.sup.- that is generated from XO and NO (Beckman, J. Dev.
Physiol., 15, 53-59 (1991); Zhu et al., Arch. Biochem. Biophys.,
298, 452-457 (1992); Ischiropoulos et al., Arch. Biochem. Biophys.,
298, 431-437 (1992); and Beckman et al. (1990), supra). The
reaction rate constant for NO and O.sub.2 is reported to be
5.6.times.10.sup.7 M.sup.-1 S.sup.-1, with the product being the
potent oxidant (OONO.sup.-) (Saran et al., Free Radic. Res.
Commun., 10, 221-226 (1990)). This anion has been speculated to
play a critical role in potentiating the toxic effects of NO,
although OONO.sup.- is rapidly converted at physiological pH to
nitrate. The quenching of O.sub.2.sup.- reduction of
ferricytochrome c by DEA/NO can be explained by the scavenging of
the O.sub.2.sup.- by NO to form peroxynitrite anion. However, any
peroxynitrite anion which might be formed under the above
conditions does not induce cytotoxicity as shown in FIG. 1A.
DEA/NO (1 mM) alone and its breakdown products diethylamine (1 mM)
and nitrite (1 mM) preincubated with HX/XO did not inhibit the
activity of XO as measured by the production of superoxide
(cytochrome c reduction) or uric acid production.
FIG. 1B is a graph of log of surviving fraction versus time (min),
wherein the lines represent the exposure of cells to hydrogen
peroxide in the absence (control, O) and presence of the DEA/NO
breakdown products diethylamine (.box-solid.) and nitrite (.brket
open-st.). FIG. 1B shows that the decomposition products of DEA/NO
did not protect cells from hydrogen peroxide or HX/XO-induced
cytotoxicity.
Monitoring of UV absorption changes at 305 nm also indicated that
uric acid production was not inhibited by the presence of DEA/NO.
Accordingly, substrate turnover is not inhibited either reversibly
or irreversibly. The presence of DEA/NO, however, inhibited the SOD
sensitive ferricytochrome c reduction. This suggests that either
reduction of oxygen to form superoxide was inhibited or NO
scavenged the HX/XO-generated superoxide to form peroxynitrite
anion, which was then rapidly converted to nitrate.
FIG. 2A is a graph of log of surviving fraction versus hydrogen
peroxide concentration (.mu.M) for a 1 hr exposure, wherein the
lines represent control (O), 1 mM DEA/NO (.circle-solid.), 1 mM
DEA/NO with 1 hr release (.quadrature.), and 1 mM DEA/NO with 16 hr
release (.tangle-solidup.). FIG. 2A shows that 1 mM DEA/NO provided
nearly complete protection against hydrogen peroxide cytotoxicity.
V79 cells exposed to a solution of 1 mM DEA/NO allowed to release
NO for 1 hr prior to addition of hydrogen peroxide exhibited only
modest protection and was not as effective as DEA/NO added just
prior to hydrogen peroxide addition. However, 1 mM DEA/NO incubated
for 16 hr in medium prior to addition of hydrogen peroxide enhanced
the cytotoxicity of hydrogen peroxide. Similarly, cells treated for
one hour with hydrogen peroxide followed by a 1 hr treatment with 1
mM DEA/NO did not treat oxygen free radical mediated tissue
damage.
FIG. 2B is a graph of log of surviving fraction versus hydrogen
peroxide concentration (.mu.M), wherein the lines represent control
(O), diethylamine (.box-solid.), and nitrite (.circle-solid.). FIG.
2B shows that diethylamine (1 mM) had no effect on hydrogen
peroxide cytotoxicity, while nitrite (1 mM) potentiated hydrogen
peroxide cytotoxicity. In vivo, however, very little nitrite is
generated and it is rapidly excreted form the body.
Chemical controls demonstrated that the rate of formation of NO due
to the decomposition of DEA/NO was not altered by the presence of
hydrogen peroxide. Conversely, hydrogen peroxide was not consumed
in the presence of DEA/NO. These results show that the
decomposition of DEA/NO did not consume hydrogen peroxide or HX/XO
nor affect XO substrate turnover and that the NO-generating
compounds are mediating the cell protection and must be present
during hydrogen peroxide exposure in order to be protective.
EXAMPLE 2
This example describes the protection against peroxide-mediated
damage of neuronal function in ventral mesencephalic cells by
nitric-oxide containing compounds that spontaneously release nitric
oxide under physiological conditions without requiring the presence
of oxygen.
The ventral tegmental mesencephalon was dissected from 14 day old
embryos (precisely timed pregnant Sprague Dawley rats;
Zivic-Miller, Allison Park, Pa.) under sterile conditions and
mechanically dissociated into complete culture medium. The culture
medium consisted of a 1:1 mixture of modified minimum essential
medium and nutrient mixture F-12 supplemented with 6 mg/ml
D-gluose, 2 mM glutamine, 0.5 U/ml penicillin G, 0.5 mg/ml
streptomycin (all from Gibco, Grand Island, N.Y.) and 15% equine
serum (Hyclone, Logan, Utah). Cells were plated into multiwell
plates that were previously coated with poly-D-lysine (15 .mu.g/ml)
and laminin (10 .mu.g/ml) at a density of 40,000 cells/cm.sup.2
(Costar). The cells were maintained for 5-7 days at 37.degree. C.
in an atmosphere of 95% air and 5% CO.sub.2 saturated with H.sub.2
O. On the fifth day of culture 1 .mu.M
cytosine-.beta.-D-arabinofuranoside was added to inhibit glial cell
growth.
Uptake of dopanine by dopaminergic neurons was assayed as follows.
The cells in each well were washed three times with 1 ml PBS
containing 6 mg/ml glucose and thereafter Dulbecco's modified
essential medium (Quality Biological, Inc., Farmingham, Mass.)
containing 6 mg/ml D-glucose, 50 .mu.M ascorbic acid, and
5.times.10.sup.-8 M [.sup.3 H]dopamine (NEN, Boston, Mass.;
specific activity of 45 .mu.Ci/mmol) was added and incubated for 15
min at 37.degree. C. [.sup.3 H]dopamine uptake was stopped by
aspirating the incubation solution and washing the cells three
times with ice-cold PBS containing 6 mg/ml D-glucose. The cells
were removed by washing the wells with an equal volume of 0.2N NaOH
and 0.2N HCl containing 0.02% Triton X-100. The residual
intracellular radioactivity in the cells was determined by
scintillation spectroscopy.
Accordingly, ventral mesencephalic cell cultures were exposed to
100 .mu.M H.sub.2 O.sub.2 for 1 hr in the presence and absence of
100 .mu.M DEA/NO and evaluated for their ability to take up
dopamine.
FIG. 3 is a graph of .sup.3 H-dopamine concentration (pmol [.sup.3
H-DA]) versus H.sub.2 O.sub.2 concentration ([H.sub.2 O.sub.2 ]
.mu.M), wherein lines represent dopamine uptake by ventral
mesencephalic cell cultures in the presence (.box-solid.) and
absence (.tangle-solidup.) of DEA/NO. FIG. 3 shows that hydrogen
peroxide significantly reduced the ability of the cells to take up
dopamine in the absence of DEA/NO, whereas cells exposed to
hydrogen peroxide in the presence of DEA/NO retained nearly 100% of
their function.
In a separate study, cells were cultured for 7 days and then
exposed to 50 or 100 .mu.M hydrogen peroxide for 60 min or to 0.04
units/ml HX/XO for 5 or 10 min in the absence or presence of 100
.mu.M DEA/NO. The cells were washed with PBS containing 6 g/l
D-glucose, culture medium was added, and the cells were incubated
for 18 hr. The ability of the cells (6 wells of 450,000 cells/well
per group) to take up .sup.3 [H]-dopamine was measured. These cells
were exposed to ten times less hydrogen peroxide or HX/XO than the
V79 cells described above. The results are shown in Table I.
TABLE I ______________________________________ [.sup.3 H] Dopamine
Uptake Addition to Time of (pmol/well/15 min) Incubation Medium
Exposure Control 100 .mu.M DEA/NO
______________________________________ None 2.9 .+-. 0.20 2.7 .+-.
0.11 50 .mu.M H.sub.2 O.sub.2 60 min 0.63 .+-. 0.15* 2.8 .+-. 0.11
100 .mu.M H.sub.2 O.sub.2 60 min 0.15 .+-. 0.014* 2.6 .+-. 0.22
0.04 U/ml HX/XO 5 min 1.1 .+-. 0.21* 2.6 .+-. 0.15 0.04 U/ml HX/XO
10 min 0.36 .+-. 0.21* 2.3 .+-. 0.10
______________________________________ * p < 0.01 when compared
with nontreated group (Student's tTest)
As shown in Table I, when 100 .mu.M DEA/NO was added together with
the hydrogen peroxide or HX/XO, complete protection was observed.
Since radiolabeled dopamine uptake can be used as a measure of
neurite viability, it can be inferred that NO protects neurons from
damage induced by reactive oxygen species.
When cells were exposed to hydrogen peroxide for 1 hr, varicosities
formed in the neurites and somas swelled. Exposure to HX/XO for as
short as 5 min elicited similar morphological changes in
mesencephalic neurons. In contrast, cells exposed to 100 .mu.M
DEA/NO for 1 hr failed to cause morphological changes, i.e.,
DEA/NO-treated cells resembled non-treated cells. Cells exposed to
hydrogen peroxide or HX/XO in the presence of 100 .mu.M DEA/NO did
not demonstrate abnormalities in the neurites and somas.
EXAMPLE 3
This example describes the initiation of reperfusion-induced
leukocyte adhesion and microvascular permeability by diminished
nitric oxide release.
Male Sprague-Dawley rats weighing 200-250 g apiece were maintained
on a purified laboratory diet and fasted for 24 hours prior to each
experiment. The animals were initially anesthetized with
pentobarbital (65 mg/kg body weight) and then a tracheotomy was
performed to facilitate breathing during subsequent
experimentation. The right carotid artery was cannulated and system
arterial pressure was measured with a Statham P23A pressure
transducer (Oxnard, Calif.) connected to the carotid artery
cannula. Systemic blood pressure and heart rate were continuously
recorder with a Grass physiologic recorded (Grass Instruments). The
left jugular vein and superior mesenteric artery were also
cannulated for drug administration.
Rats were placed in a supine position on an adjustable plexiglass
microscope stage and the mesentery was prepared for microscopic
observation as described previously (Asako et al.,
Gastroenterology, 103, 146-152 (1992); Kurose et al., Circ. Res.,
in press (1993)). Briefly, the mesentery was draped over a
non-fluorescent coverslip that allowed for observation of a 2
cm.sup.2 segment of tissue. The exposed bowel wall was covered with
Saran Wrap (Dow Chemical Co.) and the mesentery was superfused with
bicarbonate-buffered saline (pH 7.4, 37.degree. C.) that was
bubbled with a mixture of 5% CO.sub.2 and 90% N.sub.2.
An inverted microscope (TMD-25, Diaphoto, Nikon, Tokyo, Japan) with
a 40x objective lens (Fluor, Nikon) was used to observe the
mesenteric microcirculation. The mesentery was transilluminated
with a 12 V-100 W direct current-stabilized light source. A video
camera (VK-C150, Hitachi, Ibaragi, Japan) mounted on the microscope
projected the image onto a color monitor (PVM-2030, Sony, Tokyo,
Japan) and the images were recorded using a videocassette record
(NV8950, Panasonic, Tokyo, Japan). A video time-date generator
(WJ810, Panasonic) projected the time, date and stopwatch function
onto the monitor.
Single unbranched venules with diameters ranging between 25 and 35
.mu.m and length greater than 150 .mu.m were selected for study.
Venular diameter (DV) was measured either on- or off-line using a
video caliper (Microcirculation Research Institute, Texas A&M
University, College Station, Tex.). Red blood cell centerline
velocity (VRBC) was measured in venules using an optical Dopper
velocimeter (Microcirculation Research Institute, Texas A&M
University, College Station, Tex.). The velocimeter was calibrated
against a rotating glass disk coated with red blood cells. Venular
blood flow was calculated from the product of mean red blood cell
velocity (V.sub.mean =centerline velocity/1.6; Davis, Microvasc.
Res., 34, 223-230 (1987)) and microvascular cross-sectional area,
assuming cylindrical geometry. Wall shear rate (.gamma.) was
calculated based on the Newtonian definition .gamma.=8(V.sub.mean
/D).
The number of adherent leukocytes was determined off-line during
playback of videotape images. A leukocyte was considered to be
adherent to venular endothelium if it remained stationary for a
period equal to or greater than 30 seconds (Granger et al., Am. J.
Physiol., 257, G683-G688 (1989)). Adherent cells were expressed as
the number per 100 .mu.m length of venule. The number of emigrated
leukocytes was also determined off-line during playback of
videotape images. Any interstitial leukocytes present in the
mesentery at the onset of the experiment were subtracted from the
total number of leukocytes that accumulated during the course of
the experiment. Leukocyte emigration was expressed as the number
per field of view surrounding the venule. Platelet-leukocyte
aggregates, which were visible within postcapillary venules, were
quantitated and expressed as the number of aggregates crossing a
fixed point within the venule over a 5 min period. Mast cells in
the microvascular beds of the rat mesenteries were visualized by
using 0.1% toluidine blue. The number of normal and degranulated
mast cells was determined and the percentage of degranulated mast
cells was calculated.
Albumin leakage across mesenteric venules was quantified by
administering 50 mg/kg of FITC-labeled bovine albumin (Sigma
Chemical Co., St. Louis, Mo.) intravenously 15 min before each
experiment (Kurose et al., supra). Fluorescence intensity
(excitation wavelength of 420-490 nm; emission wavelength of 520
nm) was detected using a silicon intensified target camera
(C-2400-08, Hamamatsu Photonics, Skizuoka, Japan). The fluorescence
intensity of FITC-albumin within 3 segments of the venule under
study (Iv) and in 3 contiguous areas of perivenular interstitium
(Ii) area were measured at various times after administration of
FITC-albumin using a computer-assisted digital imaging processor
(Macintosh, Apple Co.). An index of vascular albumin leakage was
determined from the ratio of Ii:Iv at specific times during the
reperfusion phase.
After all parameters measured on-line were in a steady state,
images from the mesenteric preparation were recorded on videotape
for 10 min. Immediately thereafter, the superior mesenteric artery
was ligated with a snare created from polyethylene tubing. The
mesentery was made ischemic for zero (sham ischemia) or 20 min.
After the ischemic period, the ligation was gently removed. In some
experiments, either sodium nitroprusside (SNP, 100 .mu.M, Sigma
Chemical), spermine-NO (SpNO, 100 .mu.M), SIN-1 (100 .mu.M),
spermine (SP, 100 .mu.M, Sigma Chemical), or NG-nitro-L-arginine
methyl ester (L-NAME, 100 .mu.M, Sigma Chemical) was added to the
superfusate and the same protocol was employed. Spermine-NO and
SIN-1, which are NO donors, were obtained from the National
Institutes of Health, Bethesda, Md.
Plasma levels of nitrite and nitrate were determined by a
modification of the method of Green et al. (Anal. Biochem., 126,
131-138 (1982)) and Yamada et al. (Gastroenterology, 104, 759-771
(1993)). Briefly, 400 .mu.l of distilled water were added to 100
.mu.l of heparinized plasma. Protein was precipitated by the
addition of 25 .mu.l 30% ZnSO.sub.4. Five minutes after the
addition of ZnSO.sub.4, the precipitant was removed by
centrifugation. Five hundred microliters of the supernatant were
added to 25 .mu.l of E. coli-derived nitrate reductase, 20 .mu.l of
2.5M HEPES, and 50 .mu.l of 3M NH.sub.4 formate (pH 7.4), and
incubated with the reductase for 1 hr at 37.degree. C. to reduce
nitrate to nitrite (Bartholomew, Food Chem. Toxicol., 22, 541-543
(1984)). Nitrite in the incubated sample was quantified by the
method of Stuehr et al. (Sartor et al., Gastroenterology, 89,
587-595 (1985)) using the Griess reagent (1% sulfanilamide/0.1%
naphthylethylenediamine dihydrochloride/2.5% H.sub.3 PO.sub.4).
Nitrite concentrations were calculated from a standard curve using
sodium nitrite (Sigma Chemical) as the standard. The data were
analyzed using standard statistical analysis, i.e., one-way
analysis of variance and Scheffe's (post-hoc) test. All values were
reported as mean .+-. standard error from 6 rats and statistical
significance was set at p<0.05.
In untreated (control) rats, the RBC velocity and wall shear rate
in mesenteric venules were 3.12.+-.0.18 mm/sec and 535.+-.6
sec.sup.-1, respectively, under control conditions. During
occlusion of the superior mesenteric artery (SMA), blood flow
ceased within mesenteric venules. Ischemic periods up to 20 min
duration were associated with significant and sustained
reperfusion, i.e., RBC velocity (1.94.+-.0.13 mm/sec) and wall
shear rate (328.+-.16 sec.sup.-1) were restored toward normal
values following release of the SMA occlusion. Longer durations of
ischemia (>30 min) were not associated with a consistent
reperfusion response with flow rarely occurring to a significant
extent after release of the SMA occlusion. Consequently,
measurements of leukocyte-endothelial cell adhesion and albumin
leakage were obtained only in mesenteric venules exposed to 20 min
ischemia. NO donors, i.e., SNP, SpNO, and SIN-1, but not SP or
L-NAME, reversed the decreases in RBC velocity and wall shear rate
after the reperfusion. These data are summarized in Table II.
TABLE II ______________________________________ Venular diameter,
RBC velocity and wall shear rate at 30 min after reperfusion
Diameter RBC velocity Wall Shear Rate Treatment (.mu.m) (mm/sec)
(l/sec) ______________________________________ Control 29.2 .+-.
1.8 3.12 .+-. 0.18 535 .+-. 6 I/R 29.6 .+-. 1.4 1.94 .+-. 0.13* 328
.+-. 16* SNP 29.4 .+-. 1.1 2.68 .+-. 0.16.dagger. 456 .+-.
22.dagger. SIN1 29.0 .+-. 1.1 2.64 .+-. 0.23 457 .+-. 38.dagger.
SPNO 31.8 .+-. 2.1 3.06 .+-. 0.28.dagger. 480 .+-. 27.dagger. SP
29.8 .+-. 1.4 2.32 .+-. 0.19 391 .+-. 28 L-NAME 29.2 .+-. 1.5 1.84
.+-. 0.09 316 .+-. 10 ______________________________________ *p
< 0.05 vs. Control group .dagger.p < 0.05 vs. I/R untreated
group
FIGS. 4A-C are bar graphs of NO donors and NO synthesis inhibitor
versus number of adherent leukocytes per 100 .mu.m, emigrated
leukocytes per field, and % albumin leakage, respectively, for 20
min ischemia and 30 min reperfusion with data shown for 10 min and
30 min reperfusion. Error bars represent standard deviations from
the mean. The number of adherent and emigrated leukocytes were
significantly elevated at 10 min after reperfusion and increased
progressively thereafter. In animals subjected to 20 min of sham
ischemia and 30 min of reperfusion, leukocyte adherence was
2.6.+-.0.8 per 100 .mu.m with 1.4.+-.0.7 emigrated leukocytes per
field and an albumin leakage index of 8.3.+-.1.6%. Corresponding
values obtained in mesenteric preparations exposed to 20 min of
ischemia and 30 min reperfusion were 18.4.+-.1.0 per 100 .mu.m,
8.8.+-.0.8 per field, and 48.1.+-.4.0%, respectively. However, no
significant changes in leukocyte adherence were noted in animals
receiving either SP- or L-NAME (see FIG. 4A). A similar pattern of
effectiveness in reducing leukocyte emigration (FIG. 4B) was
observed with the different NO donors, i.e., SNP, SPNO, and SIN-1
reduced the number of emigrated leukocytes by 29-57%, 64-71%, and
68-75%, while SP- and L-NAME had no effect. FIG. 4C illustrates
that the large increase in albumin leakage induced by ischemia
reperfusion was significantly attenuated by SNP (46-63%), SpNO
(70%), and SIN-1 (60-71%) at both 10 min and 30 min after
reperfusion. SP- and L-NAME had no effect on ischemia reperfusion
induced albumin leakage.
FIGS. 5A and B are graphs of % albumin leakage versus adherent
leukocytes per 100 .mu.m and emigrated leukocytes per field,
respectively. FIG. 5 illustrates the dependence of ischemia
reperfusion-induced albumin leakage in single venules on the number
of adherent (FIG. 5A) and emigrated (FIG. 5B) leukocytes. All
values were derived from the 30 min values presented in FIG. 4.
Albumin leakage was highly correlated with both leukocyte adherence
(r=0.800, p<0.05) and leukocyte emigration (r=0.746, p<0.05).
Albumin leakage was greater in regions of the venule which
exhibited a high level of leukocyte adherence/emigration than in
regions exhibiting little or no adherence/emigration.
FIG. 6 is a bar graph of compounds versus leukocyte-platelet
aggregates per 5 min. Error bars represent standard deviations from
the mean. FIG. 6 summarizes the effects of NO donors on ischemia
reperfusion induced formation of platelet-leukocyte aggregates.
Although aggregates were never observed during control conditions,
12.2.+-.1.4 aggregates per 5 min were observed in venules exposed
to 20 min of ischemia and 30 min of reperfusion. Aggregate
formation was reduced in animals treated with either SNP, SpNO or
SIN-1, but not with SP- or L-NAME.
FIG. 7 is a bar graph of compounds versus % degranulated mast
cells. Error bars represent standard deviations from the mean. FIG.
7 summarizes the effects of NO donors on ischemia reperfusion
induced degranulation of microvascular mast cells. Degranulated
mast cells were less than 5% of the total mast cells observed along
postcapillary venules of control rats 30 minutes after reperfusion.
Degranulated mast cells increased to approximately 35% after 20 min
ischemia followed by 30 min reperfusion. SNP, SpNO, and SIN-1
significantly inhibited ischemia-reperfusion induced degranulation
of microvascular mast cells, while SP- or L-NAME alone had no
effect.
FIG. 8 is a bar graph of nitrite/nitrate concentration (.mu.pM)
versus carotid artery and superior mesenteric vein (SMV) before and
after ischemia reperfusion (I/R). Error bars represent standard
deviations from the mean. Nitrite and nitrate concentration in the
superior mesenteric vein was 25.82.+-.1.76 .mu.mole. After ischemia
reperfusion, the nitrite and nitrate concentration decreased to
15.67.+-.2.73 .mu.mole. Nitrite and nitrate concentration in the
carotid artery, however, showed no significant alteration after
ischemia reperfusion (before: 20.34.+-.4.21; after:
15.51.+-.1.79).
EXAMPLE 4
This example demonstrates the effects of nitric oxide-containing
compounds on the mucosa and microvasculature in cats which have
undergone transplants of the small intestine during hypothermic
ischemia.
Donor cats were fasted for 18-24 hours and were anesthetized by
intramuscular injection of approximately 75 mg ketamine
hydrochloride and 0.5 mg acepromazine maleate followed by
intravenous anesthesia with pentobarbital sodium through a right
jugular vein cannula. The cats were mechanically ventilated with a
Harvard respirator after completion of tracheotomies.
A midline laparotomy was performed, the colon removed, and a 10-20
g segment of distal ileum was isolated. Inflow and outflow rubber
cannulas were put in place and secured. The remainder of the small
intestine was resected and removed from the operative field. The
cats were then administered 10,000 units of heparin intravenously.
A large lymphatic vessel in the mesenteric pedicle was cannulated
and secured with 4-0silk ligatures.
The lumen of the small intestine specimen was flushed with 200-300
ml of cold Ringer's lactate at 50 cm H.sub.2 O. Both the superior
mesenteric artery and vein were cannulated and the artery was
flushed with 100 ml of cold Ringer's lactate at 35 cm H.sub.2
O.
The proximal mesenteric pedicle was ligated and the harvested
segment was removed for placement on a plexiglass platform and
moistened with cold Ringer's lactate. The harvested small intestine
was placed in a humidified, airtight plexiglass chamber and
refrigerated at 4.degree. C. for 6 hours.
The recipient operation was identical in procedure to the donor
model up to the laparotomy. At this point, both renal arteries were
ligated with 4-0 silk ligatures to prevent renal excretion of
.sup.51 Cr-EDTA (New England Nuclear, Boston, Mass.). .sup.51
Cr-EDTA is widely used to examine subtle changes in the intestinal
mucosa. EDTA is a small molecule with a molecular weight of 359 and
its chromium chelate will rapidly equilibrate with the
extracellular compartment after intravenous administration. The
rate-limiting barrier for blood-to-lumen movement of this molecule
is the epithelial cell monolayer of the intestinal mucosa, which is
also independent of changes in the endothelial cell layer of the
microvasculature (Kubes (1992), supra; Crissinger et al., J.
Intern. Med. Suppl., 1, 145-154 (1990)).
Heparin (10,000 units) was injected intravenously and the vena cava
and aorta were cannulated with silastic cannulas. The laparotomy
incision was closed with the inferior aspect open to allow
placement of a flow probe/Statham 23 A transducer to form the
arterial circuit. The venous circuit also included a pressure
transducer. A Grass physiological recorder was used upon
reperfusion of the intestinal segment to monitor arterial, venous,
and capillary pressure. Capillary pressure was measured using a
previously described venous occlusion technique (Granger et al.,
Am. J. Physiol., 244, G341-344 (1983)). The donor intestinal
segment was covered with clear plastic wrap to minimize evaporative
water loss while the temperature was maintained at
38.degree.-40.degree. C. with an infrared heat lamp and
thermometer.
On reperfusion of the intestinal graft, the lumen was perfused with
warm (38.degree. C.) Ringer's lactate at a rate of 1.0 ml/min,
while the effluent was collected at 5 min intervals for 120
minutes. Five hundred .mu.Ci .sup.51 Cr-EDTA was injected
intravenously after approximately 20-30 minutes on determination of
a well-functioning graft. By allowing 30 min to elapse after
injection, .sup.51 Cr-EDTA was allowed to equilibrate in the
tissue. All 5 min samples of luminal perfusate were centrifuged at
3700 rpm at 4.degree. C. for 20 min. The supernate was saved and
weighed. .sup.51 Cr-EDTA activity of the supernate was measured in
a LKB CompuGamma spectrometer (model 1282, LKB Instruments,
Gaithersburg, Md.). The .sup.51 Cr-EDTA clearance was calculated
and expressed as ml/min/100 g tissue weight according to the
following formula: ##EQU1##
Lymph flow and concentration were obtained every 15 min during
reperfusion using the cannulated mesenteric lymph vessel. Using a
200 .mu.l calibrated pipette, lymph flow (J.sub.L) was observed.
Lymph (C.sub.L) and plasma (C.sub.p) protein concentrations were
measured with an American Optical Refractometer. Prior to vascular
flushing during the harvest procedure, control values for C.sub.L,
C.sub.p, and J.sub.L were obtained. All calculated intestinal
values were normalized per 100 g tissue. Lymph protein clearance
was calculated using the formula:
Lymph protein clearance=(C.sub.L Q.sub.L)/C.sub.P.
The animals were then divided into three different groups. The
first group did not receive any therapeutic intervention and was
divided into one control group and one group subjected to 6 hours
of hypothermic ischemia. The control group involved an intestinal
specimen merely for in situ control values and did not undergo
ischemia or luminal flushing with Ringer's lactate.
The second group underwent the identical procedure as the untreated
6-hr hypothermic ischemia group. However, the intestinal lumen was
perfused with 0.1 mmol spermine NO in Ringer's lactate. A fresh
solution of 0.1 mmol spermine NO as luminal perfusate was used
every 30 minutes secondary to the short half-life of 39 minutes
(Maragos et al., J. Med. Chem., 34 (11), 3242-3247 (1991)). The
perfusate was kept at 38.degree. C. in a monitored warm water
bath.
A subgroup of the second group involved an identical experimental
procedure except 0.1 mmol spermine base (Sigma) in Ringer's lactate
was used as a control to compare versus spermine NO.
The third group was identical to the 6 hr untreated hypothermic
ischemia group except 0.5 mmol SIN-1 in Ringer's lactate was used
as luminal perfusate every 30 minutes. The light-sensitive SIN-1
solution was kept covered with aluminum foil and maintained at
38.degree. C. in a warm water bath.
Using conventional statistical methods, independent t-tests were
performed. All statistical values were reported as means .+-. S.D.
with p<0.05.
Intestinal blood to lumen .sup.51 Cr-EDTA clearance
(ml/min.times.100 g) was measured during 120 minutes of
reperfusion. The clearance from blood to lumen is a measure of
mucosal permeability. FIG. 9 is a bar graph of .sup.51 Cr-EDTA
clearance (ml/min.times.100 g) versus time (min) which shows the
.sup.51 Cr-EDTA clearance in 6-hr hypothermic ischemia test
animals, including control, untreated, SpNO-treated, and
SIN-1-treated, every 15 min for 120 min. Error bars represent the
mean .+-. S.D. FIG. 9 shows that mucosal permeability decreased in
those animals treated with spermine NO and SIN-1. A statistically
significant decrease in .sup.51 Cr-EDTA clearance was noted at 75
and 90 min with spermine NO and at 60 and 90 min for SIN-1.
Secondary to the mucosal barrier injury, water absorption normally
decreases with ischemia reperfusion. FIG. 10 is a graph of water
absorption (ml/min.times.100 g) versus time (min) for control,
untreated, SpNO, and SIN-1 treated animals. Error bars represent
the mean .+-. S.D. FIG. 10 shows that water absorption increased
significantly during 120 min of reperfusion in animals treated with
spermine NO as opposed to untreated animals. SIN-1 administration
significantly increased water absorption at 90 and 120 min.
Other measures of vascular permeability are lymph flow and lymph
protein clearance. FIGS. 11 and 12 are bar graphs of lymph flow and
lymph protein clearance (ml/min.times.100 g), respectively, versus
time (min) for control, untreated, SpNO-treated, and SIN-1-treated
animals. Error bars represent the mean .+-. S.D. Increased lymph
flow was observed for SpNO-treated animals. This increased lymph
flow was accompanied by increased lymph protein clearance, which is
a measure of vascular permeability.
Capillary pressure was also measured. FIG. 13 is a bar graph of mm
Hg versus time (min) for control, untreated, SpNO-treated, and
SIN-1-treated animals. Error bars represent the mean .+-. S.D.
Significant elevation in capillary pressure paralleling increased
lymph flow and lymph protein clearance was observed for
SpNO-treated animals. No significant changes were noted in
capillary pressure for SIN-1-treated animals. Total vascular
resistance, however, was comparable for all groups throughout
reperfusion.
All references, including publications, patents, and patent
applications, cited herein are hereby incorporated by reference to
the same extent as if each individual document were individually
and specifically indicated to be incorporated by reference and were
set forth in its entirety herein.
While this invention has been described with emphasis upon
preferred embodiments, it will be obvious to those of ordinary
skill in the art that the preferred embodiments may be varied. It
is intended that the invention may be practiced otherwise than as
specifically described herein. Accordingly, this invention includes
all modifications encompassed within the spirit and scope of the
appended claims.
* * * * *