U.S. patent application number 10/563682 was filed with the patent office on 2006-08-17 for use of nitrite salts for the treatment of cardiovascular conditions.
Invention is credited to Richard Cannon III, Mark T. Gladwin, AlanN Schechter.
Application Number | 20060182815 10/563682 |
Document ID | / |
Family ID | 36815926 |
Filed Date | 2006-08-17 |
United States Patent
Application |
20060182815 |
Kind Code |
A1 |
Gladwin; Mark T. ; et
al. |
August 17, 2006 |
Use of nitrite salts for the treatment of cardiovascular
conditions
Abstract
It has been surprisingly discovered that administration of
pharmaceutically-acceptable salts of nitrite is useful in the
regulation of the cardiovascular system. It has also been
surprisingly discovered that nitrite is reduced to nitric oxide in
vivo, and that the nitric oxide produced thereby is an effective
vasodilator. These effects surprisingly occur at nitrite doses that
do not produce clinically significant methemoglobinemia. These
discoveries now enable methods to prevent and treat conditions
associated with the cardiovascular system, for example, high blood
pressure, pulmonary hypertension, cerebral vasospasm and tissue
ischemia-reperfusion injury. These discoveries also provide methods
to increase blood flow to tissues, for example, to tissues in
regions of low oxygen tension.
Inventors: |
Gladwin; Mark T.;
(Washington, DC) ; Cannon III; Richard; (Potomac,
MD) ; Schechter; AlanN; (Bethesda, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET
SUITE #1600
PORTLAND
OR
97204-2988
US
|
Family ID: |
36815926 |
Appl. No.: |
10/563682 |
Filed: |
July 9, 2004 |
PCT Filed: |
July 9, 2004 |
PCT NO: |
PCT/US04/21985 |
371 Date: |
January 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60485959 |
Jul 9, 2003 |
|
|
|
60511244 |
Oct 14, 2003 |
|
|
|
Current U.S.
Class: |
424/718 |
Current CPC
Class: |
A61K 33/00 20130101;
A61K 31/519 20130101; Y02A 50/411 20180101; Y02A 50/30 20180101;
A61K 45/06 20130101 |
Class at
Publication: |
424/718 |
International
Class: |
A61K 33/00 20060101
A61K033/00 |
Claims
1. A method for inducing vasodilation and/or increasing blood flow
in a subject, comprising administering to the subject an effective
amount of a pharmaceutically-acceptable salt of nitrite for a
sufficient period of time to induce vasodilation and/or increase
blood flow in the subject.
2. The method of claim 1, wherein the pharmaceutically-acceptable
salt of nitrite reacts in the presence of hemoglobin in the subject
to release nitric oxide.
3. The method of claim 1, wherein the effective amount of the
pharmaceutically-acceptable salt of nitrite: induces production in
the subject of no more than about 25% methemoglobin; induces
production in the subject of no more than about 20% methemoglobin;
induces production in the subject of no more than about 10%
methemoglobin; induces production in the subject of no more than
about 8% methemoglobin; or induces production in the subject of no
more than about 5% methemoglobin.
4. The method of claim 1, wherein the effective amount of the
pharmaceutically-acceptable salt of nitrite induces production in
the subject of no more than about 3% methemoglobin.
5. The method of claim 1, comprising administering sodium nitrite
by injection at about 36 mmoles per minute for at least five
minutes into the forearm brachial artery of the subject.
6. The method of claim 1, wherein the effective amount of the
pharmaceutically-acceptable salt of nitrite is administered to a
circulating concentration in the subject of about 0.6 to 240
.mu.M.
7. The method of any one of claims 1-6, wherein the
pharmaceutically-acceptable salt of nitrite comprises as the cation
sodium, potassium, or arginine.
8. The method of claim 7, wherein the nitrite is administered as
sodium nitrite.
9. The method of any of claims 1-8, wherein the administration of
the nitrite is parenteral, oral, bucal, rectal, ex vivo, or
intraocular.
10. The method of any of claims 1-8, wherein the administration of
the nitrite is peritoneal, intravenous, intraarterial,
subcutaneous, inhaled, intramuscular, or into a cardiopulmonary
bypass circuit.
11. The method of any one of claims 1-10, wherein the subject is a
mammal.
12. The method of claim 11, wherein the subject is a human.
13. The method of any one of claims 1-12, wherein the nitrite is
administered in combination with at least one additional agent.
14. The method of claim 13, wherein the additional agent is one or
more selected from the list consisting of penicillin, hydroxyurea,
butyrate, clotrimazole, arginine, or a phosphodiesterase
inhibitor.
14. The method of claim 14, wherein the phosphodiesterase inhibitor
is sildenafil.
15. The method of any one of claims 1-13, wherein the subject has
elevated blood pressure, and the method is a method for treating at
least one vascular complication associated with the elevated blood
pressure.
16. The method of any one of claims 1-13, wherein the subject has a
hemolytic condition, and the method is a method for treating at
least one vascular complication associated with the hemolytic
condition.
17. The method of claim 15 or 16, wherein the at least one vascular
complication is one or more selected from the group consisting of
pulmonary hypertension, systemic hypertension, peripheral vascular
disease, trauma, cardiac arrest, general surgery, organ
transplantation, cutaneous ulceration, acute renal failure, chronic
renal failure, intravascular thrombosis, angina, an
ischemia-reperfusion event, an ischemic central nervous system
event, and death.
18. The method of claim 17, wherein the hemolytic condition is one
or more selected from the group consisting of sickle cell anemia,
thalassemia, hemoglobin C disease, hemoglobin SC disease, sickle
thalassemia, hereditary spherocytosis, hereditary elliptocytosis,
hereditary ovalcytosis, glucose-6-phosphate deficiency and other
red blood cell enzyme deficiencies, paroxysmal nocturnal
hemoglobinuria (PNH), paroxysmal cold hemoglobinuria (PCH),
thrombotic thrombocytopenic purpura/hemolytic uremic syndrome
(TTP/HUS), idiopathic autoimmune hemolytic anemia, drug-induced
immune hemolytic anemia, secondary immune hemolytic anemia,
non-immune hemolytic anemia caused by chemical or physical agents,
malaria, falciparum malaria, bartonellosis, babesiosis, clostridial
infection, severe haemophilus influenzae type b infection,
extensive burns, transfusion reaction, rhabdomyolysis
(myoglobinemia), transfusion of aged blood, transfusion of
hemoglobin, transfusion of red blood cells, cardiopulmonary bypass,
coronary disease, cardiac ischemia syndrome, angina, iatrogenic
hemolysis, angioplasty, myocardial ischemia, tissue ischemia,
hemolysis caused by intravascular devices, and hemodialysis.
19. The method of any one of claims 1-13, wherein the subject has a
condition associated with decreased blood flow to a tissue, and the
method is a method to increase blood flow to the tissue of the
subject.
20. The method of claim 19, wherein the decreased blood flow to the
tissue is caused directly or indirectly by at least one condition
selected from the group consisting of: sickle cell anemia,
thalassemia, hemoglobin C disease, hemoglobin SC disease, sickle
thalassemia, hereditary spherocytosis, hereditary elliptocytosis,
hereditary ovalcytosis, glucose-6-phosphate deficiency and other
red blood cell enzyme deficiencies, paroxysmal nocturnal
hemoglobinuria (PNH), paroxysmal cold hemoglobinuria (PCH),
thrombotic thrombocytopenic purpura/hemolytic uremic syndrome
(TTP/HUS), idiopathic autoimmune hemolytic anemia, drug-induced
immune hemolytic anemia, secondary immune hemolytic anemia,
non-immune hemolytic anemia caused by chemical or physical agents,
malaria, falciparum malaria, bartonellosis, babesiosis, clostridial
infection, severe haemophilus influenzae type b infection,
extensive burns, transfusion reaction, rhabdomyolysis
(myoglobinemia), transfusion of aged blood, transfusion of
hemoglobin, transfusion of red blood cells, cardiopulmonary bypass,
coronary disease, cardiac ischemia syndrome, angina, iatrogenic
hemolysis, angioplasty, myocardial ischemia, tissue ischemia,
hemolysis caused by intravascular devices, hemodialysis, pulmonary
hypertension, systemic hypertension, cutaneous ulceration, acute
renal failure, chronic renal failure, intravascular thrombosis, and
an ischemic central nervous system event.
21. The method of claim 20, wherein the tissue is an ischemic
tissue.
22. The method of any one of claims 19-21, wherein the tissue is
one or more tissues selected from the group consisting of neuronal
tissue, bowel tissue, intestinal tissue, limb tissue, lung tissue,
central nervous tissue, or cardiac tissue.
23. The method of claim 15, wherein the elevated blood pressure
comprises elevated blood pressure in the lungs.
24. The method of claim 23, wherein the subject has neonatal
pulmonary hypertension.
25. The method of claim 23, wherein the subject has primary and/or
secondary pulmonary hypertension.
26. The method of any of any one of claims 23-26, wherein the
pharmaceutically-acceptable salt of nitrite is nebulized.
27. The method of claim 26, wherein the pharmaceutically-acceptable
salt of nitrite is administered to a circulating concentration in
the subject of: no more than about 100 .mu.M; no more than about 50
.mu.M; no more than about 20 .mu.M; no more than about 16 .mu.M; or
less than about 16 .mu.M.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/485,959, filed Jul. 9, 2003, and No. 60/511,244,
filed Oct. 14, 2003, both of which are incorporated herein by
reference in their entirety.
BACKGROUND OF THE DISCLOSURE
[0002] The last decade has seen an increase in the understanding of
the critical role nitric oxide as a blood vessel dilator
contributing to the regulation of blood flow and cardiovascular
homeostasis. Nitric oxide may be oxidized in blood to nitrite
(NO.sub.2.sup.-), an anion considered to be an inert metabolic end
product of such nitric oxide oxidation. In vivo plasma levels of
nitrite have been reported to range from 150 to 1000 nM, and the
nitrite concentration in aortic ring tissue has been reported to be
in excess of 10,000 nM Rodriguez et al., Proc Natl Acad Sci USA,
100, 336-41, 2003; Gladwin et al., Proc Natl Acad Sci USA, 97,
9943-8, 2000; and Rassaf et al., Nat Med, 9, 481-3, 2003). This
potential storage pool for NO is in excess of plasma
S-nitrosothiols, which have been reported to be less than 10 nM in
human plasma (Rassaf et al., Nat Med, 9, 481-3, 2003; Rassaf et
al., Free Radic Biol Med, 33, 1590-6, 2002; Rassaf et al., J Clin
Invest, 109, 1241-8, 2002; and Schechter et al., J Clin Invest,
109, 1149-51, 2002). Mechanisms have been proposed for the in vivo
conversion of nitrite to NO, for example, by enzymatic reduction by
xanthine oxidoreductase or by non-enzymatic
disproportionation/acidic reduction (Millar et al., Biochem Soc
Trans, 25, 528S, 1997; Millar et al, FEBS Lett, 427, 225-8, 1998;
Godber et al., J Biol Chem, 275, 7757-63, 2000; Zhang et al.,
Biochem Biophys Res Commun, 249, 767-72, 1998 [published erratum
appears in Biochem Biophys Res Commun 251, 667, 1998]; Li et al., J
Biol Chem, 276, 24482-9, 2001; Li et al., Biochemistry, 42, 1150-9,
2003; Zweier et al., Nat Med, 1, 804-9, 1995; Zweier et al.,
Biochem Biophys Acta, 1411, 250-62, 1999; and Samouilov et al.,
Arch Biochem Biophys, 357:1-7, 1998).
[0003] Arterial-to-venous gradients of nitrite across the human
forearm at rest and during regional NO synthase inhibition have
been observed, with increased consumption of nitrite occurring with
exercise (Gladwin et al., Proc Natl Acad Sci USA, 97, 9943-8, 2000;
Gladwin et al., Proc Natl Acad Sci USA, 97, 11482-11487, 2000; and
Cicinelli et al., Clin Physiol, 19:440-2, 1999). Kelm and
colleagues have reported that large artery-to-vein gradients of
nitrite form across the human forearm during NO synthase inhibition
(Lauer et al., Proc Natl Acad Sci USA, 98, 12814-9, 2001). Unlike
the more simple case of oxygen extraction across a vascular bed,
nitrite may be both consumed, as evidenced by artery-to-vein
gradients during NO synthase inhibition and exercise, and produced
in the vascular bed by endothelial nitric oxide synthase-derived NO
reactions with oxygen.
[0004] At high concentrations, nitrite bas been reported to be a
vasodilator in vitro (Ignarro et al., Biochim Biophys Acta, 631,
221-31, 1980; Ignarro et al., J Pharmacol Exp Ther, 218, 739-49,
1981; Moulds et al., Br J Clin Pharmacol, 11, 57-61, 1981; Gruetter
et al., J Pharmacol Exp Ther, 219, 181-6, 1981; Matsunaga et al., J
Pharmacol Exp Ther, 248, 687-95, 1989; and Laustiola et al.,
Pharmacol Toxicol, 68, 60-3, 1991). The levels of nitrite shown to
vasodilate in vitro have always been in excess of 100,000 nM (100
.mu.M) and usually at millimolar concentrations.
[0005] Consistent with the high concentrations of nitrite required
to vasodilate in vitro, when Lauer and colleagues infused nitrite
into the forearm circulation of human subjects, they reported no
vasodilatory effects, even with concentrations of 200 .mu.M in the
forearm (Lauer et al., Proc Natl Acad Sci USA, 98, 12814-9, 2001).
Lauer et al. reported that a "complete lack of vasodilator activity
of intraartierial infusions of nitrite clearly rules out any role
for this metabolite in NO delivery" and concluded that
"physiological levels of nitrite are vasodilator-inactive."
Furthermore, Rassaf and colleagues also failed to find a
vasodilatory effect in humans following infusion of nitrite (Rassaf
et al, J Clin Invest, 109, 1241-8, 2002). Thus, in vivo studies
have concluded that physiological levels of nitrites do not serve
as a source for NO, and that physiological levels of nitrites do
not have a role in regulating blood pressure.
[0006] Historically, nitrite has been used as a treatment for
cyanide poisoning. High concentrations are infused into a subject
suffering cyanide poisoning in order to oxidize hemoglobin to
methemoglobin, which will bind cyanide. These high concentrations
of nitrite produce clinically significant methemoglobinemia,
potentially decreasing oxygen delivery. While these high
concentrations of nitrite have been shown to decrease blood
pressure in humans, the amount of methemoglobin formed precluded a
use for nitrite in the treatment of other medical conditions.
[0007] Therefore, the state of the art was that nitrite was not a
significant vasodilator at concentrations below 100 .mu.M in vitro,
and even when infused into humans at concentrations of 200 .mu.M in
the forearm. It was also the state of the art that nitrite was not
converted to nitric oxide in the human blood stream.
SUMMARY OF THE DISCLOSURE
[0008] It has been surprisingly discovered that administration of
pharmaceutically-acceptable salts of nitrite is useful in the
regulation of the cardiovascular system. It has also been
surprisingly discovered that nitrite is reduced to nitric oxide in
vivo, and that the nitric oxide produced thereby is an effective
vasodilator. These effects surprisingly occur at doses that do not
produce clinically significant methemoglobinemia. These discoveries
now enable methods to prevent and treat conditions associated with
the cardiovascular system, for example, high blood pressure,
pulmonary hypertension, cerebral vasospasm and tissue
ischemia-reperfusion injury. These discoveries also provide methods
to increase blood flow to tissues, for example, to tissues in
regions of low oxygen tension. It is particularly surprising that
the nitrite does not need to be applied in an acidified condition
in order for it to be effective in regulating the cardiovascular
system, and more particularly to act as a vasodilator in vivo.
[0009] It has been demonstrated by the inventors that nitrite can
serve as a vasodilator in humans at much lower concentrations (as
low as 0.9 .mu.M) than have been used in the past for cyanide
poisoning. The mechanism is believed to involve a reaction of
nitrite with deoxygenated hemoglobin and red blood cells, to
produce the vasodilating gas nitric oxide. This potent biological
effect is observed at doses of nitrite that do not produce
clinically significant methemoglobininemia (for instance, less than
20%, more preferably less than 5% methemoglobin in the
subject).
[0010] It has been discovered that nitrite is converted to nitric
oxide in vivo, and that the nitric oxide produced thereby is an
effective vasodilator. Further, it has been surprisingly discovered
that administration of nitrite, for instance a
pharmaceutically-acceptable salt of nitrite, to a subject causes a
reduction in blood pressure and an increase in blood flow to
tissues, for example, to tissues in regions of low oxygen tension.
These discoveries now enable useful methods to regulate the
cardiovascular system, for instance to prevent and treat
malconditions associated with the cardiovascular system, for
example, high blood pressure, or organs, tissues, or systems
suffering a lack of or inadequate blood flow. Non-limiting examples
of contemplated malconditions include stroke, heart disease, kidney
disease and failure, eye damage including hypertensive retinopathy,
diabetes, and migraines.
[0011] In one example embodiment, the present disclosure provides a
method for decreasing a subject's blood pressure or increasing
blood flow, including in a particular embodiment administering to
the subject sodium nitrite at about 36 .mu.moles per minute into
the forearm brachial artery.
[0012] The present disclosure additionally provides a method for
increasing blood flow to a tissue of a subject, including
administering to the subject an effective amount of
pharmaceutically-acceptable nitrite, such as a salt thereof, so as
to increase blood flow to a tissue of the subject. The blood flow
may be specifically increased in tissues in regions of low oxygen
tension. The present disclosure also provides a method for
decreasing a subject's blood pressure, comprising administering to
the subject an effective amount of pharmaceutically-acceptable
nitrite so as to decrease the subject's blood pressure.
[0013] The present disclosure further provides a method for
treating a subject having a condition associated with elevated
blood pressure or reduced blood flow, including administering to
the subject an effective amount of pharmaceutically-acceptable
nitrite so as to treat at least one vascular complication
associated with the elevated blood pressure.
[0014] Also provided is a method for treating a subject having a
hemolytic condition, including administering to the subject an
effective amount of pharmaceutically-acceptable nitrite so as to
treat at least one vascular complication associated with the
hemolytic condition.
[0015] The disclosure further provides a method for treating a
subject having a condition associated with elevated blood pressure
in the lungs, e.g. pulmonary hypertension, including administering
to the subject an effective amount of pharmaceutically-acceptable
nitrite. In some embodiments, this includes treating a subject
having neonatal pulmonary hypertension. In some embodiments, this
includes treating a subject having primary and/or secondary
pulmonary hypertension. In some embodiments for treating subject s
having a condition associated with elevated blood pressure in the
lungs, the nitrite is nebulized.
[0016] Also contemplated herein are methods for treating,
ameliorating, or preventing other conditions of or associated with
blood flow, including vasospasm, stroke, angina, revascularization
of coronary arteries and other arteries (peripheral vascular
disease), transplantation (e.g., of kidney, heart, lung, or liver),
treatment of low blood pressure (such as that seen in shock or
trauma, surgery and cardiopulmonary arrest) to prevent reperfusion
injury to vital organs, cutaneous ulcers (e.g., with topical,
non-acidified nitrite salt), Raynauds phenomenon, and treatment of
hemolytic conditions (such as sickle cell, malaria, TTP, and HUS)
and other conditions listed herein.
[0017] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is a graph, depicting hemodynamic and metabolic
measurements at baseline and during exercise in 18 subjects. FIG.
1A shows effects on each of the indicated values without inhibition
of NO synthesis. FIG. 1B shows effects with inhibition of NO
synthesis. Key: MAP--mean arterial pressure, mmHg; FBF--forearm
blood flow, mL/min/100 mL; O.sub.2 saturation, %; pO.sub.2--venous
oxyhemoglobin saturation, partial pressure of oxygen, mmHg; pH,
units; *=p<0.05 vs. Baseline 1 or 2, respectively; **=p<0.01
vs. Baseline 1 or 2, respectively; .dagger.=p<0.05 vs. Baseline
1; .dagger..dagger.=p<0.01 vs. Initial Exercise.
[0019] FIG. 2 is a graph, depicting effects of infusion of sodium
nitrite in bicarbonate-buffered normal saline into the brachial
arteries of 18 healthy subjects. FIG. 2A shows effects on each of
the indicated values without inhibition of NO synthesis. FIG. 2B
shows effects with inhibition of NO synthesis. Key as for FIG. 1,
plus: Nitrite--venous nitrite, .mu.M; NO-heme--venous
iron-nitrosyl-hemoglobin, .mu.M; and MetHb--venous methemoglobin,
%; +=p<0.01 vs. Initial Exercise.
[0020] FIG. 3 is a series of graphs, illustrating the effects of
infusion of low-dose sodium nitrite into the brachial arteries of
10 healthy subjects at baseline and during exercise, without and
with inhibition of NO synthesis. FIG. 3A shows forearm blood flow
at baseline and following a five-minute infusion of NaNO.sub.2.
FIG. 3B shows forearm blood flow with and without low-dose nitrite
infusion at baseline and during L-NMMA infusion with and without
exercise stress. FIG. 3C shows venous levels of nitrite from the
forearm circulation at the time of blood flow measurements. FIG. 3D
shows venous levels of S-nitroso-hemoglobin (S--NO) and
iron-nitrosyl-hemoglobin (Hb-NO) at baseline and following nitrite
infusion during exercise stress.
[0021] FIG. 4 is a pair of graphs, showing formation of
NO-hemoglobin adducts. FIG. 4A shows formation of
iron-nitrosyl-hemoglobin and S-nitroso-hemoglobin, comparing
baseline, with nitrite infusion, and nitrite infusion with
exercise. FIG. 4B compares formation of NO-hemoglobin adducts with
hemoglobin-oxygen saturation in the human circulation, during
nitrite infusion.
[0022] FIG. 5A shows NO release following nitrite injections into
solutions of PBS ("PBS"), deoxygenated red blood cells
("deoxy-RBC"), and oxygenated red blood cells ("oxy-RBC"). FIG. 5B
shows the rate of NO formation from nitrite mixed with PBS (first
bar in each set), and oxygenated and deoxygenated red blood cells
(second and third bar in each set, respectively).
DETAILED DESCRIPTION OF THE DISCLOSURE
[0023] I. TABLE-US-00001 ANOVA analysis of variance deoxy-RBC
deoxygenated red blood cells FBF forearm blood flow L-NMMA
L-NG-monomethyl-arginine NO nitric oxide NOS nitric oxide synthase
MAP mean arterial pressure MetHb methemoglobin oxy-RBC oxygenated
red blood cells PBS phosphate buffered saline pO.sub.2 (or
Po.sub.2) partial oxygen pressure S--NO S-nitroso-hemoglobin
II. Terms
[0024] Unless otherwise noted, terms used herein should be accorded
their standard definitions and conventional usage. For example, one
of skill in the art can obtain definitions for the terms used
herein in dictionaries and reference textbooks, for example:
Stedman's Medical Dictionary (26.sup.th Ed., Williams and Wilkins,
Editor M. Spraycar, 1995); The New Oxford American Dictionary
(Oxford University Press, Eds E. Jewell and F. Abate, 2001);
Molecular Cloning: A Laboratory Manual (Sambrook et al., 3.sup.rd
Ed., Cold Spring Harbor Laboratory Press, 2001); and Hawley's
Condensed Chemical Dictionary, 11.sup.th Ed. (Eds. N. I. Sax and R.
J. Lewis, Sr., Van Nostrand Reinhold, New York, N.Y., 1987);
Molecular Biology and Biotechnology: a Comprehensive Desk Reference
(VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8)).
[0025] In order to facilitate review of the various embodiments,
the following explanations of specific terms are provided:
[0026] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals.
[0027] Cerebral ischemia or ischemic stroke: A condition that
occurs when an artery to or in the brain is partially or completely
blocked such that the oxygen demand of the tissue exceeds the
oxygen supplied. Deprived of oxygen and other nutrients following
an ischemic stroke, the brain suffers damage as a result of the
stroke.
[0028] Ischemic stroke can be caused by several different kinds of
diseases. The most common problem is narrowing of the arteries in
the neck or head. This is most often caused by atherosclerosis, or
gradual cholesterol deposition. If the arteries become too narrow,
blood cells may collect in them and form blood clots (thrombi).
These blood clots can block the artery where they are formed
(thrombosis), or can dislodge and become trapped in arteries closer
to the brain (embolism).
[0029] Another cause of stroke is blood clots in the heart, which
can occur as a result of irregular heartbeat (for example, atrial
fibrillation), heart attack, or abnormalities of the heart valves.
While these are the most common causes of ischemic stroke, there
are many other possible causes. Examples include use of street
drugs, traumatic injury to the blood vessels of the neck, or
disorders of blood clotting.
[0030] Ischemic stroke is by far the most common kind of stroke,
accounting for about 80% of all strokes. Stroke can affect people
of all ages, including children. Many people with ischemic strokes
are older (60 or more years old), and the risk of stroke increases
with older ages. At each age, stroke is more common in men than
women, and it is more common among African-Americans than white
Americans. Many people with stroke have other problems or
conditions which put them at higher risk for stroke, such as high
blood pressure (hypertension), heart disease, smoking, or
diabetes.
[0031] Hypoxia: Deficiency in the amount of oxygen reaching body
tissues.
[0032] Injectable composition: A pharmaceutically acceptable fluid
composition comprising at least one active ingredient, for example,
a salt of nitrite. The active ingredient is usually dissolved or
suspended in a physiologically acceptable carrier, and the
composition can additionally comprise minor amounts of one or more
non-toxic auxiliary substances, such as emulsifying agents,
preservatives, pH buffering agents and the like. Such injectable
compositions that are useful for use with the compositions of this
disclosure are conventional; appropriate formulations are well
known in the art.
[0033] Ischemia: A vascular phenomenon in which a decrease in the
blood supply to a bodily organ, tissue, or part is caused, for
instance, by constriction or obstruction of one or more blood
vessels. Ischemia sometimes results from vasoconstriction or
thrombosis or embolism. Ischemia can lead to direct ischemic
injury, tissue damage due to cell death caused by reduced oxygen
supply.
[0034] Ischemia/reperfusion injury: In addition to the immediate
injury that occurs during deprivation of blood flow,
ischemic/reperfusion injury involves tissue injury that occurs
after blood flow is restored. Current understanding is that much of
this injury is caused by chemical products and free radicals
released into the ischemic tissues.
[0035] When a tissue is subjected to ischemia, a sequence of
chemical events is initiated that may ultimately lead to cellular
dysfunction and necrosis. If ischemia is ended by the restoration
of blood flow, a second series of injurious events ensue producing
additional injury. Thus, whenever there is a transient decrease or
interruption of blood flow in a subject, the resultant injury
involves two components--the direct injury occurring during the
ischemic interval and the indirect or reperfusion injury that
follows. When there is a long duration of ischemia, the direct
ischemic damage, resulting from hypoxia, is predominant. For
relatively short duration ischemia, the indirect or reperfusion
mediated damage becomes increasingly important. In some instances,
the injury produced by reperfusion can be more severe than the
injury induced by ischemia per se. This pattern of relative
contribution of injury from direct and indirect mechanisms has been
shown to occur in all organs.
[0036] Methemoglobin: The oxidized form of hemoglobin in which the
iron in the heme component has been oxidized from the ferrous (+2)
to the ferric (+3) state. This renders the hemoglobin molecule
incapable of effectively transporting and releasing oxygen to the
tissues. Normally, there is about 1% of total hemoglobin in the
methemoglobin form.
[0037] Methemoglobinemia: A condition in which a substantial
portion of the hemoglobin in the blood of a subject is in the form
of methemoglobin, making it unable to carry oxygen effectively to
the tissues. Methemoglobinemia can be an inherited disorder, but it
also can be acquired through exposure to chemicals such as nitrates
(nitrate-contaminated water), aniline dyes, and potassium chlorate.
It is not the presence of methemoglobin but the amount that is
important in the clinical setting. The following provides rough
indications of symptoms associated with different levels of
methemoglobin in the blood: <1.7%, normal; 10-20%, mild cyanosis
(substantially asymptomatic, though it can result in "chocolate
brown" blood); 30-40%, headache, fatigue, tachycardia, weakness,
dizziness; >35%, symptoms of hypoxia, such as dyspnea and
lethargy; 50-60%, acidosis, arrhythmias, coma, convulsions,
bradycardia, severe hypoxia, seizures; >70% usually results in
death.
[0038] Nitrite: The inorganic anion NO.sub.2 or a salt of nitrous
acid (NO.sub.2.sup.-). Nitrites are often highly soluble, and can
be oxidized to form nitrates or reduced to form nitric oxide or
ammonia. Nitrite may form salts with alkali metals, such as sodium
(NaNO.sub.2, also known as nitrous acid sodium salt), potassium and
lithium, with alkali earth metals, such as calcium, magnesium and
barium, with organic bases, such as amine bases, for example,
dicyclohexylamine, pyridine, arginine, lysine and the like. Other
nitrite salts may be formed from a variety of organic and inorganic
bases. In particular embodiments, the nitrite is a salt of an
anionic nitrite delivered with a cation, which cation is selected
from sodium, potassium, and arginine. Many nitrite salts are
commercially available, and/or readily produced using conventional
techniques.
[0039] Parenteral: Administered outside of the intestine, for
example, not via the alimentary tract. Generally, parenteral
formulations are those that will be administered through any
possible mode except ingestion. This term especially refers to
injections, whether administered intravenously, intrathecally,
intramuscularly, intraperitoneally, or subcutaneously, and various
surface applications including intranasal, intradermal, and topical
application, for instance.
[0040] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the compounds herein disclosed.
[0041] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of rannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0042] Peripheral Vascular Disease (PVD): A condition in which the
arteries that carry blood to the arms or legs become narrowed or
occluded. This interferes with the normal flow of blood, sometimes
causing pain but often causing no readily detectable symptoms at
all.
[0043] The most common cause of PVD is atherosclerosis, a gradual
process in which cholesterol and scar tissue build up, forming
plaques that occlude the blood vessels. In some cases, PVD may be
caused by blood clots that lodge in the arteries and restrict blood
flow. PVD affects about one in 20 people over the age of 50, or 8
million people in the United States. More than half the people with
PVD experience leg pain, numbness or other symptoms, but many
people dismiss these signs as "a normal part of aging" and do not
seek medical help. The most common symptom of PVD is painful
cramping in the leg or hip, particularly when walking. This
symptom, also known as "claudication," occurs when there is not
enough blood flowing to the leg muscles during exercise, such that
ischemia occurs. The pain typically goes away when the muscles are
rested.
[0044] Other symptoms may include numbness, tingling or weakness in
the leg. In severe cases, people with PVD may experience a burning
or aching pain in an extremity such as the foot or toes while
resting, or may develop a sore on the leg or foot that does not
heal. People with PVD also may experience a cooling or color change
in the skin of the legs or feet, or loss of hair on the legs. In
extreme cases, untreated PVD can lead to gangrene, a serious
condition that may require amputation of a leg, foot or toes.
People with PVD are also at higher risk for heart disease and
stroke.
[0045] A "pharmaceutical agent" or "drug" refers to a chemical
compound or other composition capable of inducing a desired
therapeutic or prophylactic effect when properly administered to a
subject.
[0046] Preventing or treating a disease: "Preventing" a disease
refers to inhibiting the full development of a disease. "Treatment"
refers to a therapeutic intervention that ameliorates a sign or
symptom of a disease or pathological condition after it has begun
to develop.
[0047] Purified: The term purified does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified nitrite salt preparation is one in which the
specified nitrite salt is more enriched than it is in its
generative environment, for instance within a biochemical reaction
chamber. Preferably, a preparation of a specified nitrite salt is
purified such that the salt represents at least 50% of the total
nitrite content of the preparation. In some embodiments, a purified
preparation contains at least 60%, at least 70%, at least 80%, at
least 85%, at least 90%, at least 95% or more of the specified
compound, such as a particular nitrite salt.
[0048] Reperfusion: Restoration of blood supply to tissue that is
ischemic, due to decrease in blood supply. Reperfusion is a
procedure for treating infarction or other ischemia, by enabling
viable ischemic tissue to recover, thus limiting further necrosis.
However, it is thought that reperfusion can itself further damage
the ischemic tissue, causing reperfusion injury.
[0049] Subject: Living multi-cellular organisms, including
vertebrate organisms, a category that includes both human and
non-human mammals.
[0050] Therapeutic: A generic term that includes both diagnosis and
treatment.
[0051] Therapeutically effective amount of [a vasodilator]: A
quantity of compound, such as a nitrite salt, sufficient to achieve
a desired effect in a subject being treated. For instance, this can
be the amount necessary to treat or ameliorate relatively high
blood pressure, or to measurably decrease blood pressure over a
period of time, or to measurably inhibit an increase in blood
pressure, in a subject.
[0052] An effective amount of a vasodilator may be administered in
a single dose, or in several doses, for example daily, during a
course of treatment. However, the effective amount will be
dependent on the compound applied, the subject being treated, the
severity and type of the affliction, and the manner of
administration of the compound. For example, a therapeutically
effective amount of an active ingredient can be measured as the
concentration (moles per liter or molar-M) of the active ingredient
(such as a pharmaceutically-acceptable salt of nitrite) in blood
(in vivo) or a buffer (in vitro) that produces an effect.
[0053] By way of example, as described herein it is now shown that
pharmaceutically-acceptable salts of nitrite (such as sodium
nitrite) are effective as vasodilators at calculated dosages of
about 0.6 to about 200 .mu.M final concentration of nitrite in the
circulating blood of a subject, which level can be determined
empirically or through calculations. Specific levels can be
reached, for instance, by providing less than about 200 mg or less
nitrite in a single dose, or a dose provided over a period of time
(e.g., by infusion or inhalation). For instance, other dosages may
be 150 mg, 100 mg, 75 mg, 50 mg or less. Specific example dosages
of nitrite salts are provided herein, though the examples are not
intended to be limiting. Exact dosage amounts will vary by the size
of the subject being treated, the duration of the treatment, the
mode of administration, and so forth.
[0054] Particularly beneficial therapeutically effective amounts of
a vasodilator, such as a pharmaceutically-acceptable nitrite salt
(e.g., sodium nitrite), are those that are effective for
vasodilation or increasing blood flow, but not so high that a
significant or toxic level of methemoglobin is produced in the
subject to which the vasodilator is administered. In specific
embodiments, for instance, no more than about 25% methemoglobin is
produced in the subject. More preferably, no more than 20%, no more
than 15%, no more than 10%, no more than 8% or less methemoglobin
is produced, for instance as little as 5% or 3% or less, in
response to treatment with the vasodilator.
[0055] The compounds discussed herein have equal application in
medical and veterinary settings. Therefore, the general term
"subject being treated" is understood to include all animals (for
example, humans, apes, laboratory animals, companion animals, etc.)
that are or may be suffering from an aberration in blood pressure,
such as hypertension.
[0056] Vasoconstriction. The diminution of the caliber or
cross-sectional area of a blood vessel, for instance constriction
of arterioles leading to decreased blood flow to a body part. This
can be caused by a specific vasoconstrictor, an agent (for instance
a chemical or biochemical compound) that causes, directly or
indirectly, constriction of blood vessels. Such an agent can also
be referred to as a vasohypertonic agent, and is said to have
vasoconstrictive activity. A representative category of
vasoconstrictors is the vasopressor (from the term pressor, tending
to increase blood pressure), which term is generally used to refer
to an agent that stimulates contraction of the muscular tissue of
the capillaries and arteries.
[0057] Vasoconstriction also can be due to vasospasm, inadequate
vasodilatation, thickening of the vessel wall, or the accumulation
of flow-restricting materials on the internal wall surfaces or
within the wall itself. Vasoconstriction is a major presumptive or
proven factor in aging and in various clinical conditions including
progressive generalized atherogenesis, myocardial infarction,
stroke, hypertension, glaucoma, macular degeneration, migraine,
hypertension and diabetes mellitus, among others.
[0058] Vasodilation. A state of increased caliber of the blood
vessels, or the act of dilation of a blood vessel, for instance
dilation of arterioles leading to increased blood flow to a body
part. This can be caused by a specific vasodilator, an agent (for
instance, a chemical or biochemical compound) that causes, directly
or indirectly, dilation of blood vessels. Such an agent can also be
referred to as a vasohypotonic agent, and is said to have
vasodilative activity.
[0059] Vasospasm: Another cause of stroke occurs secondary to spasm
of blood vessels supplying the brain. This type of stroke typically
follows a subarchnoid aneurismal hemorrhage with a delayed
development of vasospasm within 2-3 weeks of the bleeding event. A
similar type of stroke may complicate sickle cell disease.
[0060] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
III. Overview of Several Embodiments
[0061] It has been surprisingly discovered that administration of
pharmaceutically-acceptable salts of nitrite is useful in the
regulation of the cardiovascular system. It has also been
surprisingly discovered that nitrite is reduced to nitric oxide in
vivo, and that the nitric oxide produced thereby is an effective
vasodilator. These effects surprisingly occur at doses that do not
produce clinically significant methemoglobinemia. These discoveries
now enable methods to prevent and treat conditions associated with
the cardiovascular system, for example, high blood pressure,
pulmonary hypertension, cerebral vasospasm and tissue
ischemia-reperfusion injury. These discoveries also provide methods
to increase blood flow to tissues, for example, to tissues in
regions of low oxygen tension. It is particularly surprising that
the nitrite does not need to be applied in an acidified condition
in order for it to be effective in regulating the cardiovascular
system, and more particularly to act as a vasodilator in vivo.
[0062] Accordingly, the present disclosure provides in one
embodiment a method for decreasing a subject's blood pressure,
including administering to the subject sodium nitrite at about 36
.mu.moles per minute or less into the forearm brachial artery or
intravenously.
[0063] The present disclosure also provides a method for decreasing
a subject's blood pressure, including administering to the subject
an effective amount of pharmaceutically-acceptable nitrite so as to
decrease (or lower, or reduce) the subject's blood pressure.
Another embodiment is a method for treating a subject having a
condition associated with elevated blood pressure, including
administering to the subject an effective amount of
pharmaceutically-acceptable nitrite so as to treat at least one
vascular complication associated with the elevated blood pressure.
Also provided is a method for treating a subject having a hemolytic
condition, including administering to the subject an effective
amount of pharmaceutically-acceptable nitrite so as to treat at
least one vascular complication associated with the hemolytic
condition.
[0064] The present disclosure additionally provides a method for
increasing blood flow to a tissue of a subject, including
administering to the subject an effective amount of
pharmaceutically-acceptable nitrite so as to increase blood flow to
a tissue of the subject. Also provided is a method for producing an
amount of NO in a subject effective the decrease the subject's
blood pressure, including administering a
pharmaceutically-acceptable nitrite to the subject.
[0065] The present disclosure further provides a pharmaceutical
composition comprising an effective amount of a
pharmaceutically-acceptable nitrite and a carrier.
[0066] In some embodiments, the vascular complication is one or
more selected from the group consisting of pulmonary hypertension
(including neonatal pulmonary hypertension, primary pulmonary
hypertension, and secondary pulmonary hypertension), systemic
hypertension, cutaneous ulceration, acute renal failure, chronic
renal failure, intravascular thrombosis, an ischemic central
nervous system event, and death.
[0067] In some embodiments, nitrite is administered to neonates to
treat pulmonary hypertension.
[0068] In some embodiments, the hemolytic condition includes one or
more selected from: sickle cell anemia, thalassemia, hemoglobin C
disease, hemoglobin SC disease, sickle thalassemia, hereditary
spherocytosis, hereditary elliptocytosis, hereditary ovalcytosis,
glucose-6-phosphate deficiency and other red blood cell enzyme
deficiencies, paroxysmal nocturnal hemoglobinuria (PNH), paroxysmal
cold hemoglobinuria (PCH), thrombotic thrombocytopenic
purpura/hemolytic uremic syndrome (TIP/HUS), idiopathic autoimmune
hemolytic anemia, drug-induced immune hemolytic anemia, secondary
immune hemolytic anemia, non-immune hemolytic anemia caused by
chemical or physical agents, malaria, falciparum malaria,
bartonellosis, babesiosis, clostridial infection, severe
haemophilus influenzae type b infection, extensive burns,
transfusion reaction, rhabdomyolysis (myoglobinemia), transfusion
of aged blood, cardiopulomonary bypass, and hemodialysis.
[0069] In some embodiments, the decreased blood flow to the tissue
is caused directly or indirectly by at least one of the following
conditions: sickle cell anemia, thalassemia, hemoglobin C disease,
hemoglobin SC disease, sickle thalassemia, hereditary
spherocytosis, hereditary elliptocytosis, hereditary ovalcytosis,
glucose-6-phosphate deficiency and other red blood cell enzyme
deficiencies, paroxysmal nocturnal hemoglobinuria (PNH), paroxysmal
cold hemoglobinuria (PCH), thrombotic thrombocytopenic
purpura/hemolytic uremic syndrome (TTP/HUS), idiopathic autoimmune
hemolytic anemia, drug-induced immune hemolytic anemia, secondary
immune hemolytic anemia, non-immune hemolytic anemia caused by
chemical or physical agents, malaria, falciparum malaria,
bartonellosis, babesiosis, clostridial infection, severe
haemophilus influenzae type b infection, extensive burns,
transfusion reaction, rhabdomyolysis (myoglobinemia), transfusion
of aged blood, transfusion of hemoglobin, transfusion of red blood
cells, cardiopulmonary bypass, coronary disease, cardiac ischemia
syndrome, angina, iatrogenic hemolysis, angioplasty, myocardial
ischemia, tissue ischemia, hemolysis caused by intravascular
devices, hemodialysis, pulmonary hypertension, systemic
hypertension, cutaneous ulceration, acute renal failure, chronic
renal failure, intravascular thrombosis, and an ischemic central
nervous system event.
[0070] In some embodiments, the tissue is an ischemic tissue. In
some embodiments, the administration is parenteral, oral bucal,
rectal, ex vivo, or intraocular. In some embodiments, the
administration is peritoneal intravenous, intraarterial,
subcutaneous, inhaled, or intramuscular. In some embodiments, the
nitrite is administered to the subject in an environment of low
oxygen tension, or acts in an area of the subject's body that
displays relatively low oxygen tension. In some embodiments, the
nitrite is administered as a pharmaceutically-acceptable salt of
nitrite, such as, for instance, sodium nitrite, potassium nitrite,
or arginine nitrite. In some embodiments, the nitrite is
administered in combination with at least one additional active
agent. It is specifically contemplated that, in certain
embodiments, that the subject is a mammal, for instance, a
human.
[0071] The disclosure further provides a method for treating a
subject having a condition associated with elevated blood pressure
in the lungs, e.g. pulmonary hypertension, including administering
to the subject an effective amount of pharmaceutically-acceptable
nitrite. In some embodiments, this includes treating a subject
having neonatal pulmonary hypertension. In some embodiments, this
includes treating a subject having primary and/or secondary
pulmonary hypertension. In some embodiments for treating subjects
having a condition associated with elevated blood pressure in the
lungs, the nitrite is nebulized.
[0072] Thus, there is provided herein a method for inducing
vasodilation and/or increasing blood flow in a subject, which
method involves administering to the subject an effective amount of
a pharmaceutically-acceptable salt of nitrite for a sufficient
period of time to induce vasodilation and/or increase blood flow in
the subject. Non-limiting examples of pharmaceutically acceptable
salts of nitrite include sodium nitrite, potassium nitrite, and
arginine nitrite. In examples of the provided methods, the
pharmaceutically-acceptable salt of nitrite reacts in the presence
of hemoglobin in the subject to release nitric oxide.
[0073] It is a specific advantage of methods provided herein that
the effective amount of the pharmaceutically-acceptable salt of
nitrite administered to the subject does not induce toxic levels of
methemoglobin, and in many embodiments does not induced formation
of clinically significant amounts of methemoglobin in the subject.
Therefore, contemplated herein are methods in which the effective
amount of the pharmaceutically-acceptable salt of nitrite, when
administered to the subject, induces production in the subject of
no more than about 25% methemoglobin; no more than about 20%
methemoglobin; no more than about 10% methemoglobin; no more than
about 8% methemoglobin; or no more than about 5% methemoglobin.
Beneficially, examples of the provided methods induce production of
even less than 5% methemoglobin, for instance no more than about 3%
methemoglobin, less than 3%, less than 2%, or even less than
1%.
[0074] In one specific example of a method for inducing
vasodilation and/or increasing blood flow in a subject, sodium
nitrite is administered by injection at about 36 .mu.moles per
minute for at least five minutes into the forearm brachial artery
of the subject.
[0075] The effective amount of the pharmaceutically-acceptable salt
of nitrite is administered, in various embodiments, to a
circulating concentration in the subject of about 0.6 to 240 .mu.M,
measured locally to the site of administration or generally in the
subject. It is noted that the local level of nitrite is expected to
be higher than the general circulating level particularly in short
delivery regimens; in long term delivery regimens, such as delivery
using a pump or injector, or by inhalation, the system-wide or
general nitrite level is expected to near the level measured near
the administration site.
[0076] Administration of the pharmaceutically-acceptable nitrite
can be, for instance, parenteral, oral, bucal, rectal, ex vivo, or
intraocular in certain embodiments. In various embodiments, it is
also contemplated that the administration of the nitrite can be
peritoneal, intravenous, intraarterial, subcutaneous, inhaled,
intramuscular, or into a cardiopulmonary bypass circuit.
Combinations of two or more routes of administration are also
contemplated.
[0077] In various embodiments of the method for inducing
vasodilation and/or increasing blood flow in a subject, the subject
is a mammal. It is particularly contemplated that the subject can
be a human.
[0078] Combination therapy methods are contemplated, wherein the
nitrite is administered in combination with at least one additional
agent. By way of non-limiting examples, the additional agent is one
or more selected from the list consisting of penicillin,
hydroxyurea, butyrate, clotrimazole, arginine, or a
phosphodiesterase inhibitor (such as sildenafil).
[0079] In another embodiment of the method for inducing
vasodilation and/or increasing blood flow in a subject, the subject
has elevated blood pressure, and the method is a method for
treating at least one vascular complication associated with the
elevated blood pressure, or the subject has a hemolytic condition,
and the method is a method for treating at least one vascular
complication associated with the hemolytic condition. Optionally,
the subject may have both elevated blood pressure and a hemolytic
condition.
[0080] In examples of the methods provided herein, the at least one
vascular complication is one or more selected from the group
consisting of pulmonary hypertension, systemic hypertension,
peripheral vascular disease, trauma, cardiac arrest, general
surgery, organ transplantation, cutaneous ulceration, acute renal
failure, chronic renal failure, intravascular thrombosis, angina,
an ischemia-reperfusion event, an ischemic central nervous system
event, and death.
[0081] In examples of the methods in which the subject has a
hemolytic condition, the hemolytic condition is one or more
selected from the group consisting of sickle cell anemia,
thalassemia, hemoglobin C disease, hemoglobin SC disease, sickle
thalassemia, hereditary spherocytosis, hereditary elliptocytosis,
hereditary ovalcytosis, glucose-6-phosphate deficiency and other
red blood cell enzyme deficiencies, paroxysmal nocturnal
hemoglobinuria (PNH), paroxysmal cold hemoglobinuria (PCH),
thrombotic thrombocytopenic purpura/hemolytic uremic syndrome
(TTP/HIS), idiopathic autoimmune hemolytic anemia, drug-induced
immune hemolytic anemia, secondary immune hemolytic anemia,
non-immune hemolytic anemia caused by chemical or physical agents,
malaria, falciparum malaria, bartonellosis, babesiosis, clostridial
infection, severe haemophilus influenzae type b infection,
extensive burns, transfusion reaction, rhabdomyolysis
(myoglobinemia), transfusion of aged blood, transfusion of
hemoglobin, transfusion of red blood cells, cardiopulmonary bypass,
coronary disease, cardiac ischemia syndrome, angina, iatrogenic
hemolysis, angioplasty, myocardial ischemia, tissue ischemia,
hemolysis caused by intravascular devices, and hemodialysis.
[0082] In yet another embodiment of the method for inducing
vasodilation and/or increasing blood flow in a subject, the subject
has a condition associated with decreased blood flow to a tissue,
and the method is a method to increase blood flow to the tissue of
the subject. For instance, in examples of this method, the
decreased blood flow to the tissue is caused directly or indirectly
by at least one condition selected from the group consisting of:
sickle cell anemia, thalassemia, hemoglobin C disease, hemoglobin
SC disease, sickle thalassemia, hereditary spherocytosis,
hereditary elliptocytosis, hereditary ovalcytosis,
glucose-6-phosphate deficiency and other red blood cell enzyme
deficiencies, paroxysmal nocturnal hemoglobinuria (PNH), paroxysmal
cold hemoglobinuria (PCH), thrombotic thrombocytopenic
purpura/hemolytic uremic syndrome (TTP/HUS), idiopathic autoimmune
hemolytic anemia, drug-induced immune hemolytic anemia, secondary
immune hemolytic anemia, non-immune hemolytic anemia caused by
chemical or physical agents, malaria, falciparum malaria,
bartonellosis, babesiosis, clostridial infection, severe
haemophilus influenzae type b infection, extensive burns,
transfusion reaction, rhabdomyolysis (myoglobinemia), transfusion
of aged blood, transfusion of hemoglobin, transfusion of red blood
cells, cardiopulmonary bypass, coronary disease, cardiac ischemia
syndrome, angina, iatrogenic hemolysis, angioplasty, myocardial
ischemia, tissue ischemia, hemolysis caused by intravascular
devices, hemodialysis, pulmonary hypertension, systemic
hypertension, cutaneous ulceration, acute renal failure, chronic
renal failure, intravascular thrombosis, and an ischemic central
nervous system event.
[0083] It is specifically contemplated in examples of this method
that the tissue is an ischemic tissue, for instance one or more
tissues selected from the group consisting of neuronal tissue,
bowel tissue, intestinal tissue, limb tissue, lung tissue, central
nervous tissue, or cardiac tissue.
[0084] Also provided are methods for inducing vasodilation and/or
increasing blood flow in a subject having elevated blood pressure,
wherein the elevated blood pressure comprises elevated blood
pressure in the lungs. By way of example, it is contemplated that
such subject in some instances has neonatal pulmonary hypertension,
or primary and/or secondary pulmonary hypertension.
[0085] In examples of embodiments where the elevated blood
pressure, or need for increased blood flow, in the subject
comprises elevated blood pressure or need for increased blood flow
in the lungs, the pharmaceutically-acceptable salt of nitrite is
nebulized.
[0086] By way of example, in various embodiments the
pharmaceutically-acceptable salt of nitrite is administered to a
circulating concentration in the subject of no more than about 100
.mu.M; no more than about 50 .mu.M; no more than about 20 .mu.M; no
more than about 16 .mu.M; or less than about 16 .mu.M.
IV. Sodium Nitrite as an in Vivo Vasodilator
[0087] Nitrite anions are present in concentrations of about
150-1000 nM in the plasma and about 10 .mu.M in aortic tissue. This
represents the largest vascular storage pool of nitric oxide (NO),
provided physiological mechanisms exist to reduce nitrite to NO.
The vasodilator properties of nitrite in the human forearm and the
mechanisms extant for its bioactivation have been investigated and
results are reported herein. Sodium nitrite was infused at about 36
.mu.moles per minute into the forearm brachial artery of 18 normal
volunteers, resulting in a regional nitrite concentration of about
222 .mu.M and an immediate about 175% increase in resting forearm
blood flow. Increased blood flow was observed at rest, during NO
synthase inhibition and with exercise, and resulted in increased
tissue perfusion, as demonstrated by increases in venous
hemoglobin-oxygen saturation, partial pressure of oxygen, and pH.
Systemic concentrations of nitrite increased to about 16 .mu.M and
significantly reduced mean arterial blood pressure. In an
additional six subjects, the dose of nitrite was reduced about
2-logs and infused at 360 nmoles per minute, resulting in a forearm
nitrite concentration of about 2 .mu.M and an about 22% increase in
blood flow.
[0088] Nitrite infusions were associated with the formation of
erythrocyte iron-nitrosyl-hemoglobin, and to a lesser extent,
S-nitroso-hemoglobin across the forearm vasculature. The formation
of NO-modified hemoglobin appears to result from the nitrite
reductase activity of deoxyhemoglobin, linking tissue hypoxia and
nitrite bioactivation.
[0089] These results indicate that physiological levels of blood
and tissue nitrite represent a major bioavailable pool of NO that
contributes to vaso-regulation and provides a mechanism for hypoxic
vasodilation via reaction of vascular nitrite with deoxygenated
heme proteins. Substantial blood flow effects of nitrite infusion
into the brachial artery of normal human subjects results from
forearm nitrite concentrations as low as about 0.9 .mu.M.
[0090] By way of example, as described herein it is now shown that
pharmaceutically-acceptable salts of nitrite (such as sodium
nitrite) are effective as vasodilators at calculated dosages of
about 0.6 to about 200 .mu.M final concentration of nitrite in the
circulating blood of a subject. Specific circulating levels
(locally or generally in the subject) can be reached, for instance,
by providing less than about 200 mg or less nitrite in a single
dose, or a dose provided over a period of time (e.g., by infusion
or inhalation). For instance, other dosages may be 150 mg, 100 mg,
75 mg, 50 mg or less. Specific example dosages of nitrite salts are
provided herein, though the examples are not intended to be
limiting. Exact dosage amounts will vary by the size of the subject
being treated, the duration of the treatment, the mode of
administration, and so forth.
[0091] Infusion rates can be calculated, for any given desired
target circulating concentration, by using the following equation:
Infusion rate (.mu.M/min)=target concentration (.mu.mol/L, or
.mu.M).times.Clearance (L/min) where Clearance
(L/min)=0.015922087.times.weight of the subject (kg) .uparw.0.8354
The rate of clearance has been calculated based on empirical
results, including those reported herein.
[0092] For instance, when sodium nitrite is infused into a human
forearm at 36 micromoles (.mu.Mol) per minute, the concentration
measured coming out of forearm is about 222 .mu.M and about 16
.mu.M in whole body, after 15 minutes infusion. The background
level of circulating nitrite in mammals is low, around 150-500
nanoM.
[0093] Particularly beneficial therapeutically effective amounts of
a vasodilator, such as a pharmaceutically-acceptable nitrite salt
(e.g., sodium nitrite), are those that are effective for
vasodilation or increasing blood flow, but not so high that a
significant or toxic level of methemoglobin is produced in the
subject to which the vasodilator is administered. In specific
embodiments, for instance, no more than about 25% methemoglobin is
produced in the subject. More preferably, no more than 20%, no more
than 15%, no more than 10%, no more than 8% or less methemoglobin
is produced, for instance as little as 5% or 3% or less, in
response to treatment with the vasodilator.
[0094] By way of specific example, nitrite can be infused at
concentrations less than 40 .mu.Mol per minute intravenously or
intraarterially, or given by mouth. Importantly, doses used are
less than those used for the treatment of cyanide poisoning, which
are designed to induce clinically significant methemoglobinemia.
Surprisingly, the doses described herein for the
treatment/prevention of cardiovascular conditions produce
significant and beneficial clinical effects without clinically
significant methemoglobin production.
[0095] Relatively complex inorganic/organic nitrite compounds and
nitrate compounds have been utilized clinically to treat disorders,
including angina. These drugs (e.g, glyceryl trinitrate) suffer
from tolerance (requiring increases in dosage in order to maintain
the same effect), however, and are distinct vasodilators compared
to nitrite. For example, the former require cellular thiols for
metabolism, whereas nitrite or the nitrite salts discussed herein
(e.g., sodium nitrite) do not.
V. A Mechanism of Iron-Nitrosyl- and S-Nitroso-Hemoglobin Formation
in Vivo
[0096] The levels of both iron-nitrosyl- and S-nitroso-hemoglobin
formed in vivo in this study are striking. During a transit time of
less than 10 seconds through the forearm circulation during
exercise, infused nitrite (200 .mu.M regional concentration)
produced approximately 750 nM iron-nitrosyl-hemoglobin and 200 nM
SNO-Hb. The formation of both NO-hemoglobin adducts was inversely
correlated with hemoglobin-oxygen saturation, which fell during
exercise stress, measured from the antecubital vein by co-oximetry
(for iron-nitrosyl-hemoglobin r=-0.7, P<0.0001; for
S-nitroso-hemoglobin r=-0.45, P=0.04; FIG. 4B). Addition of 200
.mu.M nitrite to whole blood at different oxygen tensions (0-100%)
recapitulated the in vivo data with increasing concentrations of
iron-nitrosyl hemoglobin being formed at lower oxygen tensions (for
iron-nitrosyl-hemoglobin r=-0.968, P<0.0001; for
S-nitroso-hemoglobin r=-0.45, P=0.07), strongly suggesting that the
NO and SNO formation was dependent on the reaction of nitrite with
deoxyhemoglobin.
[0097] These data are consistent with the reaction of nitrite with
deoxyhemoglobin to form NO and iron-nitrosyl-hemoglobin (Doyle et
al., J Biol Chem, 256, 12393-12398, 1981). Nitrite is first reduced
to form NO and methemoglobin with a rate constant of 2.9
M.sup.-1sec.sup.-1 (measured at 25.degree. C., pH 7.0). This
reaction will be pseudo-first order, governed by the amounts (20
mM) of intra-erythrocytic hemoglobin, and limited by the rate of
nitrite uptake by the erythrocyte membrane. NO then binds to
deoxyhemoglobin to form iron-nitrosyl-hemoglobin, escapes the
erythrocyte, or reacts with other higher oxides, such as NO.sub.2,
to form N.sub.2O.sub.3 and S-nitroso-hemoglobin.
Equation Series 1 NO.sub.2.sup.- (nitrite)+HbFe.sup.II
(deoxyhemoglobin)+H.sup.+.fwdarw.HbFe.sup.III
(methemoglobin)+NO+OH.sup.-NO+HbFe.sup.II
(deoxyhemoglobin).fwdarw.HbFe.sup.IINO
(iron-nitrosyl-hemoglobin)
[0098] The formation of significant amounts of S-nitroso-hemoglobin
in vivo during nitrite infusion was also observed. Luschinger and
colleagues (Proc Natl Acad Sci USA, 100, 461-6, 2003) recently
proposed that nitrite reacts with deoxyhemoglobin to make
iron-nitrosyl-hemoglobin, with subsequent "transfer" of the NO to
the cysteine 93 to form S-nitroso-hemoglobin mediated by
reoxygenation and quaternary T to R transition of hemoglobin.
However, a direct transfer of NO from the heme to the thiol
requires NO oxidation to NO+ and such "cycling" has not been
reproduced by other research groups. Fernandez and colleagues have
recently suggested that nitrite catalyzes the reductive
nitrosylation of methemoglobin by NO, a process that generates the
intermediate nitrosating species dinitrogen teraoxide
(N.sub.2O.sub.3) (Inorg Chem, 42, 24, 2003). However, nitrite
reactions with hemoglobin provide ideal conditions for NO and
S-nitrosothiol generation along the oxygen gradient as nitrite
reacts with deoxyhemoglobin to form NO and with oxyhemoglobin to
form nitrogen dioxide (NO.sub.2) radical. NO.sub.2 participates in
radical-radical reactions (k=10.sup.9 M.sup.-1sec.sup.-1) with NO
to form N.sub.2O.sub.3 and S-nitrosothiol. Additional chemistry of
nitrite with hemoglobin produces reactive oxygen metabolites (such
as superoxide and hydrogen peroxide; Watanabe et al., Acta Med
Okayama 35, 173-8, 1981; Kosaka et al., Biochzim Biophys Acta 702,
237-41, 1982; and Kosaka et al., Environ Health Perspect 73,
147-51, 1987). Chemistry involving such NO radical-oxygen radical
reactions provides competitive pathways for S-nitrosothiol
formation in the presence of high affinity NO sinks, such as
hemoglobin.
VI. Physiological Considerations
[0099] The last decade has seen an increase in the understanding of
the critical role nitric oxide (NO) plays in vascular homeostasis.
The balance between production of NO and scavenging of NO
determines NO bioavailability, and this balance is carefully
maintained in normal physiology. The homeostatic, vasoregulatory
system is apparently fine-tuned to scavenge excess NO to limit
gross endocrine actions while allowing for sufficient local NO
necessary for regional tonic vasodilation. However, rapid NO
scavenging by cell-free hemoglobin disrupts this balance (Reiter et
al, Nat Med 8, 1383-1389, 2002). Under normal physiological
conditions, hemoglobin is rapidly and effectively cleared by the
hemoglobin scavenger system. However, chronic hemolytic conditions,
such as sickle cell disease, result in the daily release of
substantial quantities of hemoglobin into the vasculature,
suggesting that cell-free hemoglobin may have major systemic
effects on NO bioavailability. A current focus of research attempts
to explain and treat the vascular complications common to many
chronic hemolytic conditions, such as pulmonary hypertension,
cutaneous ulceration and acute and chronic renal failure.
Similarly, a number of clinical diseases and therapies such as
acute hemolytic crises, hemolysis during cardiopulmonary bypass
procedures, transfusion of aged blood, and myoglobinuria following
muscle infarction are often complicated by acute pulmonary and
systemic hypertension, acute renal failure, intravascular
thrombosis, ischemic central nervous system events and/or
death.
[0100] It is demonstrated herein that nitrite produces vasodilation
in humans associated with nitrite reduction to NO by
deoxyhemoglobin. Remarkably, systemic levels of 16 .mu.M resulted
in systemic vasodilation and decreased blood pressure, and regional
forearm levels of only 1-2 .mu.M significantly increased blood flow
at rest and with exercise stress. Furthermore, conversion of
nitrite to NO and S-nitrosothiol was mediated by reaction with
deoxyhemoglobin, providing a mechanism for hypoxia-regulated
catalytic NO production by the erythrocyte or endothelial/tissue
heme proteins. While high concentrations of hemoglobin in red
cells, coupled with the near diffusion-limited reaction rates
(.about.10 M.sup.-1s.sup.-1) of NO with hemoglobin, seem to
prohibit NO from being exported from the red blood cell, the data
presented herein argue to the contrary. While not intending to be
limiting, perhaps unique characteristics of the erythrocyte
membrane, with a submembrane protein and methemoglobin-rich
microenvironment, and the relative lipophilic nature of NO, allow
compartmentalized NO production at the red blood cell membrane.
This, coupled with the small yields of NO necessary for
vasodilation, could account for the export of NO despite these
kinetic constraints. It is further proposed that in vivo chemistry
for the conversion of nitrite to NO and S-nitrosothiol by reaction
with deoxyhemoglobin and methemoglobin provides a mechanism for
hypoxia-regulated catalytic NO production by the erythrocyte or
endothelial tissue heme proteins.
[0101] Three factors uniquely position nitrite, rather than
S-nitrosothiol, as the major vascular storage pool of NO: 1)
Nitrite is present in substantial concentrations in plasma,
erythrocytes and in tissues (Rodriguez et al., Proc Natl Acad Sci
USA 100:336-341, 2003). 2) Nitrite is relatively stable, because it
is not readily reduced by intracellular reductants, as are
S-nitrosothiols (Gladwin et al., J Biol Chem 21:21, 2002) and its
reaction rate with heme proteins is 10,000 times less than that of
authentic NO. 3) Nitrite is only converted to NO by reaction with
deoxyhemoglobin (or presumably deoxy-myoglobin, -cytoglobin, and
-neuroglobin) and its "leaving group" is the met(ferric)heme
protein which will not scavenge and inactivate NO Doyle et al., J
Biol Chem 256:12393-12398, 1981). Therefore, this pool provides the
ideal substrate for NO generation during hypoxia, providing a novel
mechanism for hypoxic vasodilation.
[0102] Because a deoxyhemoglobin-nitrite reductase system would
result in NO formation in deoxygenating blood, such a system links
hemoglobin oxygenation status to NO generation, the principle
previously ascribed to S-nitroso-hemoglobin (Jia et al., Nature
380:221-226, 1996). Hemoglobin possesses anionic binding cavities
that retain nitrite (Gladwin et al., J Biol Chem 21:21, 2002) and
nitrite is taken up by erythrocytes through the anion exchange
protein (AE1 or Band 3) or through the membrane as nitrous acid (a
pH dependent process that accelerates nitrite uptake during tissue
hypoxia (Shingles et al., J Bioenerg Biomembr 29:611-616, 1997; May
et al., Am I Physiol Cell Physiol 279:C1946-1954, 2000). Such
nitrite would provide a steady source of NO, NO.sub.2 and
S-nitrosothiol generation that would occur preferentially in
hypoxic vascular territories. Because the AE1 protein binds both
deoxyhemoglobin and methemoglobin and may channel nitrite, AE1
could serve to localize catalytic NO and S-nitrosothiol generation
at the erythrocyte membrane, where the relatively lipophilic NO,
NO.sub.2 and N.sub.2O.sub.3 could react in the vicinal lipid
bilayer (FIG. 5). The erythrocyte membrane is lined by an unstirred
outer diffusion barrier and an inner methemoglobin rich protein
matrix that might further promote such NO and NO.sub.2 chemistry
(Coin et al., J Biol Chem 254:1178-1190, 1979; Liu et al., J Biol
Chem 273:18709-18713, 1998; Han et al., Proc Nad Acad Sci USA
99:7763-7768, 2002).
[0103] This model is consistent with the in vitro observations of
Pawloski and colleagues (Pawloski et al., Nature 409:622-626,2001)
showing that S-nitrosation of hemoglobin and AE1 occurs in the
erythrocyte membrane after treatment of deoxygenated red blood
cells with NO solutions (which contain significant--more than 50
.mu.M--contaminating nitrite; Fernandez, et al. Inorg Chem 42:2-4,
2003). Further, N.sub.2O.sub.3 generated at the membrane could
directly nitrosate the abundant intra-erythrocytic glutathione,
eliminating the requirement of transnitrosation reactions with
S-nitroso-hemoglobin and thus facilitating rapid export of low
molecular weight S-nitrosothiol by simple diffusion across the
erythrocyte membrane (FIG. 5). A nitrite-hemoglobin chemistry
supports a role for the red cell in oxygen-dependent NO homeostasis
and provides a mechanism for the observations of multiple research
groups that red blood cells and plasma "loaded" with NO, by
exposure to NO in high concentration in solution or to NO gas or
donors (in equilibria with high concentrations of nitrite), can
export NO and induce vasodilation in vitro and in vivo (Rassaf et
al., J Clin Invest 109:1241-1248, 2002; Fox-Robichaud et al., J
Glitz Invest 101:2497-2505, 1998; McMahon et al., Nat Med 3:3,
2002; Cannon et al., J Clin Invest 108:279-287, 2001; Gladwin et
al., J Biol Chem 21:21, 2002; Gladwin et al., Circulation
107:271-278, 2003; Schechter et al, N Engl J Med 348:1483-1485,
2003).
[0104] In addition to the reaction of nitrite with deoxyhemoglobin,
reactions with deoxy-myoglobin, -cytoglobin and -neuroglobin or
with other endothelial cell heme proteins may also be important.
Such chemistry would occur between tissue nitrite and
deoxy-myoglobin in vascular and skeletal muscle, thus contributing
to hypoxic vasodilation and hypoxic potentiation of NO donors. The
P.sub.50 of these globin monomers is approximately 3-5 mm Hg,
placing their equilibrium deoxygenation point in the range of
tissue PO.sub.2 (0-10 mm Hg) during metabolic stress, such as
exercise. Such a low oxygen tension reduces oxygen availability as
substrate for NO synthesis, however, the tissue nitrite stores
could then be reduced to NO and 5-nitrosothiol, thus sustaining
critical vasodilation.
VII. Methods of Use
[0105] Therapeutic application of nitrite now can be used to
provide selective vasodilation in a subject, and particularly to
hypoxemic and ischemic tissue in the subject, and will be useful to
treat hemolytic conditions such as sickle cell disease, where free
hemoglobin released during hemolysis scavenges NO and disrupts
NO-dependent vascular function. Nitrite is expected to not only
inhibit the ability of free hemoglobin to scavenge NO by oxidizing
it to methemoglobin, but also to generate NO in tissue beds with
low oxygen tension. Thus, the applied nitrite will preferentially
release nitric oxide at areas of low oxygen tension, thereby
providing localized vasodilation and/or increased blood flow.
[0106] Nitrites can be administered to a subject to increase blood
flow to a tissue of the subject, for example, to increase blood
flow to a tissue, for instance a tissue with low oxygen tension; to
cause vasodilation; to decrease a subject's blood pressure; to
treat a subject having a condition associated with elevated blood
pressure; to treat a hemolytic condition; to treat vascular
complications associated with treatments or conditions that cause
hemolysis; to treat pulmonary hypertension, cerebral vasospasm, or
low blood flow to organs (such as ischemia reperfusion injury to
organs including brain, heart, kidney, and liver); and/or to treat
organs before and after transplantation.
[0107] The vasodilator properties of nitrite and the mechanisms for
its bioactivation were investigated as described herein. Sodium
nitrite infused at 36 .mu.moles per minute into the forearm
brachial artery of 18 normal volunteers resulted in a regional
nitrite concentration of 222 .mu.M and, surprisingly, a 175%
increase in resting forearm blood flow. Increased blood flow was
observed at rest, during NO synthase inhibition and with exercise.
The nitrite infusion also surprisingly resulted in increased tissue
perfusion, as demonstrated by increases in venous hemoglobin-oxygen
saturation, partial pressure of oxygen, and pH. Increased systemic
concentrations of nitrite (16 .mu.M) significantly reduced mean
arterial blood pressure.
[0108] In an additional ten subjects, the dose of nitrite was
reduced 2-logs, resulting in a forearm nitrite concentration of 2
.mu.M at rest and 0.9 .mu.M during exercise (FIG. 3). These
concentrations of nitrite surprisingly significantly increased
blood flow at rest and during NO synthase inhibition, with and
without exercise.
[0109] Nitrite infusions were associated with the rapid formation
of erythrocyte iron-nitrosyl-hemoglobin, and to a lesser extent,
S-nitroso-hemoglobin across the forearm vasculature. Formation of
these NO-Hb adducts was inversely proportional to the oxyhemoglobin
saturation. Additionally, vasodilation of rat aortic rings and the
formation of both NO gas and NO-modified hemoglobin from the
nitrite reductase activity of deoxyhemoglobin and deoxygenated
erythrocytes was observed, a result that links tissue hypoxia,
hemoglobin allostery, and nitrite bioactivation. These results
indicate that physiological levels of blood and tissue nitrite are
a major bioavailable pool of NO that contributes to vaso-regulation
and provide a mechanism for hypoxic vasodilation via reaction of
vascular nitrite with deoxygenated heme proteins in tissue and/or
the erythrocyte.
[0110] The findings described herein that administration of nitrite
reduces blood pressure and increases blood flow are unexpected and
surprising because published reports to date teach the person of
ordinary skill in the art that pharmacological levels of nitrites
(below about 100-200 .mu.M), when administered to subjects, lack
intrinsic vasodilatory properties (Lauer et al., Proc Natl Acad Sci
USA, 98:12814-9, 2001).
[0111] It is also believed that pharmaceutically acceptable salts
of nitrite can be infused into patients with hemolytic disease,
such as sickle cell disease, to improve blood flow, limit
ischemia-reperfusion tissue injury, and oxidize cell-free plasma
Hb. These effects should be useful in the treatment of sickle cell
vaso-occlusive pain crisis, stroke (brain ischemia) and the acute
chest syndrome.
VII. Formulations and Administration
[0112] Nitrites, including their salts, are administered to a
subject in accordance to methods provided herein, in order to
decrease blood pressure and/or increase vasodilation in a subject.
Administration of the nitrites in accordance with the present
disclosure may be in a single dose, in multiple doses, and/or in a
continuous or intermittent manner, depending, for example, upon the
recipient's physiological condition, whether the purpose of the
administration is therapeutic or prophylactic, and other factors
known to skilled practitioners. The administration of the nitrites
may be essentially continuous over a preselected period of time or
may be in a series of spaced doses. The amount administered will
vary depending on various factors including, but not limited to,
the condition to be treated and the weight, physical condition,
health, and age of the subject. Such factors can be determined by a
clinician employing animal models or other test systems that are
available in the art.
[0113] To prepare the nitrites, nitrites are synthesized or
otherwise obtained and purified as necessary or desired. In some
embodiments of the disclosure, the nitrite is a
pharmaceutically-acceptable salt of nitrite, for example, sodium
nitrite. In some embodiments of the disclosure, the nitrite is not
ethyl nitrite. In some embodiments of the disclosure, the sodium
nitrite is not on a medical devise, for example, not on a stent. In
some embodiments of the disclosure, the nitrite is not in the form
of a gel. The nitrites can be adjusted to the appropriate
concentration, and optionally combined with other agents. The
absolute weight of a given nitrite included in a unit dose can
vary. In some embodiments of the disclosure, the nitrite is
administered as a salt of an anionic nitrite with a cation, for
example, sodium, potassium, or arginine.
[0114] One or more suitable unit dosage forms including the nitrite
can be administered by a variety of routes including topical, oral
(for instance, in an enterically coated formulation), parenteral
(including subcutaneous, intravenous, intramuscular and
intraperitoneal), rectal, dermal, transdermal, intrathoracic,
intrapulmonary and intranasal (respiratory) routes.
[0115] The formulations may, where appropriate, be conveniently
presented in discrete unit dosage forms and may be prepared by any
of the methods known to the pharmaceutical arts. Such methods
include the step of mixing the nitrite with liquid carriers, solid
matrices, semi-solid carriers, finely divided solid carriers or
combinations thereof, and then, if necessary, introducing or
shaping the product into the desired delivery system. By
"pharmaceutically acceptable" it is meant a carrier, diluent,
excipient, and/or salt that is compatible with the other
ingredients of the formulation, and not deleterious or unsuitably
harmful to the recipient thereof. The therapeutic compounds may
also be formulated for sustained release, for example, using
microencapsulation (see WO 94/07529, and U.S. Pat. No.
4,962,091).
[0116] The nitrites may be formulated for parenteral administration
(e.g., by injection, for example, bolus injection or continuous
infusion) and may be presented in unit dose form in ampoules,
pre-filled syringes, small volume infusion containers or in
multi-dose containers. Preservatives can be added to help maintain
the shelve life of the dosage form. The nitrites and other
ingredients may form suspensions, solutions, or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
the nitrites and other ingredients may be in powder form, obtained
by aseptic isolation of sterile solid or by lyophilization from
solution, for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water, before use.
[0117] These formulations can contain pharmaceutically acceptable
carriers and vehicles that are available in the art. It is
possible, for example, to prepare solutions using one or more
organic solvent(s) that is/are acceptable from the physiological
standpoint, chosen, in addition to water, from solvents such as
acetone, ethanol, isopropyl alcohol, glycol ethers such as the
products sold under the name "Dowanol," polyglycols and
polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain
acids, ethyl or isopropyl lactate, fatty acid triglycerides such as
the products marketed under the name "Miglyol," isopropyl
myristate, animal, mineral and vegetable oils and
polysiloxanes.
[0118] It is possible to add other ingredients such as
antioxidants, surfactants, preservatives, film-forming, keratolytic
or comedolytic agents, perfumes, flavorings and colorings.
Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole,
butylated hydroxytoluene and .alpha.-tocopherol and its derivatives
can be added.
[0119] The pharmaceutical formulations of the present disclosure
may include, as optional ingredients, pharmaceutically acceptable
carriers, diluents, solubilizing or emulsifying agents, and salts
of the type that are available in the art. Examples of such
substances include normal saline solutions such as physiologically
buffered saline solutions and water. Specific non-limiting examples
of the carriers and/or diluents that are useful in the
pharmaceutical formulations of the present disclosure include water
and physiologically acceptable buffered saline solutions, such as
phosphate buffered saline solutions. Merely by way of example, the
buffered solution can be at a pH of about 6.0-8.5, for instance
about 6.5-8.5, about 7-8.
[0120] The nitrites can also be administered via the respiratory
tract. Thus, the present disclosure also provides aerosol
pharmaceutical formulations and dosage forms for use in the methods
of the disclosure. In general, such dosage forms include an amount
of nitrite effective to treat or prevent the clinical symptoms of a
specific condition. Any attenuation, for example a statistically
significant attenuation, of one or more symptoms of a condition
that has been treated pursuant to the methods of the present
disclosure is considered to be a treatment of such condition and is
within the scope of the disclosure.
[0121] For administration by inhalation, the composition may take
the form of a dry powder, for example, a powder mix of the nitrite
and a suitable powder base such as lactose or starch. The powder
composition may be presented in unit dosage form in, for example,
capsules or cartridges, or, e.g., gelatin or blister packs from
which the powder may be administered with the aid of an inhalator,
insufflator, or a metered-dose inhaler (see, for example, the
pressurized metered dose inhaler (MDI) and the dry powder inhaler
disclosed in Newman, S. P. in Aerosols and the Lung, Clarke, S. W.
and Davia, D. eds., pp. 197-224, Butterworths, London, England,
1984).
[0122] Nitrites may also be administered in an aqueous solution,
for example, when administered in an aerosol or inhaled form. Thus,
other aerosol pharmaceutical formulations may include, for example,
a physiologically acceptable buffered saline solution. Dry aerosol
in the form of finely divided solid compound that is not dissolved
or suspended in a liquid is also useful in the practice of the
present disclosure.
[0123] For administration to the respiratory tract, for example,
the upper (nasal) or lower respiratory tract, by inhalation, the
nitrites can be conveniently delivered from a nebulizer or a
pressurized pack or other convenient means of delivering an aerosol
spray. Pressurized packs may include a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
Nebulizers include, but are not limited to, those described in U.S.
Pat. Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol
delivery systems of the type disclosed herein are available from
numerous commercial sources including Fisons Corporation (Bedford,
Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal
Co. (Valencia, Calif.). For intra-nasal administration, the
therapeutic agent may also be administered via nose drops, a liquid
spray, such as via a plastic bottle atomizer or metered-dose
inhaler. Typical of atomizers are the Mistometer (Wintrop) and the
Medihaler (Riker). The nitrites may also be delivered via an
ultrasonic delivery system. In some embodiments of the disclosure,
the nitrites may be delivered via an endotracheal tube. In some
embodiments of the disclosure, the nitrites may be delivered via a
face mask.
[0124] The present disclosure further pertains to a packaged
pharmaceutical composition such as a kit or other container. The
kit or container holds a therapeutically effective amount of a
pharmaceutical composition of nitrite and instructions for using
the pharmaceutical composition for treating a condition.
IX. Combination Therapies
[0125] Furthermore, the nitrite may also be used in combination
with one or more other therapeutic agents, for example, pain
relievers, anti-inflammatory agents, antihistamines, and the like,
whether for the conditions described herein or some other
condition. By way of example, the additional agent is one or more
selected from penicillin, hydroxyurea, butyrate, clotrimazole,
arginine, or a phosphodiesterase inhibitor (such as
sildenafil).
[0126] It is believed that any therapy used with or suggested for
use in combination with NO therapy could be used with the nitrite
therapies described herein.
[0127] The following example is provided to illustrate certain
particular features and/or embodiments. This example should not be
construed to limit the invention to the particular features or
embodiments described.
EXAMPLE 1
Nitrite has Vasodilatory Properties in Vivo
[0128] This example provides a demonstration that nitrite,
administered by infusion to the forearm of human subjects, is an
effective vasodilator.
Methods
Human Subjects Protocol.
[0129] The protocol was approved by the Institutional Review Board
of the National Heart, Lung and Blood Institute, and informed
consent was obtained from all volunteer subjects. Nine men and nine
women, with an average age of 33 years (range 21-50 years),
participated in the study. An additional 10 subjects returned
three-six months later for a second series of experiments with low
dose nitrite infusion. Volunteers had a normal hemoglobin
concentration, and all were in excellent general health without
risk factors for endothelial dysfunction (fasting blood sugar
>120 mg/dL, low-density lipoprotein cholesterol >130 mg/dL,
blood pressure >145/95 mmHg, smoking within two years,
cardiovascular disease, peripheral vascular disease, coagulopathy,
or any other disease predisposing to vasculitis or Raynaud's
phenomenon). Subjects with G6PD deficiency, known cytochrome B5
deficiency or a baseline methemoglobin level>1% were excluded
(no screened subjects met these exclusion criteria). Lactating and
pregnant females were excluded (one subject with positive HCG
levels was excluded). No volunteer subject was allowed to take any
medication (oral contraceptive agents allowed), vitamin
supplements, herbal preparations, nutriceuticals or other
"alternative therapies" for at least one month prior to study and
were not be allowed to take aspirin for one week prior to
study.
Forearm Blood Flow Measurements
[0130] Brachial artery and antecubital vein catheters were placed
into the arm, with the intra-arterial catheter connected to a
pressure transducer for blood pressure measurements and an infusion
pump delivering normal saline at 1 mL/min. After 20 minutes of
rest, baseline arterial and venous blood samples were obtained and
forearm blood flow measurements were made by strain gauge
venous-occlusion plethysmography, as previously reported (Panza et
al., Circulation, 87, 1468-74, 1993). A series of 7 blood flow
measurements were averaged for each blood flow determination. A
series of measurements termed Parts I and II were performed in
randomized order to minimize a time effect on the forearm blood
flow response during nitrite infusion.
Measurement of Blood Flow and Forearm Nitrite Extraction During NO
Blockade and Repetitive Exercise
[0131] Part I: Following 20 minutes of 0.9% NaCl (saline) solution
infusion at 1 mL/min into the brachial artery, arterial and venous
blood samples were obtained for the assays described below and
forearm blood flow measured. Exercise was performed by repetitive
hand-grip at one-third of the predetermined maximum grip strength
using a hand-grip dynamometer (Technical Products Co.) (Gladwin et
al., Proc Natl Acad Sci USA, 97, 9943-8, 2000; Gladwin et al., Proc
Natl Acad Sci USA, 97, 11482-11487, 2000; Cannon et al., J Clin
Invest, 108, 279-87, 2001). Each contraction lasted for 10 seconds
followed by relaxation for 5 seconds. Following 5 minutes of
exercise, forearm blood flow measurements were obtained during
relaxation phases of exercise, and arterial and venous samples
collected. Following a 20-minute rest period with continued
infusion of saline into the brachial artery, repeated baseline
blood samples and forearm blood flow measurements were obtained.
L-NMMA was then infused at a rate of 1 mL/min (8 .mu.mol/min) into
the brachial artery. Following 5 minutes of L-NMMA infusion,
forearm blood flow was measured, and arterial and venous blood
samples obtained. Forearm exercise was then initiated in that arm
during continued L-NMMA infusion. Forearm blood flow was measured
and blood samples obtained after 5 minutes of exercise during
continued L-NMMA infusion (FIG. 1).
[0132] Part II: After a 30 minute rest period with continued
infusion of saline, baseline measurements were obtained, the saline
infusion was then stopped, and infusion of nitrite (NaNO.sub.2 36
.mu.mol/ml in 0.9% saline) at 1 ml/min was started. Sodium nitrite
for use in humans was obtained from Hope Pharmaceuticals (300 mg in
10 ml water) and 286 mg was diluted in 100 ml 0.9% saline by the
Pharmaceutical Development Service to a final concentration of 36
.mu.mol/ml. For the final 9 subjects studied, 0.01-0.03 mM sodium
bicarbonate was added to the normal saline, so as to titrate pH to
7.0-7.4. The nitrite solution was light protected and nitrite
levels and free NO gas in solution measured by reductive
chemiluminescence after all experiments (Gladwin et al., J Biol
Chem, 21, 21, 2002). Only 50.5.+-.40.5 nM NO was present in nitrite
solutions and was unaffected by bicarbonate buffering. There was no
correlation between NO levels in nitrite solutions and blood flow
effects of nitrite (r=-0.23; P=0.55). After 5 minutes of nitrite
infusion, forearm blood flow measurements and blood samples were
obtained, with brief interruption of the nitrite infusion to obtain
the arterial sample. With continued nitrite infusion, exercise was
performed as described previously, with forearm blood flow
measurements and blood samples obtained as described above. The
nitrite infusion was stopped and saline infusion re-started during
the subsequent 30-minute rest period. Following second baseline
measurements, the nitrite infusion was re-initiated, along with
L-NMMA at 8 .mu.mol/min. Five minutes later, forearm blood flow
measurements were performed and blood samples obtained followed by
5 minutes of exercise with continuation of nitrite and L-NMMA
infusions. Final forearm blood flow measurements and blood samples
obtained. At all time points during part II, blood samples were
obtained from the contralateral arm antecubital vein for
determination of methemoglobin and systemic levels of NO-modified
hemoglobin (FIGS. 2, 3, and 4). The total dose of sodium nitrite
infused was 36 .mu.mol/min.times.15 minutes.times.2 infusions=1.08
mmol=75 mg (MW NaNO.sub.2=69).
[0133] In additional studies in 10 subjects the same stages of
Parts I and II protocol were followed with infusion of low dose
nitrite (NaNO.sub.2 0.36 .mu.mol/ml in 0.9% saline, infused at 1
ml/min).
[0134] Arterial and venous pH, pO.sub.2, and pCO.sub.2, were
measured at the bedside using the i-STAT system (i-STAT
Corporation, East Windsor, N.J.) and methemoglobin concentration
and hemoglobin oxygen saturation measured by co-oximetry.
Measurement of Red Blood Cell S-Nitroso-Hemoglobin and
Iron-Nitrosyl-Heinoglobin.
[0135] S-nitroso-hemoglobin is unstable in the reductive red blood
cell environment and rapidly decays in a temperature and redox
dependent fashion, independent of oxygen tension (Gladwin et al., J
Biol Chem, 21:21, 2002). To stabilize the S-nitroso-hemoglobin for
measurement, the red blood cell must be rapidly oxidized with
ferricyanide. Before and during nitrite infusions, blood was drawn
from both the brachial artery and antecubital vein and the whole
blood immediately (at the bedside to eliminate processing time)
lysed 1:10 in an NO-hemoglobin "stabilization solution" of PBS
containing 1% NP-40 (to solubilize membranes), 8 mM NEM (to bind
free thiol and prevent artefactual S-nitrosation), 0.1 mM DTPA (to
chelate trace copper), and 4 mM ferricyanide and cyanide (to
stabilize S-nitrosohemoglobin and prevent artefactual ex-vivo
iron-nitrosylation during processing). The samples were desalted
across a 9.5 mL bed volume Sephadex G25 column to eliminate nitrite
and excess reagents and partially purify hemoglobin (99% hemoglobin
preparation). The hemoglobin fraction was quantified by the method
of Drabkin, and hemoglobin fractions reacted with and without
mercuric chloride (1:5 HgCl.sub.2:heme ratio--used to differentiate
S-nitrosothiol which is mercury labile versus iron-nitrosyl which
is mercury stable) and then in 0.1 M HCL/0.5% sulfanilamide (to
eliminate residual nitrite; Marley et al., Free Radic Res, 32, 1-9,
2000). The samples were then injected into a solution of tri-iodide
(I.sub.3.sup.-) in-line with a chemiluminescent nitric oxide
analyzer (Sievers, Model 280 NO analyzer, Boulder, Colo.). The
mercury stable peak represents iron-nitrosyl-hemoglobin. This assay
is sensitive and specific for both S-nitroso-hemoglobin and
iron-nitrosyl-hemoglobin to 5 nM in whole blood (0.00005% S--NO per
heme) (Gladwin et al., J Biol Chem, 21, 21, 2002).
[0136] Analysis was initially performed using red blood cell
pellet, however, despite placing the sample in ice and immediately
separating plasma from erythrocyte pellet, NO formed in the venous
blood ex vivo. To measure the true in vivo levels, whole blood was
mixed at the bedside 1:10 in the "NO-hemoglobin stabilization
solution". Plasma S-nitroso-albumin formation was negligible during
nitrite infusion so this bedside whole blood assay was used to
limit processing time and thus more accurately characterize the in
vivo chemistry. In a series of validation experiments, both
S-nitroso-hemoglobin and iron-nitrosyl-hemoglobin were stable in
the "NO-hemoglobin stabilization solution" for 20 minutes at room
temperature with no artifactual formation or decay of NO-modified
species (n=6).
Chemiluminescent Detection of NO Gas Released from Deoxyhemoglobin
and Deoxygenated Erythrocytes Following Nitrite Addition.
[0137] To determine whether free NO radical can form from the
reaction of nitrite and deoxyhemoglobin, 100 and 200 .mu.M nitrite
was mixed with 5 mL of 660 and 1000 .mu.M deoxygenated erythrocytes
in a light protected reaction vessel purged with helium or oxygen
(both 21% and 100%) in-line with a chemiluminescent NO analyzer
(Seivers, Boulder, Colo.). After allowing equilibration for 5
minutes, nitrite was injected and the rate of NO production
measured. Nitrite was injected into PBS as a control and into 100
.mu.M hemoglobin to control for the hemolysis in the 660 and 1000
.mu.M deoxygenated erythrocyte solutions. At the end of all
experiments the visible absorption spectra of the supernatant and
erythrocyte reaction mixture was analyzed and hemoglobin
composition deconvoluted using a least-squares algorithm. There was
less than 100 .mu.M hemolysis in the system, no hemoglobin
denaturation, and significant formation of
iron-nitrosyl-hemoglobin. The NO production from erythrocyte
suspensions exceeded that produced from the hemolysate control,
consistent with NO export from the erythrocyte.
Statistical Analysis.
[0138] An a priori sample size calculation determined that 18
subjects would be necessary for the study to detect a 25%
improvement in forearm blood flow during nitrite infusion when
forearm NO synthesis had been inhibited by L-NMMA compared with
normal saline infusion control values (alpha=0.05, power=0.80).
Two-sided P values were calculated by paired t-test for the
pair-wise comparisons between baseline and L-NMMA infusion values,
between baseline and exercise values, and between nitrite and
saline control values at comparable time-points of the study.
Repeated measures ANOVA were performed for artery-to-vein gradients
of NO species during basal, L-NMMA infusion, and exercise
conditions. Measurements shown are mean.+-.SEM.
Results and Discussion
[0139] Eighteen healthy subjects (9 males, 9 females; age range 21
to 50 years) were enrolled in a physiological study to determine if
nitrite is a vasodilator and to examine nitrite's in vivo
chemistry. Part I of the protocol was designed to measure the
normal hemodynamic and metabolic responses to exercise and to
inhibition of NO synthesis within the forearm as a control for Part
II of the protocol, in which these interventions were performed
during nitrite infusion. Initial baseline measurements included a
mean blood pressure of 85.6.+-.3.7 mm Hg and forearm blood flow of
4.0.+-.0.3 ml/min per 100 mL tissue (FIG. 1A). Repetitive hand-grip
forearm exercise increased blood flow approximately 600% over
resting values, and significantly decreased ipsilateral venous
hemoglobin oxygen saturation, p0.sub.2, and pH, consistent with
increased oxygen consumption and CO.sub.2 generation. Following a
20-minute rest period, repeat hemodynamic measurements showed an
approximate 10% higher forearm blood flow, but no change in
systemic blood pressure or forearm venous hemoglobin oxygen
saturation, PO.sub.2 and pH values compared with the initial
baseline values (FIG. 1B). The NO synthase inhibitor L-NMMA was
then infused into the brachial artery at 8 .mu.mol/min for 5
minutes, significantly reducing forearm blood flow by approximately
30% and significantly reducing venous hemoglobin oxygen saturation,
P0.sub.2 and pH values. Repeated forearm exercise during continued
L-NMMA infusion increased blood flow, but to a significantly lower
peak value compared with exercise alone (P<0.001). In addition,
hemoglobin oxygen saturation, P0.sub.2 and pH were significantly
lower during exercise with L-NMMA than with exercise without
regional NO synthase inhibition (P<0.001, P<0.005 and
P=0.027, respectively). Mean arterial blood pressure was unchanged
during all components of Part I of the protocol.
[0140] FIG. 1 depicts hemodynamic and metabolic measurements at
baseline and during exercise, without (FIG. 1A) and with (FIG. 1B)
inhibition of NO synthesis in 18 subjects. Mean arterial pressure
(MAP), forearm blood flow (FBF), and venous oxyhemoglobin
saturation, partial pressure of oxygen (pO.sub.2), and pH are shown
for all experimental conditions. These interventions and
measurements (part I of the protocol) served as a control for Part
II of the protocol, in which these interventions were performed
during nitrite infusion.
[0141] To determine whether nitrite has vasoactivity in humans, in
Part II of the protocol sodium nitrite in bicarbonate-buffered
normal saline (final concentration 36 .mu.mol/ml) was infused into
the brachial arteries of these 18 subjects to achieve an estimated
intravascular concentration of approximately 200 .mu.M (Lauer et
al., Proc Natl Acad Sci USA, 98, 12814-9, 2001). Following repeat
baseline measurements and infusion of sodium nitrite at 1 mL/min
for 5 minutes, nitrite levels in the ipsilateral antecubital vein
increased from 3.32.+-.0.32 to 221.82.+-.57.59 .mu.M (FIG. 2A).
Forearm blood flow increased 175% over resting values; venous
hemoglobin oxygen saturation, p0.sub.2 and pH levels significantly
increased over pre-infusion values, consistent with increased
perfusion of the forearm.
[0142] Systemic levels of nitrite were 16 .mu.M as measured in the
contralateral arm and were associated with a systemic effect of
decreased mean blood pressure of approximately 7 mm Hg. Consistent
with immediate NO generation from nitrite during an
arterial-to-venous transit, iron-nitrosylated-hemoglobin in the
ipsilateral antecubital vein increased from 55.7.+-.11.4 to
693.4.+-.216.9 nM during the nitrite infusion. During forearm
exercise with continuation of the nitrite infusion, blood flow
increased further, with evidence of metabolic stress by virtue of
reduction in forearm venous hemoglobin oxygen saturation, p0.sub.2
and pH levels from baseline values. Venous nitrite levels declined,
consistent with increased blood flow to the forearm diluting the
concentration of infused nitrite. Despite decreasing forearm
nitrite concentrations during exercise, iron-nitrosyl-hemoglobin
levels increased (FIG. 2A).
[0143] Following cessation of nitrite infusion and substitution of
saline as the intra-arterial infusate for 30 minutes, repeat
baseline measurements showed persistent elevations in systemic
levels of nitrite, iron-nitrosyl-hemoglobin and methemoglobin (FIG.
2B) over values obtained prior to the infusion of nitrite almost
one hour before. In addition, persistence of a vasodilator effect
was also apparent, as forearm blood flow was significantly higher
(4.79.+-.0.37 versus 3.94.+-.0.38 mL/min per 100 mL tissue,
P=0.003) and systemic blood pressure significantly lower
(82.1.+-.3.7 versus 89.2.+-.3.5 mm Hg, P=0.002) than initial
pre-nitrite infusion values. During re-infusion into the brachial
artery of sodium nitrite 36 .mu.mol/ml, combined with L-NMMA 8
.mu.mol/min in order to again inhibit regional synthesis of NO,
similar vasodilator effects of nitrite on resting and exercise
forearm blood flow were seen as during nitrite infusion without
L-NMMA (FIG. 2B). This stands in contrast to the vasoconstrictor
effect of NO synthase inhibition with L-NMMA observed in Part I of
the protocol (FIG. 1B). Venous nitrite and iron-nitrosyl-hemoglobin
levels followed similar patterns during NO inhibition as during the
initial nitrite infusion.
[0144] FIG. 2 depicts the effects of infusion of sodium nitrite
(NaNO.sub.2) in bicarbonate-buffered normal saline (0.9%; final
concentration 36 .mu.mol/ml) into the brachial arteries of 18
healthy subjects at 1 ml/in for 5 minutes at baseline and continued
during exercise. FIG. 2A depicts the effects without inhibition of
NO synthesis. FIG. 2B depicts the effects with inhibition of NO
synthesis. Values for mean arterial blood pressure (MAP), forearm
blood flow (FBF), venous oxyhemoglobin saturation, partial pressure
of oxygen (pO.sub.2) and pH, venous nitrite, venous
iron-nitrosyl-hemoglobin and venous methemoglobin are shown for all
experimental interventions.
[0145] As a test of the physiological relevance of vascular nitrite
as a vasodilator, nitrite concentrations were decreased by 2-logs
to 400 nmol/mL. An infusion of 1 mL/min for five minutes in 10
subjects significantly increased forearm blood flow in all ten
subjects from 3.49.+-.0.24 to 4.51.+-.0.33 ml/ml per 100 mL tissue
(FIG. 3A; P=0.0006). Blood flow significantly increased at rest and
during NO synthase inhibition with and without exercise (FIG. 3B;
P<0.05 during all conditions). Mean venous nitrite levels
increased from 176.+-.17 nM to 2564.+-.462 nM following a
five-minute infusion and exercise venous nitrite levels decreased
to 909.+-.113 nM (secondary to dilutional effects of increased flow
during exercise; FIG. 3C). Again, the vasodilator effects of
nitrite were paralleled with an observed formation of both
iron-nitrosyl-hemoglobin and S-nitroso-hemoglobin across the
forearm circulation (FIG. 3D; described below). These data indicate
that basal levels of nitrite, from 150-1000 .mu.M in plasma to
10,000 nM in vascular tissue, contribute to resting vascular tone
and hypoxic vasodilation.
[0146] FIG. 3 depicts the effects of infusion of low-dose sodium
nitrite in bicarbonate-buffered normal saline into the brachial
arteries of 10 healthy subjects at baseline and during exercise,
without and with inhibition of NO synthesis. FIG. 3A depicts
forearm blood flow at baseline and following a five-minute in
fusion of NaNO.sub.2 (0.36 .mu.mol/ml in 0.9% saline, infused at 1
ml/min). FIG. 3B depicts forearm blood flow with and without
low-dose nitrite infusion at baseline and during L-NMMA infusion
with and without exercise stress. FIG. 3C depicts venous levels of
nitrite from the forearm circulation at the time of blood flow
measurements. FIG. 3D depicts venous levels of S-nitroso-hemoglobin
(S--NO) and iron-nitrosyl-hemoglobin (Hb-NO) at baseline and
following nitrite infusion during exercise stress.
[0147] The vasodilatory property of nitrite during basal blood flow
conditions, when tissue pO.sub.2 and pH are not exceedingly low,
was unexpected. These results indicate that the previously
hypothesized mechanisms for nitrite reduction, nitrite
disproportionation and xanthine oxidoreductase activity, both of
which require extremely low pO.sub.2 and pH values not typically
encountered in normal physiology, are complemented in vivo by
additional factors that serve to catalyze nitrite reduction. While
ascorbic acid and other reductants, present in abundance in blood,
can provide necessary electrons for nitrous acid reduction, such
that the reaction might occur at physiologically attainable pH
levels, it is herein reported that deoxyhemoglobin effectively
reduces nitrite to NO, within one half-circulatory time. This
mechanism provides a graded production of NO along the
physiological oxygen gradient, tightly regulated by hemoglobin
oxygen desaturation.
Intravascular Formation of NO and S-Nitrosothiol by Reaction of
Nitrite with Intraerythrocytic Deoxyhemoglobin
[0148] Before and during nitrite infusions, blood was drawn from
both the brachial artery and antecubital vein and the whole blood
immediately (at the bedside to eliminate processing time) lysed
1:10 in an NO-hemoglobin "stabilization solution" and the
iron-nitrosyl-hemoglobin and S-nitroso-hemoglobin content
determined by tri-iodide-based reductive chemiluminescence and
electron paramagnetic resonance spectroscopy as described in
Methods. The baseline levels of S-nitroso-hemoglobin and
iron-nitrosyl-hemoglobin were at the limits of detection (<50 nM
or 0.0005% NO per heme) with no artery-to-vein gradients. Following
nitrite infusion in Part II of the protocol venous levels of both
iron-nitrosyl-hemoglobin and S-nitroso-hemoglobin rose strikingly
(FIG. 4A). The formation of both NO-hemoglobin adducts occurred
across the vascular bed, a half-circulatory time of less than 10
seconds. The rate of NO formation, measured as iron-nitrosyl and
S-nitroso-hemoglobin and quantified by subtraction of the arterial
from the venous levels with the difference being multiplied by
blood flow, increased greatly during exercise, despite a
significant decrease in the venous concentration of nitrite
secondary to increasing blood flow diluting the regional nitrite
concentration (FIG. 4A; P=0.006 for iron-nitrosyl-hemoglobin and
P=0.02 for S-nitroso-hemoglobin by repeated measures ANOVA).
[0149] FIG. 4A depicts formation of iron-nitrosyl-hemoglobin (black
squares) and S-nitroso-hemoglobin (red circles) during nitrite
infusion at baseline, during nitrite infusion and during nitrite
infusion with exercise, quantified by subtraction of the arterial
from the venous levels and multiplying the result by blood flow.
The formation of both NO-hemoglobin adducts was inversely
correlated with hemoglobin-oxygen saturation in the human
circulation during nitrite infusion (for iron-nitrosyl-hemoglobin
r=-0.7, p<0.0001, for S-nitroso-hemoglobin r=-0.45, p=0.04)
(FIG. 4B). Hemoglobin oxygen saturation was measured from the
antecubital vein by co-oximetry. Asterix in all figures signify
p<0.05 by paired t test or repeated measures analysis of
variance.
[0150] To determine whether free NO radical can form from the
reaction of nitrite and deoxyhemoglobin, 100 and 200 .mu.M nitrite
was reacted with deoxygenated erythrocytes (5 mL volume containing
a total of 660 and 1000 .mu.M in heme) in a light protected
reaction vessel purged with helium in-line with a chemiluminescent
NO analyzer (Seivers, Boulder, Colo.). As shown in FIGS. 5A and 5B,
the injection of nitrite into a solution of deoxygenated
erythrocytes resulted in the liberation of NO into the gas phase.
There was no release from nitrite in buffer control under the same
conditions, and significantly less NO was released upon nitrite
addition to oxygenated erythrocytes (21% and 100% oxygen). The
observed rate (determined by the assessment of the area under the
curve of increased steady-state NO generation following nitrite
injection calculated over 120 seconds) of NO production in the 5 mL
reaction volume was consistent with 47 pM NO production per second
(corresponding to an estimated 300 to 500 pM NO production per
second in whole blood). While NO formation rates in this
experimental system may not be extrapolated to rates of NO
formation in vivo, the experiments are consistent with two
important concepts: 1) A fraction of free NO can escape
auto-capture by the remaining heme groups; this is likely only
possible because nitrite is only converted to NO by reaction with
deoxyhemoglobin and its "leaving group" is the met(ferric)heme
protein which will limit scavenging and inactivation of NO (Doyle
et al., J Biol Chem, 256, 12393-12398, 1981); and 2) The rate of NO
production is increased under anaerobic conditions, consistent with
a nitrite-deoxyhemoglobin reaction.
[0151] While in the foregoing specification this invention has been
described in relation to certain preferred embodiments thereof, and
many details have been set forth for purposes of illustration, it
will be apparent to those skilled in the art that the invention is
susceptible to additional embodiments, and that certain of the
details described herein may be varied considerably without
departing from the basic principles of the invention.
* * * * *