U.S. patent application number 14/028850 was filed with the patent office on 2014-01-16 for nitrite and nitrite-metheme therapy to detoxify stroma-free hemoglobin based blood substitutes.
This patent application is currently assigned to The Government of the United States of America as represented by the Secretary of the Department of. The applicant listed for this patent is The Government of the United States of America as represented by the Secretary of the Department of, The Government of the United States of America as represented by the Secretary of the Department of, University of Alabama at Birmingham, Wake Forest University. Invention is credited to Mark T. Gladwin, Jeffrey Kerby, Daniel B. Kim-Shapiro, Rakesh P. Patel.
Application Number | 20140017342 14/028850 |
Document ID | / |
Family ID | 40344566 |
Filed Date | 2014-01-16 |
United States Patent
Application |
20140017342 |
Kind Code |
A1 |
Gladwin; Mark T. ; et
al. |
January 16, 2014 |
NITRITE AND NITRITE-METHEME THERAPY TO DETOXIFY STROMA-FREE
HEMOGLOBIN BASED BLOOD SUBSTITUTES
Abstract
This disclosure relates to methods of using nitrite to detoxify
stroma-free hemoglobin based blood substitutes. In particular,
methods are described for using a blood substitute comprised of
about equimolar amounts of nitrite and hemoglobin (e.g.,
nitrite-metHb) to treat, prevent, or ameliorate diseases of the
blood in a subject, or as a blood replacement in a subject.
Inventors: |
Gladwin; Mark T.;
(Pittsburgh, PA) ; Kim-Shapiro; Daniel B.;
(Winston-Salem, NC) ; Patel; Rakesh P.; (Hoover,
AL) ; Kerby; Jeffrey; (Birmingham, AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Government of the United States of America as represented by
the Secretary of the Department of
Wake Forest University
University of Alabama at Birmingham |
Rockville
Winston-Salem
Birmingham |
MD
NC
AL |
US
US
US |
|
|
Assignee: |
The Government of the United States
of America as represented by the Secretary of the Department
of
Rockville
MD
Wake Forest University
Winston-Salem
NC
University of Alabama at Birmingham
Birmingham
AL
|
Family ID: |
40344566 |
Appl. No.: |
14/028850 |
Filed: |
September 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12675347 |
Feb 25, 2010 |
8551536 |
|
|
PCT/US2008/074856 |
Aug 29, 2008 |
|
|
|
14028850 |
|
|
|
|
60969530 |
Aug 31, 2007 |
|
|
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Current U.S.
Class: |
424/718 |
Current CPC
Class: |
A61K 31/555 20130101;
A61K 33/26 20130101; Y02A 50/387 20180101; Y02A 50/30 20180101;
A61P 7/00 20180101; A61K 33/00 20130101; A61K 31/555 20130101; A61K
2300/00 20130101; A61K 33/26 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/718 |
International
Class: |
A61K 33/00 20060101
A61K033/00; A61K 31/555 20060101 A61K031/555 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under
Contract No. HL058091 awarded by the National Institutes of Health.
The United States government has certain rights in the invention.
Claims
1. A pharmaceutical composition, comprising a ferric (Fe.sup.III)
heme-containing molecule and nitrite, wherein the molar ratio of
nitrite to the ferric heme-containing molecule in the composition
is between about 1:2 and about 2:1, and wherein the pharmaceutical
composition is formulated for infusion into a subject.
2. The pharmaceutical composition of claim 1, wherein the
heme-containing molecule is hemoglobin, methemoglobin, cross-linked
hemoglobin or cross-linked methemoglobin.
3. The pharmaceutical composition of claim 1, wherein the
heme-containing molecule is a protein that binds oxygen.
4. The pharmaceutical composition of claim 1, wherein the molar
ratio of nitrite to the ferric heme-containing molecule is greater
than about 1:1.
5. The pharmaceutical composition of claim 1, wherein the molar
ratio of nitrite to the ferric heme-containing molecule is less
than about 1:1.
6. The pharmaceutical composition of claim 1, wherein the molar
ratio of nitrite to the ferric heme-containing molecule is about
1:1.
7. The pharmaceutical composition of claim 1, further comprising a
pharmaceutically acceptable carrier, an adjuvant, or a combination
thereof.
8. The pharmaceutical composition of claim 1, wherein the
pharmaceutical composition is contained in an i.v. bag.
9. A method of treating a subject having or predisposed to hypoxia,
hypoxaemia, ischemia or anoxia, comprising administering to the
subject a therapeutically effective amount of the pharmaceutical
composition of claim 1, thereby treating the subject.
10. The method of claim 9, wherein the subject has or is
predisposed to anemia, bleeding disorder, trauma, injury, burn,
coagulopathy, ectopic pregnancy, favism, gastrointestinal bleeding,
hemolytic uremic syndrome, hemophilia, microcytosis, ulcer,
bleeding in surgery, bleeding in pregnancy, hemorrhage,
rhabdomyolysis, hemorrhagic shock, sickle cell anemia,
hemoglobinopathy spherocytosis, thalassemia, and/or yellow
fever.
11. The method of claim 9, wherein the subject has lost blood
during a surgical procedure.
12. The method of claim 9, wherein the subject is a human.
13. The method of claim 9, wherein the subject is a non-human
animal.
14. A method of replacing blood in a subject, comprising
co-infusing into the subject a first pharmaceutical composition
comprising a ferric (Fe.sup.III) heme-containing molecule and
nitrite at a ratio of less than 1:1; and a second pharmaceutical
composition comprising oxyhemoglobin, wherein the first
pharmaceutical composition and the second pharmaceutical
composition are co-infused into the subject at a ratio of less than
1:1.
15. The method of claim 14, wherein the first pharmaceutical
composition consists essentially of a ferric (Fe.sup.III)
heme-containing molecule and nitrite at a ratio of less than
1:1.
13. The method of claim 14, wherein the first pharmaceutical
composition, or the second pharmaceutical composition, or both the
first pharmaceutical composition and the second pharmaceutical
composition are contained in an i.v. bag.
14. The method of claim 14, wherein the subject has or is
predisposed to anemia, bleeding disorder, trauma, injury, burn,
coagulopathy, ectopic pregnancy, favism, gastrointestinal bleeding,
hemolytic uremic syndrome, hemophilia, microcytosis, ulcer,
bleeding in surgery, bleeding in pregnancy, hemorrhage,
rhabdomyolysis, hemorrhagic shock, sickle cell anemia,
hemoglobinopathy spherocytosis, thalassemia, and/or yellow
fever.
15. The method of claim 14, wherein the ferric (Fe.sup.III)
heme-containing molecule is methemoglobin.
16. The method of claim 14, wherein the subject has lost blood
during a surgical procedure.
17. The method of claim 14, wherein the subject is a human.
18. The method of claim 14, wherein the subject is a non-human
animal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. application Ser. No.
12/675,347, filed Feb. 25, 2010, which is the U.S. National Stage
of International Application No. PCT/US2008/074856, filed Aug. 29,
2008, published in English under PCT Article 21(2), which claims
the benefit of U.S. Provisional Application No. 60/969,530, filed
Aug. 31, 2007. The above-listed applications are herein
incorporated by reference in their entirety.
FIELD
[0003] This disclosure relates to methods of producing non-toxic
stroma-free hemoglobin-based blood substitutes for use in a
subject. In particular, the disclosure relates to methods of
delivering nitrite with hemoglobin as, for instance, a
nitrite-methemoglobin intermediate, with other components of a
stroma-free hemoglobin based blood substitute in order to reduce or
eliminate vasoconstrictive properties inherent in hemoglobin based
blood substitutes.
BACKGROUND
[0004] There has long been an urgent need in the medical community
for a non-toxic blood substitute suitable for transfusion into a
patient. In addition to trauma victims and surgical patients,
patients suffering from diseases such as hemophilia and sickle-cell
anemia are in need of frequent transfusions. One in twenty
Americans will need a blood transfusion at some point in their
lives, and each year approximately eight million volunteer donors
make approximately 14 million blood donations. Several shortcomings
of donated blood have contributed to the urgent demand for a
cell-free blood substitute, including: the need to match blood
types; concerns regarding disease transmission; morally- or
religiously-based objections to blood transfusion; requirement for
freezing of blood units; and limited storage lifetime. There are
currently no clinically utilized oxygen-carrying blood substitutes
for humans.
[0005] Hemoglobin is the molecule found within red blood cells that
chelates molecular oxygen in the lungs and transports it throughout
the body. The idea of using hemoglobin-containing solutions as a
cell-free blood substitute has always been appealing. However,
clinical studies consistently revealed that cell-free hemoglobin
was associated with vasoconstriction leading to bradycardia,
hypertension and renal failure. Infusion of these products
increases the risk of myocardial infarction and death in human
clinical trials. Many of these toxicities have now been attributed
to the reaction with and scavenging of endogenous nitric oxide
(NO), an important blood vessel dilator elaborated by the lining
cells (endothelium) of the blood vessels. A major focus of recent
research has been to attempt to modify hemoglobin in such a way
that mitigates its NO scavenging and toxicity. For example,
hemoglobin has been polymerized, intra-molecularly cross-linked,
and conjugated to polyethyleneglycol (PEG). This work has resulted
in some improvements in cell-free hemoglobin that increase
circulating half-lives, modulate oxygen affinities and ameliorate
renal toxicity. However, vascular toxicity associated with
cell-free hemoglobin remains a concern and is thought to be largely
mediated by scavenging of nitric oxide and development of
hypertension, inflammation and platelet aggregation. Of great
clinical benefit would be the ability to resuscitate trauma or
surgical patients with a cell-free hemoglobin solution that
improves oxygen delivery without causing hypertension.
[0006] Recent studies reveal that the ubiquitous circulating anion
nitrite (NO.sub.2.sup.-) is a vasodilator and intrinsic signaling
molecule (Gladwin et al., Proc. Natl. Acad. Sci. USA
97:11482-11487, 2000; Cosby et al., Nat. Med. 9:1498-1505, 2003;
Gladwin et al., Nature Chemical Biology 1:308-314, 2005; Bryan et
al., Nature Chemical Biology 1:290-297, 2005; Modin et al., Acta
Physiologica Scandinavica 171:9-16, 2001). The vasodilator activity
of nitrite is associated with an allosterically controlled
heme-based reduction of nitrite to nitric oxide (NO) by
deoxygenated hemoglobin (deoxyHb) (Huang et al., J. Biol. Chem.
280:31126-31131, 2005; Huang, et al., J. Clin. Invest.
115:2099-2107, 2005). Nitrite infusions into the human circulation
increase blood flow at near-physiological concentrations (Cosby et
al., Nat. Med. 9:1498-1505, 2003). This vasodilation is temporally
associated with increases in red cell heme iron-nitrosylated
hemoglobin (HbFe.sup.II--NO, designated as {FeNO}.sup.7 using the
Enemark-Feltham notation; Enemark & Feltham, Coordination
Chemistry Reviews 13:339-406, 1974) and to a lesser extent
S-nitrosated hemoglobin (SNO-Hb, hemoglobin nitrosated at the
.beta.-93 cysteine; Cosby et al., Nat. Med. 9:1498-1505, 2003).
SUMMARY
[0007] It unexpectedly has been found that co-administration of
inorganic nitrite and cell-free hemoglobin in about equimolar
concentrations (or lower concentrations of nitrite relative to
hemoglobin) constitutes an oxygen carrying plasma expander, without
vasoconstrictive properties in vivo. Thus, described herein is
co-administration of cell-free hemoglobins with nitrite (free or as
nitrite-methemoglobin complex) as a cell-free hemoglobin solution
that improves oxygen delivery without causing hypertension.
Moreover, regimes are provided that allow administration of
therapeutics by first responders at the site of injury (e.g., in
the case of trauma patients at the site of accidents by ambulance
crews, or with soldiers on the battle field), which will be of
great clinical benefit for maintaining organ perfusion in patients.
Both cell-free hemoglobins and nitrite solutions would fulfill this
function since sterile solutions of each can be prepared, stored
and transported, and administered either intravenously or
intraperitoneally.
[0008] Also described is a method of producing a cell-free blood
substitute. The method includes contacting a ferric heme-containing
molecule, such as methemoglobin (Fe.sup.III-nitrite), with nitrite,
wherein the molar ratio of hemoglobin to nitrite is about 1:1 or
less, and thus forming nitrite-bound methemoglobin, wherein the
nitrite-containing molecules produces an intermediate with nitrogen
dioxide like electronic properties. NO in the vasculature or
produced from the reduction of additional nitrite by
deoxyhemoglobin can then react with this intermediate to form
dinitrogen trioxide (N.sub.2O.sub.3), which is a potent vasodilator
that ameliorates the toxicity of the stroma free hemoglobin. This
reaction converts the nitrite-methemoglobin back into ferrous
hemoglobin (deoxyhemoglobin), which can rebind oxygen in the lung.
Thus this novel chemistry will both deliver oxygen, deliver
N.sub.2O.sub.3 and NO, and redox cycle to rebind oxygen. In this
embodiment, the nitrite-methemoglobin is co-infused with
oxyhemoglobin in molar ratios of less than 1:1 to ensure both
oxygen delivery and NO delivery. In another embodiment, a
composition comprising nitrite and hemoglobin in a molar ratio of
less than 1:2 is administered as a cell-free blood substitute to a
subject. By way of example, the subject may be afflicted with
anemia, bleeding disorders, burns, coagulopathy, ectopic pregnancy,
favism, gastrointestinal bleeding, hemolytic uremic syndrome,
hemophilia, microcytosis, ulcer, hemorrhage, rhabdomyolysis,
hemorrhagic shock, sickle cell anemia, spherocytosis, thalassemia,
yellow fever, or another disease or condition that would benefit
from blood or plasma supplementation.
[0009] In another embodiment, the nitrite is bound to another
ferric heme protein such as nitrite bound to a ferric derivative of
a hemoglobin based blood substitute, or another hemoprotein (e.g.,
myoglobin, cytoglobin, neuroglobin) or a porphyrin compound.
[0010] The reaction of nitrite with deoxygenated hemoglobin
generates vasodilatory NO and thus has the potential to replete the
NO that is scavenged by the stroma-free hemoglobin-based blood
substitutes. In addition, the newly discovered properties of
nitrite-methemoglobin provide a new chemical pathway to NO
generation that can be used to limit and reverse the toxicity of
stroma-free hemoglobin based blood substitutes.
[0011] The foregoing and other features and advantages of the
disclosure 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
[0012] FIGS. 1A-1F are a series of graphs showing that
nitrite-MetHb is the intermediate in the deoxyhemoglobin-nitrite
reaction. (FIG. 1A) Disappearance of deoxyhemoglobin and formation
of iron-nitrosyl-hemoglobin and total MetHb (sum of MetHb and
nitrite-MetHb) over time in the reaction of 250 .mu.M
deoxyhemoglobin with 125 .mu.M nitrite at pH 7.4 and 37.degree. C.
All Hb concentrations are reported on a heme basis, so 1 mM Hb
reported here is 250 .mu.M in Hb tetramers (ignoring Hb dimer
formation). Reaction progress was monitored by absorption
spectroscopy. (FIG. 1B) Concentrations of various heme species over
time for the reaction shown in (FIG. 1A). Heme species include
deoxyhemoglobin, MetHb, iron-nitrosyl-hemoglobin, nitrite-MetHb,
and NO-MetHb. Total MetHb is also plotted labeled as Methemoglobin
and nitrite-methemoglobin and is equal to the sum of these two
species. (FIG. 1C) Comparison of chi squared values (sum squares of
residual spectra obtained by spectral deconvolution) from the
experiment in (FIG. 1A) fitted using four combination sets of
standard spectra, which include deoxyhemoglobin, MetHb,
iron-nitrosyl-hemoglobin, and additional standard spectra of either
nitrite-MetHb alone, NO-MetHb alone, both species, or neither
species. (FIG. 1D) The disappearance of deoxyhemoglobin and
formation of iron-nitrosyl-hemoglobin and total MetHb (sum of MetHb
and nitrite-MetHb) over time in the reaction of 450 .mu.M
deoxyhemoglobin with 5 mM nitrite at pH 7.4 and 37.degree. C. (FIG.
1E) Concentrations of various heme species over time for the
reaction shown in (FIG. 1D). Note that the plot of "Methemoglobin
and Nitrite-methemoglobin" is equal to the sum of these two species
that are also plotted separately. (FIG. 1F) Comparison of chi
square values from the experiment in (FIG. 1D) fitted using four
combination sets of standard spectra, which include
deoxyhemoglobin, MetHb, iron-nitrosyl-hemoglobin, and additional
standard spectra of either nitrite-MetHb alone, NO-MetHb alone,
both species, or neither species.
[0013] FIGS. 2A-2F are a series of graphs showing the EPR silence
of MetHb-NO.sub.2.sup.- and its paradoxical dissociation constant.
(FIG. 2A) At pH 7.4, nitrite (0, 0.1, 0.5, 1, and 5 mM) was added
to 84.8 .mu.M MetHb and the low field EPR measured at 4 K. Samples
were frozen for EPR five minutes after nitrite addition. The
concentrations of remaining MetHb, measured by double integration
of the MetHb signal, were 47.7, 27.4, 14.6, and 2.9 .mu.M
respectively (as indicated adjacent to each tracing). No low spin
MetHb EPR signals at lower g values were detected (inset). The EPR
silence of MetHb-NO.sub.2.sup.- was confirmed in three separate
MetHb preparations and in two different laboratories (Hogg and
Kim-Shapiro labs). The average dissociation constants of
MetHb-NO.sub.2.sup.-, calculated from the change in the g=6 MetHb
EPR signals secondary to nitrite binding, were 75 .mu.M, 271 .mu.M,
284 .mu.M, and 285 .mu.M for 0.1, 0.5, 1, and 5 mM of added
nitrite. (FIG. 2B) An analogous set of experiments performed at pH
6.5 demonstrated a more significant EPR silencing effect. The
measured concentration of MetHb remaining after the addition of 0,
0.1, 0.5, 1, and 5 mM nitrite were 75.5 .mu.M, 12.5 .mu.M, 3 .mu.M,
2.2 .mu.M, and 2.1 .mu.M (indicated on figure next to curves). The
average dissociation constants of MetHb-NO.sub.2.sup.- obtained by
analysis of the change in g=6 MetHb EPR signal secondary to nitrite
binding were 7 .mu.M, 18 .mu.M, 28 .mu.M, and 145 .mu.M for the
0.1, 0.5, 1, and 5 mM of added nitrite. (FIG. 2C) The kinetics of
nitrite (4 mM) association with MetHb (50 .mu.M) at pH 7.4 was
measured by stopped-flow absorption spectroscopy. The observed rate
of nitrite binding was 0.10 s.sup.-1 for the reaction shown here
and averaged 0.12.+-.0.01 s.sup.-1 between 6 trials. Inset depicts
raw data. (FIG. 2D) The kinetics of nitrite (4 mM) association with
MetHb (50 .mu.M) at pH 6 was measured by stopped-flow absorption
spectroscopy. The observed rate of nitrite binding was 0.87
s.sup.-1 for the reaction shown here and averaged 0.76.+-.0.11
s.sup.-1 between 6 trials. Inset depicts raw data. (FIG. 2E) The
kinetics of nitrite dissociation from MetHb was measured by
stopped-flow absorption spectroscopy for the reaction of 2.5 mM
nitrite-MetHb with 2.5 mM potassium cyanide at pH 7.4. The rate of
nitrite dissociation was 0.051 s.sup.-1 for the reaction shown here
and averaged 0.053.+-.0.002 s.sup.-1 between 3 trials. Inset
depicts raw data. (FIG. 2F) The kinetics of nitrite dissociation
from MetHb was measured by stopped-flow absorption spectroscopy for
the reaction of 2.5 mM nitrite-MetHb with 2.5 mM potassium cyanide
at pH 6. The rate of nitrite dissociation was 0.15 s.sup.-1 for the
reaction shown here and averaged 0.153.+-.0.001 s.sup.-1 between 3
trials. Inset depicts raw data.
[0014] FIGS. 3A-3C show molecular models of selected OLYP/STO-TZP
results for an Fe.sup.III-nitro (FIG. 3A) and two
Fe.sup.III-O-nitrito (FIGS. 3B and 3C) models. Each of these models
was restricted to C.sub.S symmetry, which allowed the separate
optimization of both the .sup.2A' and .sup.2A'' states, which
correspond the unpaired electron occupying one or the other of the
two d.sub..pi. (d.sub.xz or d.sub.yz) orbitals. The three diagrams
in the top rows present selected optimized bond distances (.ANG.,
in black), Mulliken spin populations (in magenta) and charges (in
green in parentheses) for the .sup.2A' states of the three models.
In the middle row are shown the corresponding spin density plots,
where the excess spin density is shown in cyan. Shown against the
oval insets at the bottom of the figure are the energies of the
various states studied (in kcal/mol), relative to the .sup.2A'
state of model A. Observe that although the O-nitrito forms B and C
are higher in energy than the A form, the .sup.2A' and .sup.2A''
states are very similar in energy for all three forms.
[0015] FIGS. 4A-4F are a series of graphs showing the reductive
nitrosylation of nitrite-MetHb catalyzed by NO. Formation of
iron-nitrosyl-hemoglobin by reductive nitrosylation of
nitrite-MetHb (rapid) and NO-MetHb (slow) was monitored by
absorption spectroscopy and spectral deconvolution. (FIG. 4A) 1 mM
NO was added to a pre-equilibrated solution of 30 .mu.M
deoxygenated MetHb and 5 mM nitrite. (FIG. 4B) The observed change
in iron-nitrosyl-hemoglobin concentration for the reaction shown in
(A) was compared to the theoretical iron-nitrosyl-hemoglobin
concentration as predicted by fitting the concentration of
HbFe.sup.II--NO to a single exponential that yielded an observed
rate of 0.0086 s.sup.-1. (FIG. 4C) 50 .mu.M NO was added to a
pre-equilibrated solution of 30 .mu.M deoxygenated MetHb and 5 mM
nitrite. (FIG. 4D) The observed change in iron-nitrosyl-hemoglobin
concentration for the reaction shown in (c) was compared to the
theoretical iron-nitrosyl-hemoglobin concentration as predicted by
fitting the concentration of HbFe.sup.II--NO to a single
exponential, which yielded an observed rate of 0.018 s.sup.-1.
(FIG. 4E) The observed rates of iron-nitrosyl-hemoglobin formation
as a function of variable nitrite concentrations. These reactions
were carried out with 30 .mu.M deoxygenated MetHb, 1 mM NO, and a
range of nitrite concentrations at pH 7.4. The line shown is a fit
to the data. The inset shows the same data re-plotted on a
different scale so that nitrite catalysis is more apparent. The
slope (k.sub.nitrite) is 0.13 M.sup.-1s.sup.-1, similar to that
reported previously. (Fernandez & Ford, J Am Chem Soc
125:10510-10511, 2003). The rate of reductive nitrosylation that
was observed when no nitrite is added is higher than that reported
previously (Fernandez & Ford, J Am Chem Soc 125:10510-10511,
2003) due, perhaps, to the fact that the experiments were performed
at a higher temperature (25.degree. C. vs. 37.degree. C.) and less
NO (1.8 mM vs. 1 mM (from 500 .mu.M ProliNO)) was used. (FIG. 4F)
The observed rates of iron-nitrosyl-hemoglobin formation as a
function of variable NO concentrations. These reactions were
carried out with 30 .mu.M deoxygenated MetHb, 5 mM nitrite, and a
range of NO concentrations at pH 7.4. The line shown is a fit to
the data.
[0016] FIGS. 5A-5F are a series of graphs showing nitrite mediated
nitrosothiol formation. (FIG. 5A) Time course of GSNO formation in
the reaction of 1 mM nitrite and 1 mM GSH in PBS, pH 6.5 or in PBS,
pH 7.4 at room temperature. Injections were made into a purge
vessel in-line with the nitric oxide analyzer at regular intervals
to detect GSNO by the 2C-assay. The figure represents raw
chemiluminescence data of individual injections made at indicated
time intervals for one of 5 repeats of the time course studies.
Notably, no signals were detected when samples were pretreated with
5 mM HgCl.sub.2 for 3 minutes. NEM was found to have no effect on
the size of the signal when tested on a sample incubated for 30
minutes. (FIG. 5B) Formation of SNO-Hb in the reaction of 300 .mu.M
deoxygenated Hb with 1 mM nitrite and 1 mM GSH, at pH 6.5 for 30
minutes at room temperature followed by treatment with
SNO-stabilizing solution for 1 hour (modified 2C assay). The figure
represents one of three repeats of the experiment and shows the raw
data of individual injections either directly (-Hg) or treated with
5 mM HgCl.sub.2 for 3 minutes (+Hg). (FIG. 5C) Formation of SNO-Hb
in the reaction of 5 mM deoxygenated hemoglobin with 1 mM nitrite
and 1 mM GSH, at pH 6.5 for 30 minutes at room temperature followed
by treatment with SNO-stabilizing solution for 1 hour (modified 2C
assay). The figure represents raw data of individual injections
either directly (-Hg) or treated with 5 mM HgCl.sub.2 for 3 minutes
(+Hg). FIGS. 5B and 5C show the presence of mercury stable peaks
that could be due to N-nitroso, O-nitroso, or C-nitroso (Feelisch
et al., Faseb J. 16:1775-1785, 2002). (FIG. 5D) 1 mM GSH was
reacted with 1 mM nitrite in either oxygenated or deoxygenated PBS
and at either pH 6.5 or pH 7.4 at room temperature. These results
are an average.+-.standard error of GSNO concentration measured by
the 2C assay (n=5). (FIG. 5E) Hemoglobin (300 .mu.M) in PBS with
100 .mu.M DTPA at pH 6.5 or pH 7.4 was reacted with 1 mM nitrite
and 1 mM GSH for 30 minutes at room temperature under different
oxygen saturations: 100% oxygenated Hb (oxyHb), 51.+-.4%
deoxygenated Hb (partially deoxyHb) or 97.+-.2% deoxygenated Hb
(deoxyHb). The samples were treated with SNO-stabilizing solution
(2-fold dilution) for 1 hour (modified 2C assay). The results
represent the average SNO-Hb measured from three trials.+-.one
standard deviation. Prior to addition of SNO-stabilization
solution, low molecular weight species were separated from Hb by
Centricon filters. Addition of NEM to the filtrate did not affect
the measured concentrations of GSNO (n=3). The concentration of
GSNO measured was 0.51.+-.0.04 .mu.M, 0.046.+-.0.08 .mu.M and
0.11.+-.0.1 .mu.M for oxyHb, partially deoxyHb and deoxyHb
respectively at pH 6.5, and 0.21.+-.0.13 .mu.M, 0 .mu.M and
0.05.+-.0.08 .mu.M for oxyHb, partially deoxyHb and deoxyHb
respectively at pH 7.4. When the above reaction was repeated
without DTPA, the average SNO-Hb measured for deoxyHb was
4.35.+-.0.44 .mu.M at pH 6.5 and 0.91.+-.0.23 .mu.M at pH 7.4. It
was generally found that detected nitrosation was 1-3 times smaller
when DTPA was not included in the incubations. When the reaction
was repeated without GSH at pH 6.5, the average SNO-Hb measured was
4.32.+-.0.51 .mu.M for 98.+-.2% deoxyHb. (FIG. 5F) Hemoglobin (5
mM) was reacted with 1 mM nitrite and 1 mM GSH in PBS with 100
.mu.M DTPA, at either pH 6.5 or pH 7.4, 30 minutes. These reactions
were carried out at two oxygen tensions: 100% oxygenated Hb (oxyHb)
and 98.+-.1% deoxygenated Hb (deoxyHb), followed by 6-fold dilution
in SNO-stabilization solution for 1 hour (modified 2C assay, n=3).
The average concentration of SNO-Hb was 0 .mu.M (oxyHb) and
24.44.+-.3.52 .mu.M (deoxyHb) at pH 6.5 and 0 .mu.M (oxyHb) and
8.94.+-.2.49 .mu.M (deoxyHb) at pH 7.4. There was no GSNO formed
based on measurements on the filtrate obtained using Centricon
filters.
[0017] FIGS. 6A-6E are a series of graphs showing that NO-catalyzed
reduction of nitrite-MetHb generates gas-phase N.sub.2O.sub.3.
(FIG. 6A) Experimental set-up used to detect the formation and
release of N.sub.2O.sub.3 into the gas phase. MetHb was
pre-equilibrated with nitrite prior to the addition of NO. Any
N.sub.2O.sub.3 released into the gas phase would subsequently flow
into the upstream trap vessel, where it could nitrosylate GSH in
the trap and form GSNO. Control reactions that excluded NO,
nitrite, or both, were carried out in parallel. The first
("reaction") vessel was purged with helium and maintained under
positive pressure to avoid oxygen leak into the system. (FIG. 6B)
Detection of GSNO by reductive chemiluminescence. Prior to
injection into tri-iodide (see Materials and Methods) aliquots of
the trap vessel solution were pre-treated with either acidified
sulfanilamide alone (.+-.) or with mercuric chloride followed by
acidified sulfanilamide (.sup..dagger-dbl.). The difference between
the sulfanilamide and mercuric chloride/sulfanilamide peaks
measures GSNO in those samples, while the absolute value of the
mercuric chloride peak measures the amount of other nitrogen oxide
species (such as HbFe.sup.II-NO). (FIG. 6C) Comparison of average
concentrations of GSNO formed in the trap vessel of the reactions
of 375 nmoles MetHb and 50 nmoles NO with and without 5 .mu.moles
nitrite. 3.82.+-.2.92 nmoles GSNO was detected in the presence of
nitrite, compared to 0.2.+-.0.08 nmoles GSNO when nitrite was
excluded from the reaction. (FIG. 6D) Comparison of average HbS-NO
concentrations formed in the purge vessel of reactions in (D).
1.16.+-.0.31 nmoles HbS-NO was detected in the presence of nitrite,
compared to 1.44.+-.0.16 nmoles HbS-NO when nitrite was excluded
from the reaction. (FIG. 6E) Comparison of average Hb-NO
concentrations formed in the purge vessel of reactions in (d).
4.65.+-.1.12 nmoles Hb-NO was detected in the presence of nitrite,
compared to 3.02.+-.0.74 nmoles Hb-NO when nitrite was excluded
from the reaction. Asterisk (*) denotes p value less than 0.05 for
the paired analysis of mean daily experiments (n=9 sets of
experiments) by the Wilcoxon matched pairs test.
[0018] FIGS. 7A-7B are two graphs showing the evidence for
N.sub.2O.sub.3 mediated nitrosation via nitrite bound MetHb. (FIG.
7A) DeoxyHb (300 .mu.M) was reacted with nitrite (1 mM) for 30
minutes at pH 6.5 in the presence or absence of KCN (5 mM). SNO-Hb
was subsequently measured using the modified 2C assay. Error bars
represent one standard deviation from the mean (n=3). When this
experiment was repeated with 2.5 mM KCN, SNO-Hb was measured to be
1.6.+-.0.5 .mu.M, compared to 4.3.+-.1.4 .mu.M when the reaction
was repeated in parallel without KCN. (FIG. 7B) DeoxyHb (300 .mu.M)
was reacted with nitrite (1 mM) for 30 minutes at pH 6.5 in either
PBS or 1 M phosphate buffer and SNO-Hb was measured by the modified
2C assay. MetHb (300 .mu.M) was also reacted with nitrite (5 mM),
generating MetHb-NO.sub.2.sup.-, with subsequent addition of
ProliNO (1 mM) in either PBS or 1 M phosphate buffer. After 5
minutes the SNO-Hb was measured by the modified 2C assay. Error
bars represent one standard deviation from the mean (n=3). When the
MetHb-NO.sub.2.sup.-/ProliNO experiment was repeated in Tris buffer
(no phosphate) slightly less SNO-Hb (2.2.+-.0.9 .mu.M) was made
than when this was done in PBS. No SNO-Hb was measured when the
MetHb (300 .mu.M) was incubated with nitrite (1 mM) in pH 6.5 PBS
buffer (no NO added) during a thirty minute anaerobic
incubation.
[0019] FIGS. 8A-8C are a series of graphs showing the absence of a
stable HbFe.sup.III--NO intermediate. (FIG. 8A) CO assay. Two 10
.mu.L, injections of partially NO saturated buffer (10 .mu.M NO)
followed by a single 10 .mu.L, injection of HbFe.sup.III--NO
(MetHbNO) into the NOA reservoir purged with a 1:1 mix of argon and
CO. This sample was generated by incubation of 1 mM MetHb with 30
.mu.M NO buffer followed by immediate injection into the NOA purge
vessel. A second injection of this sample is also shown. Ferrous
deoxyHb (100 .mu.M final concentration) was added to a new reaction
solution directly after addition of NO and immediately injected
(MetHbNO+100 .mu.M DeoxyHb). (FIG. 8B) EPR spectra for a sample
obtained after adding NO to MetHb (MetHb+NO) and after subsequently
adding ferrous deoxygenated Hb. The Fe.sup.IINO-Hb spectrum in the
"MetHb+NO" sample is secondary to the reductive nitrosylation
reaction, while the spectrum in the "MetHb+NO+DeoxyHb" sample is
most likely due to additional reductive nitrosylation and transfer
of NO from EPR silent HbFe.sup.IIINO to Fe.sup.II hemes. (FIG. 8C)
Progress of the reaction of deoxyHb (1 mM) with nitrite (250 .mu.M)
over 3 hours. Injections of 25 .mu.M reaction aliquots into the
pure CO assay were made at indicated time points. Long periods of
time between consecutive injections were cut from the data
shown.
[0020] FIGS. 9A-9C are a series of graphs showing the lack of an
oxygen transfer mechanism. (FIG. 9A) EPR spectrum of pure
Hb.sup.14NO demonstrating triplet hyperfine splitting resulting
from a mixture of 3 mM deoxyHb (treated with 15 mM dithionite) and
5 mM .sup.14Nitrite. After 6 minutes, this was followed by
treatment with 10 mM Sodium dodecyl sulphate to bring out hyperfine
structure and frozen for EPR. (FIG. 9B) EPR spectrum of pure
Hb.sup.15NO demonstrating doublet hyperfine splitting resulting
from a mixture of 3 mM deoxyHb (treated with 15 mM dithionite) and
5 mM .sup.15Nitrite. After 6 minutes this was followed by treatment
with 10 mM Sodium dodecyl sulphate and frozen for EPR. (FIG. 9C) A
representative EPR spectrum (from n=3) of reaction of 5 mM .sup.15
nitrite and 50 .mu.M .sup.14NO added to 30 .mu.M deoxygenated
MetHb, for 5 min, followed by 10 mM Sodium dodecyl sulphate. The
spectrum was fit to the basis spectra of Hb.sup.14NO and
Hb.sup.15NO (fit). The .beta.-nitrosyl component of the basis
spectra were subtracted out for the fitting. The average percentage
of Hb.sup.14NO was found to be 69.+-.5%. (The hyperfine splitting
demonstrates mixture of .sup.14N and .sup.15N in the reaction
product (HbFe.sup.II-.sup.14NO and HbFe.sup.II-.sup.15NO)).
[0021] FIG. 10 is a schematic showing a model of nitrite/Hb
mediated N.sub.2O.sub.3 export and nitrosation. Hemoglobin
deoxygenation occurs preferentially at the sub-membrane of the red
blood cell as it traverses the arteriole. Nitrite reacts with
deoxygenated Hb (deoxyHb) to make MetHb and NO. Much of this NO
binds to hemes of deoxyHb or undergoes dioxygenation forming
nitrate and MetHb from oxygenated Hb (OxyHb). MetHb binds nitrite
to form an adduct with some Fe(II)-NO.sub.2 character
(Hb-NO.sub.2.). This species reacts quickly with NO, forming
N.sub.2O.sub.3 which can diffuse out of the red cell, later forming
NO and effecting vasodilation and/or forming nitrosothiols (SNO).
Low molecular weight nitrosothiols may contribute to exportable
vasodilatory activity. The figure is not drawn to scale. Not all
reactions (such as hydrolysis of N.sub.2O.sub.3) are shown.
[0022] FIG. 11 is a graph showing that SNO-Hb is formed when 5 mM
Hb is reacted with varying amounts of nitrite for 30 minutes at
room temperature.
[0023] FIG. 12 is a graph showing the effect of KCN on
nitrite-mediated SNO-Hb formation in 1 M phosphate buffer. The
formation of SNO-Hb was measured during the reaction of 300 .mu.M
deoxygenated Hb (99.11.+-.0.1% deoxy), 100 .mu.M DTPA with 1 mM
nitrite, in 1M phosphate buffer in the absence (-KCN) or presence
(+KCN) of 5 mM KCN at pH 6.5 for 30 minutes at room temperature,
followed by treatment with SNO-stabilizing solution for 1 hour
(modified 2C assay). SNO-Hb was analyzed using the modified 2C
assay. The figure represents one of three repeats of the experiment
and shows the raw data of individual injections either directly
(-KCN, +KCN) or treated with 5 mM HgCl.sub.2 for 3 minutes
(-KCN+Hg). SNO-Hb measured was 5.35.+-.0.57 .mu.M in the absence of
KCN, and 0.70.+-.0.02 .mu.M in the presence of 5 mM KCN. The
average SNO-Hb measured in three separate experiments was
5.7.+-.0.3 .mu.M in the absence of KCN, and 0.70.+-.0.04 .mu.M in
the presence of 5 mM KCN. No SNO-Hb was detected in injections of
+KCN treated with 5 mM HgCl.sub.2 for 3 minutes.
[0024] FIGS. 13A-13B are graphs showing the detection of
HbFe.sup.III--NO by the 3C assay. (FIG. 13A) 3C assay detects
HbFe.sup.II--NO in the reaction of deoxyHb with sodium nitrite.
5.71 mM deoxyHb (78.3% deoxy) was reacted with 0.985 mM GSH and
0.592 mM sodium nitrite for 30 minute at room temperature and
injected into a purge vessel in line with the NOA, and
HbFe.sup.II-NO was measured using the 3C assay with and without
treatment with 5 mM HgCl.sub.2. The peaks are HgCl.sub.2 stable.
(FIG. 13B) 3C assay detects pure HbFe.sup.II--NO. Partially
nitrosylated Hb (120 .mu.M Hb with 52% of hemes nitrosylated by
excess NO buffer) was injected into the NOA purge vessel with and
without prior treatment with HgCl.sub.2.
[0025] FIGS. 14A-14B are two graphs showing the modified 2C assay.
(FIG. 14A) GSNO standard. GSNO (3 .mu.M) measured by direct
injection into 2C assay, with (+Hg) or without (-Hg) treatment with
5 mM HgCl.sub.2. (FIG. 14B) SNO-Hb standard. The SNO-Hb standard
was diluted into excess Hb to a final concentration of 5 mM heme
and 15 .mu.M SNO, treated with SNO-stabilization solution, passed
through two consecutive G-25 columns, and finally analyzed by the
2C assay.
[0026] FIGS. 15A-15C are a series of graphs showing the human
hemoglobin absorption spectroscopy standard spectra. (FIG. 15A)
Standard reference spectra used as a basis for deconvoluting and
fitting data obtained by absorption spectroscopy measurements of
hemoglobin reaction kinetics. (FIG. 15B) Selected absorbance
spectra of the reaction depicted in FIGS. 1A-C (250 .mu.M deoxyHb
and 125 .mu.M nitrite) taken at the indicated time points during
the reaction. Raw spectra were corrected for scatter by subtracting
the absorbance at 700 nm. Times are shown in minutes. (FIG. 15C)
Comparison of raw spectroscopic data obtained by UV-Vis
spectroscopy during the reaction shown in FIGS. 1A-C to theoretical
spectra calculated based on the concentrations of heme species
derived by least-squares deconvolution and the standard spectra of
these species (shown in FIG. 1A). The only assumption made in these
deconvolutions is that no colored species have been left out that
are likely to be present in the reaction. In some ways, this method
is equivalent to determining (for example) the concentration of
MetHb in a mixture of MetHb and OxyHb by using the known extinction
coefficients of the two species and the absorbance of the mixture
at two wavelengths. This two wavelength procedure results in two
linear equations (one at each wavelength) and two unknowns (the
concentration of MetHb and OxyHb). In the method described herein,
a similar linear equation is applied at each wavelength and solved
for the concentration of each species. The system of linear
equations is over-determined, so a least squares fit is performed
to obtain the best value for the concentration of each species at
each time point.
[0027] FIG. 16 is a graph showing data from a dog study in which
hemolysis was induced using infusions of free water into the blood
stream. Nitrite was infused at the same time. The effect of
low-level hemolysis on decreasing cardiac output secondary to
vasoconstriction and NO scavenging is shown. The addition of
nitrite therapeutically reverses this effect.
[0028] FIG. 17 is a graph showing the effect of resuscitating an
animal with cell-free hemoglobin (stroma-free hemoglobin based
oxygen carrier blood substitutes) with and without nitrite
addition. After giving the hemoglobin, blood pressure improves but
overshoots, while nitrite keeps the blood pressure normal. Sodium
nitrite reduces final blood pressure following stroma-free
hemoglobin resuscitation in a murine model of controlled
hemorrhage, shock and resuscitation. The graph shows mean arterial
pressure recorded in anesthetized mice treated at 90 minutes with
i.v. bolus of sodium nitrite (final circulating concentrations 2
.mu.M).
[0029] FIGS. 18A-18B are two graphs showing that exact ratios of
nitrite and hemoglobin will generate NO and inhibit mitochondrial
respiration, thereby demonstrating the effect of nitrite and
cell-free hemoglobin on NO generation. (FIG. 18A) Mitochondria (2
mg/ml) were stimulated to respire in the presence of no treatment,
nitrite (18 .mu.M), oxygenated hemoglobin (20 .mu.M), or nitrite
(18 .mu.M) and hemoglobin (20 .mu.M). Removal of the lid from the
sealed chamber is denoted by the arrow. Time to inhibition was
measured from removal of the lid to time the trace deviated from
zero percent oxygen. In these experiments, the oxygen trace
deviates from zero once the mitochondria stop respiring due to the
exhaustion of substrate or inhibition by NO produced by reactions
of hemoglobin with nitrite. (FIG. 18B) Quantification of several
traces similar to those shown in (FIG. 18A) with different levels
of hemoglobin. Inhibition of mitochondrial respiration (secondary
to NO production) occurred most rapidly with nitrite and low levels
of hemoglobin. The time to inhibition appears to be dependent on
the rate of NO production from reactions of nitrite with
deoxyhemoglobin and the rate of NO consumption by excess
oxyhemoglobin. All data is mean.+-.SEM of at least 3 independent
experiments (*p<0.01 compared to nitrite alone).
[0030] FIGS. 19A-19H is a series of graphs showing the
cardiovascular effects of nitrite in non-hemolyzing animals. (FIG.
19A) Serial plasma nitrite levels (.mu.M) in non-hemolyzing
animals. In animals receiving a six hour infusion of 5% dextrose
(D5W), a sodium nitrite infusion of 27.5 mg/h (open circles) led to
a rapid rise and then a sustained plasma nitrite concentration
(range: 15-21 .mu.M) compared to a placebo infusion of 0.9% NaCl
(closed circles). (FIGS. 19B-19H) In non-hemolyzing animals, sodium
nitrite increased cardiac index (CI) and decreased systemic
vascular resistance index (SVRI), pulmonary vascular resistance
(PVRI), mean arterial pressure (MAP), mean pulmonary arterial
pressure (PAM), central venous pressure (CVP), and pulmonary artery
occlusion pressure (PAOP) compared to placebo. Intravenous nitrite
enhanced cardiac performance (CI) by arterial vasodilation (SVRI,
PVRI, MAP, PAM) and caused venodilation (CVP).
[0031] FIGS. 20A-20B are two graphs showing the effects of nitrite
on the components of cardiac index in non-hemolyzing animals.
Cardiac index (FIG. 20A) and its components (FIG. 20B) have been
transformed into the log scale to demonstrate the individual
contribution of heart rate (HR) and stroke volume index (SVI) to CI
in additive fashion (normal scale: CI=SVI.times.HR; log scale: log
CI=log SVI+log HR) (Rowland et al., Pediatr. Cardiol. 21:429-432,
2000). In animals receiving D5W and nitrite, the nitrite-induced
increase in cardiac index is mediated predominantly through an
increase in SVI and to a lesser extent by a chronotropic effect.
Over time, the decrease in heart rate causes further increases in
SVI by increasing diastolic filling time in the ventricles leading
to higher end-diastolic volumes. Furthermore, the higher
end-diastolic volumes translate into higher end-diastolic pressures
which may explain the increase in PAOP over time (FIG. 19H).
[0032] FIGS. 21A-21G are a series of graphs showing the
cardiovascular effects of nitrite during intravascular hemolysis.
The cardiovascular effects of nitrite (27.5 mg/hr) at different
levels of cell-free plasma hemoglobin (zero, <25 .mu.M, and
>25 .mu.M) are shown (FIGS. 21A-21G). For each parameter, the
isolated effect of nitrite is displayed after controlling for
animal variability and the independent effects of hemolysis. The
depicted value represents the mean change in the parameter from
time zero to 1.5, 3, 4.5 and 6 h for all animals within the
specified hemolysis group (x-axis, zero=closed circles, <25
.mu.M=open circles, and >25 .mu.M=closed triangles). According
to previous experiments, if nitrite functioned purely as an NO
donor, then there should progressive attenuation of the
vasodilatory effects of nitrite with increasing levels of
hemolysis; the NO generated from nitrite should be progressively
scavenged by the increasing levels of cell-free plasma hemoglobin
(Minneci et al., J. Clin. Invest. 115:3409-3417, 2005). In these
experiments, the effect of nitrite was dependent on the level of
intravascular hemolysis (p=0.01 for a differing effect of nitrite
at low level hemolysis compared to zero and high level hemolysis
across the 7 physiologic variables combined). A consistent U-shaped
relationship between the physiologic effects of nitrite and the
levels of cell-free plasma hemoglobin was detected. At low levels
of hemolysis (Hb concentration<25 .mu.M), the vasodilatory
effects of nitrite were potentiated, whereas with higher levels of
hemolysis (cell-free plasma Hb>25 .mu.M), the expected
inhibition of the vasodilatory effects of nitrite were
observed.
[0033] FIGS. 22A-22F are a series of graphs showing nitrite levels
and plasma hemoglobin composition during intravascular hemolysis.
(FIGS. 22A, 22D) Intravascular hemolysis occurred at varying rates
in animals receiving water and nitrite infusions. In the low level
hemolysis group (Hb<25 .mu.M; FIG. 22A), the average peak
cell-free plasma hemoglobin level was 20 .mu.M; in the high level
hemolysis group (Hb>25 .mu.M; FIG. 22D), the average peak
cell-free plasma hemoglobin level was 142 .mu.M. Animals receiving
D5Wand nitrite represent the zero hemolysis control group with all
measured cell-free plasma hemoglobin levels<5 .mu.M. (FIGS. 22B,
22E) Total plasma hemoglobin composition in the low (FIG. 22B) and
high (FIG. 22E) hemolysis groups (open circles=methemoglobin;
closed circles=oxyhemoglobin). In animals receiving D5W and nitrite
(zero hemolysis), 81% of the measured cell-free plasma hemoglobin
was oxyhemoglobin (depicted as red reference lines) and 19% was
methemoglobin. With increasing hemoglobin concentrations, the rate
of methemoglobin formation increased from zero to 3 hours
(p=0.0001) producing higher levels of methemoglobin from 3 to 6
hours (p=0.0001) in animals with higher levels of hemolysis
compared to animals with lower levels of hemolysis. These results
can be explained by the fact that the overall reactions of nitrite
with oxy- and deoxy-hemoglobin are second order during their lag
phases such that increasing hemoglobin concentrations lead to
increasing rates of reaction. (FIGS. 22C, 22F) In both the low and
high level hemolysis groups, plasma nitrite levels were similar and
were maintained within a range of 16-21 .mu.M throughout the six
hour experiment.
[0034] FIGS. 23A-23F are a series of graphs showing the effects of
nitrite and intravascular hemolysis on cardiovascular responses to
sodium nitroprusside. The physiologic effects of sodium
nitroprusside (a direct NO donor) were dependent on the level (or
dose) of hemolysis and the presence of sodium nitrite (p=0.09, 0.05
and 0.009 for interaction between level of hemolysis and nitrite on
the effect of sodium nitroprusside for CI, SVRI, and PVRI
respectively). The depicted value represents the mean percent
change in the parameter for all doses of nitroprus side for all
animals within the specified hemolysis group (zero=closed circles,
<25 .mu.M=open circles, and >25 .mu.M=closed triangles). As
expected for a direct NO donor, in animals not receiving nitrite,
sodium nitroprusside-induced increases in CI and decreases in SVRI
and PVRI were progressively inhibited by increasing levels of
hemolysis (FIGS. 23A, 23C, 23E). Compared to the non-hemolyzing
animals not receiving nitrite (zero-hemolysis, no nitrite), the
nonhemolyzing animals receiving nitrite (zero-hemolysis, nitrite)
demonstrated blunted effects of sodium nitroprusside on CI, SVRI
and PVRI suggesting a decreased vasodilator effect of the donated
nitric oxide in the presence of nitrite (A vs. B, C vs. D, and E
vs. F). In the animals receiving nitrite, the effects of sodium
nitroprusside on CI, SVRI, and PVRI were accentuated with low
levels of hemolysis (Hb<25 .mu.M, nitrite) and attenuated with
high levels of hemolysis (Hb>25 .mu.M, nitrite) compared to
non-hemolyzing animals (zero hemolysis, nitrite) (FIGS. 23B, 23D,
23F).
DETAILED DESCRIPTION
I. Abbreviations
[0035] ABG arterial blood gas
[0036] Aquo-metHb water-Methemoglobin
[0037] cGMP cyclic guanosine monophosphate
[0038] CI cardiac index
[0039] CO cardiac output
[0040] CVP central venous pressure
[0041] D5W 5% dextrose
[0042] DeoxyHb deoxygenated hemoglobin
[0043] DFT Density Functional Theory
[0044] DTPA diethylenetriamine-pentaacetic acid
[0045] EPR electron paramagnetic resonance spectroscopy
[0046] GSH glutathione
[0047] GSNO S-nitrosoglutathione
[0048] Hb hemoglobin
[0049] HbFe.sup.II--NO ferrous iron-nitrosylated hemoglobin
[0050] HbFe.sup.III--NO ferric-iron-nitrosyl hemoglobin
[0051] Hb-NO nitrosyl hemoglobin
[0052] HbS-NO thio-nitrosyl hemoglobin
[0053] Hct hematocrit
[0054] HR heartrate
[0055] HUS hemolytic uremic syndrome
[0056] i.v. intravenous
[0057] KCN potassium cyanide
[0058] MAP mean arterial pressure
[0059] MetHb methemoglobin
[0060] MetHbNO ferric hemoglobin
[0061] MetHb-NO.sub.2.sup.- nitrite-bound methemoglobin
[0062] NEM N-ethylmaleimide
[0063] Nitrite-MetHb nitrite-bound methemoglobin
[0064] NOA nitric oxide analyzer
[0065] NO-metHb nitroxyl methemoglobin
[0066] NO.sub.x molecular species of nitrogen and oxygen
[0067] OLYP Handy/Cohen local exchange functional
[0068] OxyHb oxygenated hemoglobin
[0069] PAM pulmonary arterial pressure
[0070] PAOP pulmonary artery occlusion pressure
[0071] PBS phosphate-buffered saline
[0072] PCWP pulmonary capillary wedge pressure
[0073] PEG polyethylene glycol
[0074] PVRI pulmonary vascular resistance index
[0075] RBC red blood cell
[0076] SNO-Hb s-nitrosated hemoglobin
[0077] STO-TZP density functional theory calculations
[0078] SVRI systemic vascular resistance index
[0079] TTP thrombotic thrombocytopenic purpura
II. Terms
[0080] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0081] In order to facilitate review of the various embodiments of
the invention, the following explanations of specific terms are
provided:
[0082] Anemia: a deficiency of red blood cells (RBCs) and/or
hemoglobin. Anemia is the most common disorder of the blood, and it
results in a reduced ability of blood to transfer oxygen to the
tissues. Since all human cells depend on oxygen for survival,
varying degrees of anemia can have a wide range of clinical
consequences. The three main classes of anemia include excessive
blood loss (acutely such as a hemorrhage or chronically through
low-volume loss), excessive blood cell destruction (hemolysis) or
deficient red blood cell production (ineffective
hematopoiesis).
[0083] The term "anemia" refers to all types of clinical anemia,
including but not limited to: microcytic anemia, iron deficiency
anemia, hemoglobinopathies, heme synthesis defect, globin synthesis
defect, sideroblastic defect, normocytic anemia, anemia of chronic
disease, aplastic anemia, hemolytic anemia, macrocytic anemia,
megaloblastic anemia, pernicious anemia, dimorphic anemia, anemia
of prematurity, Fanconi anemia, hereditary spherocytosis,
sickle-cell anemia, warm autoimmune hemolytic anemia, cold
agglutinin hemolytic anemia.
[0084] In severe cases of anemia, or with ongoing blood loss, a
blood transfusion may be necessary. Doctors may use any of a number
of clinically accepted criteria to determine that a blood
transfusion is necessary to treat a subject with anemia. For
instance, the currently accepted Rivers protocol for early
goal-directed therapy for sepsis requires keeping the hematocrit
above 30.
[0085] Anoxia: a pathological condition in which the body as a
whole or region of the body is completely deprived of oxygen
supply.
[0086] Bleeding disorders: a general term for a wide range of
medical problems that lead to poor blood clotting and continuous
bleeding. Doctors also refer to bleeding disorders by terms such
as, for example, coagulopathy, abnormal bleeding and clotting
disorders.
[0087] Burns: any extremity experienced by the skin caused by heat,
cold, electricity, chemicals, friction or radiation.
[0088] Cell-free or Stroma-free blood substitute: a composition
lacking erythrocytes and other whole cell components of blood used
to replace whole blood in a subject. An excellent "blood
substitute" is one which mimics the oxygen-carrying capacity of
hemoglobin, which requires no cross-matching or compatibility
testing, with a long shelf life, which exhibits a long
intravascular half life (over days and weeks), and is free of side
effects and pathogens. Three main types of blood substitutes are in
development: hemoglobin-based oxygen carriers, perfluorocarbon
emulsions, and encapsulated hemoglobin in lipid vesicles.
[0089] The general task of blood within the frame of classic
transfusion medicine is to supply oxygen to tissue (oxygen
transport from lung to tissue, oxygen release and picking up carbon
dioxide). All of this is accomplished by hemoglobin (Hb), the
oxygen carrier protein contained within red cells. Early attempts
to develop blood substitutes were focused on simple cell-free
solutions of hemoglobin. Early studies conducted in experimental
animals showed that infusion of free hemoglobin caused a
substantial increase in oncotic pressure because of its
hyperosmolarity, coagulopathy, and hypertensive properties.
[0090] One significant problem and source of free hemoglobin's
hypertensive properties was the affinity of Hb for nitric oxide
(NO). NO produced by endothelial cells affects smooth muscle cells
of the vessel wall and modulates the vascular tone toward
vasodilatation. Cell-free Hb scavenges NO and shifts vasomotor tone
toward vasoconstriction. Cell-free hemoglobin-induced
vasoconstriction leads to serious side effects during transfusion
of a subject manifested as an increase in systemic and pulmonary
artery pressure without normalizing cardiac output or restoring
intravascular volume. Decreases in the cardiac index impair optimum
oxygen delivery and outweigh the advantage of an oxygen-carrying
solution. Severe vasoconstriction complications caused the
termination of clinical trials of unmodified cell-free hemoglobin
as a blood substitute.
[0091] Modified Hb molecules have been produced in an attempt to
overcome other limitations of hemoglobin for use in a blood
substitute, for example the penetration of Hb molecules into the
interstitial space of the subendothelial layers of blood vessel
walls and the sensitization of peripheral .alpha.-adrenergic
receptors. Successful modifications include purification,
cross-linkage, and polymerization. Administration of these modified
hemoglobins leads to vasoconstrictive effects that may increase
systemic and pulmonary vascular resistance with resultant decreases
in cardiac index. Clinical trials with these modified hemoglobins
in healthy volunteers showed dose-dependent moderate or severe
abdominal pain and increases in mean arterial pressure. The current
state of the art is that there are no cell-free blood substitutes
approved for clinical use in the United States.
[0092] Coagulopathy: a medical term for a defect in the body's
mechanism for blood clotting.
[0093] Ectopic pregnancy: a complication of pregnancy in which the
fertilized ovum is implanted in any tissue other than the uterine
wall.
[0094] Favism: the common name of glucose-6-phosphate dehydrogenase
(G6PD) deficiency; an X-linked recessive hereditary disease
featuring non-immune hemolytic anemia in response to a number of
causes.
[0095] Gastrointestinal bleeding: every form of hemorrhage (loss of
blood) in the gastrointestinal tract, from the pharynx to the
rectum.
[0096] Heme-containing molecule: any molecule comprising a heme
prosthetic group. The heme prosthetic group that of an iron atom
contained in the center of a large heterocyclic organic ring called
a porphyrin. Some, but not all, porphyrins contain iron. By way of
example, heme-containing molecules include (but are not limited to)
cytoglobin, neuroglobin, hemoglobin, Hemoglobin S, F, A2 zeta and
other hemoglobins, porphyrin compounds, and mutant globins, such as
hemoglobins, with modified oxygen affinity, size, viscosity, redox
potential, and/or heme pocket geometry.
[0097] Hemoglobin: the iron-containing oxygen-transport
metalloprotein in the red blood cells of the blood in vertebrates
and other animals. In humans, the hemoglobin molecule is an
assembly of four globular protein subunits. Each subunit is
composed of a protein chain tightly associated with a non-protein
heme group. Each protein chain arranges into a set of alpha-helix
structural segments connected together in a globin fold
arrangement, so called because this arrangement is the same folding
motif used in other heme/globin proteins such as myoglobin. This
folding pattern contains a pocket which strongly binds the heme
group.
[0098] The heme group consists of an iron (Fe) ion (charged atom)
held in a heterocyclic ring, known as a porphyrin. The iron ion,
which is the site of oxygen binding, bonds with the four nitrogens
in the center of the ring, which all lie in one plane. The iron is
also bound strongly to the globular protein via the imidazole ring
of a histidine residue below the porphyrin ring. A sixth position
can reversibly bind oxygen, completing the octahedral group of six
ligands. Oxygen binds in an "end-on bent" geometry where one oxygen
atom binds Fe and the other protrudes at an angle. When oxygen is
not bound, a very weakly bonded water molecule fills the site,
forming a distorted octahedron. The iron ion may either be in the
Fe.sup.II or Fe.sup.III state, but ferrihemoglobin (methemoglobin)
(Fe.sup.III) cannot bind oxygen. In binding, oxygen temporarily
oxidizes Fe to (Fe.sup.III), so iron must exist in the +2 oxidation
state in order to bind oxygen. The body reactivates hemoglobin
found in the inactive (Fe.sup.III) state by reducing the iron
center.
[0099] In adult humans, the most common hemoglobin type is a
tetramer (which contains 4 subunit proteins) called hemoglobin A,
consisting of two .alpha. and two .beta. subunits non-covalently
bound, each made of 141 and 146 amino acid residues, respectively.
This is denoted as .alpha.2.beta.2. The subunits are structurally
similar and about the same size. Each subunit has a molecular
weight of about 17,000 daltons, for a total molecular weight of the
tetramer of about 68,000 daltons. The four polypeptide chains are
bound to each other by salt bridges, hydrogen bonds, and
hydrophobic interactions.
[0100] Oxyhemoglobin is formed during respiration when oxygen binds
to the heme component of the protein hemoglobin in red blood cells.
This process occurs in the pulmonary capillaries adjacent to the
alveoli of the lungs. The oxygen then travels through the blood
stream to be delivered to cells where it is utilized in aerobic
glycolysis and in the production of ATP by the process of oxidative
phosphorylation.
[0101] Deoxyhemoglobin is the form of hemoglobin without bound
oxygen. The absorption spectra of oxyhemoglobin and deoxyhemoglobin
differ. The oxyhemoglobin has significantly lower absorption of the
660 nm wavelength than deoxyhemoglobin, while at 940 nm its
absorption is slightly higher.
[0102] Hemolysis: the breaking open of red blood cells and the
release of hemoglobin into the surrounding fluid.
[0103] Hemolytic uremic syndrome (HUS): a disease characterized by
microangiopathic hemolytic anemia, acute renal failure and a low
platelet count (thrombocytopenia). The classic childhood case of
hemolytic uremic syndrome occurs after bloody diarrhea caused by E.
coli O157:H7, a strain of E. coli that expresses verotoxin (also
called Shiga toxin). The toxin enters the bloodstream, attaches to
renal endothelium and initiates an inflammatory reaction leading to
acute renal failure and disseminated intravascular coagulation. The
fibrin mesh destroys red blood cells and captures thrombocytes,
leading to a decrease of both in full blood count. Adult HUS has
similar symptoms and pathology but is an uncommon outcome of the
following: HIV; antiphospholipid syndrome (associated with Lupus
erythematosus and generalized hypercoagulability); postpartum renal
failure; malignant hypertension; scleroderma; and cancer
chemotherapy (mitomycin, cyclosporine, cisplatin and bleomycin). A
third category is referred to as Familial hemolytic uremic
syndrome. It represents 5-10% of hemolytic uremic syndrome cases
and is due to an inherited deficiency leading to uncontrolled
complement system activation.
[0104] Hemophilia: the name of several hereditary genetic illnesses
that impair the body's ability to control coagulation.
[0105] Hemorrhage: the loss of blood from the circulatory system.
Bleeding can occur internally, where blood leaks from blood vessels
inside the body, or externally, either through a natural opening
such as vagina, mouth or rectum, or through a break in the
skin.
[0106] The average human has around 7 to 8% of their body weight
made up of blood. This equates to an average of around 5 liters of
blood (5.3 quarts) in a 70 kg (154 lbs.) man. The circulating blood
volume is approximately 70 ml/kg of ideal body weight. Thus the
average 70 kg male has approximately 5000 ml (5.3 quarts) of
circulating blood. Loss of 10-15% of total blood volume can be
endured without clinical sequelae in a healthy person, and blood
donation typically takes 8-10% of the donor's blood volume. The
technique of blood transfusion is used to replace severe quantities
of lost blood.
[0107] Hemorrhage generally becomes dangerous, or even fatal, when
it causes hypovolemia (low blood volume) or hypotension (low blood
pressure). In these scenarios various mechanisms come into play to
maintain the body's homeostasis. These include the
"retro-stress-relaxation" mechanism of cardiac muscle, the
baroreceptor reflex and renal and endocrine responses such as the
renin-angiotensin-aldosterone system.
[0108] Hemorrhage is broken down into four classes by the American
College of Surgeons' Advanced Trauma Life Support:
[0109] Class I Hemorrhage involves up to 15% of blood volume. There
is typically no change in vital signs and fluid resuscitation is
not usually necessary.
[0110] Class II Hemorrhage involves 15-30% of total blood volume. A
patient is often tachycardic (rapid heartbeat) with a narrowing of
the difference between the systolic and diastolic blood pressures.
The body attempts to compensate with peripheral vasoconstriction.
Volume resuscitation with crystalloids (Saline solution or Lactated
Ringer's solution) is all that is typically required. Atypically,
blood transfusion may be required.
[0111] Class III Hemorrhage involves loss of 30-40% of circulating
blood volume. The patient's blood pressure drops, the heart rate
increases, peripheral perfusion, such as capillary refill worsens,
and the mental status worsens. Fluid resuscitation with crystalloid
and blood transfusion are usually necessary.
[0112] Class IV Hemorrhage involves loss of >40% of circulating
blood volume. The limit of the body's compensation is reached and
aggressive resuscitation is required to prevent death.
[0113] Hemorrhagic shock: a condition of reduced tissue perfusion,
resulting in the inadequate delivery of oxygen and nutrients that
are necessary for cellular function. Hypovolemic shock, the most
common type, results from a loss of circulating blood volume from
clinical etiologies, such as penetrating and blunt trauma,
gastrointestinal bleeding, and obstetrical bleeding.
[0114] Hypoxaemia: an abnormal deficiency in the concentration of
oxygen in arterial blood.
[0115] Hypoxia: a pathological condition in which the body as a
whole (generalized hypoxia) or region of the body (tissue hypoxia)
is deprived of adequate oxygen supply.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] Microcytosis: a blood disorder characterized by the presence
of microcytes (abnormally small red blood cells) in the blood.
[0121] 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.
[0122] Nitrosation: a process of converting organic compounds into
nitroso compounds.
[0123] 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.
[0124] Pharmaceutically acceptable carrier: The pharmaceutically
acceptable carriers useful in this disclosure are conventional.
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. 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.
[0125] 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.
[0126] Rhabdomyolysis: The rapid breakdown of skeletal muscle
tissue due to traumatic injury, including mechanical, physical or
chemical. The principal result is a large release of the creatine
phosphokinase enzymes and other cell byproducts into the blood
system and acute renal failure due to accumulation of muscle
breakdown products, several of which are injurious to the
kidney.
[0127] Sickle cell anemia: A group of genetic disorders caused by
sickle hemoglobin. In many forms of the disease, the red blood
cells change shape upon deoxygenation because of polymerization of
the abnormal sickle hemoglobin. This process damages the red blood
cell membrane, and can cause the cells to become stuck in blood
vessels. This deprives the downstream tissues of oxygen and causes
ischemia and infarction, which may cause organ damage, such as
stroke.
[0128] Spherocytosis: An auto-hemolytic anemia characterized by the
production of red blood cells (or erythrocytes) that are
sphere-shaped, rather than donut-shaped.
[0129] Subject: Living multi-cellular organisms, including
vertebrate organisms, a category that includes both human and
non-human mammals.
[0130] Thalassemia: An inherited autosomal recessive blood disease.
In thalassemia, the genetic defect results in reduced rate of
synthesis of one of the globin chains that make up hemoglobin.
Reduced synthesis of one of the globin chains causes the formation
of abnormal hemoglobin molecules, and this in turn causes the
anemia which is the characteristic presenting symptom of the
thalassemias.
[0131] Therapeutically effective amount: A quantity of compound or
composition, for instance, cell-free hemoglobin based blood
substitute detoxified by treatment with nitrite, sufficient to
achieve a desired effect in a subject being treated. For instance,
this can be the amount necessary to inhibit or to measurably reduce
anemia or other symptom associated with a blood disorder. It can
also be the amount necessary to restore normal vascular tone and
oxygenation to a subject suffering from hemorrhage.
[0132] Ulcer: An open sore of the skin, eyes or mucous membrane,
often caused, but not exclusively, by an initial abrasion and
generally maintained by an inflammation, an infection, and/or
medical conditions which impede healing.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] Yellow fever: An acute viral disease that is a cause of
hemorrhagic illness, particularly in many African and South
American countries.
[0137] 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
[0138] Provided herein in one embodiment is a pharmaceutical
composition, comprising a ferric (Fe.sup.III) heme-containing
molecule and nitrite. In examples of this composition, the molar
ratio of nitrite to the heme-containing molecule is defined, for
instance the molar ratio is less than 1:1, more than 1:1, or about
1.1. Example heme-containing molecules include (but are not limited
to) hemoglobin, methemoglobin, cross-linked hemoglobin,
cross-linked methemoglobin, a protein (or protein fragment, such as
a protein domain) that binds oxygen, or a combination or mixture of
two or more thereof.
[0139] Other example compositions further comprise a
pharmaceutically acceptable carrier, an adjuvant, or two or a
combination of two or more thereof.
[0140] Also provided is a method of producing a cell-free blood
substitute, which method comprises contacting a heme-containing
molecule with nitrite, wherein the molar ratio of heme-containing
molecule and nitrite in the composition is between about 1:2 and
about 2:1, is above 1:1, is below 1:1, or is about 1:1; and forming
nitrite-bound heme-containing molecules, wherein the
nitrite-containing molecules produce dinitrogen trioxide.
[0141] Other embodiments provide methods of treating a subject
having or predisposed to hypoxia, hypoxaemia, ischemia or anoxia,
which methods comprise administering to the subject a
therapeutically effective amount of a ferric (Fe.sup.III)
heme-containing molecule and nitrite composition as described
herein, thereby treating the subject.
[0142] Yet other embodiments are methods of replacing blood in a
subject, which methods comprise administering to the subject a
therapeutically effective amount of a ferric (Fe.sup.III)
heme-containing molecule and nitrite composition as described
herein, thereby replacing blood in the subject.
[0143] By way of example, the subject to which a composition or
preparation described herein is to be administered includes a
subject that has or is predisposed to anemia, bleeding disorder,
trauma, injury, burn, coagulopathy, ectopic pregnancy, favism,
gastrointestinal bleeding, hemolytic uremic syndrome, hemophilia,
microcytosis, ulcer, bleeding in surgery, bleeding in pregnancy,
hemorrhage, rhabdomyolysis, hemorrhagic shock, sickle cell anemia,
hemoglobinopathy spherocytosis, thalassemia, and/or yellow fever.
Alternatively, the subject has lost blood during a surgical
procedure. In some examples, the subject is a human; in others, the
subject is a non-human animal.
[0144] Yet additional embodiments and examples are provided
herein.
IV. Detailed Description of Particular Embodiments
[0145] A. Production of Molecular Dinitrogen Trioxide
(N.sub.2O.sub.3) Via Reaction of NO and a Nitrite-Bound
Methemoglobin Intermediate Recent studies reveal that the
ubiquitous circulating anion nitrite (NO.sub.2) is a vasodilator
and intrinsic signaling molecule (Gladwin et al., Proc. Natl. Acad.
Sci. USA 97:11482-11487, 2000; Cosby et al., Nat. Med. 9:1498-1505,
2003; Gladwin et al., Nature Chemical Biology 1:308-314, 2005;
Bryan et al., Nature Chemical Biology 1:290-297, 2005; Modin et
al., Acta Physiologica Scandinavica 171:9-16, 2001). The
vasodilator activity of nitrite is associated with an
allosterically controlled heme-based reduction of nitrite to nitric
oxide (NO) by deoxygenated hemoglobin (deoxyHb) (Huang et al., J.
Biol. Chem. 280:31126-31131, 2005; Huang et al., J. Clin. Invest.
115:2099-2107, 2005). Nitrite infusions into the human circulation
increase blood flow at near physiological concentrations. (Cosby et
al., Nat. Med. 9:1498-14505, 2003). This vasodilation is temporally
associated with increases in red cell heme iron-nitrosylated
hemoglobin (HbFe.sup.II--NO designated as {FeNO}.sup.7 using the
Enemark-Feltham notation; Enemark & Feltham (1974) Coordination
Chemistry Reviews 13:339-406) and to a lesser extent S-nitrosated
hemoglobin (SNO-Hb, hemoglobin nitrosated at the .beta.-93
cysteine; Cosby et al., Nat. Med. 9:1498-1505, 2003).
[0146] While the in vitro incubation of nitrite with deoxygenated
red cells and hemoglobin solutions produces vasodilation, tissue
NO-dependent cGMP accumulation, gas phase NO generation, and
NO-dependent inhibition of mitochondrial oxygen consumption, the
mechanism of NO escape from the red cell following nitrite
reduction by hemoglobin remains elusive (Cosby et al., Nat. Med.
9:1498-1505, 2003; Crawford et al., Blood 107:566-574, 2006; Hunter
et al., Nat. Med. 10:1122-1127, 2004). Indeed, a major challenge to
the nitrite reductase hypothesis and other erythrocyte-NO export
theories is explaining how the NO can escape heme autocapture
(Gladwin et al., Proc. Natl. Acad. Sci. USA 97:11482-11487, 2000).
Nitric oxide reacts with both deoxy- and oxyhemoglobin extremely
rapidly with bimolecular rate constants between 10.sup.7-10.sup.8
M.sup.-1s.sup.-1 (Gladwin et al., Proc. Natl. Acad. Sci. USA
97:11482-11487, 2000; Doyle & Hoekstra, J. Inorg. Biochem.
14:351-358, 1981; Eich et al., Biochemistry-US 35:6976-6983, 1996;
Herold et al., Biochemistry-US 40:3385-3395, 2001; Cassoly &
Gibson, J. Mol. Biol. 91:301-313, 1975; Morris & Gibson, J.
Biol. Chem. 255:8050-8053, 1980; Huang et al., Biophys. J.
85:2374-2383, 2003; Kim-Shapiro, Free Radic. Biol. Med. 36:402-412,
2004). Modeling calculations have shown that only 0.1 pM NO would
be produced outside a red blood cell at steady state, even at
supra-physiological nitrite levels, unless additional mechanisms
exist to limit the scavenging reactions of NO with hemoglobin
(Jeffers et al., Comp. Biochem. Physiol. A-Mol. Integr. Physiol.
142:130-135, 2005). A similar paradox could be seen in the
cardiomyocyte where nitrite inhibits cellular respiration during
hypoxia via a nitrite reductase activity of deoxymyoglobin, despite
the fact that the high concentrations of myoglobin in the
cardiomyocyte could be expected to inhibit NO-dependent signaling
(Shiva et al., Circ. Res. 100:654-661, 2007). The possibility was
therefore considered that the nitrite-hemoglobin reactions are
either compartmentalized at the red cell membrane to limit
cytoplasmic scavenging or generate gaseous NO.sub.x species such as
N.sub.2O.sub.3 which can concentrate in the membrane or in
hydrophobic membrane channels, and thereby diffuse out of the red
blood cell (Huang et al., J. Clin. Invest. 115:2099-2107, 2005;
Robinson & Lancaster, Am. J. Respir. Cell Mol. Biol.
32:257-261, 2005). N.sub.2O.sub.3 is a logical candidate for such
an intermediate as 1) it is a primary nitrosating species capable
of generating red cell S-nitrosothiols (Robinson & Lancaster,
Am. J. Respir. Cell Mol. Biol. 32:257-261, 2005; Williams
(Elsevier, Amersterdam, 2004) Nitrosation Reactions and the
Chemistry of Nitric Oxide; Wink et al., Chem. Res. Toxicol.
6:23-27, 1993; Dabora et al., Iarc Scientific Publications 311-316,
1984), which clearly form as a side product of the
nitrite-hemoglobin reaction (Cosby et al., Nat. Med. 9:1498-1505,
2003), 2) it is small and uncharged, facilitating diffusion through
the red cell membrane, and 3) it can homolyze to NO and NO.sub.2.,
allowing for export of NO (Jeffers et al., Comp. Biochem. Physiol.
A-Mol. Integr. Physiol. 142:130-135, 2005; Robinson &
Lancaster, Am. J. Respir. Cell Mol. Biol. 32:257-261, 2005).
[0147] If N.sub.2O.sub.3 is formed in the reaction of nitrite and
deoxyhemoglobin, then nitrosated products should be detectable.
During nitrite infusions in humans, in addition to the expected
products of methemoglobin (MetHb) and HbFe.sup.II--NO, SNO-Hb is
also formed (Cosby et al., Nat. Med. 9:1498-1505, 2003). The
reaction of deoxyHb and nitrite also forms S-nitrosothiols,
including SNO-Hb (Stepuro et al., Polish Journal of Pharmacology
46:601-607, 1994; Stepuro et al., Biochem.-Moscow 62:960-966, 1997;
Luchsinger et al., Proc. Natl. Acad. Sci. USA 100:461-466, 2003;
Fernandez & Ford, J Am Chem Soc 125:10510-10511, 2003; Nagababu
et al., Nitric Oxide 15:20-29, 2006). It has also been suggested
that the formation of SNO-Hb from nitrite involves the formation of
ferrous-iron-nitrosyl-hemoglobin (HbFe.sup.II--NO) followed by the
transfer of the NO group to cysteine 93 during the R-to-T
allosteric transition or via the formation of a stable
ferric-iron-nitrosyl hemoglobin (MetHb-NO or
HbFe.sup.II--NO/HbFe.sup.II--NO.sup.+, designated as {FeNO}.sup.6
using the Enemark-Feltham notation; Enemark & Feltham,
Coordination Chemistry Reviews 13:339-406, 1974). (Nagababu et al.,
Nitric Oxide 15:20-29, 2006; Angelo et al., Proc. Natl. Acad. Sci.
USA 103:8366-8371, 2006). When written as the HbFe.sup.II--NO.sup.+
resonance form, the nitrosonium is emphasized, highlighting
potential subsequent transfer to nitrosate cysteine .beta.-93.
[0148] In the current study, S-nitrosation was observed, but the
putative HbFe.sup.III--NO intermediate was undetectable. The
nitrosation that was observed cannot be explained by allosteric
intra-molecular transfer of heme bound NO to the cysteine. In
contrast, the nitrite anion was found to directly bind to MetHb,
and surprisingly, this results in an electronic configuration that
is silent when observed by electron paramagnetic resonance
spectroscopy. Based on density functional theory (DFT)
calculations, it is proposed that the nitrite bound MetHb exhibits
ferrous heme nitrogen dioxide (Fe.sup.II--NO.sub.2.) character that
reacts rapidly with NO to form N.sub.2O.sub.3. In this reaction,
deoxyhemoglobin redox cycles and catalyzes the conversion of two
nitrite ions into N.sub.2O.sub.3. These results could solve the
mystery of NO escape from the erythrocyte and explain many unusual
phenomena observed in the NO-hemoglobin field, such as the
nitrosating effects of low NO concentrations (Gow & Stamler,
Nature 391:169-173, 1998), the link between heme oxidation and
SNO-Hb formation (Gow & Stamler, Nature 391:169-173, 1998), the
identity of a mysterious EPR silent intermediate that can be
measured by gas-phase reductive chemiluminescence (Nagababu et al.,
J. Biol. Chem. 278:46349-46356, 2003), and the mechanism of
nitrite-dependent nitrosation (Nagababu et al., Nitric Oxide
15:20-29, 2006; Angelo et al., Proc. Natl. Acad. Sci. USA
103:8366-8371, 2006). These experiments therefore reveal a
fundamental novel metal and nitrite catalyzed chemical reaction
pathway to N.sub.2O.sub.3 and S-nitrosothiol, which could
constitute the basis of in vivo nitrite-dependent nitrosylation. It
should be noted that this represents a novel mechanism for both
anaerobic and metal catalyzed N.sub.2O.sub.3 formation and
S-nitrosation, and likely represents a major pathway for
NO-dependent signaling in a heme rich environment.
[0149] The details of this pathway are becoming clear. Nitrite
reacts with deoxyhemoglobin to generate MetHb and NO (Equation
1).
Nitrite(NO.sub.2.sup.-)+deoxyhemoglobin(Fe.sup.II)+H.sup.+.fwdarw.NO+met-
hemoglobin(Fe.sup.III)+OH.sup.- (1)
Nitrite also binds to MetHb to form MetHb-NO.sub.2.sup.- with a
dissociation constant of about 1 mM at neutral pH (Rodkey, Clin.
Chem. 22:1986-1990, 1976), which in fact was found in this work to
be much lower at limiting nitrite concentrations and/or lower pH
(Kd=7 .mu.M at pH 6.5).
NO.sub.2.sup.-+methemoglobin(HbFe.sup.III).fwdarw.HbFe.sup.III--NO.sub.2-
.sup.- (7)
It has been previously shown that MetHb-NO.sub.2.sup.- is formed in
the nitrite-deoxyhemoglobin reaction and herein it is shown that
this species is the major intermediate in this reaction. (Huang et
al., J. Biol. Chem. 280:31126-31131, 2005). Experimental precedence
(Nasri et al., Inorg Chem 43:2932-2942, 2004; Lim et al., J Am Chem
Soc 124:9737-9743, 2002; Copeland et al., J Inorg Biochem
100:1413-1425, 2006) as well as the DFT calculations indicate that
the HbFe.sup.III--NO.sub.2.sup.- may have either an N-bound or
O-bound nitrite. Both forms have high electron affinities and
should avidly react with NO via radical-radical like pathway that
generates N.sub.2O.sub.3 and is potentially kinetically competitive
with NO reactions with deoxyhemoglobin or oxyhemoglobin.
HbFe.sup.III--NO.sub.2.sup.-HbFe.sup.II--NO.sub.2.+NO.fwdarw.HbFe.sup.II-
+N.sub.2O.sub.3 (8)
Note that, according to the final stoichiometry of this reaction
pathway (Equation 9), hemoglobin is catalytic, functioning as an
allosterically regulated enzyme that converts two nitrite ions into
a molecule of N.sub.2O.sub.3:
2NO.sub.2.sup.-HbFe.sup.II+H.sup.+.fwdarw.HbFe.sup.II+N.sub.2O.sub.3+OH.-
sup.- (9)
[0150] Although the disappearance of the g=6 paramagnetism is
consistent with an altered electronic configuration, the extent of
the N-bound nitrite vs. O-bound nitrite and the radical character
of HbFe.sup.III--NO.sub.2.sup.- remains to be completely
determined. It is known that MetHb itself is characterized by minor
spectral changes as a function of pH, which has been attributed to
protein ionizations, likely at the imino N of the proximal
histidine (George & Hanania, Biochem. J. 52:517-523, 1952;
George & Hanania, Biochem. J. 55:236-243, 1953). A conjugate
acid (+HN) of this imino N is purported to have a pK of 5.1 so that
1 in 25 would be protonated at pH 6.5 and 1 in 200 at pH 7.4. A
protonated histidine would pull the electron towards the heme, thus
enhancing the HbFe.sup.II--NO.sub.2. character of the O-bound form.
The presence of anions has been suggested to reduce the effects of
the presence of the +HN (George & Hanania, Biochem. J.
52:517-523, 1952; George & Hanania, Biochem. J. 55:236-243,
1953), which might explain why the dissociation constant of
nitrite-MetHb increased with increasing nitrite concentrations.
Further work is required to firmly establish the mechanism of the
nitrite concentration-dependent dissociation constant. Even with
this +HN configuration, the electron may be mostly on the nitrite.
However, the degree to which HbFe.sup.III--NO.sub.2.sup.- undergoes
O-bonding and exhibits HbFe.sup.II--NO.sub.2. character is likely
responsible for the observed rapid reactions with NO. Molecular
orbital calculations support the idea that there is more than one
form of HbFe.sup.III--NO.sub.2.sup.-, and it is likely that these
will react with NO at different rates.
[0151] Nitrite mediated SNO formation has been observed in vivo
(Cosby et al., Nat. Med. 9:1498-1505, 2003; Gladwin et al., Nature
Chemical Biology 1:308-314, 2005; Bryan et al., Nature Chemical
Biology 1:290-297, 2005). Here (FIGS. 11 and 5F), it is shown that
substantial SNO-Hb is formed even when heme is excess to nitrite.
One could consider four pathways to SNO formation in the red blood
cell with associated export of vasodilatory activity: (1) The
nitrite-MetHb+NO mechanism forming N.sub.2O.sub.3 discussed here
(Equations 3 and 8), (2) The nitrite+NO-MetHb mechanism forming
N.sub.2O.sub.3 (Equation 4), (3) the nitrosonium transfer mechanism
(Equation 5), and (4) the oxygen transfer mechanism (Equation 6).
Data examining oxygen transfer suggest that Nitrite-MetHb does not
efficiently transfer oxygen so that this mechanism is unlikely to
play a role. Generally, the nitrosonium transfer mechanism is
unappealing on theoretical grounds as it seems unlikely that
NO.sup.+ would nitrosate a specific protein thiol in the presence
of 55 M water, and a directed transfer of charged NO.sup.+ through
water-free protein channel needs to be postulated. FIGS. 1 and 8
show that there is no stable HbFe.sup.III--NO intermediate, thus
limiting the potential for the nitrite+NO-MetHb and nitrosonium
transfer mechanisms. However, it is likely that HbFe.sup.III--NO is
formed transiently (lasting only a few seconds) when nitrite reacts
with deoxyhemoglobin to form MetHb and NO, as is seen in the case
of bacterial nitrite reductases (Gladwin et al., Nature Chemical
Biology 1:308-314, 2005; Averill & Tiedje, FEBS Lett. 138:8-12,
1982; Marti et al., J Phys Chem B 108:18073-18080, 2004). One might
propose that N.sub.2O.sub.3 and the subsequent nitrosation is
actually due to the reaction of this transient HbFe.sup.III--NO
with another nitrite anion that may also be present in the heme
pocket. However, data showing reduced nitrosation in the presence
of KCN (FIG. 7A) argues against this alternative mechanism as the
KCN is not likely to have a large effect on the transiently formed
HbFe.sup.III--NO. In addition, data on the rate of reductive
nitrosylation (FIG. 4) supports the importance of a
HbFe.sup.III--NO.sub.2.sup.- species. The biggest challenge to the
favored Nitrite-MetHb+NO mechanism is that this reaction must
compete with the reaction of NO and ferrous hemes in the red blood
cell. Molecular orbital calculations suggest that it may only be
one electronic configuration such as the O-bound nitrite-MetHb that
has radical character that can react quickly enough to be a viable
mechanism of N.sub.2O.sub.3 formation in a red blood cell. Although
the data strongly support the Nitrite-MetHb+NO mechanism, they do
not completely rule out the possibility that, under physiological
conditions in a red blood cell, other mechanisms (particularly that
of involving a transient NO-MetHb reacting with nitrite) may be
involved.
B. Molecular Dinitrogen Trioxide (N.sub.2O.sub.3) and Nitric Oxide
(NO) Bioactivity
[0152] The production of N.sub.2O.sub.3 by nitrite-heme reactions
facilitates export of NO bioactivity from the erythrocyte via
multiple pathways. First, N.sub.2O.sub.3 is the primary nitrosating
species capable of forming red cell S-nitrosothiols, which form as
a side product of the nitrite-hemoglobin reaction and may be
exportable. It is a small and uncharged molecule, which promotes
its concentration and diffusion through the red cell membrane.
Finally N.sub.2O.sub.3 can homolyze to NO and NO.sub.2., allowing
for NO export (Jeffers et al., Comp. Biochem. Physiol. A-Mol.
Integr. Physiol. 142:130-135, 2005).
[0153] These concerted pathways leading from nitrite-heme chemistry
to export of NO or other related, potentially vasodilatory species
out of the red cell are illustrated in FIG. 10. The lifetime of
N.sub.2O.sub.3 (1 millisecond) and its diffusion coefficient, D
(1000 .mu.m.sup.2/s), leads to the conclusion that the distance it
can diffuse (= Dt) is about 1 .mu.m. For a RBC that is 2 .mu.m
high, that means some will get out. In addition, it is quite
possible that different isomers of N.sub.2O.sub.3 have different
and perhaps longer lifetimes further facilitating potential export
(Espey et al., J. Biol. Chem. 276:30085-30091, 2001; Challis &
Kyrtopoulos, Journal of the Chemical Society-Perkin Transactions 2
1296-1302, 1978). As hemoglobin deoxygenates from artery to vein
the rate of nitrite reduction by deoxygenating hemoglobin
increases, producing more NO, which can then react rapidly with
MetHb-bound nitrite to form N.sub.2O.sub.3. The primary nitrite
reductase reaction that generates NO is allosterically regulated
and the rate is maximal as hemoglobin desaturates to 50% (at the
hemoglobin P.sub.50), resulting in maximal rates of nitrite
reduction to NO as oxygen and pH decrease (Huang et al. J. Clin.
Invest. 115:2099-2107, 2005). Remarkably, the formation of
N.sub.2O.sub.3 is also promoted as pH decreases. This may be due to
the fact that as proton concentration increases, the affinity of
nitrite binding to MetHb is enhanced, the stability of generated
N.sub.2O.sub.3 increases, and/or the initial reaction of deoxyHb is
accelerated due to its requirement of a proton. Once N.sub.2O.sub.3
has formed, it may be exported from the red blood cell and homolyze
to form NO, thus explaining nitrite mediated vasodilation (Cosby et
al., Nat. Med. 9:1498-1505, 2003; Crawford et al., Blood
107:566-574, 2006).
[0154] The efficiency of nitrite reduction and NO release would
also be significantly increased if the effective concentrations of
the reactants, i.e. nitrite and hemoglobin, are increased at the
erythrocyte sub-membrane. A putative nitrite reductase metabolon
located within the red cell lipid raft composed of deoxy- and
MetHb, an anion exchange protein (for nitrite import into the
cell), carbonic anhydrase, aquaporin, and Rh channels (Gladwin et
al., Free Radic Biol Med 36:707-717, 2004) would effectively
concentrate the NO-generating deoxyhemoglobin-nitrite reaction,
MetHb bound to band 3, and the necessary reactants (nitrite,
protons) near highly hydrophobic channels at the membrane. Because
NO and N.sub.2O.sub.3 are both lipophilic they could rapidly
diffuse out of the cell and thus limit further autocapture. FIG. 10
illustrates the enhancement of this chemistry and concentration of
N.sub.2O.sub.3 at the red cell membrane.
[0155] This mechanism helps explain and unify many of the
paradoxical observations in the NO hemoglobin field such as (1) the
vasodilatory activity of deoxygenating red cells and hemoglobin
solutions in the presence of nitrite (Cosby et al., Nat. Med.
9:1498-1505, 2003; Crawford et al., Blood 107:566-574, 2006), (2)
the nitrosating effects of low concentrations of NO (Gow &
Stamler, Nature 391:169-173, 1998; Gow et al., Proc. Natl. Acad.
Sci. USA 96:9027-9032, 1999; Herold & Rock, J. Biol. Chem.
278:6623-6634, 2003), (3) the link between heme oxidation and
SNO-Hb formation (i.e. effect of ferricyanide on increasing SNO-Hb
formation in the presence of nitrite) (Luchsinger et al., Proc.
Natl. Acad. Sci. USA 100:461-466, 2003; Bryan et al., Nitric
Oxide-Biol Ch 10:221-228, 2004; Gladwin et al., J. Biol. Chem.
277:27818-27828, 2002), (4) the identity of a mysterious EPR silent
intermediate that is measurable by gas-phase reductive
chemiluminescence (Nagababu et al., J. Biol. Chem. 278:46349-46356,
2003), (5) the mechanism of nitrite-dependent nitrosation (Nagababu
et al., Nitric Oxide 15:20-29, 2006; Nagababu et al., J. Biol.
Chem. 278:46349-46356, 2003), and (6) the faster than predicted
rate of reductive nitrosylation observed by many investigators
after addition of NO solutions to hemoglobin (Han et al., Proc.
Natl. Acad. Sci. USA 99:7763-7768, 2002). This latter effect is
related to the suggestion that enhanced formation of
HbFe.sup.II--NO compared to MetHb upon adding NO as a bolus to
mixtures of oxygenated and deoxygenated Hb is due to secondary
reductive nitrosylation of MetHb formed from NO and oxyhemoglobin
(Han et al., Proc. Natl. Acad. Sci. USA 99:7763-7768, 2002). As
nitrite is commonly present in NO solutions, the reaction of NO
with MetHb-NO.sub.2.sup.- would convert MetHb to
HbFe.sup.II--NO.
[0156] This nitrite-hemoglobin reaction also provides a novel and
kinetically appealing mechanism for S-nitrosothiol formation. If
N.sub.2O.sub.3 is formed in the nitrite hemoglobin reaction, then
in addition to NO and iron-nitrosyl-hemoglobin (HbFe.sup.II--NO),
S-nitrosated products should be detected. During nitrite infusions
in humans, both iron-nitrosyl hemoglobin (HbFe.sup.II--NO) and
SNO-Hb form in blood (Cosby et al., Nat. Med. 9:1498-1505, 2003).
It has also been shown that the reaction of deoxyHb and nitrite
forms S-nitrosothiols, including SNO-Hb (Stepuro et al., Polish
Journal of Pharmacology 46:601-607, 1994; Stepuro et al.,
Biochem.-Moscow 62:960-966, 1997; Luchsinger et al., Proc. Natl.
Acad. Sci. USA 100:461-466, 2003; Fernandez & Ford, J Am Chem
Soc 125:10510-10511, 2003; Nagababu et al., Nitric Oxide 15:20-29,
2006). The proposed mechanism for SNO-Hb formation is similar to
nitrite-catalyzed reductive nitrosylation, in which nitrite reacts
with HbFe.sup.III--NO to form N.sub.2O.sub.3 (Fernandez & Ford,
J Am Chem Soc 125:10510-10511, 2003). However, rather than
requiring HbFe.sup.III--NO to be present at steady state, the
reaction depends on HbFe.sup.III--NO.sub.2.sup.-. The existence of
this intermediate was obscured in prior studies due to the high NO
concentrations used in those studies, which effectively competed
with nitrite for binding to MetHb and thus precluded the more rapid
nitritecatalyzed reductive nitrosylation (Fernandez & Ford, J
Am Chem Soc 125:10510-15011, 2003).
[0157] These reactions form a general route for NO and
S-nitrosative signaling under physiological hypoxia, with a number
of heme-globins subserving this function at different oxygen
tensions. Hemoglobin would function as a nitrite reductase at
oxygen tensions of 60-20 mm Hg, near the hemoglobin P.sub.50, while
myoglobin, neuroglobin and cytoglobin would deoxygenate at oxygen
partial pressures below 5-10 mm Hg (Huang et al., J. Clin. Invest.
115:2099-2107, 2005). The formation of N.sub.2O.sub.3 is
kinetically appealing as it may compete with NO-heme reactions that
otherwise limit nitrosation chemistry. According to this paradigm,
nitrite is the major stable NO reservoir in blood and tissues and
forms from NO synthase during normoxia (Lauer et al., Proc. Natl.
Acad. Sci. USA 98:12814-12819, 2001; Shiva et al., Nature Chemical
Biology 2:486-493, 2006). Nitrite can then be reduced to NO and
N.sub.2O.sub.3 along the physiological oxygen and pH gradient by
the heme globins. In this context, the heme globins are
allosterically regulated enzymes, which are responsive to tissue
metabolism (oxygen and proton levels), and which catalyze the
conversion of two nitrite anions into a molecule of
N.sub.2O.sub.3.
[0158] These experiments therefore reveal fundamental novel metal
and nitrite catalyzed chemical reaction pathways that generate free
NO, N.sub.2O.sub.3 and nitrosothiol. These reactions constitute the
basis of in vivo nitrite-dependent hypoxic signal transduction and
more globally, a mechanism for NO signaling in a heme-rich
environment.
[0159] By way of example, in one embodiment two containers of cell
free hemoglobin would be prepared: one container (for instance, an
I.V. bag) would contain nitrite and ferric methemoglobin
(Fe.sup.III) (at a ratio of less than 1:1); a second container
(e.g., I.V. bag) would contain oxyhemoglobin (Fe.sup.II--O.sub.2).
The two solutions would be coinfused into a subject at ratios less
than 1 part methemoglobin-nitrite to 1 part oxyhemoglobin. After
and during the infusion, the oxyhemoglobin would deliver oxygen to
the tissue as the oxygen delivery vehicle to form deoxyhemoglobin
(Fe.sup.II). Some of this would react with excess nitrite from the
first container to form NO. The methemoglobin-nitrite from that
same container would form an intermediate (Fe.sup.II--NO.sub.2
radical); this would react with NO to form N.sub.2O.sub.3 and
Fe.sup.II (deoxyhemoglobin). The N.sub.2O.sub.3 would vasodilate
and restore NO homeostasis, and the deoxyhemoglobin would now be
able to bind oxygen again in the lung. This system thus delivers
oxygen, generates N.sub.2O.sub.3 and NO, and redox cycles to rebind
oxygen in the lung.
[0160] The composition comprising nitrite and ferric methemoglobin
(or other ferric heme protein) would be prepared by addition of
sodium nitrite to oxidized heme protein. The oxidation could occur,
for instance, by simple autooxidation or by reaction with an
oxidant like excess nitrite or ferricyanide. The ratios of nitrite
to ferric methemoglobin (or other hemoprotein) would be balanced to
form the most nitrite-methemoglobin complex. As this complex is
stabilized at decreasing pH, the pH value may be adjusted to a less
than physiological level (<pH 7.4) to maximize the formation of
intermediate. The two bags would be prepared separately and are
expected to be stable as frozen or refrigerated solutions.
[0161] The final nitrite-methemoglobin solution is infused at the
same time an oxygenated ferrous hemoglobin based blood substitute
is infused into the subject. The amount of nitrite-methemoglobin
would be delivered to reach a blood concentration of at least 5-10
.mu.M but may be as high as 2 mM. The stroma free hemoglobin based
blood substitute would be infused to reach a concentration 1-2 mM
(thus the ratio of nitrite-methemoglobin to ferrous oxyhemoglobin
would be 1:1 or less).
C. Nitrite Reductase Activity of Hemoglobin as a Systemic Nitric
Oxide Generator Mechanism to Detoxify Plasma Hemoglobin Produced
During Hemolysis
[0162] Under physiologic conditions, the experiments described in
the Examples below demonstrate that low dose sodium nitrite is a
potent arterial vasodilator that increases cardiac performance by
direct afterload reduction with mild chronotropic effects. During
hemolysis, a consistent U-shaped relationship between the effects
of nitrite and cell-free plasma hemoglobin levels was detected
across three experimental settings, suggesting an interaction
between nitrite and the level of intravascular hemoglobin.
[0163] Nitrite reacts with oxy- and deoxy-hemoglobin to form
methemoglobin and methemoglobin+nitric oxide respectively (Brooks,
Proc R Soc Med 123:368-382, 1937; Crawford et al., Blood
107:566-574, 2006; Doyle & Hoekstra, J Inorg Biochem
14:351-358, 1981; Huang et al., J Biol Chem 280:31126-31131, 2005;
Huang et al., J Clin Invest 115:2099-2107, 2005). During low level
hemolysis, these reactions will minimize the amount of
oxyhemoglobin available in the plasma that can consume NO (via the
dioxygenation reaction) and generate NO by the reaction of nitrite
with deoxyhemoglobin. The net result is accentuated vasodilation
compared to no hemolysis. At higher levels of intravascular
hemolysis, the large amounts of cell-free plasma hemoglobin
overwhelms the nitrite reductase reaction of hemoglobin and
consumes both the NO formed by nitrite reduction with hemoglobin
and endothelial derived NO. The net result is vasoconstriction
compared to low level hemolysis and no hemolysis. During a sodium
nitroprusside infusion with low level hemolysis and nitrite, the
nitrite-oxyhemoglobin reaction minimizes oxyhemoglobin
concentration and allows the donated NO from nitroprusside to cause
vasodilation. This vasodilation is further accentuated by the
production of additional NO from reactions of nitrite with
deoxyhemoglobin. At higher level hemolysis, the vasodilatory
effects of sodium nitroprusside are attenuated by the high levels
of oxyhemoglobin which consume both the NO donated from
nitroprusside and the NO generated from the reaction of nitrite
with deoxyhemoglobin. During mitochondrial experiments, maximal NO
production and accumulation occurred with nitrite and low levels of
hemoglobin because the excess heme-groups at higher levels of
hemoglobin consumed the NO generated by the nitrite-deoxyhemoglobin
reaction leading to decreased NO accumulation.
[0164] Traditional NO donors, such as sodium nitroprusside, produce
dose-dependent vasodilation that is inhibited by cell-free plasma
hemoglobin (Minneci et al., J Clin Invest 115:3409-3417, 2005). In
contrast, nitrite led to accentuated vasodilation during low level
hemolysis despite the presence of oxyhemoglobin levels sufficient
to scavenge any NO that might be formed if nitrite acted as pure NO
donor. The vasodilatory effect of nitrite clearly differs from
traditional NO donors in the presence of hemoglobin and can in part
be explained by the nitrite reductase activity of hemoglobin
(Crawford et al., Blood 107:566-574, 2006; Huang et al., J Biol
Chem 280:31126-31131, 2005; Huang et al., J Clin Invest
115:2099-2107, 2005). Generation of NO from nitrite and hemoglobin
requires both hypoxia and an acidic environment which are present
in hypoxic tissues. This allows for maximal NO generation by the
deoxyheme-nitrite allosteric reaction as hemoglobin deoxygenates
within the circulation.
[0165] The studies disclosed herein provide in vivo evidence that
hemoglobin possesses a functional nitrite reductase activity. The
notable interaction between nitrite and hemoglobin in these studies
was markedly different from the behavior of a traditional NO donor,
sodium nitroprusside. While the latter was inhibited in a dose
dependent manner, the former was potentiated by hemoglobin at
concentrations that produced NO in the in vitro mitochondrial NO
sensor experiments (see Example 4). Prior studies have examined how
NO could be generated from the nitrite reductase activity of
hemoglobin and then be able to escape heme autocapture (via
equation 1 or an analogously fast reaction with deoxyhemoglobin)
(Basu et al., Nat Chem Biol 3:785-794, 2007). It was found that
nitrite can also bind to methemoglobin to form a
nitrite-methemoglobin intermediate that possesses
nitrogendioxide-ferrous hemoglobin character. NO that forms from
nitrite reduction can react rapidly in a radical-radical reaction
with the nitrogen dioxide to form N.sub.2O.sub.3 (Basu et al., Nat
Chem Biol 3:785-794, 2007). N.sub.2O.sub.3 is an uncharged, highly
lipophilic and diffusible molecule that is more stable than
authentic NO. N.sub.2O.sub.3 can nitrosate thiols to form
vasodilatory S-nitrosothiols, can homolyze back into NO, or can
regenerate nitrite. The apparent inhibition of the nitrite effect
at higher hemoglobin concentrations in these studies indicate that
at least part of the mechanism must involve the regeneration of NO,
which can be in part scavenged by excess hemoglobin.
[0166] The levels of cell-free plasma hemoglobin in the low
hemolysis group of the studies described herein are consistent with
the levels observed during sickle cell vaso-occlusive crisis (Kaul
& Hebbel, J Clin Invest 106:411-420, 2000; Naumann et al., Am J
Clin Pathol 56:137-147, 1971; Reiter et al., Nat Med 8:1383-1389,
2002) and during other clinically relevant human hemolytic
conditions such as cardiopulmonary bypass, malarial infection,
HUS/TTP, paroxysmal nocturnal hemoglobinuria, allo-immune hemolytic
anemia and rhabdomyolysis (myoglobin) (Davis et al., J Am Soc
Nephrol 10:2396-2402, 1999; Kaul & Hebbel, J Clin Invest
106:411-420, 2000; Murakami et al., Artif Organs 21:803-807, 1997;
Naumann et al., Am J Clin Pathol 56:137-147, 1971; Pepper et al.,
Free Radic Res 21:53-58, 1994; Reiter et al., Nat Med 8:1383-1389,
2002; Shimono et al., Asaio J 43:M735-739, 1997). All of these
conditions have now been associated with progressive vasculopathy
and pulmonary hypertension and are associated with systemic NO
scavenging by plasma hemoglobin (Gladwin et al., N Engl J Med
350:886-895, 2004; Minneci et al., J Clin Invest 115:3409-3417,
2005; Rother et al., JAMA 293:1653-1662, 2005). Furthermore,
hemolysis is associated with platelet activation and inhibition of
NO-cGMP signaling in platelets (Villagra et al., Blood
110:2166-2172, 2007).
[0167] These represent processes in which the allosteric nitrite
reductase activity of hemoglobin may make nitrite an ideal
therapeutic agent to attenuate the effects of accelerated NO
scavenging by cell-free hemoglobin released during intravascular
hemolysis (Aessopos et al., Chest 107:50-53, 1995; Du et al., Am
Heart J 134:532-537, 1997; Eberhardt et al., Am J Hematol
74:104-111, 2003; Kaul et al., J Clin Invest 114:1136-1145, 2004;
Minneci et al., J Clin Invest 115:3409-3417, 2005; Nolan et al.,
Blood, 2005; Reiter et al., Nat Med 8:1383-1389, 2002). In these
clinical scenarios, the administration of low dose nitrite will
have minimal physiologic effects in normal tissues. However, in
tissues that have become hypoxic secondary to vasoconstriction from
accelerated NO scavenging by cell-free hemoglobin, low dose nitrite
may cause vasodilation by: 1) reacting with oxyhemoglobin to form
methemoglobin, thereby preventing NO scavenging, and 2) reacting
with deoxyhemoglobin to generate NO and methemoglobin. The net
effect would be hypoxic vasodilation in local tissues which have
become ischemic from the vasoconstrictive effects of accumulating
cell-free plasma hemoglobin from ongoing low level intravascular
hemolysis.
[0168] In addition to low level intravascular hemolysis, the
studies described herein indicate that nitrite has a therapeutic
role in minimizing the vascular toxicities of more severe episodes
of intravascular hemolysis (for example, cell-free plasma
hemoglobin levels >50 .mu.M) and the administration of several
types of cell-free hemoglobin based blood substitutes (for example,
cell-free plasma hemoglobin levels >600 .mu.M) (Doherty et al.,
Nat Biotechnol 16:672-676, 1998; Dou et al., Biophys Chem
98:127-148, 2002; Hess et al., J Appl Physiol 74:1769-1778, 1993;
Hess et al., Artif Cells Blood Substit Immobil Biotechnol
22:361-372, 1994; Winslow, Vox Sang 79:1-20, 2000). In these
clinical scenarios, the ability of nitrite to attenuate the
physiologic effects of cell-free plasma hemoglobin will be
overwhelmed by the accelerated NO consumption caused by the large
amounts of plasma hemoglobin. However, in these scenarios, the
affected tissues and organs will subsequently develop areas of
hypoxia and acidosis. Within these areas, there will be accelerated
reduction of nitrite by deoxygenated cell-free plasma hemoglobin
leading to local NO generation and vasodilation. Therapeutic
strategies to deliver hemoglobin-based blood substitutes will
either require increasing the molar ratio of nitrite:hemoglobin or
will require modulating the reaction kinetics by increasing the
concentration of nitrite bound to methemoglobin (to facilitate
formation of N.sub.2O.sub.3) (Basu et al., Nat Chem Biol 3:785-794,
2007), decreasing the hemoglobin oxygen affinity (so that there is
more deoxyheme to reduce nitrite) or by decreasing the redox
potential of the heme-based blood substitute (to increase the
reactivity with nitrite).
[0169] Therefore, the studies disclosed herein indicate that
nitrite will be able to limit organ damage and dysfunction during
severe hemolytic episodes and during the administration of
hemoglobin based blood substitutes.
[0170] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the invention to the particular features or
embodiments described.
EXAMPLES
Example 1
Materials and Methods
[0171] This example describes materials and methods used to carry
out Examples 2-4. Although particular examples of materials and
methods are described, one will understand that other materials and
methods also can be used.
Reagents
[0172] All buffers were made with water that had been run through a
Milli-RO system followed by a four-cartridge Mill-Q system
(Millipore) so that the resistivity of the water was greater than
18 M.OMEGA.-cm. All reagents were purchased from Sigma-Aldrich (St.
Louis, Mo.) unless otherwise indicated. Red blood cells were
obtained by repeated sedimentation/washing in PBS of freshly drawn
blood from healthy volunteers. Hemoglobin was prepared from red
cells by lysing in excess distilled water and sedimentation
followed by freezing in liquid nitrogen for storage as previously
described (Geraci et al., Biol. Chem. 17:4664-4667, 1969; Huang et
al., Biochim. Biophys. Acta 1568:252-260, 2001). MetHb was prepared
by incubation with a two-fold molar excess of potassium
ferricyanide followed by dialysis or sephadex G-25 column
filtration. SNO-Hb was prepared as described previously (Gladwin et
al., J. Biol. Chem. 277:27818-27828, 2002). HbFe.sup.II--NO was
prepared by the addition of NO saturated buffer to deoxyHb as
described previously (Huang et al., Biochim. Biophys. Acta
1568:252-260, 2001) or by addition of PROLI NONOate (Alexis
Biochemicals).
Chemiluminescence Assay Used to Measure S-Nitroso Species
[0173] Several assays were considered in performing this study: the
tri-iodide (3I) assay, the Cu-cysteine-CO assay (3C), the ascorbic
assay/cupric chloride assay and the modified 2C assay. The 31 assay
was performed as described previously (Gladwin et al., J. Biol.
Chem. 277:27818-27828, 2002) using SNO-Hb stabilization solution
(containing NEM, ferricyanide--but no cyanide, and DTPA) as
previously described (Yang et al., Free Radic. Res. 37:1-10, 2003).
In all experiments using SNO-stabilization solution, the final
concentration of these reagents were the same. The 3C assay was
performed as described (Doctor et al., Proc. Natl. Acad. Sci. USA
102:5709-5714, 2005), carefully monitoring escape of CO into the
laboratory and limiting heating of the nitric oxide analyzer NOA
(Sievers; Boulder, Colo.) hopcolite filter. The ascorbic
acid/cupric chloride chemiluminescence assay was performed as
described previously (Nagababu et al., Nitric Oxide 15:20-29,
2006). Absorption spectroscopy, either in the visible or near
infra-red (for concentrated samples), was used to determine Hb
oxygen saturation or presence of other Hb species by fitting to
basis spectra as described previously (Huang et al., J. Biol. Chem.
280:31126-31131, 2005).
[0174] Previously, the inventors have published results from a
comparison of the 3C and 31 assays on matched serial dilutions of
standard SNO-Hb samples (Huang et al., Blood 107:2602-2604, 2006).
The two assays were very consistent with each other, with a
correlation coefficient of r=0.999258, p<0.001. The 3C assay has
the advantage that no chemical treatment or Sephadex G25 column
separation is required to remove nitrite. When the 3C assay was
applied to the study of SNO-Hb formation in the Hb/nitrite
reaction, very large signals were observed. However, control
experiments using HbFe.sup.II--NO (FIG. 13) demonstrated that these
large signals were actually due to iron nitrosyl species, not
SNO-Hb. Thus, contrary to the conclusion of a previous study
(Doctor et al., Proc. Natl. Acad. Sci. USA 102:5709-5714, 2005),
the 3C assay has some sensitivity to
HbFe.sup.II--NO.sup..sctn..sctn.. However, as previously noted
(Doctor et al., Proc. Natl. Acad. Sci. USA 102:5709-5714, 2005),
the presence of nitrosothiols can be verified by subtracting the
difference between signals from samples incubated with Hg from
those that are not exposed to Hg. Although the 3C appeared to be a
workable method, the fact that it was necessary to look for a small
(possibly null) signal in the background of a relatively large one
(due to the HbFe.sup.II--NO) was less than optimal. Thus, a
modified 2C assay was developed and employed that does not detect
HbFe.sup.II--NO.
[0175] In order to avoid detection of HbFe.sup.II--NO in the 2C
assay, samples were pretreated with potassium ferricyanide (as part
of the SNO stabilization solution--see above) oxidizing the Hb to
MetHb, which both eliminates HbFe.sup.II--NO and also makes the use
of CO unnecessary as MetHb does not capture NO effectively. The
modified 2C assay used here to measure SNO is a variation of the 2C
assay previously described (Fang et al., Biochem. Biophys. Res.
Commun. 252:535-540, 1998). The modification involves treatment
with SNO stabilization solution (10 mM NEM, 4 mM ferricyanide, 100
.mu.M DTPA, final concentrations; Yang et al., Free Radic. Res.
37:1-10, 2003) for 0.5 to 1 hour. The ferricyanide oxidizes both
HbFe.sup.II and HbFe.sup.II--NO to MetHb to prevent autocapture of
NO released from SNO-Hb and eliminates the iron-nitrosyl-hemoglobin
signal; the NEM blocks free thiols to prevent artifactual SNO-Hb
formation. After incubation with SNO stabilization solution, the
sample was run through two G-25 Sephadex columns to remove excess
ferricyanide. Validation of the modified 2C assay included
verification that the assay does not give a signal for
HbFe.sup.II--NO or NEM blocked Hb. Similar tests were performed for
the 3C and 31 assays and in all cases HbFe.sup.II--NO was made by
adding NO saturated buffer to excess Hb so that the Hb becomes
partially nitrosylated (with no free NO). NEM treatment was
performed on oxygenated samples and verified using the Ellman's
reagent for free thiols (Ellman et al., Biochem. Pharmacol.
7:88-95, 1961). In order to ensure that no false positive SNO-Hb
signal is given from HbFe.sup.II--NO, the modified 2C assay was
performed on HbFe.sup.II--NO prepared by adding NO buffer to excess
deoxyHb. HbFe.sup.II--NO did not produce a significant signal
whether the samples were pre-treated with NEM before deoxygenation
or not. It was also observed that EPR signals from HbFe.sup.II--NO
completely disappear after incubation with SNO stabilization
solution. FIG. 14 shows typical signals obtained from standard
samples using the modified 2C assay.
[0176] Nitrosation of small molecular weight molecules incubated
together with nitrite and Hb or RBCs (such as GSH) was assayed by
filtering out the protein using a Sentricon filter (Millipore,
Billerica, Mass.). The filtrate was then injected directly into the
NOA using the 2C assay.
Absorption Spectroscopy
[0177] Spectra were taken on either Cary 50 spectrometers or a
Hewlett Packard 8453 UV-Vis spectrophotometer. Oxygen leakage into
the system was prevented by application of positive helium, argon
or nitrogen pressure without a channel for gas escape. Spectral
deconvolution was performed by a least squares fit using basis
spectra as described previously (Huang et al., J. Biol. Chem.
280:31126-31131, 2005). At each time point, the measured absorption
spectrum is fit to a linear combination of normalized pure species,
the basis spectra. Basis spectra used are presented in FIG. 15. No
kinetic models are invoked in this procedure. Stopped-flow
absorption was carried out using an OLIS RSM 1000 spectrometer
coupled to a Molecular Kinetics three syringe mixer. Kinetics from
stopped-flow were analyzed by singular value decomposition and
global analysis fitting to a single exponential process.
Electron Paramagnetic Resonance Spectroscopy
[0178] EPR spectroscopy was performed as described previously
(Azarov et al., J. Biol. Chem. 280:39024-39032, 2005).
HbFe.sup.II--NO was measured by EPR using a Bruker EMX 10/12
spectrometer operating at 9.4 GHz, 5-G modulation, 10.1-milliwatt
power, 655.36-ms time constant, and 167.77-s scan or 327.68-ms time
constant and 83.89-s scan over 600 G at 110 K. MetHb was measured
by EPR (at low field using 15-G modulation, 10.1-milliwatt power,
81.92-ms time constant, and 41.94-s scan over 700 G) at 4 K using
liquid helium. The concentrations of Hb species measured by EPR
were determined by performing the double integral calculation and
comparing to standard samples.
[0179] EPR spectroscopy was used to determine the dissociation
constant of nitrite bound MetHb at various concentrations of
nitrite. The dissociation constant is given by
K.sub.d=[MetHb][nitrite]/[MetHb-nitrite]. Known amounts of MetHb
([MetHb].sub.i--also confirmed by EPR) were mixed with known
amounts of nitrite ([nitrite].sub.i). EPR spectra were used to
determine [MetHb] after nitrite addition (this concentration is the
unliganded MetHb present in the expression for K.sub.d). Nitrite
bound MetHb was assumed to be completely EPR silent so
[MetHb-nitrite] was determined from the difference of
[MetHb].sub.i-[MetHb].
Generation and Detection of Gas-Phase N.sub.2O.sub.3 by Reductive
Nitrosylation
[0180] All reactions were carried out anaerobically and in the dark
at 25.degree. C., inside a purge vessel in line with the NO gas
analyzer (NOA; Sievers NO analyzer; GE Analytical Instruments).
0.001% anti-foam B emulsion in 0.1 M phosphate buffer, pH 7.4, was
purged with helium gas for a minimum of 30 minutes before the
addition of 75 .mu.M MetHb. 1 mM nitrite was added 4 minutes after
MetHb and allowed to equilibrate with the MetHb for another 4
minutes prior to injection of 5 .mu.M Proli NONOate (Alexis
Biochemicals; a 5 mM Proli NONOate stock solution in 0.01 M NaOH
was used; equivalent to 10 .mu.M NO). Parallel control reactions
were carried out that excluded either nitrite or Proli NONOate. The
reaction was stopped 20 minutes after injection of the last
reagent. All reagents were thoroughly deoxygenated prior to
injection. The trap vessel contained 10 mL of 5 mM reduced
L-glutathione and 100 .mu.M diethylenetriamine-pentaacetic acid
(DTPA) in 0.01 phosphate buffered saline (PBS), pH 7.4. When the
reaction was terminated, the trap solution was divided into 1 mL
aliquots and immediately frozen at -80.degree. C. for subsequent
analysis. One aliquot of the trap solution was analyzed by
absorbance spectroscopy to ensure absence of trap solution
contamination by MetHb from the purge vessel. The reaction solution
from the purge vessel was collected and passed through a Sephadex
G-25 column (Amersham Biosciences) to remove excess nitrite, the
hemoglobin quantified by absorbance spectroscopy, aliquoted into 1
mL fractions, and frozen at -80.degree. C. for subsequent analysis.
All aliquots were stored in dark (foil-wrapped) Eppendorf tubes
until analyzed in triplicate for their nitrosothiol and
iron-nitrosyl-heme content using ozone-based chemiluminescence
assay as previously described. Sample analysis was subsequently
performed using reductive chemiluminescence (Gladwin et al., J.
Biol. Chem. 277:27818-27828, 2002). 1 mL aliquots of each sample
were reacted with either 100 .mu.L PBS (2 minutes) followed by 100
.mu.L 5% acidified sulfanilamide in 1 N HCl (5 minutes) or with 100
.mu.L 50 mM mercuric chloride in PBS (2 min) followed by 100 .mu.L
5% acidified sulfanilamide in 1 N hydrochloric acid (5 min) prior
to injection into a purge vessel containing 6 mL tri-iodide
solution (2 g potassium iodide and 1.3 g iodine dissolved in 40 mL
water and 140 mL acetic acid) in line with the NOA.
Statistical Analysis
[0181] Data from all gas-phase N.sub.2O.sub.3 generation
experiments was analyzed using GraphPad Prism 4.0 (GraphPad
Software Inc., San Diego, Calif.) and is reported as mean.+-.S.E.M.
Wilcoxon matched pairs test was used to compare average values of
GSNO, SNOHb, and iron nitrosyl Hb with and without nitrite addition
for nine sets of experiments. Results were considered statistically
significant with p<0.05.
DFT Calculations
[0182] DFT calculations were carried out with the OLYP (Handy &
Cohen, Molecular Physics 99:403-412, 2001; Lee et al., Physical
Review B 37:785-789, 1988) generalized gradient approximation
(GGA), triple-.zeta. plus polarization Slater-type orbital basis
sets, and a fine mesh for numerical integration of the matrix
elements. The results were checked against a number of other
functionals and found not to vary to any significant extent. The
ADF 2006 (Scientific Computing and Modeling, Amsterdam) program
system was used for all calculations.
Animal Studies
[0183] Thirty-two purpose-bred beagles (12-28 months, 9-12 kg) were
studied. All procedures were performed after inducing anesthesia
with halothane (1-4%), and initiating mechanical ventilation. Upon
completion, the halogenated gas was terminated and 100% oxygen
administered until the dog emerged from anesthesia and was
extubated. Subsequently, the animal was breathing room air
spontaneously and was sedated throughout the duration of the
experiments. Continuous infusions of medetomidine (sedation; 2-5
mcg/kg/h) and fentanyl (analgesia; 2.5-20 mcg/kg/h) were initiated
post-extubation and maintained for the study duration. Animals were
monitored continuously and signs of pain and distress were
evaluated immediately and the infusions adjusted appropriately.
Nitrite Infusion
[0184] Using pilot experiments to characterize beagle-specific
pharmacokinetics of sodium nitrite, an infusion of 165 mg of sodium
nitrite over 6 hours (27.5 mg/h) was chosen to be administered
during the study to reach a targeted plasma nitrite concentration
between 15 and 20 .mu.M. Animals randomized to groups not receiving
sodium nitrite received an equivalent rate and total volume
infusion of 0.9% NaCl (normal saline) to serve as a placebo
control.
Water-Infusion Intravascular Hemolysis Model
[0185] A previously developed and validated canine model of
water-infusion induced intravascular hemolysis was used in this
study (Minneci et al., J. Clin. Invest. 115:3409-3417, 2005).
Water-induced hemolysis produces direct intravascular hemolysis
thereby maintaining the same intravascular concentration of total
hemoglobin during hemolysis, while altering the distribution of
hemoglobin between the red cell and plasma compartment. In this
model, a six-hour infusion of water (rate: 16 ml/kg/h) produces
clinically relevant levels of cell-free plasma hemoglobin (20-300
.mu.M heme), simulating an acute hemolytic episode. The extent of
hemolysis increases over time, allowing for a graded physiological
assessment of vasomotor dysfunction as plasma hemoglobin levels
rise. The final levels of plasma hemoglobin would be analogous to
those achieved following coronary bypass surgery or a hemolytic
crisis induced by paroxysmal nocturnal hemoglobinuria or acute
immune mediated hemolysis.
[0186] Control animals received an equivalent rate and total volume
infusion of 5% Dextrose (D5W) to account for any potential
hypotonic and volume effects of the water infusion on hemodynamics.
A full-factorial study design was used with four groups of animals
receiving D5W, D5W+ intravenous sodium nitrite, Water, or Water+
intravenous sodium nitrite. This design allows for determining the
physiologic effects of a sodium nitrite infusion, the physiologic
effects of intravascular hemolysis (water) and to assess for an
interaction between nitrite and hemolysis. Specifically, the
interaction statistic tests if the effects of nitrite and
hemoglobin are influenced by the nitrite reductase activity of
cell-free plasma hemoglobin. Paired experiments were performed in
twenty animals (5 per group). In the first week, all animals
underwent a baseline study and received a D5W infusion (16 ml/kg/h)
through a central venous catheter to determine the physiologic
effects of the volume load in each animal. The D5W infusion does
not cause hemolysis; it allows each animal to serve as its own
control for the effects of a hypotonic volume load in the model.
One week later, the animals underwent an intervention study and
were randomized to receive a 6-hour infusion through a central
venous catheter of either D5W (16 ml/kg/h), D5W (16
ml/kg/h)+nitrite (27.5 mg/h), Water (16 ml/kg/h) or Water (16
ml/kg/h)+nitrite (27.5 mg/h).
[0187] This paired experimental design allows for minimization of
animal-to-animal variability by calculating the change for each
measurement performed in each animal during the baseline and
intervention studies. Subsequent analyses calculate the differences
across treatment groups by subtracting the previously calculated
differences within animals (from baseline to intervention study) in
one treatment group from another treatment group (i.e. comparison
of the differences of the differences). This design allows for
analysis of the effects of hemolysis, the effects of sodium
nitrite, and detection of any interaction between the two.
[0188] Preliminary analysis demonstrated a wider range of hemolysis
than previously described secondary to the addition of salt-based
therapies (sodium nitrite or sodium chloride) that affected the
rate of hypotonic erythrocyte lysis. In these experiments, the
6-hour water infusions produced low rate hemolysis in 50% of the
animals and rapid rate hemolysis in 50% of the animals. This
created two equal sized groups of animals with either low or high
levels of cell-free plasma hemoglobin respectively. Both groups had
peak cell-free plasma hemoglobin levels that continued to be within
a clinically relevant range (20-200 .mu.M heme). Preliminary data
analyses also suggested a possible interaction between sodium
nitrite and hemolysis that was dependent on the amount of hemolysis
(heme concentration <25 .mu.M vs.>25 .mu.M). Subsequently,
the variation in hemolytic rate and this potential interaction was
accounted for by calculating the number of animals needed to
determine if there was an interaction between nitrite and hemolysis
level (assuming a 1:1 ratio of low:high rate hemolysis in animals
receiving a water infusion) and included the level of hemolysis in
the final data analysis. The necessary additional paired
experiments were then performed using the same treatment regimens
with a weighted randomization scheme to the following groups:
D5W+nitrite (n=2), water (n=5) or water+nitrite (n=5). Overall,
these studies utilized thirty-two animals.
Sodium Nitroprusside Challenge
[0189] In order to determine the vascular responsiveness to
exogenous NO in the presence and absence of hemolysis and sodium
nitrite, all animals received a 20 minute infusion of escalating
doses of sodium nitroprusside, a direct NO donor, (1, 3, 9 and 27
mcg/kg/min) at 5 minute intervals prior to concluding the study.
These experiments allowed for comparison of a "traditional" NO
donor with nitrite to determine if the observed nitrite effects
(i.e. hemoglobin-based nitrite reduction) were distinct from a pure
NO vasodilatory effect.
Data Collection
[0190] Femoral arterial (20-gauge) and external jugular venous (8
French) catheters (Maxxim Medical, Athens, Tex.) were placed
percutaneously under anesthesia using sterile technique. Mean
arterial pressure (MAP) and heart rate (HR) were obtained from the
femoral artery catheter tracing. Additionally, a pulmonary artery
thermodilution catheter (7 French, Abbott Critical Care, Chicago,
Ill.) was introduced through the external jugular vein catheter in
order to measure cardiac output (CO), pulmonary artery pressure
(PAM), pulmonary artery occlusion pressure (PAOP), and central
venous pressure (CVP). At the end of the first week's fluid control
experiments, all catheters were removed and the animals recovered.
At the end of the second week's intervention experiments, all
animals were euthanized.
[0191] Hemodynamic measurements (MAP, CVP, PAP, CO, and PCWP) and
laboratory studies (hematocrit (Hct), hemoglobin (Hb), serum
chemistries, arterial blood gas analysis (ABG),
spectrophotometric-based quantification of cell-free hemoglobin
concentration and chemiluminescence-based assays of nitric oxide
consumption and nitrite levels) were obtained at 0, 1.5, 3.0, 4.5,
and 6.0 hour time points. Hemodynamic measurements were also
obtained at the end of each dose of sodium nitroprusside.
Plasma Nitrite and Hemoglobin Assays
[0192] Plasma nitrite levels were measured by I3-based
chemiluminescent assay as previously described using the NO
analyzer (Seivers, Model 280i NO analyzer, Boulder, Colo.) (Yang et
al., Free Radic. Res. 37:1-10, 2003). Total plasma hemoglobin
concentration (expressed in terms of heme groups; division by four
gives hemoglobin concentration) was measured by visible absorbance
spectrophotometry (HP8453 UV-Vis Diode Array Spectrophotometer,
Hewlett Packard). The concentration of oxyhemoglobin and
methemoglobin were analyzed by deconvoluting the spectrum into
components from basis spectra of canine hemoglobin in PBS buffer
using a least square method as previously described, with
subtraction of background plasma scattering (Huang et al., Biochim.
Biophys. Acta. 1568:252-260, 2001).
In-Vitro Mitochondrial Respiration Experiments
[0193] Male Sprague Dawley rats (175-250 g) were used in accordance
with the ACUC of the National Heart Lung Blood Institute. Liver
mitochondria were isolated by differential centrifugation in buffer
consisting of Sucrose (250 mM), Tris (10 mM), and EGTA (1 mM), as
previously described (Shiva et al., Circ. Res. 100:654-661, 2007).
Mitochondrial respiration was measured by suspending isolated
mitochondria (2 mg/ml) in respiration buffer (120 mM KCL, 25 mM
Sucrose, 10 mM HEPES, 1 mM EGTA, 1 mM KH.sub.2PO.sub.4, 5 mM
MgCl.sub.2) in a stirred sealed chamber fit with a Clark-type
oxygen electrode (Instech Corp.) connected to a data recording
device (DATAQ systems). Mitochondria were supplemented with
succinate (15 mM) and ADP (1 mM) to stimulate respiration.
[0194] In experiments testing the effects of nitrite and
hemoglobin, sodium nitrite and human purified oxyhemoglobin
(Ignarro et al., Proc. Natl. Acad. Sci. U.S.A. 84:9265-9269, 1987)
were incubated with the mitochondria at the beginning of the
experiment (Shiva et al., Circ. Res. 100:654-661, 2007). In this
experimental system, the rate of oxygen generation from the added
hemoglobin is less than the rate of oxygen consumption by the
mitochondria so that the oxygen increase in the system after
hemoglobin addition (20 .mu.M) is not detected by the oxygen
electrode and not observed in the raw trace unless high
concentrations of hemoglobin are added, in which case a transient
increase in the oxygen level may be detected. Note that in this
system the chamber is opened to air and oxygen is diffusing into
the system as well, but the rate of oxygen diffusion into the
system is less than the rate of oxygen consumption by the
mitochondria. Only after mitochondrial inhibition do the oxygen
levels rise to detection by electrode.
Statistical Analysis for Animal Studies
[0195] Data were analyzed using an ANOVA, with main effects for
study (baseline and intervention), hemolysis (0 .mu.M (D5W), <25
.mu.M Heme, >25 .mu.M Heme), nitrite, time, and animal (Minneci
et al., J. Clin. Invest. 115:3409-3417, 2005). Two- and three-way
interactions were included in the model. Analysis of responses to
sodium nitroprusside were performed using ANOVA on percent change
in hemodynamic variables with increasing dose in the intervention
study with main effects for hemolysis, nitrite, nitroprusside dose
and animal. Two-way interactions were included in the model. All
values are depicted in the figures as mean+/-SE and all hemoglobin
concentrations are expressed in terms of heme groups.
Example 2
Catalytic Generation of N.sub.2O.sub.3 by a Concerted Nitrite
Reductase/Anhydrase Activity of Hemoglobin
[0196] This example describes the hemoglobin-catalyzed generation
of N.sub.2O.sub.3, a gaseous nitric oxide (NO) precursor with the
capacity to escape a heme-rich environment and subsequently deliver
vasodilatory NO to tissues.
Identification of Nitrite-Bound Methemoglobin
(Fe.sup.III--NO.sub.2.sup.-) During the Reaction of Nitrite with
deoxyHb
[0197] The anaerobic reaction of deoxyhemoglobin with nitrite is an
allosteric second order reaction with a bimolecular rate constant
that ranges from 0.12 M.sup.-1s.sup.-1 at the beginning of the
reaction (T-state) to a maximum of 6 M.sup.-1s.sup.-1 later in the
reaction (R-state) at 25.degree. C. and pH 7.4 (Huang et al., J.
Clin. Invest. 115:2099-2107, 2005). As predicted by Equations 1 and
2, an equal ratio of MetHb to iron-nitrosyl-hemoglobin was measured
at the end of the reaction.
Nitrite(NO.sub.2.sup.-)+deoxyhemoglobin(Fe.sup.II)+H.sup.+.fwdarw.NO+met-
hemoglobin(Fe.sup.III)+OH.sup.- (1)
NO+deoxyhemoglobin(Fe.sup.II).fwdarw.HbFe.sup.II--NO({FeNO}.sup.7)
(2)
However, others have observed that more MetHb than
iron-nitrosyl-hemoglobin is made in the course of this reaction,
particularly at low nitrite to hemoglobin ratios, which they
attributed to the formation of an NO-MetHb (Fe.sup.III--NO)
intermediate (Angelo et al., Proc. Natl. Acad. Sci. USA
103:8366-8371, 2006; Nagababu et al., J. Biol. Chem.
278:46349-46356, 2003). This intermediate possesses nitrosonium ion
character through the resonance form NO.sup.+-ferrous hemoglobin
(Fe.sup.II--NO.sup.+, designated as {FeNO}.sup.6 using the
Enemark-Feltham notation; Enemark & Feltham, Coordination
Chemistry Reviews 13:339-406, 1974) that could potentially
nitrosate thiols to make S-nitrosothiols (SNO). Consistent with
this hypothesis, the nitrite reaction with deoxyhemoglobin forms
S-nitroso-hemoglobin (SNO-Hb), although the yields and conditions
necessary for this chemistry have been elusive (Cosby et al., Nat.
Med. 9:1498-1505, 2003; Luchsinger et al., Proc. Natl. Acad. Sci.
USA 100:461-466, 2003).
[0198] As NO-MetHb has a clearly distinguishable visible spectrum,
it should be possible to determine if this species is formed as a
significant intermediate using least-squares deconvolution of
visible spectra take during the time course of the reaction between
nitrite and deoxyHb. In FIGS. 1A (2-fold excess in deoxyhemoglobin)
and 1D (11-fold excess in nitrite), using reference spectra with
and without the NO-MetHb species in the reference standard, the
derived yields and residuals were compared over time, assuming that
the smallest residual was due to inclusion of all proper heme
species in the deconvolution. Five basis spectra were used; they
included deoxyhemoglobin, MetHb, and iron-nitrosyl-hemoglobin
species, while nitrite-MetHb (formed when nitrite binds to MetHb:
MetHb-NO.sub.2.sup.- or HbFe.sup.III--NO.sub.2) and NO-MetHb
(putative intermediate) were added in isolation and together to
discern their respective contributions to the reaction
constituents.
[0199] Approximately equal product yields of
iron-nitrosyl-hemoglobin and total MetHb were observed at the low
(FIGS. 1A and 1B), equimolar, and high nitrite:hemoglobin (FIGS. 1D
and 1E) ratios, consistent with Equations 1 and 2 and previous
studies. Interestingly, a significant amount of nitrite-MetHb but
no NO-MetHb was observed when both species were included in the
regression analysis (FIGS. 1B, 1E). To confirm the presence of
nitrite-MetHb and the absence of NO-MetHb, chi-square values over
time were compared for these reactions, fitted with and without
each species in the standard reference spectra (FIGS. 1C, 1F). At
all nitrite:hemoglobin ratios, significantly lower residuals were
observed when nitrite-MetHb was included. Addition of NO-MetHb did
not further lower the chi-square values. Notably, inclusion of
NO-MetHb alone often resulted in residuals that were nearly as high
as when neither species was included. Additional experiments were
performed at room temperature and NO-MetHb was not observed when 1
mM deoxyHb was reacted with 250 .mu.M nitrite for 180 minutes. In
all these cases with four fold heme to nitrite (n=3), regression
analysis reported no NO-MetHb at any of the time-points when all
the basis spectra were used. These data suggest that nitrite-MetHb,
and not quasi-stable NO-MetHb, is an intermediate of the
deoxyhemoglobin-nitrite reaction. Similar results were observed in
the deoxymyoglobin-nitrite reaction (Shiva et al., Circ Res.
100(5):654-661, 2007). Importantly, the absence of an NO-MetHb
species in these reactions was confirmed using a novel
chemiluminescence-based approach.
Analysis of Nitrite Bound Methemoglobin
(HbFe.sup.III--NO.sub.2.sup.-)
[0200] In order to confirm the stoichiometry of reactions described
above, freeze-quench EPR measurements of products were performed
during the course of the nitrite-deoxyhemoglobin reaction.
Surprisingly, MetHb yields by EPR were consistently lower than
those measured by UV-Vis absorption spectroscopy. To further probe
this observation, increasing concentrations of nitrite were added
to MetHb and it was found that, similar to nitrite bound to
bacterial nitrite reductase and other ferric heme proteins (Day et
al., Biochemistry-US 27: 2126-2132, 1988; Young & Siegel,
Biochemistry-US 27:2790-2800, 1988), MetHb-NO.sub.2.sup.- is
effectively EPR silent (FIG. 2A). The large low field peak due to
high spin MetHb around g=6 decreased as nitrite was added and no
concomitant signal for the low spin MetHb was seen at higher
magnetic fields (see FIG. 2A inset). Interestingly, the EPR silent
species is more stable at lower pH as evidenced by the faster
disappearance of the g=6 signal at equivalent nitrite
concentrations at pH 6.5 (FIG. 2B). The tighter binding of nitrite
to MetHb at lower pH was also confirmed using stopped-flow
absorption measuring the association and dissociation rates of
nitrite bound MetHb (FIGS. 2C-2F). Thus, both absorption and EPR
spectroscopy show that nitrite binds MetHb tighter at lower pH.
This effect is not likely to be solely due to the presence of
hydroxyl vs. water as the transition between aquo-MetHb and
hydroxyl MetHb has a pk.sub.a of 8. Thus, whether water is bound or
not would not be expected to have a large differential effect
between pH 7.4 and 6.5. Some contribution may be due to other
ionizations such as at the proximal histidine (see discussion).
Interestingly, the dissociation constant measured by examination of
the g=6 EPR signal (as described in the methods section) is much
lower than that calculated using absorption spectroscopy (reported
to be around 1-5 mM; Rodkey, Clin. Chem. 22:1986-1990, 1976; Wanat
et al., J Biol Inorg Chem 7:165-176, 2002). Moreover, more
extensive EPR silencing was observed with lower nitrite
concentrations, resulting in EPR-derived dissociation constants of
nitrite-MetHb of 75 .mu.M for 100 .mu.M of added nitrite and 285
.mu.M for 5 mM added nitrite at pH 7.4 and 7 .mu.M for 100 .mu.M
added nitrite and 145 .mu.M for 5 mM of added nitrite at pH 6.5.
These data suggest that under physiological conditions in the
erythrocyte, a substantial fraction of nitrite would be bound to
MetHb rather than free in solution.
[0201] The EPR silence of MetHb-NO.sub.2.sup.- could occur due to
line broadening resulting from g-strain, as considered for other
ferric heme proteins (Day et al., Biochemistry-US 27: 2126-2132,
1988; Young & Siegel, Biochemistry-US 27:2790-2800, 1988). To
explore this possibility and other aspects of the electronic
configuration of MetHb-NO.sub.2.sup.-, density functional theory
(OLYP/TZP) calculations were performed on various six-coordinate
nitrite-bound ferric porphyrins. The proximal ligand in the
majority of these calculations is imidazole with an acetic acid
side-chain that hydrogen bonds to the NH group, while the N-bound
("nitro") or O-bound nitrite hydrogen bonds to another imidazole on
the distal side (FIG. 3). For all the models, the nitro form was
found to be more stable than the O-nitrito form by about 7
kcal/mol. However, the relative orientation of the nitrite and
proximal imidazole planes (coplanar or perpendicular) made little
difference (<1 kcal/mol) in the energy of these species,
suggesting that states with either conformation would be equally
populated. For every conformation of every species examined
(whether nitro or O-nitrito), the two alternative d.sub..pi..sup.1
states (.sup.2A' and .sup.2A'' for C.sub.S point group symmetry)
proved to be within 0.5 kcal/mol of each other; suggesting that
rapid fluctuation between these states may provide an explanation
for the lack of an EPR signal for nitrite-MetHb.
[0202] Unlike nitrate, nitrite may be a noninnocent ligand. A
recent DFT analysis (Conradie & Ghosh, Inorg Chem 45:4902-4909,
2006) emphasizes the it-accepting character of N-bound nitrite and
the high electron affinities (EAs) of ferric-nitro porphyrins.
Here, it was found that both the six-coordinate ferric-nitro and
O-nitrito models have similar EAs of about 2.0 eV, which is high
for electroneutral ferric porphyrins but not so high as to preclude
the existence of these species. As shown in FIG. 3, the O-nitrito
ligand is noninnocent in certain conformations (FIG. 3), which may
be viewed as a Fe.sup.II--NO.sub.2. character. Interestingly, the
existence of a Fe.sup.II--NO.sub.2. nature of nitrite bound to an
Fe.sup.III porphyrin has been considered previously in other
contexts (Oshea, et al., J Org Chem 61:6388-6395, 1996; Castro
& Oshea, J Org Chem 60:1922-1923, 1995). Generally, these
results help explain recent reports of an EPR silent intermediate
in the nitrite-deoxyhemoglobin reaction that is detectable by
reductive chemiluminescence (Nagababu et al., Nitric Oxide 15:20-9,
206; Nagababu et al., J. Biol. Chem. 278:46349-46356, 2003).
The Reaction of NO with Nitrite-MetHb; Turning Reductive
Nitrosylation Upside Down
[0203] It was therefore hypothesized that a highly electron-hungry
nitrite-metHb intermediate may undergo a radical-radical reaction
of the coordinated nitrite with NO leading to the formation of
N.sub.2O.sub.3. Synthetic ferric-nitro porphyrins, where the sixth
ligand is more labile than the proximal histidine in hemoglobin,
also exhibit a similar means of relieving their electron deficiency
by reacting with NO, leading to the formation of highly stable
{FeNO}.sup.6 ferric-nitro-nitrosyl complexes. (Nasri et al., Inorg
Chem 43:2932-42, 2004; Lim et al., J Am Chem Soc 124:9737-43,
2002). NO formed in the nitrite-deoxyhemoglobin reaction could
react with the Fe.sup.IINO.sub.2. intermediate at very rapid
reaction rates and potentially compete with the otherwise dominant
and inactivating reactions of NO with ferrous heme groups. This
general reaction scheme is shown in Equation 3.
HbFe.sup.III--NO.sub.2.sup.-+NO.fwdarw.HbFe.sup.II+N.sub.2O.sub.3
(3)
[0204] Reductive nitrosylation is a classical reaction of two
molecules of NO with MetHb in which the first NO molecule binds to
MetHb (forming {FeNO}.sup.6). The nitrosonium ion will react with
water or another nucleophile and the second NO binds to the newly
formed ferrous heme. While this reaction is very slow (on the order
of 0.001 s.sup.-1 when performed with high NO concentrations (1-2
mM NO); Fernandez & Ford, J Am Chem Soc 125:10510-10511, 2003),
it has been reported to occur at much faster apparent rates
(Nagababu et al., Nitric Oxide 15:20-29, 2006). Fernandez and Ford
showed that nitrite, which is a ubiquitous and abundant contaminant
of NO solutions, could catalyze the reductive nitrosylation
reaction, increasing the observed rate by approximately 4-fold in
the presence of 20 mM nitrite and 1-2 mM NO (Fernandez & Ford,
J Am Chem Soc 125:10510-10511, 2003). They suggested that nitrite
accelerated the reaction through a mechanism in which nitrite
reacts with the ferrous-nitrosonium intermediate to yield
deoxyhemoglobin and N.sub.2O.sub.3.
Fe.sup.II--NO.sup.++NO.sub.2.sup.-.fwdarw.Fe.sup.II+N.sub.2O.sub.3
(4)
However, while this mechanism could accelerate MetHb reduction by
NO and lead to N.sub.2O.sub.3 formation, it is still too slow to
compete with NO heme reactions and is difficult to reconcile with
more rapid rates of reductive nitrosylation observed by others in
the field (Nagababu et al., Nitric Oxide 15:20-9, 2006; Han et al.,
Proc. Natl. Acad. Sci. USA 99:7763-7768, 2002).
[0205] An alternative solution based on the EPR findings disclosed
herein was therefore considered. Previous experiments, including
those by Fernandez and Ford, were conducted at high NO
concentrations, such that NO may have preferentially bound to the
MetHb and thereby prevented or greatly diminished nitrite binding
to MetHb (NO binds MetHb with approximately 25 times the affinity
of nitrite; Rodkey, Clin. Chem. 22:1986-90, 1976; Cooper, Biochim.
Biophys. Acta-Bioenerg. 1411:290-309, 1999). Such competitive
binding of NO would inhibit generation of the nitrite-MetHb
complex, which may be involved in catalytic reductive
nitrosylation.
[0206] To test this hypothesis, the effect of varying the nitrite
concentration in the presence of excess NO was examined (1 mM) and
similar rates of MetHb reduction and iron-nitrosylhemoglobin
formation with increasing nitrite concentration were observed as
reported earlier by Fernandez and Ford (Fernandez & Ford, J Am
Chem Soc 125:10510-10511, 2003). FIG. 4A traces the change in all
heme species over time as measured by absorption spectroscopy,
while FIG. 4B shows the fit for HbFe.sup.II--NO formation to a
single exponential process. A summary of observed rates measured
with 1 mM NO and various nitrite concentrations is shown in FIG.
4E. The deviation of the fit from measured HbFe.sup.II--NO in FIG.
4E suggests that the process is more complicated than a single
exponential process, possibly due to a faster concurrent process in
which NO reacts with HbFe.sup.III--NO.sub.2.sup.-. This hypothesis
is confirmed by the observation that the observed reaction rate for
HbFe.sup.II--NO formation is actually significantly faster at lower
NO concentrations (FIGS. 4C, 4D and 4F). The observed rate of
formation of HbFe.sup.II--NO from the reaction of 30 .mu.M MetHb
with 1 mM NO and 5 mM nitrite is 0.0086 s.sup.-1. For the mechanism
of nitrite-mediated catalysis of reductive nitrosylation suggested
by Fernandez and Ford, the rate should decrease as the NO
concentration is decreased. In contrast, the observed rate of the
reaction actually increased when 30 .mu.M MetHb was mixed with 50
.mu.M NO and 5 mM nitrite (0.018 s.sup.-1). The calculated observed
reaction rates as a function of variable NO with 5 mM nitrite and
30 .mu.M MetHb are summarized in FIG. 4F. Notably, the observed
reaction rate increased significantly with decreasing NO
concentrations, consistent with inhibition of nitrite-MetHb
formation at high NO concentrations secondary to competitive
binding of excess NO. Examination of reaction intermediates by
spectral deconvolution (FIGS. 4A and 4C) also indicates that after
NO addition, MetHb-NO.sub.2.sup.- is consumed faster than MetHb,
consistent with a predominant and faster reaction between
nitrite-MetHb and NO.
Evidence for N.sub.2O.sub.3 Formation in the Reaction of Nitrite
and deoxyHb: Oxygen and pH Dependence of 5-Nitrosation
[0207] In order to examine if the nitrite/deoxyhemoglobin reaction
generates a nitrosating agent, a modified Cu/cysteine (2C)
reductive-chemiluminescent assay (see Materials and Methods) was
used to examine SNO formation in a large variety of conditions with
and without inclusion of glutathione (GSH), in order to determine
if S-nitrosoglutathione (GSNO) is formed. FIG. 5 shows
representative and summation data for the different conditions
studied. To assess Hb-dependent RSNO formation it was important to
first assess basal levels of RSNO formation that occur when nitrite
is incubated with GSH. The degree of nitrosation in the absence of
Hb was examined. In FIG. 5A, 1 mM GSH and 1 mM nitrite were mixed
under anaerobic conditions at pH 7.4 or pH 6.5 and GSNO was
measured by the 2C assay at the indicated time points. Within
twenty minutes of mixing, the GSNO signal was much larger at pH 6.5
than at pH 7.4. FIG. 5D summarizes GSNO formation as measured by
the nitric oxide analyzer (NOA) at the indicated time points under
oxygenated and deoxygenated reaction conditions in the presence of
1 mM GSH and 1 mM nitrite. The samples were injected directly into
a nitric oxide analyzer at the times indicated. The most GSNO was
formed at pH 6.5 under deoxygenated conditions. Under these
conditions about 200 nM GSNO is formed within five minutes of
mixing and almost 1 .mu.M GSNO accumulated after one hour. The pH
dependence of nitrosation that was observed suggests the
involvement of nitrous acid. The pK.sub.a of nitrous acid is about
3.15 so that (in the absence of any other reactions) adding 1 mM
nitrite would yield about 60 nM nitrous acid at pH 7.4 and 500 nM
nitrous acid at pH 6.5 (Williams (Elsevier, Amsterdam, 2004)
Nitrosation Reactions and the Chemistry of Nitric Oxide).
Nitrosation can occur through direct reaction of nitrous acid and a
thiol but the nature of the acid catalysis of this reaction and its
kinetics make it unlikely that this would occur in the experiments
described above (Morris & Williams, J Chem Society-Perkin
Transactions 2 513-516, 1988). Another possibility is that
nitrosation occurred via the intermediacy of N.sub.2O.sub.3 which
is in equilibrium with nitrous acid (Williams (Elsevier, Amsterdam,
2004) Nitrosation Reactions and the Chemistry of Nitric Oxide).
Since the concentration of N.sub.2O.sub.3 depends on the
concentration of nitrous acid squared, one expects nitrosation to
be about 100 times more efficient at pH 6.5 than at pH 7.4. The
stability of nitrous acid has been reported to decrease as the
oxygen tension increases (Williams (Elsevier, Amsterdam, 2004)
Nitrosation Reactions and the Chemistry of Nitric Oxide; Beake
& Moodie, J Chem Society-Perkin Transactions 2 1045-1048,
1995), suggesting a possible explanation for the enhanced
nitrosation observed under anaerobic conditions.
[0208] Having established the degree of nitrosation of GSH by
nitrite under basal conditions, the effects of Hb were examined.
FIGS. 5B and 5C show the result of nitrosothiol measurement by the
modified 2C assay following a thirty minute incubation of 1 mM
nitrite, 1 mM GSH, and either 300 .mu.M (5b) or 5 mM (5c)
deoxygenated Hb. The difference in the signal observed in the
presence and absence of Hg is proportional to the amount of
S-nitrosothiol produced. FIG. 5E summarizes the results of
experiments performed similarly to those shown in FIG. 5D (where Hb
is absent), but when 300 .mu.M Hb is added to the mixture of 1 mM
GSH and 1 mM nitrite and incubated for thirty minutes. The goal was
to measure both low and high molecular weight nitrosothiols when
both Hb and GSH were included. The low molecular weight fraction in
a portion of the sample was separated from hemoglobin using
Centricon filters to allow quantification of GSNO, and SNO-Hb was
measured by the modified 2C assay in the remaining portion. It
should be noted that GSNO reacts with deoxyHb to make NO (and hence
HbFe.sup.II--NO) and can also react with OxyHb through
trans-nitrosation so that in oxygenated and partially oxygenated
conditions, SNO-Hb may be a better assessment of accumulated
N.sub.2O.sub.3 formation (Patel et al., J. Biol. Chem.
274:15487-15492, 1999; Spencer et al., J. Biol. Chem.
275:36562-36567, 2000). As with the mixtures without Hb present,
most nitrosation was observed under deoxygenated conditions at pH
6.5, with SNO-Hb making up almost all of the nitrosated product.
Comparison with FIG. 5D reveals that the presence of deoxygenated
Hb significantly increases the yield of S-nitrosothiol from
nitrite. Under oxygenated conditions all the nitrosation occurs on
GSH, with more being formed at pH 6.5 (0.51.+-.0.04 .mu.M) than at
pH 7.4 (0.21.+-.0.13 .mu.M) and that amount being more than when
the Hb is absent (compare to FIG. 5D, where less than 0.3 .mu.M
GSNO was observed after 30 minutes at pH 6.5). These data suggest
that nitrite reactions with hemoglobin involve S-nitrosation
chemistry, with increasing S-nitrosation occurring under
deoxygenated conditions and at lower pH. Notably, inclusion of Hb
increases the total amount of nitrosated products from nitrite
compared to when Hb is absent.
[0209] A summary of results obtained when 5 mM Hb is used are shown
in FIG. 5F. The trends already illustrated in FIGS. 5C and 5D where
the most SNO is made under deoxygenated conditions at pH 6.5 are
observed under these conditions as well. The higher SNO yield at
lower pH could be due to the increased stability of N.sub.2O.sub.3
at the lower pH (Williams (Elsevier, Amsterdam, 2004) Nitrosation
Reactions and the Chemistry of Nitric Oxide) and/or the increased
nitrite binding by MetHb at the lower pH (as shown in FIG. 2B).
Remarkably, 23 .mu.M SNO-Hb is produced when 5 mM deoxygenated Hb
is mixed with 1 mM nitrite and 1 mM GSH for thirty minutes. When
the nitrite concentration is reduced to the levels obtained in
studies of nitrite infusions in humans (approximately 250 .mu.M;
Cosby et al., Nat. Med. 9:1498-505, 2003; Lauer et al., Proc. Natl.
Acad. Sci. USA 98:12814-12819, 2001), 2.5 .mu.M SNO-Hb is formed,
similar to levels measured in vivo (Cosby et al., Nat. Med.
9:1498-505, 2003; FIG. 11).
N.sub.2O.sub.3 Formation by the Reaction of NO with
Nitrite-MetHb
[0210] As shown herein (FIG. 4), NO reacts rapidly with the
Met-Hb-nitrite complex. In order to see if this reaction generates
a freely diffusible nitrosating agent, consistent with the
formation of N.sub.2O.sub.3 predicted from Equation 3, the
experimental system shown in FIG. 6A was assembled. Nitrite and
hemoglobin are placed in a purge vessel and continuously bubbled
with helium gas. The helium gas is then bubbled through a second
chamber (trap) containing GSH and DTPA, and the accumulation of
S-nitrosothiols in this second chamber is measured by reductive
chemiluminescence. FIG. 6B shows raw chemiluminescence data
indicating that addition of NO to a solution of MetHb/nitrite
causes the accumulation of synergistically higher levels of GSNO
when compared to either NO or nitrite alone. As shown in FIG. 6C,
when 375 nmoles MetHb (75 .mu.M in solution) is reacted with 50
nmoles NO (10 .mu.M in solution) after brief pre-equilibration with
5 .mu.moles nitrite (1 mM in solution), 3.82.+-.2.92 nmoles of GSNO
is detected in the trap solution. Importantly, GSNO formation was
significantly lower when nitrite was excluded from the reaction,
such that only 0.2.+-.0.08 nmoles of GSNO formed in the absence of
nitrite (p<0.02). There was minimal GSNO formation in the
absence of NO as well.
[0211] The extent of hemoglobin iron-nitrosylation and
S-nitrosation (FIG. 6D) within the reaction solution itself was
also investigated. There was no significant difference in SNO-Hb
formation whether MetHb was reacted with NO alone (1.44.+-.0.16
nmoles) or with both nitrite and NO (1.16.+-.0.3 nmoles).
Interestingly, as demonstrated in FIG. 6E, the amount of
HbFe.sup.II--NO in the reaction solution was significantly lower
when MetHb was reacted with both nitrite and NO (3.+-.0.74 nmoles)
than when MetHb was reacted with NO alone (4.65.+-.1.12 nmoles).
This suggests that while the NO, in the absence of nitrite, can
reduce MetHb via the uncatalyzed nitrite-independent reductive
nitrosylation pathway, this mechanism generates less gas-phase
N.sub.2O.sub.3 (limited GSNO in the trap vessel, see FIG. 6C) and
HbFe.sup.II--NO remains in the reaction solution. In contrast,
NO-mediated reduction of nitrite-MetHb is simultaneously producing
N.sub.2O.sub.3 which is then purged out of solution, such that the
concentration of HbFe.sup.II--NO in the reaction vessel is
significantly lower (FIG. 6D) and the concentration of GSNO in the
trap vessel is significantly higher (FIG. 6C). These results
suggest that NO reactions with nitrite-MetHb can effectively
compete with ferric heme autocapture and thus increase the
efficiency of NO.sub.x escape.
N.sub.2O.sub.3 Formation and S-Nitrosation in the deoxyHb/Nitrite
Reaction Requires Reactions with MetHb-NO.sub.2.sup.-
[0212] Nagababu et al. (Nitric Oxide 15:20-29, 2006) have recently
reported the formation of SNO-Hb in the reaction of deoxyHb and
nitrite. Using an ascorbate/Cu(II) chemiluminescence-based assay,
they detected approximately 22 .mu.M SNO-Hb formed after a sixty
minute incubation of 250 .mu.M nitrite with 1 mM deoxyHb at neutral
pH. This is much more than is observed here, even when 1 mM nitrite
is used (FIG. 5). One important difference between the methods used
by Nagababu et al. and those used here is that when the heme
reactions were stopped here with ferricyanide, the samples were
also treated with N-ethylmaleimide (NEM) to block all free thiols.
In contrast, Nagababu and colleagues did not treat with NEM to
block thiol. Indeed, when their ascorbate/Cu(II) method was used
and the samples were not treated with NEM along with the added
ferricyanide, significantly more SNO-Hb was measured than when the
NEM was added. Likewise, when NEM was left out of the SNO-Hb
stabilization solution in the modified 2C assay, much more SNO was
detected (4.1.+-.2.6 .mu.M vs. 1.7.+-.2.4 .mu.M SNO for one hour
incubation of 250 .mu.M deoxyHb with 1 mM nitrite). These results
are consistent with recent observations by Feelisch and colleagues,
who have also described artifactually high SNO-Hb formation after
treatment with ferricyanide (Bryan et al., Nitric Oxide-Biol Ch
10:221-8, 2004). It is therefore likely that ferricyanide oxidizes
deoxyhemoglobin to methemoglobin and thus increases the
concentration of MetHb-NO.sub.2.sup.- that is necessary for
N.sub.2O.sub.3 formation via the reaction of the Nitrite-MetHb
intermediate with NO generated in the nitrite-deoxyhemoglobin
reaction.
[0213] If MetHb-NO.sub.2.sup.- is involved in the formation of
SNO-Hb then the binding of cyanide (CN) to the ferric heme should
greatly reduce SNO-Hb formation. Since CN does not bind to ferrous
hemes, it should not otherwise interfere with the
nitrite-deoxyhemoglobin reaction. FIG. 7A shows that addition of
KCN to the reaction mixture of deoxyhemoglobin and nitrite
significantly lowered the SNO-Hb yield, confirming nitrite-MetHb
participation. To ensure that the effect of KCN is not due an
increase in pH, experiments like those shown in FIG. 7A were
repeated using 1 M phosphate buffer. As shown in FIG. 12, KCN
greatly reduces SNO-Hb formation without affecting pH.
[0214] The concentration of phosphate has been shown to influence
N.sub.2O.sub.3 mediated nitrosation. In most cases, increasing the
concentration of phosphate reduces N.sub.2O.sub.3 mediated
nitrosation (DeMaster et al., Biochem. Pharmacol. 53:581-585, 1997;
Singh et al., Proc. Natl. Acad. Sci. USA 93:14428-14433, 1996;
Lewis et al., J Am Chem Soc 117:3933-3939, 1995) but under some
conditions nitrosation can be enhanced (Dabora et al., Iarc
Scientific Publications 311-316, 1984). The effect of incubating
300 .mu.M deoxyHb with 1 mM nitrite for thirty minutes at pH 6.5 in
either phosphate buffered saline (PBS: 10 mM phosphate) or 1 M
phosphate (FIG. 7B) was therefore examined. The formation of SNO-Hb
was increased in the presence of phosphate, consistent with other
studies (Dabora et al., Iarc Scientific Publications 311-316,
1984). SNOHb formation was also measured in these two buffers after
addition of the NO donor ProliNO to Nitrite-MetHb
(HbFe.sup.III--NO.sub.2.sup.-; FIG. 7B). Consistent with the
hypothesis that SNO-Hb formed in the nitrite-deoxyhemoglobin
reaction relies on N.sub.2O.sub.3 generated in the reaction of NO
with MetHb-NO.sub.2.sup.-, the addition of NO to Met-NO.sub.2.sup.-
resulted in SNO-Hb formation. Moreover, the dependence of SNOHb
formation on phosphate concentration in the deoxyhemoglobin-nitrite
reaction was identical to that of the MetHb-NO.sub.2.sup.-/ProliNO
reaction, supporting a similar chemistry.
Examination of Other Proposed Mechanisms for Nitrite-Hemoglobin
Dependent Nitrosation
[0215] One proposed mechanism for SNO-Hb formation is the
intra-molecular transfer of NO generated in the deoxyHb-nitrite
reaction from the ferrous heme to the .beta.-93 cysteine following
the T-to-R conformational change upon hemoglobin oxygenation
(Luchsinger et al., Proc. Natl. Acad. Sci. USA 100:461-466, 2003;
Angelo et al., Proc. Natl. Acad. Sci. USA 103:8366-8371, 2006;
Singel & Stamler, Annu Rev Physiol 67:99-145, 2005). However,
such an allosterically-controlled intra-molecular transfer of NO
from the heme to the cysteine during oxygenation is not observed by
EPR and is not balanced electronically (Huang et al., Blood
107:2602-2604, 2006; Xu et al., Proc. Natl. Acad. Sci. USA
100:11303-11308, 2003). Moreover, this mechanism could not apply to
the observations reported herein as the most nitrosation is
observed under deoxygenated conditions--without subsequent
oxygenation.
[0216] A second proposed mechanism for SNO-Hb formation in the
nitrite-deoxyhemoglobin reaction is the formation of a MetHb bound
with NO intermediate (HbFe.sup.III--NO/HbFe.sup.II--NO.sup.+) that
could lead to nitrosation (Equation 5) (Nagababu et al., Nitric
Oxide 15:20-29, 2006; Angelo et al., Proc. Natl. Acad. Sci. USA
103:8366-8371, 2006).
HbFe.sup.IIINO+RSH.fwdarw.HbFe.sup.II+RSNO (5)
As the nitrosonium ion is not likely to survive in an aqueous
environment, the nitrosonium transfer mechanism (Equation 5) is
proposed to occur intra-molecularly to form SNO-Hb (Nagababu et
al., Nitric Oxide 15:20-29, 2006; Angelo et al., Proc. Natl. Acad.
Sci. USA 103:8366-8371, 2006). With subsequent binding of another
NO, the nitrosonium transfer mechanism also encompasses reductive
nitrosylation. One group has proposed that oxygen is necessary for
this transfer (Angelo et al., Proc. Natl. Acad. Sci. USA
103:8366-8371, 2006), while another has proposed that oxygen
inhibits the transfer (Nagababu et al., Nitric Oxide 15:20-29,
2006).
[0217] As described in FIG. 1, it was found that
MetHb-NO.sub.2.sup.- is the major spectral intermediate in the
deoxyHb/nitrite reaction, rather than MetHb-NO. However, since it
can be difficult to definitively identify minor species among many
others using absorption spectroscopy, an additional sensitive and
specific chemiluminescence-based assay for HbFe.sup.III--NO was
developed. Since NO dissociates from HbFe.sup.III--NO at a rate of
1 s.sup.-1 (Cooper, Biochim. Biophys. Acta-Bioenerg. 1411:290-309,
1999), HbFe.sup.III--NO should be detectable when injected into a
purge vessel in-line with a gas-phase chemiluminescent NO analyzer,
without the need for any additional chemistry. To prevent capture
of NO released from HbFe.sup.III--NO by any ferrous deoxyhemoglobin
present in solution, the NOA was purged with CO (mixed 1:1 with
argon). The molar excess of CO would bind any ferrous hemes in
milliseconds and thus prevent NO autocapture. FIG. 8A shows results
from the validation of this assay. Partially saturated
HbFe.sup.III--NO was made by adding 10 to 30 .mu.M NO to 1 mM
MetHb. Based on the affinity of MetHb for NO (Cooper, Biochim.
Biophys. Acta-Bioenerg. 1411:290-309, 1999), it was calculated
that, for these conditions, approximately 80% of the added NO
should be bound to the MetHb with the rest being free in solution.
Within thirty seconds after NO addition to the MetHb, the sample
was injected into the NOA. Overall, 78.+-.39% recovery of the added
NO was observed, indicating that most of the NO came from
HbFe.sup.III--NO. This signal decayed on the order of minutes as a
second injection of the HbFe.sup.III--NO produced a barely
perceptible peak (FIG. 8A). The decay of the HbFe.sup.III--NO
signal was due to reductive nitrosylation, that generated
HbFe.sup.II--NO, which is not detectable in the CO-based
chemiluminescence assay. These results were verified using EPR
(FIG. 8B).
[0218] After this validation was performed, the stability of an
HbFe.sup.III--NO "intermediate" was tested in the presence of
excess deoxyhemoglobin which is a necessary reactant in the
deoxyhemoglobin-nitrite reaction. Based on the million-fold higher
affinity of ferrous deoxyhemoglobin for NO compared to MetHb, and
the fact that NO is released from MetHb at a rate of 1 s.sup.-1
(Cooper, Biochim. Biophys. Acta-Bioenerg. 1411:290-309, 1999), one
would not expect HbFe.sup.III--NO to be stable in the presence of
deoxyhemoglobin. To test this, 100 .mu.M of ferrous deoxyHb was
added to a new MetHb-NO sample followed by immediate injection of
the mixture into the CO gas purging NOA system. The deoxyHb was
added immediately after the NO, followed by injection into the NOA
within thirty seconds. As shown in FIG. 8A, the mixture did not
produce a signal in the NOA, indicating that all NO on the MetHb
was quickly transferred to the added ferrous hemes. Finally, in
order to examine whether any HbFe.sup.III--NO accumulates in the
nitrite-deoxyhemoglobin reaction, aliquots of a mixture of 1 mM
deoxyHb and 250 .mu.M nitrite were injected into the CO-purging NO
analyzer at 10 seconds, and 1, 10, 30, 60, 90 and 180 minutes after
the reaction began in pH 7.4 buffer. In all cases (n=3), no signal
was observed in the NOA (representative data shown in FIG. 8C),
indicating that HbFe.sup.III--NO does not form as a stable or
quasi-stable intermediate.
[0219] Another possible mechanism for SNO formation and reductive
nitrosylation involves oxygen transfer (Equation 6).
HbFe.sup.III--NO.sub.2.sup.-+NO.fwdarw.NO.sub.2.+HbFe.sup.II--NO
(6)
Oxygen transfer from nitrite to a variety of substrates including
NO has been demonstrated for iron(III) porphyrins (Castro &
Oshea, J Org Chem 60:1922-1923, 1995). In the context of
nitrite-MetHb, oxygen transfer would directly lead to
iron-nitrosyl-Hb and the NO.sub.2 radical. A second NO could react
with the NO.sub.2 radical to form N.sub.2O.sub.3 with subsequent
nitrosation. Alternatively, the NO.sub.2 radical could oxidize
cysteinyl thiol to form a thiol radical and a second NO could then
react to form SNO-Hb. A similar route to SNO could take place when
the NO.sub.2 radical is released from N.sub.2O.sub.3.
[0220] A possible role of the oxygen transfer mechanism in the
above-described Hb experiments was tested in two ways. Firstly, it
was examined whether incubation of Nitrite-MetHb led to formation
of iron-nitrosyl-Hb by following the reaction spectroscopically.
This was done with several compounds shown to be efficient oxygen
trapping substrates (Castro & Oshea, J Org Chem 60:1922-1923,
1995) including CO and dimethyl sulfide. In these experiments, no
iron-nitrosyl-Hb formation was observed. In addition, there was no
evidence for formation of dimethyl sulfoxide by gas chromatography
after incubation of metHb-nitrite with dimethyl sulfide. Secondly,
iron-nitrosyl-Hb formation was studied when .sup.15N labeled
nitrite bound to MetHb was combined with .sup.14N nitric oxide. If
the oxygen transfer mechanism (Equation 6) occurs, the iron
nitrosyl would be entirely HbFe.sup.II--.sup.15NO, as it is derived
from the nitrite. If, on the other hand, the Nitrite-MetHb+NO
reaction to form N.sub.2O.sub.3 occurs (Equation 3), one would get
HbFe.sup.II--.sup.14NO when the bound NO comes from that added to
the nitrite-MetHb which could be mixed with some
HbFe.sup.II--.sup.15NO if the bound NO comes from the
N.sub.2O.sub.3. When treated to bring out the hyperfine structure,
HbFe.sup.II--.sup.14NO produces a triplet whereas
HbFe.sup.II--.sup.15NO produces a doublet in EPR (FIG. 9). Using
isotope labeled nitrite, no evidence was found for oxygen transfer
in the mechanism for reductive nitrosylation (and hence SNO
formation) when NO is added to nitrite-MetHb (FIG. 9).
Example 3
Use of Inorganic Nitrite or Nitrite-Methemoglobin in a Cell-Free
Blood Substitute
[0221] This example demonstrates use of inorganic nitrite to
detoxify a cell-free blood substitute to be administered to a
subject in need of plasma expansion, tissue oxygenation, or
treatment of another condition without concomitant vasoconstriction
or resulting pathologies.
Nitrite Prevents Decreased Cardiac Output Resulting from Low-Level
Hemolysis
[0222] Hemolysis is the rupturing of the erythrocyte membrane, and
the subsequent release of free hemoglobin into the blood. Hemolysis
results in a host of complications similar or identical to those
resulting from transfusion with cell-free hemoglobin. As in
cell-free hemoglobin transfusion, hemolysis introduces free
hemoglobin into the blood where it scavenges nitric oxide and
thereby causes vasoconstriction. Therefore, treatments that
antagonize the vasoconstrictive effects of hemolysis by inhibiting
the scavenging of nitric oxide by free hemoglobin are expected to
be useful treatments for the detoxification of cell-free hemoglobin
based blood substitutes.
[0223] Hemolysis was induced in dogs by infusing free water into
the blood stream. The resulting osmotic pressure ruptured
erythrocytes in vivo, releasing free hemoglobin. Consequently,
cardiac output was reduced secondary to the primary effects of NO
scavenging and vasoconstriction, but co-infusion with nitrite
therapeutically reversed these effects (FIG. 16). This result
demonstrates the therapeutic efficacy of nitrite in preventing NO
scavenging and vasoconstriction by free hemoglobin in vivo.
Blood Substitute Comprising Nitrite Maintains Mean Arterial Blood
Pressure During Trauma Hemorrhagic Shock and Resuscitation
[0224] The toxicity of cell-free hemoglobin as a blood substitute
is observed following resuscitation of a subject experiencing
controlled hemorrhage as a restoration of mean arterial blood
pressure above the normal range. Nitric oxide scavenging and
vasoconstriction narrow the vasculature, thereby elevating blood
pressure in the subject.
[0225] In a murine model of controlled hemorrhage, shock, and
resuscitation, administration of a cell-free hemoglobin based
oxygen carrier blood substitute indeed elevates mean arterial blood
pressure in the resuscitated subject to more than 95 mm Hg, where
approximately 75 mm Hg is the normal range (FIG. 17). Subjects
receiving an i.v. bolus of nitrite experienced restoration of mean
arterial blood pressure to the normal range (FIG. 17). This result
demonstrates the utility of nitrite in the detoxification of
cell-free hemoglobin based blood substitutes. In some embodiments,
the detoxified blood substitute may be used as here, to resuscitate
a subject following hemorrhage and shock.
Blood Substitute Comprising Nitrite Provides Superior Blood
Oxygenation
[0226] It is an advantage of cell-free hemoglobin based blood
substitutes detoxified by nitrite that free hemoglobin in the blood
substitute catalyzes the generation of nitric oxide. Unexpectedly,
this effect is only observed within the narrow range of nearly
exact molar ratios of hemoglobin to nitrite. This result is shown
in FIG. 18, where nearly exact ratios of nitrite and hemoglobin
generate NO, and thereby inhibit mitochondrial respiration. Under
conditions where there is almost half as much hemoglobin as
nitrite, or more than twice as much, the effect is diminished (FIG.
18B). Without hemoglobin, or with a more than 5-fold molar excess,
the effect is completely ablated.
Example 4
Hemodynamic Responses to Nitrite Infusion in a Canine Model
[0227] This example describes effects of sodium nitrite infusion in
canines. Hemodynamic responses in study animals, including
hemolysis, arterial pressure and vascular resistance, are
described.
Physiologic Effects of Intravenous Sodium Nitrite
[0228] While many groups have now confirmed that sodium nitrite is
a potent vasodilator in vivo, no group has characterized more
specifically its activity in vivo as a relative arterial versus
venous vasodilator or its effects on inotropy and chronotropy.
Intravenous infusion of sodium nitrite (27.5 mg/h) rapidly
increased plasma nitrite levels to a steady state concentration
(range: 15-21 .mu.M) that was maintained throughout the duration of
the 6-hour infusion (FIG. 19A). In animals receiving a D5W
infusion, sodium nitrite increased cardiac index (CI; p=0.001) and
decreased systemic vascular resistance index (SVRI; p=0.04),
pulmonary vascular resistance index (PVRI; p=0.001), mean systemic
arterial pressure (MAP; p=0.08), mean pulmonary arterial pressure
(PAM; p=0.09), central venous pressure (CVP; p=0.01), and pulmonary
artery occlusion pressure (PAOP; p=0.65) compared to placebo
(normal saline) (FIGS. 19B-H). These physiologic effects suggest
that low dose sodium nitrite is a more potent arterial vasodilator
than a venodilator, and that nitrite increases cardiac performance
by direct afterload reduction.
[0229] Supporting evidence for the vasodilatory effects of nitrite
can be derived by examining the components of CI in the log scale
(FIG. 20) (Rowland et al., Pediatr. Cardiol. 21:429-432, 2000). In
this format, the individual contribution of each component of Clare
additive (normal scale: CI=SVI.times.HR; log scale: log CI=log
SVI+log HR). This transformation demonstrates that the
nitrite-induced increase in CI is mediated predominantly through a
sustained increase in stroke volume index (SVI) and to a lesser
extent by a chronotropic effect. This transformation also accounts
for the rise in PAOP during the last three hours of the study (FIG.
19H). The decrease in heart rate over time (an effect of
anesthesia/analgesia seen in all groups in this model) (FIG. 20B)
increases diastolic filling time in the ventricles leading to
higher end-diastolic volumes and pressures that translate into
increases in PAOP and further increases in SVI. These data imply
that nitrite enhances cardiac performance by afterload reduction
through an arterial vasodilatory mechanism. These data also
indicate that isolated measures of MAP in animal studies may fail
to sensitively assess the magnitude of nitrite-dependent
vasodilation because of the rise in cardiac index.
Physiologic Effects of Sodium Nitrite During Intravascular
Hemolysis In previous intravascular hemolysis experiments (Minneci
et al., J. Clin. Invest. 115:3409-3417, 2005) and in the current
experiments, cell-free plasma hemoglobin increased systemic and
pulmonary arterial pressures, systemic and pulmonary vascular
resistance, and pulmonary arterial occlusion pressure (Table 1;
p=0.04, 0.14, 0.06, 0.42, and 0.21 respectively for the interaction
of hemolysis level and the mean change in each physiologic variable
during baseline and intervention studies). If nitrite functioned
purely as an NO donor medication, one would expect the
vasoconstrictive effects of intravascular hemolysis to attenuate
the vasodilatory effects of nitrite because any NO generated from
nitrite would be readily scavenged by the cell-free plasma
hemoglobin. However, in these experiments, the physiologic effects
of nitrite were not simply inhibited by increasing levels of
hemolysis.
TABLE-US-00001 TABLE 1 Physiological effects of intravascular
hemolysis (Mean change during 6 hour intervention study) SVRI PVRI
(dynes/ (dynes/ Level of MAP PAM sec/ sec/ PAOP hemolysis (mmHg)
(mmHg) *cm.sup.-5) *cm.sup.-5) (mmHg) zero (n = 5) 6.7 3.0 15.1 1.3
2.9 <25 .mu.M (n = 4) 7.9 4.2 28.1 3.0 2.8 >25 .mu.M (n = 4)
16.4 4.8 33.9 2.3 3.8 MAP: mean systemic arterial pressure; PAM:
mean pulmonary arterial pressure; SVRI: systemic vascular
resistance index; PVRI: pulmonary vascular resistance index; PAOP:
pulmonary artery occlusion pressure
[0230] In fact, the effect of nitrite was dependent on the level of
intravascular hemolysis in an unusual way (FIG. 21). A consistent
U-shaped relationship was detected between the physiologic effects
of nitrite and the levels of cell-free plasma hemoglobin suggesting
an interaction between the effects of nitrite and the amount of
intravascular hemolysis (FIG. 21; p=0.01 for a differing effect of
nitrite at low level hemolysis compared to zero and high level
hemolysis across the 7 physiologic variables combined). At low
levels of hemolysis (Hb<25 .mu.M), the vasodilatory effects of
nitrite are apparently potentiated, whereas with higher levels of
hemolysis (Hb>25 .mu.M), the expected inhibition of the
vasodilatory effects of nitrite are observed. These results suggest
that there are two reactions that regulate the availability of NO
at the smooth muscle: the reaction of hemoglobin with nitric oxide
and an opposing reaction of nitrite with deoxyhemoglobin that
generates NO. The results described herein demonstrate that at low
levels of hemoglobin, the physiologic effects of the latter
reaction are detected; however with increasing hemoglobin
concentration, the former reaction dominates. These effects are
examined more closely and compared with the NO donor sodium
nitroprusside in additional experiments described below.
Nitrite Levels and Hemoglobin Species Formed During Intravascular
Hemolysis
[0231] In animals receiving nitrite, plasma nitrite levels were
similar and were maintained within a range of 16-20 .mu.M
throughout the six hour experiment (FIG. 22 and FIG. 19A).
Intravascular hemolysis occurred at varying rates (FIG. 22).
Animals receiving D5W and nitrite represent the zero hemolysis
control group with all measured cell-free plasma hemoglobin levels
<5 .mu.M. In animals receiving water and nitrite infusions with
low levels of hemolysis (Hb<25 .mu.M), the average peak
cell-free plasma hemoglobin level was 20 .mu.M. In animals
receiving water and nitrite infusions with high levels of
hemolysis, the average peak cell-free plasma hemoglobin level was
142 .mu.M. In animals receiving D5W and nitrite (zero hemolysis),
81% of the measured cell-free plasma hemoglobin was oxyhemoglobin
(FIG. 22, values depicted as a red reference line in FIG. 22B and
FIG. 22E), consistent with observations in normal volunteers and
sickle cell patients that plasma hemoglobin is maintained largely
in the reduced or ferrous-oxygen bound state
(HbFe.sup.+2--O.sub.2). In hemolyzing animals, oxyhemoglobin
accounted for 71% and 69% of the measured cell-free plasma
hemoglobin in animals with low and high levels of hemolysis
respectively (FIG. 22).
[0232] During nitrite infusions at low levels of hemolysis, the
nitrite reacted with hemoglobin to form approximately 30%
methemoglobin (FIG. 22; values for the D5W+nitrite zero-hemolysis
control are depicted as a blue reference line in FIG. 22B and FIG.
22E). This reaction likely reflects two reactions of nitrite: the
reaction of nitrite with oxyhemoglobin to form methemoglobin and
nitrate (NO.sup.2-+Hb0.sub.2. MetHb+NO.sup.3-) and the reaction of
nitrite with deoxyhemoglobin to form methemoglobin and NO. The
former reaction will decrease NO scavenging and the latter reaction
will contribute to NO generation. Note that paradoxically there is
sufficient oxyhemoglobin at the end of 6 hours to almost completely
scavenge and inhibit any NO that might form, yet the nitrite
remains vasoactive and potentiated by low levels of hemoglobin
(FIG. 21). As shown in FIG. 22B and FIG. 22E, with increasing
hemoglobin concentrations, the rate of methemoglobin formation
increases from zero to 3 hours (p=0.0001) producing higher levels
of methemoglobin from 3 to 6 hours (p=0.0001) in animals with
higher levels of hemolysis compared to animals with lower levels of
hemolysis. This is because the overall reactions of nitrite and
hemoglobin are second order during their lag phases, meaning that
as hemoglobin concentration increases the rate of the reactions
increase. Again, the amount of oxyhemoglobin at the end of the
reaction is sufficient to almost completely scavenge any NO that
might be formed if nitrite acted as a pure NO donor.
Effects of Nitrite and Level of Hemolysis on Hemodynamic Responses
to Sodium Nitroprusside, an Infused NO Donor
[0233] Sodium nitroprus side was administered to all animals to
determine the physiologic effects of a direct NO donating agent in
the setting of hemolysis with and without sodium nitrite. The
physiologic effects of sodium nitroprusside were dependent on the
level (or dose) of hemolysis and the presence of nitrite. As
expected, in animals that did not receive nitrite, sodium
nitroprusside-induced increases in CI and decreases in SVRI and
PVRI were progressively inhibited by increasing levels of
hemolysis, suggesting progressive consumption of the donated NO by
increasing levels of cell-free plasma hemoglobin during
intravascular hemolysis (FIG. 23). In contrast, the effects of
sodium nitroprusside at the three levels of hemolysis were
different in animals receiving nitrite compared to the animals not
receiving nitrite (FIG. 23). Compared to the nonhemolyzing animals
not receiving nitrite (zero-hemolysis, no nitrite), the
non-hemolyzing animals receiving nitrite (zero-hemolysis, nitrite)
demonstrated blunted effects of sodium nitroprusside on CI, SVRI
and PVRI suggesting a decreased vasodilator effect of the donated
NO in the presence of nitrite without hemolysis.
[0234] If the effect of nitrite on the response to sodium
nitroprusside during hemolysis was additive (i.e. same effect at
all levels of hemolysis), then the demonstrated relationship should
be a similar linear relationship to the one demonstrated in the
animals not receiving nitrite, but starting at a smaller magnitude
percent change due to the decreased vasodilator effect of the
donated NO from nitroprus side in the presence of nitrite
(comparing zero hemolysis no nitrite to zero hemolysis+nitrite).
However, in the animals receiving nitrite, the effects of sodium
nitroprusside on CI, SVRI, and PVRI were accentuated with low
levels of hemolysis (Hb<25 .mu.M, nitrite) and then attenuated
with high levels of hemolysis (Hb>25 .mu.M, nitrite) compared to
non-hemolyzing animals (zero hemolysis, nitrite) (FIGS. 23; p=0.09,
0.05 and 0.009 for the interaction demonstrating a different
relationship between level of hemolysis and nitrite on the effect
of sodium nitroprus side for CI, SVRI, and PVRI respectively).
Animals with low level hemolysis demonstrated a similar or greater
percent change on the physiologic variables than zero hemolysis
(instead of the expected smaller effect in an additive model) and
animals with higher level hemolysis demonstrated blunted
physiologic responses. This interaction is consistent with the
U-shaped physiologic effects of nitrite demonstrated during the
6-hour hemolysis study; compared to the effect in animals with zero
hemolysis, the physiologic effect of nitrite is accentuated with
low level hemolysis and then attenuated at higher levels of
hemolysis. This interaction may be explained by the additional
nitrite reduction reaction with hemoglobin contributing to
vasodilation.
[0235] Nitrite reacts with oxy- and deoxy-hemoglobin to form
methemoglobin and methemoglobin+NO respectively (Cosby et al., Nat.
Med. 9:1498-1505, 2003). These nitrite reactions may lead to
enhanced vasodilation by sodium nitroprus side in the setting of
low levels of hemolysis by: 1) minimizing the amount of
oxyhemoglobin available in the plasma to consume the donated NO
from sodium nitroprusside and 2) by directly causing vasodilation
secondary to the NO generated by the reaction of nitrite with
deoxyhemoglobin. At higher levels of intravascular hemolysis, the
nitrite reduction reaction with hemoglobin may be overwhelmed by
the large amounts of cell-free plasma hemoglobin that consume any
NO formed from the reaction. Consequently the donated NO from
sodium nitroprusside and the generated NO from the reaction of
nitrite with deoxyhemoglobin are consumed by the excess
oxyhemoglobin in the plasma.
Confirmatory In Vitro Mitochondrial Respiration Experiments
[0236] In vitro mitochondrial respiration experiments were
performed with nitrite and cell-free hemoglobin levels similar to
those obtained in vivo to confirm that the effects of nitrite on
vasoactivity during hemolysis are dependent on the reaction of
nitrite and deoxyhemoglobin to generate NO. In these experiments,
mitochondria serve as NO sensors because NO avidly binds to
cytochrome-C oxidase to inhibit respiration. In this experimental
system, mitochondria suspended in a closed chamber respire until
the chamber becomes anoxic (oxygen trace reads zero). Removal of
the chamber lid allows oxygen diffusion into the chamber; however
the trace remains at zero due to rapid oxygen consumption by the
respiring mitochondria. The oxygen trace deviates from zero only
once the mitochondria stop respiring due to the exhaustion of
substrate or inhibition (FIG. 18A). Time to inhibition (oxygen
reaccumulation) should be dependent on the rate of NO production
from reactions of nitrite with deoxyhemoglobin and the rate of NO
consumption by excess oxyhemoglobin.
[0237] With the addition of nitrite (18 .mu.M) and low levels of
hemoglobin (10-20 .mu.M), mitochondrial respiration was inhibited
in comparison to mitochondria with nitrite or hemoglobin alone. The
shortest time to inhibition was observed with nitrite and 20 .mu.M
hemoglobin, above which increasing concentrations of hemoglobin
resulted in longer times to inhibition (FIG. 18B). These
mitochondrial inhibition experiments demonstrate a U-shaped
relationship between nitrite and hemoglobin level consistent with
the results of the in vivo experiments described above. The animal
experiments suggest an interaction between the effects of nitrite
and the level of hemolysis such that low levels of hemolysis
accentuate the vasodilatory effects of nitrite.
[0238] These mitochondrial experiments demonstrate that NO
generation and accumulation from nitrite reduction by hemoglobin is
maximal at low levels of hemolysis and decreases with higher levels
of hemolysis. These results suggest that the in vivo accentuated
vasodilatory effects of nitrite during low levels of hemolysis may
be mediated by the generation of NO from the reduction of nitrite
by hemoglobin.
Example 5
Administration of a Cell-Free Blood Substitute Detoxified by
Nitrite to a Human Subject
[0239] This example describes that a cell-free blood substitute can
be detoxified by nitrite and used for treating oxygen deficiency or
replacing lost blood in a human subject.
Patient Selection
[0240] In one embodiment, the human subject is a human diagnosed
with hypoxia, hypoxemia, ischemia, anoxia or another disease for
which treatment includes increasing blood oxygenation by
administration of a blood substitute, and wherein the human subject
is, has been, or will be treated with transfusion of whole blood or
a blood substitute. In another embodiment, the human subject is
afflicted or is predisposed to being afflicted with a disease or
condition treatable by transfusion of whole blood or a blood
substitute, for example, anemia, bleeding disorders, burns,
coagulopathy, ectopic pregnancy, favism, gastrointestinal bleeding,
hemolytic uremic syndrome, hemophilia, microcytosis, ulcer,
hemorrhage, rhabdomyolysis, hemorrhagic shock, sickle cell anemia,
spherocytosis, thalassemia, or yellow fever. In a further
embodiment, the human subject is undergoing, or has undergone, a
surgical procedure wherein a clinically dangerous amount of blood
has been lost, or wherein a clinically dangerous amount of blood
may be lost. In such embodiments, the human subject may develop
shock immediately after blood loss occurs, shortly after blood loss
occurs, or a longer period of time after blood loss occurs. In some
embodiments, the human subject may need to be resuscitated.
[0241] In most embodiments, the human subject is under the care of
a physician. The physician can identify the presence of a disease
or condition treatable by transfusion of whole blood or a blood
substitute in the subject according to any methods disclosed above
or known to one skilled in the art. A representative method of
treatment for such diseases is by administration of a cell-free
hemoglobin based blood substitute detoxified by nitrite. The
physician can also assess the severity of blood loss in a human
subject according to methods known to one skilled in the art, and
determine the necessity of blood replacement. A representative
method for blood replacement in such subjects is by administration
of a cell-free hemoglobin based blood substitute detoxified by
nitrite.
Administration of Cell Free Hemoglobin Based Blood Substitute
Detoxified by Nitrite to a Human Subject
[0242] A therapeutically effective amount of a cell-free hemoglobin
based blood substitute detoxified by nitrite is administered to the
human subject. In some embodiments, the blood substitute is
detoxified by pretreatment with nitrite. In other embodiments,
nitrite is co-administered with the blood substitute to detoxify
the blood substitute. The cell-free hemoglobin based blood
substitute is administered according to any method known to one
skilled in the art. For example, in some embodiments the blood
substitute is administered intravenously. In other embodiments, the
blood substitute is administered intraarterially. In further
embodiments, the blood substitute is administered according to any
technique appropriate for transfusion of whole blood.
[0243] For example, two i.v. bags of cell free hemoglobin would be
prepared: one bag would contain ferric methemoglobin (Fe.sup.III)
with nitrite (at a ratio of less than 1:2); a second bag would
contain oxyhemoglobin (Fe.sup.II--O.sub.2). The two solutions would
be coinfused into a subject at ratios less than 1 part
methemoglobin-nitrite to 1 part oxyhemoglobin. After and during the
infusion, the oxyhemoglobin would deliver oxygen to the tissue as
the oxygen delivery vehicle to form deoxyhemoglobin (Fe.sup.II).
Some of this would react with excess nitrite from the first bag to
form NO. The methemoglobin-nitrite from that same bag would form an
intermediate (Fe.sup.II--NO.sub.2 radical); this would react with
NO to form N.sub.2O.sub.3 and Fe.sup.II (deoxyhemoglobin). The
N.sub.2O.sub.3 would vasodilate and restore NO homeostasis, and the
deoxyhemoglobin would now be able to bind oxygen again in the lung.
This system thus delivers oxygen, generates N.sub.2O.sub.3 and NO,
and redox cycles to rebind oxygen in the lung.
Patient Recovery and Outcome Assessment
[0244] The physician can then assess the therapeutic efficacy of
the cell-free hemoglobin based blood substitute detoxified by
nitrite in increasing blood oxygenation in the human subject
according to any of the methods disclosed above, or according to
methods known to one skilled in the art, wherein a reduction of
symptoms associated with hypoxia in the human subject indicates the
effectiveness of the blood substitute in treating pathological
blood deoxygenation in the subject.
[0245] In some embodiments, the human subject is treated with the
cell-free hemoglobin based blood substitute detoxified by nitrite
until the human subject exhibits relief from hypoxia, for example a
lessening of one or more hypoxic symptoms or a cure, or inhibition
of the development (for instance, prevention) of hypoxia. In such
embodiments, treatment with the blood substitute can be
discontinued at that point, or it can be continued to an endpoint
according to the direction of a physician. It is also possible for
the blood substitute to be administered to the human subject during
the subject's surgical procedure, or following the surgical
procedure. A physician uses methods known to one skilled in the art
to assess vascular tone and blood oxygenation during the procedure
and during the administration of the blood substitute. Blood
substitute is administered according to a regime designed to
restore and/or maintain a desirable vascular tone and level of
blood oxygenation.
[0246] A unique aspect of adding nitrite or nitrite-methemoglobin
to hemoglobin based blood substitutes is that the latter treatments
(on their own) are associated with myocardial infarctions (heart
attacks). Nitrite has potent effects at limiting myocardial
infarction and will thus serve to limit this specific toxicity of
the hemoglobin based blood substitutes. In addition, when nitrite
or nitrite-methemoglobin are administered particularly in settings
of civilian or military trauma with hemorrhage or organ injury, the
cytoprotective effects of nitrite are expected to improve organ
function and survival following resuscitation with hemoglobin-based
blood substitutes.
[0247] This disclosure describes production of cell free blood
substitutes. The disclosure further provides methods of preparing
and using such compositions, as well as the advantages provided by
compositions described herein. It will be apparent that the precise
details of the methods described may be varied or modified without
departing from the spirit of the described invention. We claim all
such modifications and variations that fall within the scope and
spirit of the claims below.
* * * * *