U.S. patent application number 14/795115 was filed with the patent office on 2015-10-29 for five-coordinate neuroglobin and use thereof as a blood substitute.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education, Wake Forest University. Invention is credited to Mark T. Gladwin, Daniel B. Kim-Shapiro, Mauro Tiso.
Application Number | 20150306183 14/795115 |
Document ID | / |
Family ID | 43354589 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150306183 |
Kind Code |
A1 |
Gladwin; Mark T. ; et
al. |
October 29, 2015 |
FIVE-COORDINATE NEUROGLOBIN AND USE THEREOF AS A BLOOD
SUBSTITUTE
Abstract
Described herein is the finding that a mutant form of human
neuroglobin (H64L) with a stable five-coordinate geometry reduces
nitrite to nitric oxide approximately 2000-times faster than the
wild type neuroglobin. Five-coordinate neuroglobin is also capable
of binding and releasing oxygen. Based on these findings, the use
of five-coordinate neuroglobin as a blood substitute is described
herein. Particularly provided is a method of replacing blood and/or
increasing oxygen delivery to tissues in a subject by administering
to the subject a therapeutically effective amount of neuroglobin
with a stable five-coordinate geometry. In some cases,
five-coordinate neuroglobin is administered in combination with
another therapeutic agent or composition, such as a second blood
replacement product (for example, a hemoglobin-based oxygen
carrier), a blood product (such as red blood cells, serum or
plasma) or whole blood.
Inventors: |
Gladwin; Mark T.;
(Pittsburgh, PA) ; Kim-Shapiro; Daniel B.;
(Winston-Salem, NC) ; Tiso; Mauro; (Bethesda,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education
Wake Forest University |
Pittsburgh
Winston-Salem |
PA
NC |
US
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
Wake Forest University
Winston-Salem
NC
|
Family ID: |
43354589 |
Appl. No.: |
14/795115 |
Filed: |
July 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12817085 |
Jun 16, 2010 |
9114109 |
|
|
14795115 |
|
|
|
|
61187527 |
Jun 16, 2009 |
|
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Current U.S.
Class: |
424/530 ;
424/529; 424/531; 424/533; 514/13.4 |
Current CPC
Class: |
A61K 35/14 20130101;
A61P 7/06 20180101; A61P 7/00 20180101; A61P 7/08 20180101; A61K
35/18 20130101; A61P 17/02 20180101; A61P 9/10 20180101; Y02A
50/387 20180101; A61P 9/12 20180101; A61K 38/42 20130101; A61K
9/0019 20130101; A61K 38/41 20130101; A61K 45/06 20130101; A61K
35/16 20130101 |
International
Class: |
A61K 38/41 20060101
A61K038/41; A61K 45/06 20060101 A61K045/06; A61K 9/00 20060101
A61K009/00 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support grant number
HL058091 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of replacing blood in a subject, comprising
administering to the subject a therapeutically effective amount of
neuroglobin with a stable five-coordinate geometry, thereby
replacing blood in the subject.
2. The method of claim 1, wherein the subject has or is at risk of
developing a disease, disorder or injury associated with a
deficiency in red blood cells and/or hemoglobin, or associated with
a reduction in oxygen delivery to tissues.
3. The method of claim 2, wherein the disease, disorder or injury
comprises a bleeding disorder, a bleeding episode, anemia, shock,
ischemia, hypoxia, anoxia, hypoxaemia, a burn, an ulcer, ectopic
pregnancy, microcytosis, rhabdomyolysis, hemoglobinopathy,
spherocytosis, hemolytic uremic syndrome, thalassemia,
disseminating intravascular coagulation, stroke or yellow
fever.
4. The method of claim 3, wherein the bleeding episode results from
anticoagulant overdose, aneurysm, blood vessel rupture, surgery,
traumatic injury, gastrointestinal bleeding, pregnancy, hemorrhage
or infection.
5. The method of claim 3, wherein the bleeding disorder comprises
hemophilia A, hemophilia B, hemophilia C, Factor VII deficiency,
Factor XIII deficiency, a platelet disorder, a coagulopathy,
favism, thrombocytopenia, vitamin K deficiency or von Willebrand's
disease.
6. The method of claim 3, wherein the anemia comprises microcytic
anemia, iron deficiency anemia, 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 or cold
agglutinin hemolytic anemia.
7. The method of claim 3, wherein shock comprises septic shock,
hemorrhagic shock or hypovolemic shock.
8. The method of claim 1, wherein the subject suffers from or is at
risk of suffering from myocardial infarction, stroke,
ischemia-reperfusion injury, pulmonary hypertension or
vasospasm.
9. The method of claim 1, wherein the stable five-coordinate
neuroglobin is human neuroglobin.
10. The method of claim 9, wherein the human neuroglobin is
recombinant human neuroglobin.
11. The method claim 1, wherein the amino acid sequence of the
stable five-coordinate neuroglobin is at least 95% identical to SEQ
ID NO: 9 and comprises a leucine at amino acid residue 64.
12. The method of claim 11, wherein the amino acid sequence of the
stable five-coordinate neuroglobin comprises SEQ ID NO: 9.
13. The method of claim 11, wherein the amino acid sequence of the
stable five-coordinate neuroglobin consists of SEQ ID NO: 9.
14. The method of claim 1, wherein the stable five-coordinate
neuroglobin is administered to the subject intravenously.
15. The method of claim 1, further comprising administering to the
subject a second blood replacement product, a blood product or
whole blood.
16. The method of claim 15, wherein the second blood replacement
product comprises a hemoglobin-based oxygen carrier, artificial red
blood cells or an oxygen releasing compound.
17. The method of claim 15, wherein the blood product comprises
packed red blood cells, plasma or serum.
18. The method of claim 1, wherein the subject is a human.
19. The method of claim 1, 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.
12/817,085, filed Jun. 16, 2010, which claims the benefit of U.S.
Provisional Application No. 61/187,527, filed on Jun. 16, 2009. The
above-referenced applications are incorporated herein by reference
in their entirety.
FIELD
[0003] This disclosure concerns neuroglobin with a stable
five-coordinate geometry and its use as a cell-free blood
substitute.
BACKGROUND
[0004] A phylogenic analysis of the heme-globin family of proteins
indicates that the well-characterized proteins hemoglobin and
myoglobin were antedated by neuroglobin, which existed already 800
million years ago (Hankeln et al., J Inorg Biochem 99:110-119,
2005; Brunori and Vallone, Cell Mol Life Sci 64:1259-1268, 2007).
Neuroglobin (Ngb) sequences remained highly conserved throughout
mammalian evolution, suggesting a strongly selected vital
functionality (Burmester et al., IUBMB Life 56:703-707, 2004). This
heme containing, monomeric, 16.9 kDa protein shares 21-25% sequence
homology with myoglobin and hemoglobin. However, unlike myoglobin
and hemoglobin, it possesses a bis-histidine six-coordinate heme
geometry, such that the proximal and distal histidines in the heme
pocket are directly bonded to the heme iron (both Fe.sup.+2 or
Fe.sup.+3 oxidation states) (Dewilde et al., J Biol Chem
276:38949-38955, 2001). Indeed, at equilibrium the concentration of
the five-coordinate neuroglobin is very low, reported from 0.1 up
to 5% (Uzan et al., Biophys J 87:1196-1204, 2004). Binding of
oxygen or other gas ligands, such as nitric oxide (NO) or carbon
monoxide, to the heme iron occurs upon displacement of the 6.sup.th
coordination bond with the distal histidine 64 residue (Capece et
al., Proteins 75(4):885-894, 2009; Kriegl et al., Proc Natl Acad
Sci USA 99:7992-7997, 2002). Despite this structural difference
with myoglobin, neuroglobin displays comparable .alpha.-helix
globin folding and high oxygen affinity (P.sub.50 about 1-2 mmHg at
20.degree. C.) (Kiger et al., IUBMB Life 56:709-719, 2004; Giuffre
et al., Biochem Biophys Res Commun 367:893-898, 2008). However, the
low tissue concentration of neuroglobin and the rapid
auto-oxidation of the oxygen bound species suggest neuroglobin has
not evolved to store and supply oxygen, leading to a number of
different hypotheses about the physiological function of this
conserved heme-globin (Brunori and Vallone, Cell Mol Life Sci
64:1259-1268, 2007; Burmester and Hankeln, J Exp Biol
212:1423-1428, 2009).
[0005] Despite uncertainty about the molecular functionality of
neuroglobin, expression of this protein produces cytoprotective
effects in vitro and in vivo, limiting neuronal cell death during
glucose deprivation and hypoxia and limiting the volume of brain
infarction in stroke models (Greenberg et al., Curr Opin Pharmacol
8:20-24, 2008; Khan et al., Proc Natl Acad Sci USA 103:17944-17948,
2006; Wang et al., Stroke 39:1869-1874, 2008; Sun et al., Proc Natl
Acad Sci USA 98:15306-15311, 2001). An understanding of the
functionality of neuroglobin could provide a paradigm shift in both
biology and therapeutics, because many heme proteins in plants,
bacteria, invertebrates and vertebrates are both highly conserved
and exist in equilibrium between dominant six-coordinate geometry
and the lower frequency five-coordinate state. Examples of these
six-coordinate heme-proteins include cytoglobin, cytochrome c,
Drosophila melanogaster hemoglobin, and the plant hemoglobins
(Weiland et al., J Am Chem Soc 126:11930-11935, 2004; Nadra et al.,
Proteins 71:695-705, 2008; Garrocho-Villegas et al., Gene
398:78-85, 2007).
[0006] Over the last five years, groups have examined the ability
of deoxygenated hemoglobin and myoglobin to react with and reduce
nitrite to NO (Huang et al., J Clin Invest 115:2099-2107, 2005;
Shiva et al., Circ Res 100:654-661, 2007). It has been proposed
that this reaction serves a function similar to the bacterial
nitrite reductases, in which a coupled electron and proton transfer
to nitrite generates NO.
Fe.sup.+2+NO.sub.2.sup.-+H.sup.+.fwdarw.Fe.sup.+3+NO.+OH.sup.-
(equation 1)
[0007] In the heart, myoglobin can reduce nitrite to NO to regulate
hypoxic mitochondrial respiration and enhance the cellular
resilience to prolonged ischemia, analogous to the cytoprotective
effects of neuroglobin (Shiva et al., Circ Res 100:654-661, 2007).
Studies using the myoglobin knockout mouse have now confirmed that
myoglobin is necessary for nitrite-dependent NO and cGMP generation
in the heart, nitrite-dependent cytoprotection after
ischemia/reperfusion and nitrite-dependent control of hypoxic
cellular respiration (Hendgen-Cotta et al., Proc Natl Acad Sci USA
105:10256-10261, 2008). It is therefore apparent that both
myoglobin and neuroglobin may have roles in limiting cell death
after ischemia-reperfusion injury. Of relevance to neuroglobin, it
has recently been discovered that the mitochondrial protein
cytochrome c can reduce nitrite to NO more rapidly than either
hemoglobin or myoglobin, but only when it assumes the
five-coordinate conformation (Basu et al., J Biol Chem
283:32590-32597, 2008). This conformation only occurs during the
interaction with anionic phospholipids or upon oxidation or
nitration of protein residues, suggesting a post-translational
tertiary structure regulation of nitrite reduction and NO
generation.
[0008] Interestingly, human neuroglobin contains two surface
cysteines (C46 and C55) that form a disulfide bridge upon oxidation
(Hamdane et al., J Biol Chem 278:51713-51721, 2003). Disulfide bond
formation is accompanied by a decrease in the distal histidine
binding affinity to heme iron (K.sub.His, has been shown to
decrease from .about.3000 to 280, values calculated as
k.sub.on/k.sub.off are dimensionless) (Hamdane et al., Micron
35:59-62, 2004). This in turn increases the sub-population of
five-coordinate neuroglobin and increases the affinity for
endogenous ligands such as oxygen (P.sub.50 shift from about 9 to 1
mmHg) (Hamdane et al., J Biol Chem 278:51713-51721, 2003). Nicolis
et al. reported that the oxidized disulfide-bridged neuroglobin
also exhibits a higher affinity for nitrite than the thiol reduced
form (Nicolis et al., Biochem J 407:89-99, 2007).
SUMMARY
[0009] Disclosed herein is the surprising finding that stable
five-coordinate neuroglobin is capable of very rapidly converting
nitrite to NO. Five-coordinate neuroglobin is also capable of
binding and releasing oxygen. Based on these important features,
the use of five-coordinate neuroglobin as a blood substitute is
provided herein. Many of the previously described blood substitutes
are associated with cardiovascular complications due to NO
scavenging, thus five-coordinate neuroglobin represents a
therapeutic compound with the potential to alleviate the toxicity
associated with previous blood substitutes.
[0010] Provided herein is a method of replacing blood and/or
increasing oxygen delivery to tissues in a subject. In some
embodiments, the method includes administering to the subject a
therapeutically effective amount of neuroglobin with a stable
five-coordinate geometry. The subject to be treated, for example,
is any subject in need of increasing blood volume and/or increasing
oxygen and/or NO delivery to tissues. In some embodiments, the
subject has or is at risk of developing a disease, disorder or
injury associated with a deficiency in red blood cells and/or
hemoglobin, or associated with a reduction in oxygen delivery to
tissues. In some embodiments, the subject to be treated suffers
from or is at risk of suffering from a disease or condition
associated with decreased blood flow, such as myocardial
infarction, stroke, ischemia-reperfusion injury, pulmonary
hypertension or vasospasm.
[0011] In some embodiments of the methods disclosed herein, stable
five-coordinate neuroglobin is recombinant human neuroglobin. In
particular examples, five-coordinate neuroglobin is H64L
neuroglobin.
[0012] In some embodiments, the method further includes
administering to the subject a second blood replacement product, a
blood product or whole blood.
[0013] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIGS. 1A-1F: Anaerobic reaction of deoxyneuroglobin with
nitrite in the absence and in the presence of dithionite. (FIG. 1A)
Selected visible spectra of the reaction between 10 .mu.M deoxyNgb
and 10 mM nitrite at 1 minute intervals. (FIG. 1B) Time-dependent
changes of deoxyNgb, iron-nitrosyl-Ngb and total met-Ngb
concentration during the reaction. (FIG. 1C) Selected visible
spectra of the reaction between 10 .mu.M deoxyNgb and 10 mM nitrite
in the presence of 3 mM dithionite at 1 minute intervals. (FIG. 1D)
Time-dependent changes of deoxyNgb, iron-nitrosyl-Ngb and total
met-Ngb concentration during the reaction in the presence of 3 mM
dithionite. (FIG. 1E) Plot of observed rate constants (k.sub.obs)
versus nitrite concentration; the second-order bimolecular rate
constant obtained from the linear fit of the data is 0.12.+-.0.02
M.sup.-1 sec.sup.-1. (FIG. 1F) Effect of pH on the nitrite
reductase reaction rates. Inset: BRC is linear with the proton
concentration and it extends through the zero point (line shows
linear regression analysis of the data). All measurements were made
in 100 mM phosphate buffer and 25.degree. C. as described in
Example 1.
[0015] FIGS. 2A-2F: Redox state of cysteines 46 and 55 modulates
nitrite reductase reactivity. (FIG. 2A) Model of the wild-type
human neuroglobin structure with indicated reduced cysteines C46,
C55 and C120. (FIG. 2B) Determination of the number of reduced
cysteines by the 4-PDS assay (see Example 1). (FIG. 2C) Comparison
of the decrease of deoxy-Ngb and the formation of iron-nitrosyl Ngb
over time for wild-type Ngb with oxidized (SS) and reduced (SH)
thiol, C46A and C55A mutant Ngb. (FIG. 2D) Observed nitrite
reductase rate constants versus determined redox potentials. The
midpoint redox potential of the thiol/disulfide couple in wild-type
Ngb is -194.+-.3 mV. (FIG. 2E) Comparison of the NMR spectrum of
wild type and C55A mutant met-Ngb. (FIG. 2F) Nitrite binding
affinity constant for wild-type, DTT cysteines reduced and C55A
mutant Ngb.
[0016] FIGS. 3A-3E: Kinetics of nitrite reaction with mutant H64L
Ngb. (FIG. 3A) and (FIG. 3B) Spectrophotometric analysis of the
anaerobic reaction of 10 .mu.M H64L deoxy-Ngb with 100 .mu.M
nitrite at pH 7.4, 25.degree. C. and 3 mM dithionite. (FIG. 3C)
Plot of k.sub.obs versus nitrite concentration (10 .mu.M-1 mM) for
H64L Ngb-mediated reduction of nitrite and formation of Ngb
Fe(II)NO at pH 7.4 and 25.degree. C. The bimolecular rate constant
derived from the linear fit of the data is 259.+-.8 M.sup.-1
s.sup.-1. (FIG. 3D) Effect of different pH on the nitrite reductase
rates. Inset: BRC is linear with the proton concentration. (FIG.
3E) Comparison of representative traces of Ngb wild-type (with
reduced and oxidized surface thiols) and mutants H64L and C55A. The
absorbance decreases of the Soret peak (425 nm) are plotted as the
percentage of the total absorbance change for human Ngb H64L
measured at 25.degree. C., pH 7.4.
[0017] FIGS. 4A-4D: Electron paramagnetic resonance (EPR)
spectroscopy. (FIG. 4A) and (FIG. 4C) EPR spectra showing Fe(II)-NO
build-up following addition of indicated amount of nitrite. (FIG.
4B) and (FIG. 4D) The rate of formation of iron-nitrosyl-heme
(Fe.sup.+2-NO) species measured by EPR. The concentrations were
determined by performing the double integral calculation and
comparing to standard samples.
[0018] FIGS. 5A-5C: Nitrite reduction by deoxyneuroglobin generates
NO gas. (FIG. 5A) Representative chemiluminescence traces of NO
detection in gas phase released during the anaerobic reaction of
nitrite with buffer only or 20 .mu.M deoxyNgb wild type, H64L or
C55A. (FIG. 5B) Quantification of the rate of NO detected per
minute. (FIG. 5C) The nitric oxide signal measured during
incubation of 30 .mu.M H64L deoxyNgb and increasing concentrations
of nitrite.
[0019] FIGS. 6A-6E: Deoxyneuroglobin nitrite reduction mediates
intracellular NO signaling. (FIG. 6A) Traces of oxygen consumption
by isolated mitochondria showing nitrite dependent inhibition of
respiration; the early rise in oxygen tension indicates
NO-dependent inhibition of cellular respiration which is maximal
for cyanide. (FIG. 6B) Comparison of percentage of extent of
inhibition (cyanide defined as 100% inhibition) as measured in
(FIG. 6A) for isolated mitochondria. (FIG. 6C) Quantification of
expression of GFP only, wild type Ngb and H64L mutant Ngb in
lentivirus transfected and cloned SHSYSY cells by Western blot of
4-15% SDS-polyacrylamide gradient gel. (FIG. 6D) Mean extent of
hypoxic inhibition of cellular respiration by incubation of SHSYSY
cells expressing GFP, wild type Ngb or H64L Ngb with 20 .mu.M
nitrite (*P<0.01, ** P<0.05, compared with control). (FIG.
6E) Intracellular NO signaling mediated by deoxyneuroglobin nitrite
reduction determined as cGMP formation in SHSY5Y neuronal cells (*
P<0.01).
[0020] FIGS. 7A-7C: Visible standard reference spectra of
neuroglobin and myoglobin proteins utilized for deconvolution.
Visible spectra of deoxy-, oxy-, met-, and iron-nitrosyl-human
wild-type Ngb (FIG. 7A), H64L Ngb (FIG. 7B) and myoglobin (FIG.
7C). Spectra were normalized at 700 nm and utilized for
least-squares analysis of multi-component spectra.
[0021] FIGS. 8A-8B: Anaerobic reaction of myoglobin (Mb) with
nitrite in the absence and in the presence of dithionite. (FIG. 8A)
Time-dependent changes of deoxy-Mb, iron nitrosyl-Mb and total
met-Mb concentration during the reaction of 50 .mu.M deoxy-Mb with
2.5 mM nitrite. (FIG. 8B) Time-dependent changes of deoxy-Mb, iron
nitrosyl-Mb and total met-Mb concentration during the reaction of
50 .mu.M deoxy-Mb with 2.5 mM nitrite in the presence of 2 mM
dithionite. Myoglobin reacts with nitrite with a BRC of 2.9.+-.0.2
M.sup.-1 sec.sup.-1 in 100 mM phosphate buffer containing mM EDTA,
pH 7.4 and at 25.degree. C.
SEQUENCE LISTING
[0022] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand.
The Sequence Listing is submitted as an ASCII text file, created on
Jul. 7, 2015, 8.42 KB, which is incorporated by reference
herein.
[0023] In the accompanying sequence listing:
[0024] SEQ ID NOs: 1-6 are the nucleotide sequences of
oligonucleotides used for site-directed mutagenesis of
neuroglobin.
[0025] SEQ ID NOs: 7 and 8 are the nucleotide and amino acid
sequences of human neuroglobin (GENBANK.RTM. Accession No.
NM.sub.--021257, incorporated herein by reference as it appears in
the GENBANK.RTM. database on Jun. 16, 2010).
[0026] SEQ ID NO: 9 is the amino acid sequence of H64L
neuroglobin.
DETAILED DESCRIPTION
I. Introduction
[0027] Hemoglobin and myoglobin evolved from a common ancestor of
neuroglobin, a highly conserved hemoprotein of uncertain
physiological function. Neuroglobin possesses a bis-histidine
six-coordinate heme geometry, such that the proximal and distal
histidines in the heme pocket are directly bound to the heme iron.
The present disclosure describes the new finding that deoxygenated
human neuroglobin reacts with and reduces nitrite to form NO.
Remarkably, this reaction is allosterically regulated by redox
sensitive surface thiols, cysteine 55 and 46, which regulate the
open probability of heme pocket, nitrite binding and NO formation.
Using site directed mutagenesis, it was demonstrated herein that a
stable five-coordinate neuroglobin mutant (H64L) reduces nitrite to
NO approximately 2000-times faster than wild type neuroglobin,
while mutation of either C55 or C46 to alanine stabilizes the
six-coordinate structure and slows the reaction. Lentivirus
expression systems were used to confirm that the six-to-five
coordinate status of neuroglobin regulates canonical intracellular
hypoxic NO signaling pathways
[0028] These studies suggest that neuroglobin functions as a
post-translationally redox-regulated nitrite reductase that
generates NO under six-to-five coordinate heme pocket control. The
surprising ability of five-coordinate neuroglobin to rapidly
convert nitrite to NO, and its ability to bind and release oxygen,
makes five-coordinate neuroglobin a potential cell-free,
hemoglobin-based blood substitute. As many of the previously
described blood substitutes are associated with cardiovascular
complications (e.g., vasoconstriction, brachycardia, and
hypertension) due to NO scavenging, five coordinate neuroglobin
represents a novel therapeutic compound with the potential to solve
the major toxicity of current blood substitutes.
II. Abbreviations
[0029] BRC bimolecular rate constant
[0030] DTT dithiothreitol
[0031] EPR electron paramagnetic resonance
[0032] GSH reduced glutathione
[0033] GSSG oxidized glutathione
[0034] HBOC hemoglobin-based oxygen carrier
[0035] IPTG isopropyl-.beta.-D-thio-galactosidase
[0036] Mb myoglobin
[0037] Met-Ngb ferric neuroglobin
[0038] Ngb neuroglobin
[0039] NMR nuclear magnetic resonance
[0040] NO nitric oxide
[0041] PFC perfluorocarbon
[0042] RBC red blood cell
[0043] SH reduced thiol
[0044] SS oxidized thiol
[0045] UV ultraviolet
III. Terms and Methods
[0046] 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).
[0047] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0048] Anemia: A deficiency of red blood cells 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).
[0049] 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.
[0050] 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.
[0051] Anoxia: A pathological condition in which the body as a
whole or region of the body is completely deprived of oxygen
supply.
[0052] Bleeding disorder: 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. Bleeding disorders include any congenital, acquired or
induced defect that results in abnormal (or pathological) bleeding.
Examples include, but are not limited to, disorders of insufficient
clotting or hemostasis, such as hemophilia A (a deficiency in
Factor VIII), hemophilia B (a deficiency in Factor IX), hemophilia
C (a deficiency in Factor XI), other clotting factor deficiencies
(such as Factor VII or Factor XIII), abnormal levels of clotting
factor inhibitors, platelet disorders, thrombocytopenia, vitamin K
deficiency and von Willebrand's disease.
[0053] Bleeding episode: Refers to an occurrence of uncontrolled,
excessive and/or pathological bleeding. Bleeding episodes can
result from, for example, drug-induced bleeding (such as bleeding
induced by non-steroidal anti-inflammatory drugs or warfarin),
anticoagulant overdose or poisoning, aneurysm, blood vessel
rupture, surgery and traumatic injury (including, for example,
abrasions, contusions, lacerations, incisions or gunshot wounds).
Bleeding episodes can also result from diseases such as cancer,
gastrointestinal ulceration or from infection.
[0054] Blood: The fluid that circulates through the heart,
arteries, capillaries and veins (that is, the circulatory system),
and is the primary transport mechanism in the body. Blood
transports oxygen from the lungs to the body tissues and carbon
dioxide from the tissues to the lungs. Blood also transports
nutritive substances and metabolites to the tissues and removes
waste products to the kidneys and other organs for excretion. In
addition, blood plays a critical role in maintenance of fluid
balance. Blood has two primary parts--plasma (the fluid portion)
and formed elements (the solid components). The solid components of
blood include erythrocytes (red blood cells), leukocytes (white
blood cells) and platelets. As used herein, "whole blood" refers to
blood that has not had any components removed (blood that contains
both the fluid and solid components). A "blood product" refers to
one or more components of the blood, such as red blood cells, serum
or plasma.
[0055] Blood replacement product or blood substitute: A composition
used to fill fluid volume and/or carry oxygen and other blood gases
in the cardiovascular system. Blood substitutes include, for
example, volume expanders (to increase blood volume) and oxygen
therapeutics (to transport oxygen in blood). Oxygen therapeutics
include, for example, hemoglobin-based oxygen carriers (HBOC) and
perfluorocarbons (PFCs).
[0056] 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 which is free of side effects and pathogens.
[0057] 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. Studies conducted in animal systems showed
that infusion of cell-free hemoglobin caused a substantial increase
in oncotic pressure because of its hyperosmolarity, coagulopathy,
and hypertensive properties.
[0058] 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.
[0059] Modified Hb molecules have been produced in an attempt to
overcome other limitations of Hb 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 for humans in the United States.
[0060] Burns: Any extremity experienced by the skin caused by heat,
cold, electricity, chemicals, friction or radiation.
[0061] 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.
[0062] Cerebral ischemia or ischemic stroke: A condition that
occurs when an artery to or in the brain is partially or completely
blocked such that the oxygen demand of the tissue exceeds the
oxygen supplied. Deprived of oxygen and other nutrients following
an ischemic stroke, the brain suffers damage as a result of the
stroke.
[0063] Ischemic stroke can be caused by several different kinds of
diseases. The most common problem is narrowing of the arteries in
the neck or head. This is most often caused by atherosclerosis, or
gradual cholesterol deposition. If the arteries become too narrow,
blood cells may collect in them and form blood clots (thrombi).
These blood clots can block the artery where they are formed
(thrombosis), or can dislodge and become trapped in arteries closer
to the brain (embolism).
[0064] Another cause of stroke is blood clots in the heart, which
can occur as a result of irregular heartbeat (for example, atrial
fibrillation), heart attack, or abnormalities of the heart valves.
While these are the most common causes of ischemic stroke, there
are many other possible causes. Examples include use of street
drugs, traumatic injury to the blood vessels of the neck, or
disorders of blood clotting.
[0065] Ischemic stroke is by far the most common kind of stroke,
accounting for about 80% of all strokes. Stroke can affect people
of all ages, including children. Many people with ischemic strokes
are older (60 or more years old), and the risk of stroke increases
with older ages. At each age, stroke is more common in men than
women, and it is more common among African-Americans than white
Americans. Many people with stroke have other problems or
conditions which put them at higher risk for stroke, such as high
blood pressure (hypertension), heart disease, smoking, or
diabetes.
[0066] Coagulopathy: A medical term for a defect in the body's
mechanism for blood clotting.
[0067] Ectopic pregnancy: A complication of pregnancy in which the
fertilized ovum is implanted in any tissue other than the uterine
wall.
[0068] 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.
[0069] Gastrointestinal bleeding: Refers to any form of hemorrhage
(loss of blood) in the gastrointestinal tract, from the pharynx to
the rectum.
[0070] 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.
[0071] 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 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.
[0072] 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.
[0073] 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.
[0074] Hemoglobin-based oxygen carrier (HBOC): A transfusable fluid
of purified, recombinant and/or modified hemoglobin that functions
as an oxygen carrier and can be used as a blood substitute. A
number of HBOCs are known and/or in clinical development. Examples
of HBOCs include, but are not limited to, DCLHb (HEMASSIST.TM.;
Baxter), MP4 (HEMOSPAN.TM.; Sangart), pyridoxylated Hb
POE-conjugate (PHP)+catalase & SOD (Apex Biosciences),
O-R-PolyHbAo (HEMOLINK.TM.; Hemosol), PolyBvHb (HEMOPURE.TM.;
Biopure), PolyHb (POLYHEME.TM.; Northfield), rHb1.1 (OPTRO.TM.;
Somatogen), PEG-Hemoglobin (Enzon), OXYVITA.TM. and HBOC-201
(Greenburg and Kim, Crit Care 8(Suppl 2):S61-S64, 2004; to Lintel
Hekkert et al., Am J Physiol Heart Circ Physiol 298:H1103-H1113,
2010; Eisenach, Anesthesiology 111:946-963, 2009).
[0075] Hemolysis: The breaking open of red blood cells and the
release of hemoglobin into the surrounding fluid.
[0076] 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); post-partum
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.
[0077] Hemophilia: The name of several hereditary genetic illnesses
that impair the body's ability to control coagulation.
[0078] 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 the vagina, mouth or rectum, or through a break in the
skin.
[0079] 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.
[0080] 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.
[0081] Hemorrhage is broken down into four classes by the American
College of Surgeons' Advanced Trauma Life Support:
[0082] 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.
[0083] 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 crystaloids (saline solution or Lactated
Ringer's solution) is all that is typically required. Atypically,
blood transfusion may be required.
[0084] 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 crystaloid
and blood transfusion are usually necessary.
[0085] 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.
[0086] 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.
[0087] Hypoxaemia: An abnormal deficiency in the concentration of
oxygen in arterial blood.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] Microcytosis: A blood disorder characterized by the presence
of microcytes (abnormally small red blood cells) in the blood.
[0094] Neuroglobin: A member of the vertebrate globin family,
believed to be involved in cellular oxygen homeostasis. Neuroglobin
is an intracellular hemoprotein expressed in the central and
peripheral nervous system, cerebrospinal fluid, retina and
endocrine tissues. Neuroglobin is a monomer that reversibly binds
oxygen with an affinity higher than that of hemoglobin. It also
increases oxygen availability to brain tissue and provides
protection under hypoxic or ischemic conditions, potentially
limiting brain damage. Neuroglobin is of ancient evolutionary
origin, and is homologous to nerve globins of invertebrates. In
some embodiments herein, neuroglobin is human neuroglobin (for
example, with the amino acid sequence of SEQ ID NO: 8). In some
embodiments, neuroglobin is a mutant form of neuroglobin that
causes the protein to retain a stable five-coordinate geometry. In
particular examples, the mutant neuroglobin comprises a mutation at
residue 64 (H64L; the amino acid sequence of which is set forth
herein as SEQ ID NO: 9). Nienhaus et al. (J Biol Chem
279(22):22944-22952, 2004) describe mutant forms of mouse
neuroglobin, including H64L neuroglobin.
[0095] Nitrite: The inorganic anion .sup.-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 some cases, 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.
[0096] 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.
[0097] Peripheral Vascular Disease (PVD): A condition in which the
arteries that carry blood to the arms or legs become narrowed or
occluded. This interferes with the normal flow of blood, sometimes
causing pain but often causing no readily detectable symptoms at
all.
[0098] The most common cause of PVD is atherosclerosis, a gradual
process in which cholesterol and scar tissue build up, forming
plaques that occlude the blood vessels. In some cases, PVD may be
caused by blood clots that lodge in the arteries and restrict blood
flow. PVD affects about one in 20 people over the age of 50, or 8
million people in the United States. More than half the people with
PVD experience leg pain, numbness or other symptoms, but many
people dismiss these signs as "a normal part of aging" and do not
seek medical help. The most common symptom of PVD is painful
cramping in the leg or hip, particularly when walking. This
symptom, also known as "claudication," occurs when there is not
enough blood flowing to the leg muscles during exercise, such that
ischemia occurs. The pain typically goes away when the muscles are
rested.
[0099] Other symptoms may include numbness, tingling or weakness in
the leg. In severe cases, people with PVD may experience a burning
or aching pain in an extremity such as the foot or toes while
resting, or may develop a sore on the leg or foot that does not
heal. People with PVD also may experience a cooling or color change
in the skin of the legs or feet, or loss of hair on the legs. In
extreme cases, untreated PVD can lead to gangrene, a serious
condition that may require amputation of a leg, foot or toes.
People with PVD are also at higher risk for heart disease and
stroke.
[0100] 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.
[0101] Plasma: The fluid portion of the blood in which the formed
elements (blood cells) are suspended.
[0102] Preeclampsia: A disease of unknown cause in pregnant women,
characterized by hypertension, abnormal blood vessels in the
placenta, and protein in the urine. It often but not always occurs
with gestational diabetes or in diabetics. Additional symptoms may
include water retention, leading to swelling in the face, hands and
feet, and greater weight gain. Also called toxemia. Preeclampsia
can lead to eclampsia if not treated. The only known cure for
preeclampsia is delivery of the child.
[0103] 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.
[0104] Reperfusion: Restoration of blood supply to tissue that is
ischemic, due to decrease in blood supply. Reperfusion is a
procedure for treating infarction or other ischemia, by enabling
viable ischemic tissue to recover, thus limiting further necrosis.
However, it is thought that reperfusion can itself further damage
the ischemic tissue, causing reperfusion injury.
[0105] 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.
[0106] Serum: The clear portion of plasma that does not contain
fibrinogen, cells or any solid elements.
[0107] 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.
[0108] Spherocytosis: An auto-hemolytic anemia characterized by the
production of red blood cells (or erythrocytes) that are
sphere-shaped, rather than donut-shaped.
[0109] Subject: Living multi-cellular organisms, including
vertebrate organisms, a category that includes both human and
non-human mammals.
[0110] 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.
[0111] Therapeutically effective amount: A quantity of compound or
composition, for instance, recombinant five-coordinate neuroglobin,
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 or blood loss. It can also be the amount necessary to
restore normal vascular tone and oxygenation to a subject suffering
from hemorrhage.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] Vasospasm: Another cause of stroke; occurs secondary to
spasm of blood vessels supplying the brain. This type of stroke
typically follows a subarachnoid aneurismal hemorrhage with a
delayed development of vasospasm within 2-3 weeks of the bleeding
event. A similar type of stroke may complicate sickle cell
disease.
[0117] Yellow fever: An acute viral disease that is a cause of
hemorrhagic illness, particularly in many African and South
American countries.
[0118] 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 disclosure 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 disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references (including Accession
numbers) 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.
IV. Overview of Several Embodiments
[0119] Disclosed herein is the finding that stable five-coordinate
neuroglobin can not only bind and release oxygen, but is capable of
very rapidly converting nitrite to NO. In particular, a mutant form
of human neuroglobin, referred to as H64L neuroglobin (the amino
acid sequence of which is set forth herein as SEQ ID NO: 9) is
capable of reducing nitrite to NO approximately 2000-times faster
than the wild type. Based on these important features of
five-coordinate neuroglobin, the use of five-coordinate neuroglobin
as a blood substitute is described herein. Many of the previously
described blood substitutes are associated with cardiovascular
complications due to NO scavenging, thus five-coordinate
neuroglobin represents a new therapeutic compound with the
potential to alleviate the toxicity associated with current blood
substitutes.
[0120] Accordingly, provided herein is a method of replacing blood
and/or increasing oxygen delivery to tissues in a subject. In some
embodiments, the method includes administering to the subject a
therapeutically effective amount of neuroglobin with a stable
five-coordinate geometry, thereby replacing blood and/or increasing
oxygen delivery in the subject.
[0121] The subject to be treated, for example, is any subject in
need of increasing blood volume or increasing oxygen delivery to
tissues. In some embodiments, the subject has or is at risk of
developing a disease, disorder or injury associated with a
deficiency in red blood cells and/or hemoglobin, or associated with
a reduction in oxygen delivery to tissues. In some examples, the
disease, disorder or injury comprises a bleeding disorder, a
bleeding episode, anemia, shock, ischemia, hypoxia, anoxia,
hypoxaemia, a burn, an ulcer, ectopic pregnancy, microcytosis,
rhabdomyolysis, hemoglobinopathy, spherocytosis, hemolytic uremic
syndrome, thalassemia, disseminating intravascular coagulation,
stroke or yellow fever.
[0122] In some embodiments, the bleeding episode in the subject to
be treated with five-coordinate neuroglobin results from
anticoagulant overdose, aneurysm, blood vessel rupture, surgery,
traumatic injury, gastrointestinal bleeding, pregnancy, hemorrhage
or infection.
[0123] In some embodiments, the bleeding disorder in the subject to
be treated with five-coordinate neuroglobin comprises hemophilia A,
hemophilia B, hemophilia C, Factor VII deficiency, Factor XIII
deficiency, a platelet disorder, a coagulopathy, favism,
thrombocytopenia, vitamin K deficiency or von Willebrand's
disease.
[0124] In some embodiments, the anemia in the subject to be treated
with five-coordinate neuroglobin comprises microcytic anemia, iron
deficiency anemia, 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 or cold agglutinin hemolytic
anemia.
[0125] In some embodiments, shock in the subject to be treated with
five-coordinate neuroglobin comprises septic shock, hemorrhagic
shock or hypovolemic shock.
[0126] In some embodiments, the subject to be treated suffers from
or is at risk of suffering from a disease or condition associated
with decreased blood flow, such that increased oxygen and NO
delivery is beneficial for treatment of the subject. Examples of
diseases or conditions that can be treated using the disclosed
methods include, but are not limited to, ischemia, myocardial
infarction, stroke, ischemia-reperfusion injury, elevated blood
pressure, pulmonary hypertension (including neonatal pulmonary
hypertension, primary pulmonary hypertension, and secondary
pulmonary hypertension), systemic hypertension, cutaneous
ulceration, acute renal failure, chronic renal failure,
intravascular thrombosis, an ischemic central nervous system event,
vasospasm (such as cerebral artery vasospasm), a hemolytic
condition, peripheral vascular disease, trauma, cardiac arrest,
general surgery or organ transplantation. Diseases and conditions
that benefit from treatment that results in increased NO delivery
are described in, for example, PCT Publication No. WO 2005/004884,
the disclosure of which is herein incorporated by reference.
[0127] The five-coordinate neuroglobin can any type of neuroglobin
with a stable five-coordinate geometry that retains the capacity to
bind and release oxygen and rapidly reduce nitrite to nitric oxide.
For example, the stable five-coordinate neuroglobin can be a mutant
and/or recombinant form of neuroglobin. In some embodiments, the
amino acid sequence of the stable five-coordinate neuroglobin is at
least 85%, at least 90%, at least 95% or at least 99% identical to
SEQ ID NO: 9 and comprises a leucine at amino acid residue 64. In
particular examples, the amino acid sequence of the stable
five-coordinate neuroglobin comprises SEQ ID NO: 9, or consists of
SEQ ID NO: 9.
[0128] The neuroglobin can further be human neuroglobin or
neuroglobin from other species, such as non-human primate
neuroglobin, bovine neuroglobin or murine neuroglobin. In
particular examples, the five-coordinate neuroglobin is recombinant
human neuroglobin.
[0129] Five-coordinate neuroglobin can be administered to the
subject using any suitable route of administration. In some
embodiments, the stable five-coordinate neuroglobin is administered
to the subject intravenously. In other embodiments, the stable
five-coordinate neuroglobin is administered to the subject
intraarterially.
[0130] The subject can either be administered stable
five-coordinate neuroglobin alone or can be administered a second
therapeutic agent or composition, such as a second blood
replacement product (also referred to as a blood substitute), a
blood product or whole blood.
[0131] In some embodiments, the subject is administered a second
blood replacement product. In some examples, the second blood
replacement product comprises a hemoglobin-based oxygen carrier
(HBOC), artificial red blood cells, an oxygen releasing compound,
or other blood substitute product. A number of HBOCs are known in
the art and are described herein.
[0132] In some embodiments, the subject is administered a blood
product. In some examples, the blood product comprises packed red
blood cells, plasma or serum.
[0133] The five-coordinate neuroglobin can be administered to a
subject in a single dose (such as a single infusion), or can be
administered repeatedly as needed. The dose and dosing schedule can
be determined by a medical professional.
[0134] In some embodiments of the methods disclosed herein, the
subject is a human. In other embodiments, the subject is a
non-human animal.
V. Five-Coordinate Neuroglobin as a Blood Substitute
[0135] The "holy grail" of the transfusion medicine field has been
the prospect of developing a cell-free hemoglobin-based oxygen
carrier as a red blood cell substitute. Over the last ten years, an
estimated investment of more than one billion dollars by the U.S.
Department of Defense and pharmaceutical companies has ground to a
halt based on a previously unsuspected reaction: the scavenging
reaction of endothelial derived NO with the hemoglobins. This
scavenging of NO has adverse consequences on vascular function
because NO is a critical regulator of blood vessel homeostasis by
producing tonic vasodilation, inhibiting thrombosis and platelet
activation, and down-regulating the expression of endothelial
adhesion molecules. Therefore, the complete scavenging of NO by
infused hemoglobin solutions in clinical trials resulted in
hypertension, renal failure, myocardial infarction and possible
increases in mortality.
[0136] Second generation hemoglobin molecules have been developed
that are decorated with macromolecules to increase their molecular
size, and while these products have reduced the hypertensive
effects to some extent, the physiological perturbations of NO
depletion remain problematic. Thus, a need exists for an oxygen
carrier molecule that can bind and deliver molecular oxygen and
generate NO, rather than simply destroying it. Such a molecule
would offer the potential to solve this central problem in the
blood substitute field. The data disclosed herein indicate that
five-coordinate neuroglobin meets these criteria. Mutation of the
proximal histidine (the histidine at residue 64, numbered with
reference to SEQ ID NO: 8) produces a unique molecule that rapidly
generates NO from nitrite at enzyme-like rates, but also stably
binds and releases oxygen.
[0137] Thus, described herein is the use of stable five-coordinate
hemoglobin as a blood substitute. Provided is a method of replacing
blood and/or increasing oxygen delivery to tissues in a subject. In
some embodiments, the method includes administering to the subject
a therapeutically effective amount of neuroglobin with a stable
five-coordinate geometry, thereby replacing blood and/or increasing
oxygen delivery in the subject. Five-coordinate neuroglobin is
contemplated for use as a blood substitute for the treatment of a
number of diseases, disorders or injuries that result in a loss of
blood volume and/or a deficiency of oxygen delivery to tissues.
[0138] In many cases, the subject to be treated with
five-coordinate neuroglobin has or is at risk of developing a
disease, disorder or injury associated with a deficiency in red
blood cells and/or hemoglobin, or associated with a reduction in
oxygen delivery to tissues. Exemplary diseases, disorders and
injuries include, but are not limited to bleeding disorders (such
as hemophilia A, hemophilia B, hemophilia C, Factor VII deficiency,
Factor XIII deficiency, a platelet disorder, a coagulopathy,
favism, thrombocytopenia, vitamin K deficiency or von Willebrand's
disease), bleeding episodes (such as a bleeding episode that
results from anticoagulant overdose, aneurysm, blood vessel
rupture, surgery, traumatic injury, gastrointestinal bleeding,
pregnancy, hemorrhage or infection), anemia (such as microcytic
anemia, iron deficiency anemia, 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 or cold
agglutinin hemolytic anemia), shock (such as septic shock,
hemorrhagic shock or hypovolemic shock), ischemia, hypoxia, anoxia,
hypoxaemia, a burn, an ulcer, ectopic pregnancy, microcytosis,
rhabdomyolysis, hemoglobinopathy, spherocytosis, hemolytic uremic
syndrome, thalassemia, disseminating intravascular coagulation,
stroke or yellow fever.
[0139] In some embodiments, the subject to be treated suffers from
or is at risk of suffering from a disease or condition associated
with decreased blood flow, such as myocardial infarction, stroke,
ischemia-reperfusion injury, pulmonary hypertension or
vasospasm.
[0140] Five-coordinate neuroglobin can be administered to the
subject using any suitable route of administration, such as
intravenous or intraarterial. In addition, the subject can either
be administered stable five-coordinate neuroglobin as a single
therapeutic compound or the subject can be treated with a second
(or additional) therapeutic agent or composition. For example,
five-coordinate neuroglobin can be administered in combination with
a second blood substitute, a blood product or whole blood. As used
herein, "co-administration" of a second therapeutic composition is
not limited to administration at the same time as five-coordinate
neuroglobin or in the same composition as five-coordinate
neuroglobin, but rather includes administration prior to and
following administration of five-coordinate neuroglobin. For
example, administration of the second therapeutic agent or
composition can occur 1 hour, 2 hours, 8 hours, 12 hours, 24 hours,
2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or
4 weeks prior to or following administration of five-coordinate
neuroglobin.
[0141] In some cases, the subject is co-administered a second blood
replacement product, such as a blood expander (to increase blood
volume) or an oxygen therapeutic (such as an HBOC or PFC). In some
examples, the second blood replacement product comprises a HBOC,
artificial red blood cells or an oxygen releasing compound. A
number of HBOCs are known in the art and are described herein.
Non-limiting examples of HBOCs include DCLHb (HEMASSIST.TM.;
Baxter), MP4 (HEMOSPAN.TM.; Sangart), pyridoxylated Hb
POE-conjugate (PHP)+catalase & SOD (Apex Biosciences),
O--R-PolyHbAo (HEMOLINK.TM.; Hemosol), PolyBvHb (HEMOPURE.TM.;
Biopure), PolyHb (POLYHEME.TM.; Northfield), rHb1.1 (OPTRO.TM.;
Somatogen), PEG-Hemoglobin (Enzon), OXYVITA.TM. and HBOC-201
(Greenburg and Kim, Crit Care 8(Suppl 2):561-S64, 2004; to Lintel
Hekkert et al., Am J Physiol Heart Circ Physiol 298:H1103-H1113,
2010; Eisenach, Anesthesiology 111:946-963, 2009).
[0142] In some cases, the subject is co-administered a blood
product, such as packed red blood cells, plasma or serum.
[0143] The five-coordinate neuroglobin can be administered to a
subject in a single dose (such as a single infusion), or can be
administered repeatedly as needed. The dose and dosing schedule can
be determined by a medical professional.
[0144] The actual dosage of five-coordinate neuroglobin will vary
according to factors such as the type and severity of disease,
disorder or injury and particular status of the subject (for
example, the subject's age, size, fitness, extent of symptoms,
susceptibility factors, and the like), time and route of
administration, other drugs or treatments being administered
concurrently. Dosage regimens can be adjusted to provide an optimum
therapeutic response. A therapeutically effective amount is also
one in which any toxic or detrimental side effects of the blood
substitute and/or other therapeutic agent is outweighed in clinical
terms by therapeutically beneficial effects.
[0145] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1
Material and Methods
[0146] This example describes the experimental procedures used for
the studies described in Example 2.
Reagents and General Methods
[0147] All reagents were purchased from Sigma-Aldrich unless
otherwise specified. UV-visible spectra and kinetic data were
recorded on an HP8453 UV-Vis spectrophotometer (Hewlett-Packard)
using 1 cm path length quartz or special optical glass cuvettes.
Superdex 5200 gel filtration columns were purchased from GE
Healthcare Life Science. Horse heart myoglobin (Mb) was purified by
passing through a Sephadex.TM. G-25 gel filtration column and
elution with 100 mM potassium phosphate buffer (pH 7.4). Solutions
of sodium dithionite and nitrite were prepared and kept at
25.degree. C. with argon degassed 0.1 M phosphate buffer (pH 7.4)
under inert gas.
Standards Sample Preparation
[0148] Neuroglobin was oxidized with excess potassium ferricyanide
or reduced by incubation with 500 mM sodium dithionite; excess
reagents were removed by passing the mixture through two sequential
Sephadex.TM. G-25 desalting columns. Met-Ngb concentrations were
estimated by measuring the absorbance of the heme Soret band using
.epsilon..sub.414=129 mM.sup.-1 cm.sup.-1. Standard reference
species of recombinant Ngb for spectral deconvolution were prepared
following procedures previously described for hemoglobins (Shiva et
al., Circ Res 100:654-661, 2007; Grubina et al., J Biol Chem
282:12916-12927, 2007). Reference spectra were recorded for
deoxy-Ngb, iron-nitrosyl-Ngb, met-Ngb, and oxy-Ngb. When necessary,
anaerobic reduced Ngb samples were prepared in glovebox under a
2%-4% H2 atmosphere of catalyst-deoxygenated nitrogen, collected
directly in cuvettes and sealed with rubber septa inside the
glovebox. To reduce the intramolecular Ngb disulfide bond, Ngb
solutions were dialyzed in PBS containing 10 mM DTT dissolved in
degassed 100 mM HEPES or phosphate buffer and 0.5 mM EDTA as
previously described (Nicolis et al., Biochem J 407:89-99, 2007).
The number of accessible thiol groups per heme was measured by the
4-PDS assay (Grassetti and Murray, Arch Biochem Biophys 119:41-49,
1967).
Cloning, Expression and Purification of Recombinant Ngb
[0149] Restriction digestions, ligation, transformation, cloning,
bacterial growth and isolation of DNA fragments were performed
using standard techniques. For the expression of the 151 amino acid
polypeptide of human Ngb, the cDNA SC122910 (GENBANK.RTM. Accession
No. NM.sub.--021257; SEQ ID NO: 7) was cloned in
BL21(DE3)pLysS(pET28a). Cells were grown in LB broth containing 30
.mu.g/ml kanamycin and 25 .mu.g/ml chloramphenicol, expression was
induced with 1 mM IPTG and carried out for 4 hours at 37.degree. C.
including 8-amino-levulinic acid (0.4 mM) in the media.
Purification was carried out as previously described with minor
modifications (Burmester et al., Nature 407:520-523, 2000). To
increase purification yield, human Ngb cDNA was fused with a
6.times.His tag in the N-terminus and cloned into pET28a. Proteins
were overexpressed in E. coli strain BL21(DE3). Purification of His
tagged human Ngb was performed using Ni-NTA-agarose (Qiagen)
affinity column according to the manufacturer's instructions. His
tagged Ngb was eluted with 200 mM imidazole after washing with 20
mM imidazole. The eluted protein was dialyzed against PBS at
4.degree. C., concentrated with a 10 kD cutoff filter and stored in
aliquots at -80.degree. C. The additional amino acids at the
N-terminus of His tagged Ngb were removed using a thrombin cleavage
capture kit (Novagen). The purity of each recombinant Ngb batch
prepared was assessed by SDS-PAGE and UV-visible spectroscopy.
Mutagenesis of Recombinant Ngb
[0150] Site directed mutagenesis was performed using QuikChange.TM.
II kit (Stratagene). The oligonucleotides for mutations C46A, C55A
and H64L are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Oligonucleotides used to perform
site-directed mutagenesis of Ngb Ngb SEQ ID Mutant Sequence NO:
C46A CTCTTCCAGTACAACGCCCGCCAGTTCTCCAG 1 C46A
CTGGAGAACTGGCGGGCGTTGTACTGGAAGAG 2 C55A
TCCAGCCCAGAGGACGCTCTCTCCTCGCCTGAG 3 C55A
CTCAGGCGAGGAGAGAGCGTCCTCTGGGCTGGA 4 H64L
CTGAGTTCCTGGACCTGATCAGGAAGGTGATGC 5 H64L
GCATCACCTTCCTGATCAGGTCCAGGAACTCAG 6
[0151] The template used for C46A and C55A was pCMV-1A and for H64L
was pET28a. Clones were sequenced to confirm the desired mutations.
Expression and purification of mutant Ngb were carried out using
the same procedures as for wild type Ngb.
Anaerobic Reactions of Globins with Excess Nitrite
[0152] Reaction kinetics of known amounts of Mb or Ngb with nitrite
were monitored by absorption spectroscopy for the indicated time in
a cuvette in the presence or absence of 2-4 mM sodium dithionite.
All reactions were run at 25.degree. C. or 37.degree. C. in 0.1 M
phosphate buffer at controlled pH. Previously deoxygenated nitrite
was added, using an airtight syringe, to a sealed anaerobic cuvette
to initiate the reaction. Oxygen contamination was prevented by
application of positive argon pressure without a channel for gas
escape. Concentrations of single species during reactions were
determined by least squares deconvolution of the visible absorption
spectrum into standard reference spectra using Microsoft Excel
analysis. OxyNgb was included to confirm successful deoxygenation
before the reaction. To vary pH, deoxy-Ngb and nitrite were
prepared in phosphate buffer adjusted to the target pH values. Fast
kinetic studies were performed using an Applied Photophysics DX-17
stopped-flow instrument equipped with rapid-scanning diode array
detection. Experiments were carried out at 25.degree. C. by rapidly
mixing a solution of reduced deoxy-Ngb containing 2 mM dithionite
with a known solution of nitrite at controlled pH. To determine
bimolecular rate constants all reactions were analyzed with Pro-K
software (Applied Photophysics) using singular value decomposition
followed by fitting of the reduced data matrix to a pseudo-first
order kinetic model.
Model of the Wild-Type Human Ngb Structure
[0153] Crystallization of the wild-type human Ngb is hindered by
aggregation and precipitation problems. Mutation of the three
cysteine residues yielded a protein suitable for crystallization
studies (Pesce et al., Structure 11:1087-1095, 2003). The reported
structure (PDB 1OJ6) thus includes the mutations Cys46Gly, Cys55Ser
and Cys120Ser. To assess the possible structure of the wild type
enzyme a homology model was built using the Swiss-Model server
(Schwede et al., Nucleic Acids Res 31:3381-3385, 2003) with the
sequence of the wild type Ngb and the available human structure as
template. The coordinates of the heme molecule were copied from the
1OJ6 structure.
Determination of the Midpoint Redox Potential of the
Thiol/Disulfide Couple in Ngb
[0154] Wild-type and C55A mutant Ngb (50-60 .mu.M) were incubated
at 37.degree. C. in anaerobic glove box with solutions containing
various ratios of reduced (GSH) and oxidized (GSSG) glutathione,
with the total GSH and GSSG concentration fixed at 25 mM in 0.1 M
phosphate buffer pH=7.0. The GSH/GSSG ratio was varied to establish
a gradient of redox potentials between -130 and -250 mV, calculated
by the Nernst equation according to a midpoint reduction potential
of -240 mV (Yi et al., J Biol Chem 284:20556-20561, 2009). After at
least 1 hour incubation, glutathione was removed by passage through
a G25 column and Ngb was reacted immediately with 10 mM nitrite in
0.1 M phosphate buffer pH=7.0 as described above. The observed rate
constant determined at each glutathione ratio was fitted using the
Nernst equation and the midpoint reduction potential of the
thiol/disulfide couple of Ngb calculated.
Determination of Nitrite Binding Constants
[0155] To determine the binding constant of nitrite to metNgb, 10
.mu.M wild type and mutant Ngb in 200 mM phosphate buffer, pH 7.4,
were incubated in a cuvette at 25.degree. C. with increasing
concentrations of nitrite and the UV-visible spectra were recorded
after each increase in nitrite concentration. The constant K.sub.D
for each protein was determined by interpolation of the absorbance
data following procedures in Nicolis et al. (Biochem J 407:89-99,
2007).
NMR Spectroscopy
[0156] .sup.1H NMR spectra in .sup.1H.sub.2O were collected at 29 C
on a Bruker DRX-600 NMR spectrometer operating at 599.79 MHz with a
5 mm triple resonance probe using a water presaturation pulse
sequence with 1 s irradiation time. Samples of wild type and mutant
250-300 .mu.M met-Ngb were prepared in 0.1M phosphate buffer pH
7.4. Typically 1024 transients were averaged, using 90 degree
pulses, spectral width of 80 ppm and 16K time domain points.
Spectra are referenced indirectly through the resonance of the
water, which occurs at 4.76 ppm downfield from the methyl resonance
of DSS (2,2-dimethyl-2-silapentane-5-sulfonate).
Electron Paramagnetic Resonance Spectroscopy
[0157] Iron nitrosyl species were measured by EPR spectroscopy
using a Bruker EMX 10/12 spectrometer operating at 9.4 GHz, 5-G
modulation, 10.1-milliwatt power, 327.68-ms time constant and
163.84-s scan over 600 G at 110 K as described previously (Basu et
al., J Biol Chem 283:32590-32597, 2008; Azarov et al., J. Biol.
Chem. 280:39024-38032, 2005). The concentrations of Mb and Ngb
species were determined by performing the double integral
calculation and comparing to standard samples.
Direct Measurement of NO Release
[0158] Deoxy-Ngb (final concentration 20 .mu.M) was injected in 3
ml anaerobic 100 mM phosphate buffer, pH 7.4 in a vessel purged
with helium gas and connected in line to an NO chemiluminescence
analyzer (Sievers, GE Analytical Instruments). Once a stable
baseline was established Ngb was reacted with a known amount of
nitrite as previously described (Huang et al., J Clin Invest
115:2099-2107, 2005).
Isolation and Respiration of Isolated Mitochondria with Neuroglobin
Molecules
[0159] Mitochondria were isolated from the livers of male Sprague
Dawley rats and incubated with wild-type or mutant Ngb proteins in
a sealed, stirred chamber at 37.degree. C. State 3 respiration was
stimulated with succinate (15 mM) and ADP (1 mM) and oxygen
consumption was measured with a Clark-type oxygen electrode. To
measure inhibition of respiration in hypoxic conditions, respiring
mitochondria were allowed to consume oxygen until the chamber
became anoxic and then the chamber lid was removed to allow the
diffusion of air back into the chamber. The rate of mitochondrial
respiration was greater than the rate of oxygen entering the
chamber such that the oxygen electrode trace remained at zero while
the mitochondria were respiring. Nitrite was added to the chamber
prior to the removal of the lid and deviation of the oxygen trace
from a zero reading signified a decrease in respiration rate. All
experiments were performed under conditions where substrates were
not limiting. The extent of respiratory inhibition was quantified
by measuring the time from equilibration of the mitochondria with
air to the time when the oxygen trace deviated from zero. This time
to inhibition was expressed as a percentage of maximal inhibition,
where 100% inhibition was defined as the time to inhibition in the
presence of cyanide and the time to the exhaustion of substrates
was used as a measure of 0% inhibition. Similar experiments were
performed with SHSY5Y cells suspended in the respirometer and
treated with the uncoupler FCCP (5 .mu.M) to measure hypoxic
inhibition of cellular respiration.
Immunoblotting of Neuroglobin Expression in SHSY5Y Neuronal
Cells
[0160] Equal amounts of denatured total proteins (25 .mu.g) from
the SHSY5Y neuronal cells expressing GFP vector, wild type and H64L
mutant Ngb, were subjected to 4-15% SDS-polyacrylamide gradient
gels and immunoblotted with anti-GFP monoclonal antibody (Santa
Cruz Biotechnologies, Inc.) and scanned with the Odyssey imaging
system (LI-COR Biosciences).
Determination of cGMP in SHSY5Y Neuronal Cells
[0161] SHSY5Y cells, expressing the GFP vector, wild type Ngb or
H64L mutant Ngb were plated on CORNING.RTM. CELLBIND.RTM. Surface
100 mm culture dishes at a concentration of 5-7.5.times.10.sup.5
cells/plate and grown to 80-90% confluence. After four days of
growth, the cells were incubated for 6 hours under hypoxic
conditions (1% oxygen). Following hypoxic treatment, the cGMP
levels were measured using the cyclic GMP EIA Kit (Cayman Chemicals
catalog #581021) according to the manufacturer's instructions.
Protein levels were measured and used to normalize results.
Statistical Analysis
[0162] Each experiment was performed at least in triplicate and
values are representative of two or more independent determinations
using different batches of protein purified separately. Data were
analyzed using Origin 8.0 (OriginLab) and expressed as
mean.+-.standard deviation of the mean. Analysis for statistically
significant differences among mean values was done, when
applicable, using the Student's t-test with a value of p<0.05
considered as significant.
Example 2
Human Neuroglobin Functions as a Redox Regulated Nitrite
Reductase
[0163] This example describes the finding that a stable
five-coordinate neuroglobin mutant (H64L) reduces nitrite to NO
approximately 2000-times faster than the wild type neuroglobin, and
mutation of either C55 or C46 to alanine stabilizes the
six-coordinate structure and slows nitrite reduction.
Nitrite is Reduced to NO Via Reaction with Deoxygenated Human
Neuroglobin
[0164] In order to examine the reaction of nitrite with
neuroglobin, recombinant human neuroglobin was expressed and
purified. Spectrophotometric analysis of His-tagged or untagged
proteins confirmed the six-coordinate heme structure in both the
ferrous and ferric states of Ngb, with visible .alpha. and .beta.
peaks around the 550 nm wavelength (FIG. 7A). Ferrous deoxy-Ngb was
prepared in an anaerobic glove box as detailed in Example 1 and the
visible spectra of the reaction was recorded between 10 .mu.M
deoxy-Ngb and 10 mM nitrite at 25.degree. C. at constant intervals
in a sealed air tight cuvette under external argon pressure (FIG.
1A). The time-dependent changes of deoxy-Ngb, ferric met-Ngb and
iron-nitrosyl-Ngb (Fe.sup.+2-NO) species (FIG. 1B) were calculated
by least squares deconvolution of the reaction spectra using
standard reference spectra (FIG. 7A). In an anaerobic environment
nitrite is reduced to NO according to equation 1 and the NO
generated has very high affinity (k.sub.on=10.sup.8M.sup.-1
s.sup.-1) for the ferrous Ngb heme thus yielding iron-nitrosyl-heme
(Fe.sup.+2-NO) as a final reaction product (equation 2).
Fe.sup.+2+NO.sub.2.sup.-+H.sup.+.fwdarw.Fe.sup.+3+NO.+OH.sup.-
(equation 1)
NO.+Fe.sup.+2Fe.sup.+2--NO (equation 2)
[0165] A reaction stoichiometry consistent with the reaction of
nitrite with hemoglobin or myoglobin was observed, with two
deoxy-Ngb molecules forming one iron-nitrosyl-Ngb and one ferric
Ngb (FIG. 1B). Analysis of the instantaneous bimolecular rate
constant (BRC) over time indicated that the reaction of nitrite
with Ngb at pH 7.4 proceeds at 0.12.+-.0.02 M.sup.-1 s.sup.-1 at
25.degree. C. (0.26.+-.0.02 M.sup.-1 s.sup.-1 at 37.degree. C.). A
recent study (Petersen et al., J Inorg Biochem 102:1777-1782, 2008)
reported that the reaction of deoxy mouse neuroglobin with nitrite
in the range 7-230 .mu.M generated ferric met-Ngb in excess of
ferrous nitrosyl-Ngb at apparent second-order rate constant of
5.1.+-.0.4 M.sup.-1 s.sup.-1; however, the current experimental
conditions with human neuroglobin differ considerably.
[0166] Both Salhany and the Gladwin group have shown (Grubina et
al., J Biol Chem 283(6):3628-3638, 2008; Salhany, Biochemistry
47:6059-6072, 2008) that the reaction of nitrite with hemoglobin in
the presence of dithionite proceeds via equation 1-2, but the
ferric heme that is formed is reduced back to the ferrous form to
continue the reaction. Thus iron-nitrosyl-heme forms at the same
rate as deoxyheme is consumed and the overall stoichiometry is one
deoxy-Ngb forming one iron-nitrosyl-Ngb. Performing the reaction in
the presence of dithionite limits the auto-oxidation of the ferrous
heme prior to the reaction with nitrite and allows for facile
assessments of anaerobic reaction mechanisms and kinetics. By
complementary studies using myoglobin it was verified that the
rate-limiting step of the reaction in the presence of dithionite is
the heme iron catalyzed conversion of nitrite to NO (FIGS. 8A and
8B). Then the reaction of anaerobic nitrite and deoxy-Ngb (10 mM
and 10 .mu.M respectively) was performed as described above in the
presence of 3 mM excess dithionite at pH 7.4 in 100 mM phosphate
buffer (FIGS. 1C and 1D). The stoichiometry was consistent with one
deoxy-Ngb forming one iron-nitrosyl-Ngb and the calculated BRC was
0.11.+-.0.01 M.sup.-1 sec.sup.-1, in accordance with the BRC value
obtained in the absence of dithionite. The reactivity of deoxy-Ngb
with nitrite in the concentration range 0.25-20 mM (FIG. 1E) was
further investigated. The second-order bimolecular rate constant
derived from the linear fit of the observed rate constants versus
nitrite concentration is 0.12.+-.0.02 M.sup.-1 sec.sup.-1 in
agreement with the calculated instantaneous BRC.
Proton Dependence of the Nitrite Reductase Reaction with
Neuroglobin
[0167] It was next explored whether deoxy-Ngb dependent nitrite
reduction requires a proton (equation 1). The pH dependence of the
bimolecular rate constant of the nitrite reductase reaction near
the physiological range (pH 6.5-8.0) (FIG. 1F) was determined. It
was found that increasing concentration of protons accelerate the
reaction by 10-fold for each pH unit decrease. The slope of the
linear fit, which represents the order of rate dependence on
[H.sup.+] is 0.96, close to the ideal 1.0, and it extends through
the zero point (FIG. 1F inset) indicating the requirement for one
proton in the reaction. It was concluded that the reaction
constitutes a concerted electron and proton transfer to nitrite to
form NO analogous to bacterial nitrite reductase.
Surface Cysteines C46 and C55 Regulate the Heme Pocket Coordination
and the Rate of Nitrite Reduction to NO
[0168] The control of the six-to-five coordinate iron heme
transition is the subject of much interest in the hexa-coordinate
globin field (Nadra et al., Proteins 71:695-705, 2008; Basu et al.,
J Biol Chem 283:32590-32597, 2008; Bykova et al., Biochem Biophys
Res Commun 347:301-309, 2006; Smagghe et al., Biochemistry
45:561-570, 2006; Fago et al., J Inorg Biochem 100:1339-1343,
2006). Unlike most other globins, human Ngb displays 3 conserved
cysteines (notable exception being mouse Ngb) at positions 46, 55
and 120 located on the protein surface as shown in the wild type
thiol reduced human Ngb structure model (FIG. 2A). Investigators
have identified a role for cysteines 46 and 55 in the regulation of
the heme ligand binding equilibrium. These cysteines form an
intra-molecular disulfide bond (Wakasugi et al., J Biol Chem
278:36505-36512, 2003), which influences the position of the
E-helix containing the distal histidine (Hamdane et al., J Biol
Chem 278:51713-51721, 2003). Reduction of the disulfide bond allows
additional structural freedom in the orientation of the E-helix
(FIG. 2A), that leads to an increased proportion of molecules in
the six-coordinate state and thus reduced oxygen and nitrite
binding affinities (Hamdane et al., J Biol Chem 278:51713-51721,
2003; Nicolis et al., Biochem J 407:89-99, 2007). Using the 4-PDS
assay, the number of accessible thiols per heme in wild-type
neuroglobin, as purified and reduced by DTT, and in the C55 to
alanine mutant neuroglobin, was determined (FIG. 2B). The results
are consistent with the quantitative formation of a disulfide bond
during protein purification and the presence of the single reduced
Cys120 in the oxidized thiol form.
[0169] To determine if the rate of nitrite reduction is influenced
by the redox state of cysteines 46 and 55, the cysteines were first
reduced by incubation with 10 mM dithiothreitol (DTT) and then the
rate of nitrite reduction was measured after anaerobic DTT removal.
FIG. 2C shows that reduction of the disulfide bond slows down the
rate by about 2-fold (0.062.+-.0.005 M.sup.-1 sec.sup.-1 at
25.degree. C., pH 7.4). To directly test the hypothesis that
disulfide bridge reduction affects the nitrite reactivity of
neuroglobin, recombinant mutants with cysteine 55 or 46 replaced by
alanine (C55A and C46A), which slowed down the rate of nitrite
reduction to similar rates observed with Ngb having fully reduced
cysteines, were produced (FIG. 2C).
Physiological Redox Control of the C46-C55 Disulfide Bond Regulates
the Rate of Nitrite Reduction to NO
[0170] To determine if the formation of a disulfide bond between
Cys 46 and 55 is redox-regulated within the physiological range of
cellular redox status, wild-type and C55A mutant Ngb were incubated
with increasing ratios of reduced/oxidized glutathione that
established a gradient of ambient redox potentials. After 60
minutes incubation, the rates of nitrite reduction were measured
after removal of glutathione by passage through a G25 column (FIG.
2D). It was found that there was a sudden and substantial drop in
the observed nitrite reductase rate constants (k.sub.obs) with
decreasing redox potential only for the wild-type protein. Fitting
the data to the Nernst equation provided a midpoint reduction
potential of the C46/C55 thiol/disulfide redox couple of -194.+-.3
mV. This value is within the range of cellular redox potentials (E.
Coli cytosol E.sub.0=-280 mV (Schafer and Buettner, Free Radic Biol
Med 30:1191-1212, 2001)).
[0171] To directly examine whether the cysteines redox state causes
changes in heme pocket molecular and electronic structure, the NMR
spectrum of wild type and C55A mutant met-Ngb (FIG. 2E) was
compared. Characteristic NMR signals for the heme methyls are
visible in the spectral regions around 36 ppm, 23 ppm and 20 to 12
ppm and were assigned by comparison with the published spectra (Du
et al., J Am Chem Soc 125:8080-8081, 2003; Xu et al., J Inorg
Biochem 103:1693-1701, 2009). The two spectra are largely similar
but a few marked differences in the positions of several heme
methyl resonances (M8-B, M5-A, M1-A, M5-B) as well of several
hyperfine shifted resonances between 18 and 12 ppm (FIG. 2E, region
marked with an asterisk) were assigned. Also several unassigned
ring current shifted resonances around -2 ppm are different. It was
concluded that the thiol mutation C55A clearly affects the geometry
of the heme pocket environment.
[0172] The nitrite binding affinity constant for the oxidized and
reduced cysteines of wild type and C55A mutant met-Ngb were
determined by difference spectra titration (FIG. 2F). The
calculated dissociation constants (K.sub.D) reported in Table 2
confirmed the influence of the cysteine redox state on the nitrite
binding affinity to the heme iron. During these experiments it was
also observed that met-Ngb very slowly reacts with nitrite to
produce nitrosyl-Ngb (BMC reported in Table 2). The slow rates of
reaction produce a detectable spectroscopic effect only at high
nitrite concentrations (approaching 0.1 M) and result in an
artificial decrease of maximal absorbance difference that has
previously been assigned to a second low-affinity binding constant
(Nicolis et al., Biochem J 407:89-99, 2007).
TABLE-US-00002 TABLE 2 Nitrite dissociation constants (K.sub.d) and
bimolecular rate constants (BRC) for reactions of met-Ngb with
nitrite in the presence of dithionite Neuroglobin K.sub.d
(NO.sub.2.sup.-) BRC of nitrite ferric protein (mM) heme reduction
WT SS 6.2 .+-. 2.1 0.0005 .+-. 0.0005 WT SH 12.6 .+-. 3.3 0.0002
.+-. 0.0005 C55A 30.1 .+-. 4.5 0.0002 .+-. 0.0005 H64L 0.17 .+-.
0.08 0.032 .+-. 0.002
[0173] These experiments indicate that the redox state of cysteines
C46 and C55 regulates both the five-to-six coordinate equilibrium
and the rate of nitrite conversion to NO. Intriguingly, an
analogous effect is observed with hemoglobin, in which oxidation of
the cysteine 93 speeds up the rate of nitrite reduction to NO, and
reduction slows the rate (Crawford et al., Blood 107:566-574,
2006). This effect has been attributed to the effect of thiol
oxidation on decreasing the heme redox potential.
The Rate of Nitrite Reduction is Maximal in the Five-Coordinate
State of Neuroglobin
[0174] To test the hypothesis that a change in the equilibrium
between the five- and six-coordinate Ngb sub-populations mediates
the control of the nitrite reduction rate, we generated recombinant
Ngb with a His64 to Leu substitution (H64L). The absorbance spectra
analysis of oxygen bound and deoxygenated ferrous Ngb and ferric
Ngb (FIG. 7B) confirmed that the mutant H64L Ngb is "locked" in the
five coordinate conformation (Nienhaus et al., J Biol Chem
279:22944-22952, 2004) and has very similar spectral
characteristics to the classic five coordinate heme proteins
hemoglobin and myoglobin (for comparison, FIG. 7C). The reaction of
nitrite with deoxygenated H64L Ngb was examined in the presence of
excess dithionite similarly to experiments with wild type Ngb, but
using only 100 .mu.M nitrite (FIGS. 3A and 3B). Surprisingly, the
rate of deoxy-Ngb conversion to nitrosyl-Ngb was extremely fast,
and the BRC was approximately 2000-fold higher than the wild type
Ngb. Fast mixing stopped-flow spectroscopy was then used to
determine the rates of the reaction in the range 10-1000 .mu.M
nitrite (FIG. 3C). The observed rate constants increased linearly
with increasing nitrite concentrations and the BRC derived from the
linear least square fit was 259.+-.8 M.sup.-1 s.sup.-1 at
25.degree. C., pH 7.4. Examination of the reaction at different pH
values (FIG. 3D) indicates that the reaction requires a proton
similar to the reaction with wild type Ngb. Remarkably the rate
increases above 2,500 M.sup.-1 s.sup.-1 at pH 6.5 and 25.degree. C.
This is the fastest reaction of nitrite with a heme-globin ever
reported and confirms the hypothesis that the six-to-five
coordinate transition at the heme pocket regulates the rate of
nitrite reduction to NO.
[0175] Finally, representative traces (absorbance decreases of the
Soret peak at 425 nm) of the reaction of 1 mM nitrite were compared
with wild-type Ngb, with or without disulfide bond (SS-Ngb and
SH-Ngb respectively), H64L and C55A mutant Ngb in 0.1 M HEPES at pH
7.4. The relative percentage of the total absorbance change
occurring in the first 60 minutes of the reaction is shown in FIG.
3E (with H64L-Ngb normalized to 100%, wild-type SS-Ngb was 38%,
wild-type SH-Ngb 20%, C55A Ngb 18% respectively). The reaction of
five-coordinate H64L Ngb reached the end point in the first minute
of the reaction and is expanded in the inset of FIG. 3E.
Confirmation of Reaction Kinetics Using Electron Paramagnetic
Resonance (EPR) Spectrometry
[0176] EPR spectrometry allows for direct measurement of the
paramagnetic NO-heme (iron-nitrosyl) ligand and provides
confirmation of NO formation in this reaction. The reaction of 1 mM
nitrite with wild-type SS-Ngb, SH-Ngb and mutant H64L Ngb (40.+-.5
.mu.M) was evaluated and compared with the rate of
iron-nitrosyl-myoglobin formation (FIGS. 4A and 4B). EPR spectra
analysis confirmed that the reduction of the cysteines (stabilizing
the six-coordinate heme geometry) slowed the rate of
iron-nitrosyl-Ngb formation, while replacement of the distal
histidine with leucine (five-coordinate stabilization) dramatically
increased the rate of NO formation. In particular, experiments
using H64L mutant Ngb and 1 mM nitrite were almost complete in one
minute and to allow assessment of the reaction kinetics, lower
concentrations of Ngb (10 .mu.M) and nitrite (50 .mu.M) were
necessary (FIGS. 4C and 4D). The calculated rates of nitrosyl-Ngb
formation are similar to data obtained by absorbance
spectrometry.
Nitrite Reduction by Deoxyneuroglobin Generates NO
[0177] The reaction of nitrite with deoxy-Ngb generates NO and
ferric Ngb. Although in in vitro conditions deoxy-Ngb can recapture
the NO, it was next explored if free NO gas can escape at
measurable rates. Anaerobic Ngb (20 .mu.M) and nitrite (1 mM) were
mixed in a vessel purged with helium and carried in-line to a
chemiluminescent NO analyzer. In these conditions the anaerobic
mixture generated NO in gas phase (FIG. 5A) and the rate of NO
formation was again regulated by the cysteines 46-55 disulfide bond
and by the heme pocket six-to-five coordination equilibrium. FIG.
5B shows that the rate of NO detected was significantly decreased
in reactions with six-coordinate C55A Ngb and increased in
reactions with the five coordinate H64L Ngb, consistent with the
hypothesis of six-to-five coordinate heme pocket control of nitrite
reduction. Finally, when increasing amounts of nitrite (10, 25,
100, 500, 1000 .mu.M final concentrations) were reacted with mutant
H64L Ngb (30 .mu.M), the fastest nitrite reductase, a readily
proportional NO generation response was observed (FIG. 5C).
Nitrite Reduction by Deoxyneuroglobin Mediates Intracellular NO
Signaling
[0178] Ngb is expressed in metabolically active cells and organs
(neurons, endocrine organs, retina, etc.) and has been hypothesized
to interact with mitochondria and mediate cytoprotective responses
to ischemic stress (Liu et al., J Neurosci Res 87:164-170, 2009).
It was therefore hypothesized that the nitrite reductase activity
of Ngb may regulate two canonical intracellular signaling pathways:
1) the hypoxic inhibition of cellular respiration by NO binding to
cytochrome c oxidase, and 2) the NO-dependent activation of soluble
guanylate cyclase to increase the intracellular concentrations of
cGMP. NO binding to cytochrome c oxidase has been shown to
reversibly inhibit electron transport at low oxygen tensions, in a
process thought to contribute physiologically to hypoxic
vasodilation and to the extension of oxygen diffusion gradients
(Mason et al., Proc Natl Acad Sci USA 103:708-713, 2006; Brunori et
al., Biochim Biophys Acta 1655:365-371, 2004).
[0179] To test whether Ngb generated NO inhibits mitochondrial
respiration during hypoxia, isolated rat liver mitochondria were
placed in a sealed, stirred respirometer and substrates were added
to stimulate respiration as previously described (Shiva et al.,
Circ Res 100:654-661, 2007). Mitochondria were allowed to respire
until the ambient oxygen tension dropped below detection level. At
this point, the respirometer is opened to air oxygen and cyanide is
added to evaluate the time to complete inhibition of respiration,
as determined by the increase in oxygen tensions measured with a
Clark electrode (FIG. 6A). The extent of mitochondrial inhibition
for all experiments was then compared to the effect of cyanide. No
significant inhibition of respiration was detected when nitrite (20
.mu.M) or purified wild type Ngb (5 .mu.M) were incubated alone
with respiring mitochondria. However, when the same concentrations
of nitrite and protein reacted together, 78.+-.6% inhibition of
respiration was observed. The extent of inhibition was increased
significantly by the H64L mutant Ngb (96.+-.2% inhibition) and
decreased by the C55A mutant Ngb (62.+-.4% inhibition) (FIG. 6B).
To evaluate this in cells, the cells of the neuronal cell line
SHSY5Y were stably transfected using a lentivirus vector with
GFP-tagged wild type and H64L mutant Ngb (FIG. 6C) and were used to
perform similar experiments. One million intact SHSY5Y cells were
suspended in the respirometer and maximal respiration rate was
stimulated by addition of the uncoupler FCCP. Then nitrite was
added to cells transfected with GFP only (negative control) and
cells expressing wild type Ngb or the H64L mutant Ngb. In FIG. 6D,
the extent of respiration inhibition was compared to the cyanide
effect (complete inhibition): cells with GFP only exhibited no
significant inhibition but about 15% and 40% inhibition,
respectively, was observed for wild type and H64L Ngb.
[0180] Finally, the effect of Ngb in the activation of sGC during
hypoxic conditions was explored. Under basal conditions, SHSY5Y
cells expressed neuronal NOS, which generates NO and nitrite under
normoxic conditions, for 4 days without added exogenous nitrite
then were exposed for 6 hours to hypoxic conditions (1% oxygen). It
was found that cGMP levels were significantly increased in cells
expressing the five-coordinate H64L mutant neuroglobin (FIG. 6E).
Altogether these data demonstrate an interaction between nitrite
and deoxygenated neuroglobin that generates bioavailable NO. This
can bind to cytochrome c oxidase to inhibit hypoxic mitochondrial
respiration and can activate sGC to promote cGMP-dependent
intracellular signaling. The extent of mitochondrial inhibition and
sGC activation is dependent on the heme coordination structure of
neuroglobin and intrinsic nitrite reductase activity.
CONCLUSIONS
[0181] The molecular examination of key heme pocket and surface
thiol amino acids, using site directed mutagenesis, provides a
novel understanding of neuroglobin functionality as an enzyme with
a redox regulated six-to-five coordinate iron heme transition that
directs nitrite in the heme pocket for controlled electron and
proton transfer reactions to form NO. The results presented herein
support the provocative hypothesis that the cellular six-coordinate
heme globins, neuroglobin, cytoglobin, Drosophila melanogaster
hemoglobin, and plant hemoglobins may subserve a function as
primordial allosterically redox regulated NO synthases. The
identification of other allosteric regulators of the six-to-five
coordination of the neuroglobin heme pocket may reveal new
intracellular mechanisms for controlling NO signaling via nitrite
reduction.
Example 3
Administration of Stable Five-Coordinate Neuroglobin to a Human
Subject
[0182] This example describes that five-coordinate neuroglobin can
be used as a blood substitute for treating oxygen deficiency or
replacing lost blood in a human subject.
Patient Selection
[0183] 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.
[0184] 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 known to one
skilled in the art. A representative method of treatment for such
diseases is by administration of stable five-coordinate
neuroglobin. 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 five-coordinate neuroglobin. In some cases, the
patient is further administered a second blood substitute or is
administered whole blood or a component of blood.
Administration of Five-Coordinate Neuroglobin Blood Substitute to a
Human Subject
[0185] A therapeutically effective amount of stable five-coordinate
neuroglobin (such as H64L human neuroglobin; SEQ ID NO: 9) is
administered to the human subject. The five-coordinate neuroglobin
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.
Patient Recovery and Outcome Assessment
[0186] The physician can then assess the therapeutic efficacy of
the five-coordinate neuroglobin blood substitute in increasing
blood oxygenation in the human subject according to any method
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.
[0187] In some embodiments, the human subject is treated with the
five-coordinate neuroglobin blood substitute 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.
[0188] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
Sequence CWU 1
1
9132DNAArtificial SequenceSynthetic oligonucleotide 1ctcttccagt
acaacgcccg ccagttctcc ag 32232DNAArtificial SequenceSynthetic
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32333DNAArtificial SequenceSynthetic oligonucleotide 3tccagcccag
aggacgctct ctcctcgcct gag 33433DNAArtificial SequenceSynthetic
oligonucleotide 4ctcaggcgag gagagagcgt cctctgggct gga
33533DNAArtificial SequenceSynthetic oligonucleotide 5ctgagttcct
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3371885DNAHomo sapiensCDS(376)..(831) 7ttcccaggcc accatagcgg
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cac ggc acc gtc ctg ttt 459Trp Arg Ala Val Ser Arg Ser Pro Leu Glu
His Gly Thr Val Leu Phe 15 20 25 gcc agg ctg ttt gcc ctg gag cct
gac ctg ctg ccc ctc ttc cag tac 507Ala Arg Leu Phe Ala Leu Glu Pro
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atc agg aag gtg atg ctc gtg att gat gct gca gtg 603Phe Leu Asp His
Ile Arg Lys Val Met Leu Val Ile Asp Ala Ala Val 65 70 75 acc aat
gtg gaa gac ctg tcc tca ctg gag gag tac ctt gcc agc ctg 651Thr Asn
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ggc agg aag cac cgg gca gtg ggt gtg aag ctc agc tcc ttc tcg aca
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Glu 145 150 cgcccggcag cccccatcca tctgtgtctg tctgttggcc tgtatctgtt
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1441cctgacagag tcggtttcct ttggcggcat tccctttccc tcattcagca
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tgcaggagtg agtcttacct cccctggccc tcctttctgg 1681ctcagcctgc
agcgactgtg aggccacagc tcctcagatt cactgcccgc tgtgtgccag
1741tactcaggca gctggagaga agagaaggca gcagcagagg cccccgccct
caccccagcc 1801atctgcactt gtaccatttg ctctgtgctg actgtggtcc
tataaattca tgagaaataa 1861actggttctg tgtgcaaaaa aaaa
18858151PRTHomo sapiens 8Met Glu Arg Pro Glu Pro Glu Leu Ile Arg
Gln Ser Trp Arg Ala Val 1 5 10 15 Ser Arg Ser Pro Leu Glu His Gly
Thr Val Leu Phe Ala Arg Leu Phe 20 25 30 Ala Leu Glu Pro Asp Leu
Leu Pro Leu Phe Gln Tyr Asn Cys Arg Gln 35 40 45 Phe Ser Ser Pro
Glu Asp Cys Leu Ser Ser Pro Glu Phe Leu Asp His 50 55 60 Ile Arg
Lys Val Met Leu Val Ile Asp Ala Ala Val Thr Asn Val Glu 65 70 75 80
Asp Leu Ser Ser Leu Glu Glu Tyr Leu Ala Ser Leu Gly Arg Lys His 85
90 95 Arg Ala Val Gly Val Lys Leu Ser Ser Phe Ser Thr Val Gly Glu
Ser 100 105 110 Leu Leu Tyr Met Leu Glu Lys Cys Leu Gly Pro Ala Phe
Thr Pro Ala 115 120 125 Thr Arg Ala Ala Trp Ser Gln Leu Tyr Gly Ala
Val Val Gln Ala Met 130 135 140 Ser Arg Gly Trp Asp Gly Glu 145 150
9151PRTArtificial SequenceSynthetic polypeptide 9Met Glu Arg Pro
Glu Pro Glu Leu Ile Arg Gln Ser Trp Arg Ala Val 1 5 10 15 Ser Arg
Ser Pro Leu Glu His Gly Thr Val Leu Phe Ala Arg Leu Phe 20 25 30
Ala Leu Glu Pro Asp Leu Leu Pro Leu Phe Gln Tyr Asn Cys Arg Gln 35
40 45 Phe Ser Ser Pro Glu Asp Cys Leu Ser Ser Pro Glu Phe Leu Asp
Leu 50 55 60 Ile Arg Lys Val Met Leu Val Ile Asp Ala Ala Val Thr
Asn Val Glu 65 70 75 80 Asp Leu Ser Ser Leu Glu Glu Tyr Leu Ala Ser
Leu Gly Arg Lys His 85 90 95 Arg Ala Val Gly Val Lys Leu Ser Ser
Phe Ser Thr Val Gly Glu Ser 100 105 110 Leu Leu Tyr Met Leu Glu Lys
Cys Leu Gly Pro Ala Phe Thr Pro Ala 115 120 125 Thr Arg Ala Ala Trp
Ser Gln Leu Tyr Gly Ala Val Val Gln Ala Met 130 135 140 Ser Arg Gly
Trp Asp Gly Glu 145 150
* * * * *