U.S. patent application number 13/412620 was filed with the patent office on 2012-08-30 for nitric oxide-blocked cross-linked tetrameric hemoglobin.
This patent application is currently assigned to IKOR, INC.. Invention is credited to Ross Walden Tye.
Application Number | 20120220529 13/412620 |
Document ID | / |
Family ID | 39325230 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120220529 |
Kind Code |
A1 |
Tye; Ross Walden |
August 30, 2012 |
NITRIC OXIDE-BLOCKED CROSS-LINKED TETRAMERIC HEMOGLOBIN
Abstract
The present invention includes compositions containing
carboxamidomethylated cross-linked hemoglobin where the cysteine
moiety of the hemoglobin includes a thiol protecting group and
where the hemoglobin has a reduced ability to bind with nitric
oxide. Preferably, the hemoglobin is deoxygenated, endotoxin free,
and stroma free. The present invention also includes method of
preparation, process of preparation and the method of use including
supplementing blood volume in mammals and treating disorders in
mammals where oxygen delivery agents are of benefit.
Inventors: |
Tye; Ross Walden; (Chico,
CA) |
Assignee: |
IKOR, INC.
|
Family ID: |
39325230 |
Appl. No.: |
13/412620 |
Filed: |
March 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12287558 |
Oct 10, 2008 |
8129338 |
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13412620 |
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11713195 |
Mar 1, 2007 |
7504377 |
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12287558 |
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60853968 |
Oct 23, 2006 |
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Current U.S.
Class: |
514/13.4 |
Current CPC
Class: |
C07K 14/805 20130101;
Y02P 20/55 20151101; A61K 38/42 20130101; A61P 17/02 20180101; A61P
7/00 20180101; A61P 9/00 20180101; A61P 7/06 20180101; A61P 7/08
20180101; A61P 9/10 20180101 |
Class at
Publication: |
514/13.4 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 7/00 20060101 A61P007/00; A61P 9/10 20060101
A61P009/10; A61P 7/06 20060101 A61P007/06 |
Claims
1. A blood substitute suitable for administration to a human
patient, the blood substitute comprising: a tetrameric hemoglobin;
and a pharmaceutically acceptable carrier; wherein said tetrameric
hemoglobin comprises four bovine polypeptides covalently
cross-linked together; and wherein said tetrameric hemoglobin
includes at least one cysteine moiety chemically modified with a
thiol-protecting group such that said at least one cysteine moiety
is incapable of binding nitric oxide; and wherein said tetrameric
hemoglobin has a p50 for oxygen greater than that of whole human
blood.
2. The blood substitute of claim 1, wherein said tetrameric
hemoglobin has a molecular weight greater than 60,000 daltons.
3. The blood substitute of claim 1, wherein said blood substitute
is endotoxin and stroma free.
4. The blood substitute of claim 1, wherein said blood substitute
is non-pyrogenic.
5. The blood substitute of claim 1, wherein said blood substitute
is deoxygenated.
6. The blood substitute of claim 1, wherein said tetrameric
hemoglobin comprises four bovine polypeptides covalently
cross-linked together with bis 3',5' dibromo salicyl fumarate.
7. The blood substitute of claim 1, wherein said tetrameric
hemoglobin comprises four bovine polypeptides covalently
cross-linked together with a poly-functional agent selected from
the group consisting of: glutaraldehyde; succindialdehyde;
activated polyoxyethylene; activated dextran; .alpha.-hydroxy
aldehyde; glycolaldehyde;
N-maleimido-6-aminocaproyl-(2'-nitro,4'-sulfonic acid)-phenyl
ester; m-maleimidobenzoic acid-N-hydroxysuccinimide ester;
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
m-maleimidobenzoyl-N-hydroxysuccinimide ester;
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester;
N-succinimidyl(4-iodoacetyl)aminobenzoate; sulfosuccinimidyl
(4-iodoacetyl)aminobenzoate; succinimidyl
4-(p-maleimidophenyl)butyrate; sulfosuccinimidyl
4-(p-maleimidophenyl)butyrate;
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;
N,N'-phenylene dimaleimide; bis-imidate; acyl diazide; and aryl
dihalide.
8. The blood substitute of claim 1, wherein the thiol-protecting
group is a carboxymethyl such that the tetrameric hemoglobin is a
carboxymethylated tetrameric hemoglobin.
9. The blood substitute of claim 1, wherein the thiol-protecting is
one of: 4-pyridylmethyl; acetylaminomethyl; alkoxyalkyl;
triphenylmethyl; carboxymethyl; acetyl; benzyl; benzoyl;
tert-butoxycarbonyl; p-hydroxyphenacyl; p-acetoxybenzyl;
p-methoxybenzyl; 2,4-dinitrophenyl; isobutoxymethyl;
tetrahydropyranyl; acetamidomethyl; bezamidomethyl;
bis-carboethoxyethyl; 2,2,2-trichloroethoxycarbonyl;
tert-butoxycarbonyl; N-alkyl carbamate; and N-alkoxyalkyl
carbamate.
10. The blood substitute of claim 1, in combination with an oxygen
impermeable polymer bag in which the blood substitute is
enclosed.
11. A blood substitute suitable for administration to a human
patient, the blood substitute comprising: tetrameric hemoglobin;
and a pharmaceutically acceptable carrier; wherein said tetrameric
hemoglobin comprises four polypeptide chains derived from a
non-human source covalently cross-linked together; and wherein said
tetrameric hemoglobin includes at least one cysteine moiety
chemically modified with a thiol-protecting group such that said at
least one cysteine moiety is incapable of binding nitric oxide; and
wherein said covalently cross-linked tetrameric hemoglobin has a
p50 for oxygen greater than that of human whole blood.
12. The blood substitute of claim 11, wherein said four polypeptide
chains are bovine polypeptide chains.
13. The blood substitute of claim 11, wherein said four polypeptide
chains are porcine polypeptide chains.
14. The blood substitute of claim 11, wherein said blood substitute
is deoxygenated and non-pyrogenic.
15. The blood substitute of claim 11, in combination with an oxygen
impermeable polymer bag in which the blood substitute is
enclosed.
16. The blood substitute of claim 11, wherein said cross-linked
tetrameric hemoglobin comprises hemoglobin proteins derived from a
non-human source and cross-linked with bis 3',5' dibromo salicyl
fumarate.
17. The blood substitute of claim 11, wherein said cross-linked
tetrameric hemoglobin comprises hemoglobin proteins derived from a
non-human source and cross-linked with a poly-functional agent
selected from the group consisting of: glutaraldehyde;
succindialdehyde; activated polyoxyethylene; activated dextran;
.alpha.-hydroxy aldehyde; glycolaldehyde;
N-maleimido-6-aminocaproyl-(2'-nitro,4'-sulfonic acid)-phenyl
ester; m-maleimidobenzoic acid-N-hydroxysuccinimide ester;
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
m-maleimidobenzoyl-N-hydroxysuccinimide ester;
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester;
N-succinimidyl(4-iodoacetyl)aminobenzoate; sulfosuccinimidyl
(4-iodoacetyl)aminobenzoate; succinimidyl
4-(p-maleimidophenyl)butyrate; sulfosuccinimidyl
4-(p-maleimidophenyl)butyrate;
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;
N,N'-phenylene dimaleimide; bis-imidate; acyl diazide; and aryl
dihalide.
18. The blood substitute of claim 11, wherein the thiol-protecting
group is a carboxymethyl such that the covalently cross-linked
tetrameric hemoglobin is a covalently cross-linked
carboxymethylated tetrameric hemoglobin.
19. The blood substitute of claim 11, wherein the thiol-protecting
is one of: 4-pyridylmethyl; acetylaminomethyl; alkoxyalkyl;
triphenylmethyl; carboxymethyl; acetyl; benzyl; benzoyl;
tert-butoxycarbonyl; p-hydroxyphenacyl; p-acetoxybenzyl;
p-methoxybenzyl; 2,4-dinitrophenyl; isobutoxymethyl;
tetrahydropyranyl; acetamidomethyl; bezamidomethyl;
bis-carboethoxyethyl; 2,2,2-trichloroethoxycarbonyl;
tert-butoxycarbonyl; N-alkyl carbamate; and N-alkoxyalkyl
carbamate.
20. A blood substitute suitable for administration to a human
patient, the blood substitute comprising: tetrameric hemoglobin in
a pharmaceutically acceptable carrier; wherein said tetrameric
hemoglobin comprises four polypeptide chains derived from a
non-human source covalently cross-linked together with bis 3',5'
dibromo salicyl fumarate; and wherein said tetrameric hemoglobin
includes at least one cysteine moiety chemically modified with a
thiol-protecting group such that said at least one cysteine moiety
is incapable of binding nitric oxide; and wherein said tetrameric
hemoglobin has a p50 for oxygen greater than 27 mm Hg.
Description
CLAIM OF PRIORITY
[0001] The present application is a continuation of, and claims
priority to U.S. patent application entitled "NITRIC OXIDE-BLOCKED
CROSS-LINKED TETRAMERIC HEMOGLOBIN," Ser. No. 12/287,558 filed Oct.
10, 2008, which claims priority to U.S. patent application entitled
"NITRIC OXIDE-BLOCKED CROSS-LINKED TETRAMERIC HEMOGLOBIN," Ser. No.
11/713,195 filed Mar. 1, 2007, now U.S. Pat. No. 7,504,377, which
claims priority to U.S. Provisional Application Ser. No.
60/853,968, first author Ross Tye, filed on Oct. 23, 2006, all of
which applications are incorporated herein by reference.
INTRODUCTION
[0002] This invention relates to nitric oxide-blocked cross linked
tetrameric hemoglobins, more specifically to a
carboxamidomethylated cross linked tetrameric hemoglobin, which has
low reactivity with nitric oxide (NO), and which is cross linked to
stabilize the tetramer for in-vivo applications. Methods of
preparation and use as blood volume expansion agents and as oxygen
delivery therapy agents are also disclosed.
BACKGROUND OF THE INVENTION
[0003] One of the limitations on the use of blood in an emergency
setting is a requirement to type and cross-match the blood to
minimize the risk of transfusion reactions. Type and cross-matching
may require at least 10 minutes and a complete type and cross-match
can take up to an hour. Furthermore, the risk of HIV transmission
has been estimated to be 1 in 500,000 units of blood and the risk
of hepatitis C transmission has been estimated to be 1 in 3,000
units. The safety of blood supply and blood logistics are critical
issues in developing countries, where the risk of infectious
disease transmission as well as the risk of outdated supply is
relatively higher. Up to 25% of the blood is discarded in
developing countries because of the presence of infectious disease.
Hence, there are pressing factors to find blood substitutes or
artificial blood compositions that avoid disease transmission and
provide rapid response to improve chances of survival.
[0004] Two aspects of artificial blood use in clinical settings are
volume expansion and oxygen therapeutics. Volume expander agents
are inert, merely increasing, blood volume, and thus allow the
heart to pump fluid efficiently. Oxygen therapeutics mimic human
blood's oxygen transport ability. Oxygen therapeutics can be
divided in two categories based on transport mechanism:
perfluorocarbon based, which function by simple dissolution of
oxygen, and hemoglobin protein based, which transports oxygen by
facilitated capture and release. In hemoglobin-based products, pure
hemoglobin (Hb) separated from red blood cells (RBCs) may not be
useful for a number of reasons, including instability, induction of
renal toxicity, and unsuitable oxygen transport and delivery
characteristics when separated from red blood cells.
[0005] Hemoglobin based oxygen therapeutics have been shown to
exert various degrees of vasoactive effects both in animal and
human studies (Winslow et al., Adv Drug Del Rev 2000; 40: 131-42;
Stowell et al., Transfusion 2001; 41: 287-99; Spahn et al., News
Physiol Sci 2001; 16: 38-41; Spahn et al., Anesth Analg 1994; 78:
1000-21; Kasper et al., Anesth Analg 1996; 83: 921-7; Kasper et
al., Anesth Analg 1998; 87: 284-91; Levy et al., J Thorac
Cardiovasc Surg 2002; 124: 35-42;). This vasoactivity may be due to
the effects of these products in binding intracellular NO (Kasper
et al., Anesth Analg 1996; 83: 921-7; Dietz et al., Anesth Analg
1997; 85: 265-273; Schechter et al., N Engl J Med 2003; 348:
1483-5), endothelial release (Gulati et al., Crit. Care Med 1996;
24: 137-47), or sensitization of peripheral .alpha.-adrenergic
receptors (Gulati et al., J Lab Clin Med 1994; 124: 125-33).
Alternatively, the increased vasoconstrictive effects could be due
to increases in the rate of oxygen release, secondary to the
administration of these products, at a higher concentration than
RBCs, resulting in vasoconstriction (Winslow et al., J Intern Med
2003; 253: 508-17; McCarthy et al., Biophys Chem 2001; 92: 103-17;
Intaglietta et al., Cardiovasc Res 1996; 32: 632-43; Vandegriff et
al., Transfusion 2003; 43: 509-16).
[0006] The ability of stroma-free Hb solutions to induce blood
pressure increases has been known. It has been demonstrated that
some cross-linked Hb solutions could increase mean arterial
pressure as much as 25-30% in a dose-dependent manner within 15 min
of administration and that the effect could last as long as 5
h.
[0007] Vasoconstriction may be due to NO scavenging by the
hemoglobin based therapeutic (Katsuyama et al., Artif Cells Blood
Substit Immobil Biotechnol 1994; 22:1-7; Schultz et al., J Lab Clin
Med 1993; 122:301-308, hereby incorporated by reference in its
entirety). Vasoconstriction could be also caused by the
contamination of the hemoglobin by phospholipids and endotoxin.
Although the remaining phospholipids and endotoxin contamination
during Hb purification may cause hemodynamic effects (Macdonald et
al., Biomater Artif Cells Artif Organs 1990; 18: 263-282), it is
less likely that this contamination be the major factor explaining
the potent vasoactive effect of some of these products (Gulati et
al., Life Sci 1995; 56: 1433-1442).
[0008] NO is a smooth-muscle relaxant that functions via activation
of guanylate cyclase and the production of cGMP or by direct
activation of calcium-dependent potassium channels. The increase in
the free Hb can result in an increase in the NO binding. The
increase in the NO binding can result in transient and in repeat
dosing, sustained hemodynamic changes responding to vasoactive
substances or the lack of vasoactive regulatory substances. In some
circumstances the lack of nitric oxide may lead to blood pressure
increases and if prolonged, hypertension. It has been demonstrated
that NO may bind to the reactive sulfhydryls of Hb and may be
transported to and from the tissues in a manlier analogous to the
transport of oxygen by heme groups (Jia et al., Nature 1996;
80:221-226).
[0009] Nitric oxide along with precapillary sphincter movement are
regulators of the arteriolar perfusion of any tissue. Nitric oxide
is synthesized and released by the endothelium in the arterial
wall, where it can be bound by hemoglobin in red blood cells. When
a tissue is receiving high levels of oxygen, nitric oxide is not
released and the arterial wall muscle contracts making the vessel
diameter smaller, thus decreasing perfusion rate and cause a change
in cardiac output. When demand for oxygen increases, the
endothelium releases nitric oxide, which causes vasodilatation. The
nitric oxide control of arterial perfusion operates over the
distance that NO diffuses after release from the endothelium.
Nitric oxide is also needed to mediate certain inflammatory
responses. For example, nitric oxide produced by the endothelium
inhibits platelet aggregation. Consequently, as nitric oxide is
bound by cell-free hemoglobin, platelet aggregation may be
increased. As platelets aggregate, they release potent
vasoconstrictor compounds such as thromboxane A.sub.2 and
serotonin. These compounds may act synergistically with the reduced
nitric oxide levels caused by hemoglobin scavenging resulting in
significant vasoconstriction. In addition to inhibiting platelet
aggregation, nitric oxide also inhibits neutrophil attachment to
cell walls, which in turn may lead to cell wall damage. Because
nitric oxide binds to hemoglobin inside the red blood cell, it is
expected that nitric oxide may bind to free Hb (stroma free
crosslinked tetrameric Hb) as well.
[0010] In many formulations free Hb and stabilized hemoglobin
infusions appear to be linked to vasoconstriction of the blood
vessels, resulting in extremely high blood pressures. The
hemoglobin moiety of these products can diffuse into the
endothelial lining of the vascular wall and act as a sink in
binding and removing NO which is needed for maintaining the normal
tone of the vascular wall. This can result in vasoconstriction of
the smooth muscle cells of the vascular wall. The free Hb solution
can leak into the surrounding tissues. Also, the extent of
vasoconstriction which occurs subsequent to administration of
different molecular size hemoglobin-based therapeutic bears an
inverse relationship to the molecular size of the product used,
i.e. infusion of larger oxygen carriers results in less
vasoconstriction and hypertension (Sakai, et al. Am J Physiol 2000;
279: H908-15). The smaller sized Hb molecule may be the most
permeable and may show a higher level of vasoconstriction and
hypertension (Faivre-Fiorina et al., Am J Physiol Heart Circ
Physiol 1999; 276: H766-70). In rabbit models, transfusion of free
Hb through the ear vein has caused cerebral vasculature ischemia
and death. Therefore, it is important to minimize the impact of
administration of most free Hb on the arterial system during
administration. Vasoactive agents such as verapamil, atenocard,
sildenafil citrate, etc., may be administered to the patient prior
to free Hb infusion. This is intended to ensure that the arterial
system is minimally changed during infusion. Nitric oxide and
verapamil are preferred vasoactive agents. Slow channel calcium
blockers (or a selective inhibitor of cyclic guanosine
monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), such
as sildenafil citrate) may also be helpful in the prevention of the
severe vasoconstriction. However, a slower infusion rate may not be
possible with respect to a trauma patient when demand for volume is
acute and critical.
[0011] One mechanism of modifying the NO scavenging properties of
hemoglobin based therapeutics is blocking of NO binding sites on
these molecules. Unprotected thiol on the cysteine moiety of the
hemoglobin may bind with NO. Protection of thiol or sulfhydryl
groups in the hemoglobin molecule may prevent the binding of NO to
the hemoglobin at the thiol site and hence prevent an acute
vasoactive response of the blood vessels causing a hypertensive
reaction. The prevention of NO binding to hemoglobin based
therapeutics may also prevent interference with normal platelet
aggregation and neutrophil migration when this class of
therapeutics is administered.
[0012] Therefore, some of the desirable characteristics of
hemoglobin based oxygen delivery therapeutics are: toxicity-free,
lack of induction of harmful immunogenic response, satisfactory
oxygen carrying and delivery capacity, suitable circulatory
persistence to permit effective oxygenation of tissues, long shelf
life, capacity for storage at room temperature, absence of viral or
other pathogens to prevent disease transmission, elimination of the
requirement for blood typing, and capacity for deployment by first
responders such as, paramedics, front line military medics etc.
These characteristics provide a rapid, safe response to blood loss
and the immediate support of tissue metabolic needs, thus improving
the chances for survival.
[0013] The present invention disclosed herein provides
compositions, characteristics and methods to prepare deoxygenated,
endotoxin free, stroma free, thiol blocked, cross-linked tetrameric
hemoglobin which has low reactivity with Nitric Oxide (NO), and the
tetrameric structures is stabilized by cross-linking. In particular
a carboxamidomethylated cross linked tetrameric hemoglobin is
provided as a stable NO blocked tetrameric Hb of the invention, as
well as methods for its production. A process and methods of
preparation of stable NO-blocked tetrameric Hb of the invention are
disclosed as well as methods of use as blood volume expansion
agents and as oxygen delivery therapy agents.
SUMMARY OF THE INVENTION
[0014] The present invention relates to a proteinaceous iron
containing compound having a molecular weight distribution in the
range of about 60,000 daltons to about 500,000 daltons and having
at least one cysteine moiety wherein the cysteine moiety includes a
thiol protecting group such that the proteinaceous compound has a
reduced ability to bind nitric oxide at the cysteine site(s). In
some embodiments, the proteinaceous iron containing compound
transports oxygen with a p50 of about 20 mm Hg to about 45 mmHg. In
some embodiments, the proteinaceous iron containing compound is
incapable of binding nitric oxide at the cysteine site(s).
[0015] Another aspect of the invention relates to a composition
comprising a proteinaceous iron containing compound having a
molecular weight distribution in the range of about 60,000 daltons
to about 500,000 daltons and having at least one cysteine moiety
wherein the cysteine moiety includes a thiol protecting group such
that the proteinaceous compound has a reduced ability to bind
nitric oxide at the cysteine site(s).
[0016] Yet another aspect of the invention relates to a composition
comprising a proteinaceous iron containing compound having a
molecular weight distribution in the range of about 60,000 daltons
to about 500,000 daltons and having at least one cysteine moiety
wherein the cysteine moiety includes a thiol protecting group such
that the proteinaceous compound has a reduced ability to bind
nitric oxide at the cysteine site(s) and wherein said compound is a
cross-linked tetrameric hemoglobin.
[0017] Another aspect of the invention relates to a process for a
preparation of a tetrameric hemoglobin wherein the hemoglobin has a
thiol protecting group attached to a cysteine group of the
hemoglobin comprising: (a) removing endotoxin and other
lipopolysaccharides from a preparation containing red blood cells;
(b) lysing the red blood cells; (c) separating hemoglobin by
removing stroma from the lysed red blood cells; (d) optionally
removing oxygen from the hemoglobin; (e) adding to a hemoglobin
solution a reagent which provides a thiol protecting group for a
cysteine of the hemoglobin, and (f) separating a hemoglobin which
has a thiol protecting group attached to a cysteine.
[0018] In some embodiments of the aforementioned aspect of the
invention, the process further comprises: (a) optionally removing
oxygen from the hemoglobin which has a thiol protecting group
attached to a cysteine; and crosslinking the hemoglobin which has a
thiol protecting group attached to a cysteine of the hemoglobin,
yielding a stable NO-blocked tetrameric Hb of the invention, which
is cross-linked.
[0019] In another aspect of the invention, a method for producing
tetrameric hemoglobin is provided wherein a thiol protecting group
is attached to a cysteine in the hemoglobin, by a process
comprising: (a) removing endotoxin from a preparation containing
red blood cells; (b) lysing said red blood cells; (c) separating
hemoglobin by removing stroma from said lysed red blood cells; (d)
optionally deoxygenating said hemoglobin; (e) adding to a
hemoglobin solution a reagent which provides a thiol protecting
group for a cysteine of said hemoglobin, and (f) separating a
hemoglobin which has a thiol protecting group attached to a
cysteine of the hemoglobin.
[0020] In another aspect of the invention, a method for producing a
cross linked tetrameric hemoglobin is provided, by a process
comprising: optionally removing oxygen from the product of the
method for producting tetrameric hemoglobin; and crosslinking said
product.
[0021] Yet another aspect of the invention relates to a method for
producing a NO-blocked tetrameric hemoglobin wherein a thiol
protecting group is attached to a cysteine in the hemoglobin, by a
process comprising: (a) adding to the hemoglobin solution a reagent
which provides a thiol protecting group for a cysteine of the
hemoglobin, and (b) separating a hemoglobin which has a thiol
protecting group attached to a cysteine of the hemoglobin. In some
embodiments the hemoglobin is further cross-linked.
[0022] Another aspect of the invention relates to a method of
supplementing the blood volume of a mammal comprising administering
to the mammal a composition comprising a proteinaceous iron
containing compound having a molecular weight of about 60,000
daltons to about 500,000 daltons and having at least one cysteine
moiety wherein the cysteine moiety includes a thiol protecting
group such that the proteinaceous compound has reduced ability to
bind nitric oxide at the cysteine site(s), and further comprises a
pharmaceutically acceptable carrier. In some embodiments the
proteinaceous iron containing compound is cross-linked.
[0023] In another aspect of the invention, a method of treating a
mammal suffering from a disorder is provided, comprising
administering a composition comprising a proteinaceous iron
containing compound having a molecular weight of about 60,000
daltons to about 500,000 daltons and having at least one cysteine
moiety where the cysteine moiety includes a thiol protecting group
such that the proteinaceous compound has reduced ability to bind
nitric oxide at the cysteine site(s). In some embodiments the
proteinaceous iron containing compound is cross-linked.
[0024] In another aspect of the invention, a method is provided for
perfusing an organ comprising administering an effective amount of
the stable NO-blocked tetrameric hemoglobins of the invention,
which can further be performed in-vivo or ex-vivo.
[0025] In some embodiments, the proteinaceous iron containing
compound increases oxygen offloading capacity relative to native,
cell free hemoglobins. In some embodiments, the proteinaceous iron
containing compound increases oxygen delivery ability. In some
embodiments, the crosslinked tetrameric hemoglobin is materially
reduced in its ability to bind nitric oxide. In some embodiments
the cross linked tetrameric hemoglobin is incapable of binding
nitric oxice. In some preferred embodiments, the crosslinked
tetrameric hemoglobin transports oxygen with a p50 of about 20 mm
Hg to about 45 mm of Hg. In some embodiments the proteinaceous iron
containing compound transports oxygen with a p50 of about 20 mm Hg
to about 45 mm of Hg.
[0026] In some embodiments of the invention, the proteinaceous iron
containing compound is a thiol-protected hemoglobin. In some
embodiments, the proteinaceous iron containing compound is a
cross-linked tetrameric hemoglobin. In some embodiments, the
proteinaceous iron containing compound has been crosslinked with
his 3',5' dibromo salicyl fumarate. In some embodiments, the
hemoglobin has been modified by reaction with
pyridoxal-5'-phosphate. In some embodiments, the hemoglobin is
mammalian. In some embodiments, the hemoglobin is human hemoglobin.
In some embodiments, the hemoglobin is bovine (i.e. bovine (genus
bos) or bison (genus bison)) or porcine hemoglobin. In some
preferred embodiments, the hemoglobin is non-pyrogenic, endotoxin
free, oxygen free and stroma free, enzyme free, and with low
induction of negative immunogenic reactions.
[0027] In some preferred embodiments, oxygen is removed from
hemoglobin which may or may not have a thiol protecting group
attached to a cysteine of the hemoglobin. In some embodiments the
oxygen is removed by contactor membrane technology.
[0028] In another aspect of the invention, the proteinaceous iron
containing compound of the invention is a thiol blocked stroma free
hemoglobin that may be safely stored for extended periods. This
thiol blocked stroma free hemoglobin may be a stable intermediate
which can endure packaging, shipping and further handling to yield
another hemoglobin composition of the invention. In some
embodiments the stable intermediate is further optionally
deoxygenated, cross-linked, and purified to remove excess reagents
and byproducts of the reaction, for example, dibromo salicylic
acid. In some embodiments the stable NO blocked tetrameric
hemoglobin is packaged.
[0029] In other embodiments of the invention, the compound is
non-pyrogenic, endotoxin free, and stroma free. In some embodiments
of the invention is proteinaceous compound is of low viscosity. In
some embodiments the proteinaceous compound of the invention is
oxygen free.
[0030] In some embodiments, the reagent that provides a thiol
protecting group is selected from the group consisting of
4-pyridylmethyl chloride, alkoxyalkylchloride, dimethoxymethane,
N-(hydroxymethyl)acetamide, triphenylmethyl chloride, acetyl
chloride, acetic anhydride, haloacetamide, iodoacetate, benzyl
chloride, benzoyl chloride, di-tert-butyl dicarbonate,
p-hydroxyphenacyl bromide, p-acetoxybenzyl chloride,
p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride,
tetrahydropyran, acetamidohydroxymethane, acetone,
bis-carboethoxyethene, 2,2,2-trichloroethoxycarbonyl chloride,
tert-butoxycarbonyl chloride, alkyl isocyanate, and alkoxyalkyl
isocyanate. In some preferred embodiments, the haloacetamide is
iodoacetamide. In some embodiments, the thiol protecting group is
selected from the group consisting of 4-pyridylmethyl,
acetylaminomethyl, alkoxyalkyl, triphenylmethyl, derivatives of
carboxymethyl, carboxamidomethyl, acetyl, benzyl, benzoyl,
tert-butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl,
p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl,
tetrahydropyranyl, acetamidomethyl, benzamidomethyl,
bis-carboethoxyethyl, 2,2,2-trichloroethoxycarbonyl,
tert-butoxycarbonyl, N-alkyl carbamate, and N-alkoxyalkyl
carbamate. In some embodiments, the thiol protecting group is a
carboxamidomethyl group.
[0031] Some embodiments of the invention provide compositions
comprising the proteinaceous iron containing compound and a
pharmaceutically acceptable carrier. In some embodiments provide a
container containing a composition comprising the proteinaceous
compound of the invention, optionally comprising a pharmaceutically
acceptable carrier.
[0032] In some embodiments, the mammal suffers from acute anemia,
anemia related conditions, hypoxia of ischemia. In some
embodiments, the mammal needs volume transfusion of a blood
substitute for transport of oxygen. In some embodiments, the mammal
is in trauma and has suffered an acute volume loss.
[0033] In some embodiments of the methods of the invention,
administration is made by implant, injection or transfusion.
[0034] In other embodiments of the method of the invention, the
mammals are suffering from a disorder including anemia, anemia
related conditions, hypoxia and ischemia. The anemia and anemia
related conditions may be caused by renal failure, diabetes, AIDS,
chemotherapy, radiation therapy, hepatitis, G.I. blood loss, iron
deficiency, or menorrhagia. In some embodiments of the invention,
the method includes administering erythropoietin therapy
[0035] In some embodiments of the method of the invention, the
disorder being treated is ischemia, which is caused by burns,
stroke, emerging stroke, transient ischemic attacks, myocardial
stunning and hibernation, acute angina, unstable angina, emerging
angina, or infarct. In other embodiments of the method, the
disorder is carbon monoxide poisoning.
[0036] In other embodiments of the method of the invention, the
disorder that the mammal is treated for is recovery after surgery.
In some other embodiments of the method of the invention, the
disorder is diabetic wound healing. In yet other embodiments of the
method the disorder is sickle cell anemia, and the administration
may further be made prior to surgery. In other embodiments of the
method of the invention, the disorder is acute coronary syndrome.
In other embodiments of the method of the invention, the disorder
is cardiogenic shock.
[0037] In some embodiments of the method of the invention the
proteinaceous iron containing compound is administered to a mammal
in need of a blood transfusion. In some embodiments of the
invention, the mammal is suffering from trauma. In some embodiments
of the method, the disorder that the mammal is suffering from is
lack of oxygen delivery capacity is caused by environmental stress
or physical stress.
[0038] In other embodiments of the method of the invention, the
proteinaceous iron containing compound is administered in
combination with radiation therapy. In yet other embodiments of the
invention, the method further comprises administering to said
mammal an oxygen dependent pharmaceutical agent.
[0039] In some embodiments of the method of the invention,
administering the proteinaceous iron containing compound to said
mammal permits visualization of intravascular space in-vivo, while
maintaining oxygenation of the tissue within the viewing field.
INCORPORATION BY REFERENCE
[0040] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0042] FIG. 1 is a flow chart showing the steps of the methods as
disclosed herein.
[0043] FIG. 2 depicts the time course of a lysis experiment showing
resistance of WBC lysis.
[0044] FIG. 3 depicts the size standards used in electrophoretic
separations as disclosed within.
[0045] FIG. 4A depicts overlays of electrophoretic separations of
native hemoglobin, dXCMSFH, and size standard in the range around
20 KDa.
[0046] FIG. 4B depicts overlays of the electrophoretic separation
of native hemoglobin, dXCMSFH, and size standards for the full
electropherogram.
[0047] FIG. 5 depicts the HLPC size exclusion separation of
products of the cross linking reaction.
[0048] FIG. 6 shows oxygen affinity curves for bovine whole blood,
stroma free Hb, cross linked hemoglobin, and fresh human blood.
[0049] FIGS. 7A-D depict the cardiac output, systemic vascular
resistance, and mean arterial pressure, respectively, in a pig
safety trial.
DETAILED DESCRIPTION OF THE INVENTION
[0050] The term "endotoxin free" or its grammatical equivalents as
used herein, means a hemoglobin that has been treated to reduce
exposure to and to remove substantially or completely all endotoxin
as measured by a very sensitive assay technique such as a
tubidometric assay or a chromogenic assay. These methods are
capable of detecting less than 0.05 EU per ml. Thus, endotoxin-free
hemoglobin can have less than or the equivalent of the amount of
endotoxin present in Water for Injection (WFI).
[0051] The term "non-pyrogenic" or its grammatical equivalents as
used herein, means a hemoglobin that may be administered to a
mammal without causing IL-8 overproduction, complement activation,
platelet activation, inflammatory response or a febrile
reaction.
[0052] The term "oxygen free", "deoxygenated", or its grammatical
equivalents as used herein, means a hemoglobin that has been
treated to remove substantially or completely all oxygen bound to
the heme pocket. Oxygen-free hemoglobin thus is substantially or
completely in the higher energy "tense" or "T" configuration.
[0053] The term "stroma free" or its grammatical equivalents as
used herein, means a hemoglobin that has been treated or processed
to remove substantially or completely all stromal material, such
that the preparation no longer exhibits the immunoreactivity to red
cell surface type antigens characteristic of RBC membranes. Stroma
are the cell membrane structural proteins and removing stroma also
will remove antigens associated with the cell membrane. Stroma-free
hemoglobin therefore substantially or completely lacks the toxic
and/or pyrogenic properties associated with preparations of
hemolyzed red blood cells still containing portions of the lipid
membrane surrounding the hemoglobin protein, and thus after
molecular stabilization, this stabilized stroma free hemoglobin can
be administered to an individual without causing transfusion
reaction toxicity or inflammatory reaction.
[0054] The term "NO-blocked tetrameric Hb" refers to
endotoxin-free, stroma-free thiol blocked tetrameric hemoglobins of
the present invention. The term "stable NO-blocked tetrameric Hb"
refers to the cross-linked, endotoxin-free, stroma free, thiol
blocked hemoglobins of the present invention.
[0055] The term "dNO-blocked tetrameric Hb" refers to the
deoxygenated endotoxin-free, stroma-free, thiol blocked,
cross-linked hemoglobins of the present invention. One embodiment
of this class of compounds is "dXCMSFH", which is a specific
example of a deoxygenated endotoxin-free, stroma-free,
carboxamidomethylated cross linked hemoglobin of the present
invention.
[0056] The term "dTBSFH" refers to the deoxygenated endotoxin-free,
stroma-free, thiol-blocked uncross-linked hemoglobin of the present
invention.
[0057] The term "dCMSFH refers to the deoxygenated endotoxin-free,
stroma free, carboxamidomethylated uncross-linked hemoglobin of the
invention and is a specific example of a dTBSFH.
[0058] The term "mammal" refers to both human and non-human
animals.
[0059] The compositions and methods of the present invention relate
to a thiol-protected, cross-linked tetrameric hemoglobin (stable NO
blocked Hb) where at least one cysteine moiety in the hemoglobin
molecule includes a thiol protecting group, for example, a
carboxamidomethyl group, such that the thiol group in the cysteine
moiety is not available for binding with nitric oxide (NO).
Preferably, at least two cysteine moieties in the hemoglobin are
protected with a thiol (or sulfhydryl) protecting group such that
the thiol group in the cysteine moiety is not available for binding
with nitric oxide (NO). These NO-blocked hemoglobins disclosed
herein prevent vasoactive reactions of blood vessels when
administered. The stable NO-blocked tetrameric hemoglobins of the
invention are further cross linked to provide an oxygen carrying
capacity with a p50 of about 20 mm Hg to about 45 mm Hg and an
extended circulatory half life. Preferably, the hemoglobin is
non-pyrogenic, endotoxin free, oxygen free, and stroma free.
Therefore, thiol-protected cross-linked hemoglobins of the present
invention (a stable NO-blocked tetrameric Hb) have high oxygen
exchange capacity and are functionally superior to native
hemoglobin.
I. HEMOGLOBIN COMPOSITIONS
1. Hemoglobin Sources and Molecular Structure
[0060] Hemoglobin (or blood or RBCs which it may be isolated from)
used in the present-invention may be obtained from a variety of
mammalian sources, such as, for example, human, bovine (genus bos),
bison (genus bison), ovine (genus ovis), porcine (genus sus)
sources, other vertebrates or transgenically-produced hemoglobin.
Alternatively, the stroma-free hemoglobin used in the present
invention may be synthetically produced by a bacterial, or more
preferably, by a yeast, mammalian cell, or insect cell expression
vector system (Hoffman, S. J. et al., U.S. Pat. No. 5,028,588 and
Hoffman, et al., WO 90/13645, both herein incorporated by
reference). Alternatively, hemoglobin can be obtained from
transgenic animals; such animals can be engineered to express
non-endogenous hemoglobin (Logan, J. S. et al. PCT Application No.
PCT/US92/05000; Townes, T. M. et al., PCT Application No.
PCT/US/09624, both herein incorporated by reference in their
entirety). Preferably, the stroma-free hemoglobin used in the
present invention is isolated from bison, bovine or human
sources.
[0061] The genus bos includes, subgenus bos including bos taurus
(western cattle, including oxen and aurochs) and bos aegyptiacus;
subgenus bibos including bos frontalis (gaur, gayal or Indian
bison) and bos javanicus (banteng); subgenus novibos including bos
sauveli (kouprey or grey ox), and; subgenus poephagus including bos
grunniens (yak; also bos mutus). The bos taurus, includes similar
types from Africa and Asia such as, bos indicus, the zebu; and the
bos primigenius, the aurochs. The bos gurus includes subspecies,
bos gaurus laosiensis, bos gaurus gaurus (such as in India, Nepal)
also called "Indian bison", bos gaurus readei, bos gaurus hubbacki
(such as in Thailand, Malaysia), and bos gaurus frontalis, a
domestic gaur, or a gaur-cattle hybrid breed.
[0062] Bison is a taxonomic genus containing six species within the
subfamily bovinae. The bison may be called buffalo in Asia (such as
water buffalo) and Africa (such as African buffalo). The genus
bison includes species such as, bison latifrons (long-horned
bison), bison antiquus, bison occidentalis, bison priscus, bison
bison, bison bison bison, bison bison athabascae, bison bonasus,
bison bonasus bonasus, bison bonasus caucasicus, and bison bonasus
hungarorum. In some embodiments of the present invention, the
hemoglobin is from genus bos or bison It shall be understood that
any mammalian species may be used as a source of hemoglobin and is
within the scope of the present invention.
[0063] Bovine Hb is easier to obtain and more abundant than human
Hb. Typically human Hb extracted from outdated RBCs is used for
Hb-based artificial blood research. However, outdated RBCs are not
available in sufficient quantities to produce large amounts of
viable oxygen delivery therapeutics or blood substitutes.
[0064] Hemoglobin, whether derived from an animal, synthetic or
recombinant, may be composed of the "naturally existing" hemoglobin
protein, or may contain some or be entirely composed of, a mutant
hemoglobin protein. Preferred mutant hemoglobin proteins include
those whose mutations result in more desirable oxygen
binding/release characteristics. Examples of such mutant hemoglobin
proteins include those provided by Hoffman, S. L. et al. (U.S. Pat.
Nos. 5,028,588 and 5,776,890) and Anderson, D. C. et al. (U.S. Pat.
Nos. 5,844,090 and 5,599,907), all herein incorporated by reference
in their entirety.
[0065] Hemoglobin or haemoglobin (Hb) is a proteinaceous heme
iron-containing compound having a molecular weight of about 60,000
daltons which transports oxygen in the red blood cells of the blood
in mammals and other animals. Hemoglobin transports oxygen from the
lungs to the rest of the body, such as to the muscles, wherein it
releases part of the oxygen load. 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 individual protein chain arranges in a set of
.alpha.-helix structural segments connected together in a
"myoglobin fold" arrangement, so called because this arrangement is
the same folding motif used in the heme/globin proteins. This
folding pattern contains a pocket which is suitable to strongly
bind the heme group. A heme group consists of an iron atom held in
a heterocyclic ring, known as a porphyrin. These iron atoms are the
sites of oxygen binding. The iron atom is bonded equally to all
four nitrogens in the center of the ring, which lie in one plane.
Two additional bonds perpendicular to the plane on each side can be
formed with the iron to form the fifth and sixth positions, one
connected strongly to the protein, the other available for binding
of oxygen. The iron atom can either be in the Fe.sup.2+ or
Fe.sup.3+ state, but ferrihaemoglobin (methemoglobin) (Fe.sup.3+)
cannot bind oxygen.
[0066] In adult humans, the predominate 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..sub.2.beta..sub.2. The subunits are
structurally similar and about the same size. Each subunit has a
molecular weight of about 16,000 daltons, for a total molecular
weight of the tetramer of about 64,000 daltons. The four
polypeptide chains are bound to each other by salt bridges,
hydrogen bonds and hydrophobic interactions. There are two kinds of
contacts between the .alpha. and the .beta. chains:
.alpha..sub.1.beta..sub.1 and .alpha..sub.1.beta..sub.2. However,
adult hemoglobin may also comprise .delta. globin subunits. The
.delta. globin subunit replaces .beta. globin and pairs with
.alpha. globin as .alpha..sub.2.delta..sub.2 to form hemoglobin
A2.
[0067] Bovine Hb is structurally similar to human Hb. The bovine Hb
also contains two .alpha. chains and two .beta. chains, with
similar molecular weight distribution.
2. Protecting the Sulfhydryl Groups of the Cysteine of the
Hemoglobin
[0068] A thiol group of a cysteine moiety in a hemoglobin may bind
to nitric oxide and may result in transient or sustained changes in
hemodynamic properties of blood or vasoactive substances and may
lead to hypertensive reactions. This occurrence can be avoided by
protecting the thiol group of the cysteine moiety in the hemoglobin
such that the resulting hemoglobin is incapable of binding with
NO.
[0069] Bovine hemoglobin contains only two thiol groups (Cys 93 on
each of the beta chains) which are involved in binding NO. Hence
preferably, both thiol groups are protected with a thiol protecting
group in bovine Hb. Human hemoglobin contains six thiol groups
(.alpha. Cys 104, .beta. Cys 93, and .beta. Cys 112), and at least
two of which (Cys 93 on each of the beta chains) are involved in
binding NO. Preferably these two thiols are protected and up to six
thiol groups may be protected with a thiol protecting group in
human Hb.
[0070] The SFH of the present invention can be reacted with various
reagents to result in protection of the thiol group in the cysteine
moiety of the hemoglobin. Without limiting the scope of the present
invention, all the reagents known in the art for the protection of
a functional group such as, but not limited to, hydroxyl, thiol, or
carboxyl, are included in the present invention.
[0071] Some of the examples of the reagents include, but are not
limited to, 4-pyridylmethyl chloride, alkoxyalkylchloride,
dimethoxymethane, N-(hydroxymethyl)acetamide, triphenylmethyl
chloride, acetyl chloride, 2-chloroacetic acid, acetic anhydride,
haloacetamide such as, iodoacetamide, bromoacetamide,
chloroacetamide, or fluoroacetamide, haloacetate such as
iodoacetate, bromoacetate, chloroacetate, or fluoroacetate, benzyl
chloride, benzoyl chloride, di-tert-butyl dicarbonate,
p-hydroxyphenacyl bromide, p-acetoxybenzyl chloride,
p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride,
tetrahydropyran, acetamidohydroxymethane, acetone,
bis-carboethoxyethene, 2,2,2-trichloroethoxycarbonyl chloride,
tert-butoxycarbonyl chloride, alkyl isocyanate, and alkoxyalkyl
isocyanate. In a preferred embodiment, the reagent is
haloacetamide. In a further preferred embodiment, the reagent is
iodoacetamide. It is understood that any reagent known in the art
that can be used for carboxamidomethylation of the thiol group in
the cysteine moiety of the hemoglobin is within the scope of the
present invention.
[0072] Without limiting the scope of the present invention, all the
protecting groups known in the art for the protection of a
functional group such as, but not limited to, hydroxyl, thiol, or
carboxyl, are included in the present invention. Some of the
examples of the protecting group include, but are not limited to,
4-pyridylmethyl, acetylaminomethyl, alkoxyalkyl, triphenylmethyl,
carboxamidomethyl, acetyl, benzyl, benzoyl, tert-butoxycarbonyl,
p-hydroxyphenacyl, p-acetoxybenzyl, p-methoxybenzyl,
2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl,
acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl,
2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl
carbamate, and N-alkoxyalkyl carbamate. In a preferred embodiment,
the protecting group is carboxamidomethyl such that the protection
of the thiol group in the cysteine moiety of the hemoglobin results
in a non-pyrogenic, endotoxin free, stroma free,
carboxamidomethylated Hb (CMSFH or a stable NO blocked Hb). More
generally, protection of the thiol group in cysteine(s) of
hemoglobin results in a non-pyrogenic, endotoxin free, stroma free
thiol blocked Hb (TBSFH or an NO blocked Hb).
3. Oxygen Affinity Modulation and Stabilization by Cross Linking
within Tetrameric Hemoglobin.
[0073] Bovine Hb and human Hb differ in the way in which oxygen
affinity is modulated. In the tetrameric form of normal adult human
hemoglobin, the binding of oxygen is a cooperative process. The
binding affinity of hemoglobin for oxygen is increased by the
oxygen saturation of the molecule. As a consequence, the oxygen
binding curve of hemoglobin is sigmoidal, or S-shaped. This
positive cooperative binding may be achieved through steric
conformational changes of the hemoglobin protein complex. When one
subunit protein in hemoglobin becomes oxygenated, it induces a
conformational or structural change in the whole complex causing
the other subunits to gain an increased affinity for oxygen.
[0074] When hemoglobin binds oxygen, it shifts from the high energy
"tense" or "T" state (deoxygenated or oxygen free) to the lower
energy "relaxed" or "R" state (oxygenated). Human .alpha. and
.beta. globin genes have been cloned and sequenced (Liebhaber et
al., Proc. Natl. Acad. Sci. (U.S.A). 77:7054-58 (1980); Marotta et
al., J. Biol. Chem. 252:5040-43 (1977); and Lawn et al., Cell
21:647 (1980), all of which are incorporated by reference in their
entirety). The tetrameric structure of human T state
deoxyhemoglobin has increased stability from six ionic bonds and
while in the T state, hemoglobin is effectively prevented from
disassociating into dimers. In this conformation, the beta cleft
contact area between the two beta chains (also known as the beta
pocket, phosphate pocket, and 2,3-diphosphoglycerate binding site)
in deoxyhemoglobin is substantially different than in
oxyhemoglobin. The changed conformation of the beta cleft in the T
state is believed to explain the decreased oxygen affinity
stabilized by 2,3-diphosphoglycerate. The T state of hemoglobin is
stable and resistant to denaturation.
[0075] Inside red blood cells, the binding of
2,3-diphosphoglycerate to its binding site within human hemoglobin
decreases the hemoglobin's oxygen affinity to a level compatible
with oxygen transport and delivery in a physiologic range of pH 7.2
to 7.4. The binding of 2,3-diphosphoglycerate to hemoglobin is weak
and may require high concentrations (i.e., concentrations
approaching 1M or more) in order to modify the oxygen affinity of
hemoglobin. For example, in people acutely acclimated to high
altitudes, the concentration of 2,3-diphosphoglycerate (2,3-DPG) in
the blood is increased, which allows these individuals to release a
larger amount of oxygen to tissues under conditions of lower oxygen
tension.
[0076] Thus, when the red blood cells are ruptured to produce
stroma free hemoglobin (SFH), the 2,3-diphosphoglycerate may not be
retained in close proximity to the hemoglobin and may disassociate
from the hemoglobin. As a consequence, unless further modified,
Human SFH may exhibit a higher affinity for oxygen than does
hemoglobin in RBCs. The p50 of stroma free human hemoglobin in
solution can be approximately 12 to 17 mm Hg as compared to native,
RBC associated hemoglobin p50 of approximately 27 mm Hg. The
increased affinity of the SFH for oxygen, under physiological
conditions, may prevent high capacity release of the bound oxygen
to the tissues.
[0077] In contrast, bovine Hb, possessing a further internal salt
bridge, has its affinity for oxygen affected by the ionic strength
of the local environment. Bovine hemoglobin does not require
2,3-DPG to maintain a p50 for oxygen in the range of 30 mm Hg to 40
mm Hg. An advantage to affinity modulation by altering ionic
strength versus that induced by 2,3-DPG binding is that sufficient
concentration of ionic species is generally present in plasma while
2,3-DPG is only contained within RBCs. Thus, the oxygen affinity of
acellular bovine Hb can be modulated more easily than acellular
human Hb.
[0078] This advantage of modulation of affinity by general ionic
interaction can be built back into human Hb by reacting it with
pyridoxal-5-phosphate (PLP). PLP modifies human Hb by introducing a
negative charge near a penultimate .beta. chain histidine residue
and by removing a positive charge at the amino terminal end of the
same chain, An altered human Hb of this class can now respond more
similarly to bovine Hb to charged species in the local environment
and not solely depend on binding of 2,3-DPG to affect oxygen
affinity.
[0079] Within the RBC, the association of the .alpha. chain with
its corresponding .beta. chain is very strong and does not
disassociate under physiological conditions. The association of one
.alpha./.beta. dimer with another .alpha./.beta. dimer, however, is
fairly weak and outside of the RBC, the two dimers may disassociate
even under physiological conditions. Upon disassociation, the dimer
is filtered through the glomerulus. The rapid clearing of stroma
free hemoglobin (SFH) by the kidney is a consequence of its
quaternary molecular arrangement.
[0080] To avoid such removal of human and bovine hemoglobin alike,
cell-free hemoglobin can be conjugated or cross-linked by various
methods known in the art. One of the methods is by conjugating Hb
to another molecule such as polyethylene glycol (PEG), which forms
a hydrophilic shield around the Hb molecule and simultaneously
increases its size which in turn increases its circulatory
half-life. Hb can also be cross-linked intramolecularly to prevent
dissociation of the tetramer into .alpha..beta. dimers and/or
cross-linked intermolecularly to form polymers which also increases
the oxygen carrier's size and thus increases its circulatory
half-life. Using site-specific cross-linking reagents,
intramolecular covalent bonds may be formed, which may convert Hb
into a stable tetramer, thus preventing its dissociation into
.alpha..beta. dimers. On the other hand, the use of non-specific
cross-linkers such as glutaraldehyde may lead to non-specific
covalent bonding between amino acid residues residing within and
between Hb tetramers. This leads to the formation of hemoglobin
polymers (polyHb) of various molecular weights and oxygen
affinities. Chemical reagents with multi-aldehyde functionalities
can be used as cross-linking agents. These include molecules such
as glutaraldehyde, ring-opened raffinose and dextran. In the case
of aldehydes, the formation of covalent cross-links may be
initiated by the carbonyl group of the aldehyde reacting with an
amino group present in the Hb tetramer. Polymerization of Hb into
larger molecules may increase the intravascular half-life of the
polyHb with respect to native tetrameric Hb and prevent Hb
dissociation into .alpha..beta. dimers. PolyHb may be eventually
filtered out of the systemic circulation throughout the kidneys,
the lymphatic system, and the reticuloendothelial system (RES).
[0081] Several other chemical agents can be used to cross-link
hemoglobin .alpha./.beta. dimers and prevent their filtration by
the glomerulus into the urine, and yet maintain the oxygen
transport and delivery properties of native hemoglobin. Bis 3',5'
dibromo salicyl fumarate (DBSF) is an activated diester of fumaric
acid that has been used as a cross-linker to cross-link hemoglobin
(Tye, U.S. Pat. No. 4,529,719, hereby incorporated by reference in
its entirety). Bis 3',5' dibromo salicyl fumarate effects this
change by associating the salicyl moieties with the sites known to
bind aspirin within hemoglobin, and then effecting cross linking by
the fumarate active functionalities with the alpha and beta chains.
This maintains the two dimers in proper orientation for
cross-linking with lysine residues. Cross-linking the .alpha. or
.beta. chains to a like chain of the other half of the tetramer
forming hemoglobin can prevent disassociation of the tetramer and
yields stable hemoglobins of the invention with a oxygen carrying
capacity with a p50 of about 20 mm Hg to about 45 mm Hg, with a p50
test performed in vitro in the absence of CO.sub.2. Cross linking
is also possible between unlike chains in opposing dimeric pairs.
Thus cross linking hemoglobin can address both the issues of oxygen
affinity, by locking the conformation of the modified hemoglobin
into the T state, and the problem of rapid filtration by the
kidney.
[0082] Hemoglobin's oxygen-binding capacity may be decreased in the
presence of carbon monoxide because both gases compete for the same
binding sites on hemoglobin, carbon monoxide binding preferentially
relative to oxygen. Hemoglobin binding affinity for CO is 200 times
greater than its affinity for oxygen, meaning that small amounts of
CO may reduce hemoglobin's ability to transport oxygen. When
hemoglobin combines with CO, it forms a very bright red compound
called carboxyhemoglobin. When inspired air (i.e., for example in
the environment of tobacco smoking, cars, and furnaces) contains CO
levels as low as 0.02%, headache and nausea may occur; if the CO
concentration is increased to 0.1%, unconsciousness may follow. In
heavy smokers, up to 20% of the oxygen-active sites can be blocked
by CO. Hemoglobin also has competitive binding affinity for sulfur
monoxide (SO), nitrogen dioxide (NO.sub.2), nitric oxide (NO), and
hydrogen sulfide (H.sub.2S). The iron atom in the heme group is in
the Fe.sup.2+ oxidation state to support oxygen transport.
Oxidation to Fe.sup.3+ state converts hemoglobin into
methemoglobin, which cannot bind oxygen. Nitrogen dioxide and
nitrous oxide are capable of converting hemoglobin to
methemoglobin.
[0083] Carbon dioxide occupies a different binding site on the
hemoglobin. Hemoglobin can bind protons and carbon dioxide, causing
a conformational change in the protein and facilitating the release
of oxygen. Protons bind at various sites along the protein and
carbon dioxide binds at the .alpha.-amino group, hence forming
carbamate. Conversely, when the carbon dioxide levels in the blood
decrease (i.e., around the lungs), carbon dioxide is released,
increasing the oxygen affinity of the protein. This control of
hemoglobin's affinity for oxygen by the binding and release of
carbon dioxide is known as the Bohr effect.
[0084] As described above, the conformational change affected by
the change in proton binding to hemoglobin facilitates oxygen
offloading in tissues where the carbon dioxide concentration is
increasing, with resultant pH decrease. This creates a leftward
shift of the cooperativity curve for hemoglobin's affinity for
oxygen, yielding greater efficiency in delivery of oxygen per gram
of hemoglobin. Enhancing this shift in a modified hemoglobin may
result in an effective therapeutic intervention for patients with
poor cardiac function, thus providing more effective oxygenation
with less work required by the heart. Additionally, a hemoglobin so
modified to yield superior oxygen offloading can be useful in
treating patients subject to performance related oxygenation
deficits.
II. METHODS FOR PRODUCING CARBOXAMIDOMETHYLATED CROSS-LINKED
HEMOGLOBIN
[0085] The steps for some of the embodiments of the present
invention are depicted in FIG. 1. Without limiting the scope of the
present invention, the steps can be performed independently of each
other or one after the other. One or more steps can be deleted in
the methods of the present invention. The method of producing the
hemoglobin of the present invention can include step 101 comprising
removing plasma proteins and endotoxin from a preparation
containing red blood cells by washing; step 102 comprising lysing
the red blood cells; step 103 comprising separating hemoglobin by
removing stroma, including membranes and leucocytes, from the lysed
red blood cells; step 104 comprising removing oxygen from the
hemoglobin; step 105 comprising adding to a hemoglobin solution a
reagent which provides a thiol protecting group for a cysteine of
the hemoglobin; step 106 comprising separating the hemoglobin which
has a thiol protecting group attached to a cysteine group of the
hemoglobin; step 107 comprising cross linking the hemoglobin; and
step 108 comprising equilibrating the hemoglobin in biologicially
compatible buffer and preparing a non-pyrogenic, endotoxin free,
oxygen free, stroma free, cysteine protected, cross linked
hemoglobin. Without limiting the scope of the present invention,
the order of the steps may be changed depending on the requirements
for producing a hemoglobin according to this invention.
1. Materials and Equipment Preparation
[0086] Whole blood from bovine sources may be obtained from live or
freshly slaughtered donors. Upon collection, the blood is typically
mixed with at least one anticoagulant to prevent significant
clotting of the blood. Suitable anticoagulants for blood are as
classically known in the art and include, for example, sodium
citrate, ethylenediaminetetraacetic acid and heparin. When mixed
with blood, the anticoagulant may be in a solid form, such as a
powder, or in an aqueous solution. It is understood that the blood
solution source can be from a freshly collected sample or from an
old sample. The methods of the invention provide for the use of
expired human blood from a blood bank. Further, the blood solution
could previously have been maintained in frozen and/or liquid
state. It is preferred that the blood solution is not frozen prior
to use in this method.
[0087] Prior to introducing the blood solution to anticoagulants,
antibiotic levels in the blood solution, such as penicillin, may be
assayed. Antibiotic levels may be determined to provide a degree of
assurance that the blood sample is not burdened with an infecting
organism by verifying that the donor of the blood sample was not
being treated with an antibiotic. Alternatively, a herd management
program to monitor and insure the lack of disease in or antibiotic
presence from treatment of the cattle may be used. The blood
solution may be strained prior to or during the anticoagulation
step, for example by straining, to remove large aggregates and
particles. A 150 micron filter is a suitable strainer for this
operation.
[0088] Any of a variety of assays may be employed to demonstrate
the non-pyrogenicity of the compositions of the present invention,
for example, but are not limited to, interleukin-6 and other
cytokine induction (Pool, E. J. et al., J. Immunoassay 19:95-111
(1998), and; Poole, S. et al., Dev. Biol. Stand. 69:121-123
(1988)); human monocytoid cell line assays (Eperon, S. et al., J.
Immunol. Meth. 207:135-145 (1997), and; Taktak, Y. S. et al., J.
Pharm. Pharmacol. 43:578-582 (1991)); the limulus amoebocyte lysate
(LAL) test (Fujiwara, H. et al., Yakugaku Zasshi 110:332-40 (1990),
and; Martel F. et al., Rev Fr Transfus Immunohematol 28:237-250
(1985)) and the rabbit pyrogen test (Bleeker W. K. et al., Prog
Clin Biol Res 189:293-303 (1985); Simon, S. et al., Dev. Biol.
Stand. 34:75-84 (1977), and; Allison, E. S. et al., Clin. Sci. Mol.
Med. 45:449-458 (1973)), all references incorporated herein in
their entirety. The rabbit pyrogen test was the preferred
pyrogenicity assay until enhanced LAL-testing has replaced this
former technique. It is understood that other methods of removing
pyrogen are known in the art and are within the scope of the
present invention, including filters, absorbers, affinity
materials, etc.
[0089] Serum lipases, such as lipase A, do not inactivate
endotoxins bound to the hemoglobin molecule. Therefore, endotoxins
remain active toxins when taken up by the hepatocyte metabolizing
the hemoglobin. Friedman, H. I. et al. reported triad hepatoxicity
in a rat model consistent with this theory (See, Friedman, H. I. et
al., Lab Invest 39:167-77 (1978), and; Colpan et al., U.S. Pat. No.
5,747,663) have reported a process for reducing or removing
endotoxins from a cellular lysate solution. Wainwright et al. (U.S.
Pat. No. 5,627,266) have described an endotoxin binding protein
immobilized to a solid support and the use of this molecule in the
removal of endotoxins from solution.
[0090] In some embodiments of the present invention, the
elimination of contamination with endotoxins can be ensured by
preventing the introduction of endotoxins to the chemical processes
of the present invention. Typically, endotoxins are added
inadvertently by using endotoxin contaminated water, non-sterile
techniques, or the simple process of bacteria exposure during
collection. Measurement of endotoxins can be difficult, and
standard LAL binding assays do not work in the presence of
hemoglobin since initial collection endotoxin binds strongly to
hemoglobin. However, turbidometric, and chromogenic assays have
been validated that allow for very low limits of detection. Water
and the blood collection can be the most likely candidates for
introduction of endotoxins since increased number of steps in the
preparation of hemoglobin may increase the level of toxicity.
Preparations using dialysis and filtration methods can expose the
hemoglobin to a thousand volumes of water/buffer that may be
contaminated with endotoxin. Membrane systems may be pretreated
with NaOH or NaOCl to reduce or eliminate endotoxins. These
materials may then be flushed and cleaned from the various
devices.
It is preferred that all membranes, and equipment used to produce
the hemoglobin of the present invention be cleansed in a manner
sufficient to cause the removal or elimination of endotoxin that
may be present on such materials and equipment. Preferably, such
cleansing is accomplished by pre-washing surfaces and equipment
that may come into contact with the hemoglobin of the present
invention using a dilute solution of hemoglobin, previously
qualified as non-endotoxin bearing. Such a solution serves to bind
endotoxin and hence to remove residual endotoxin that may be
present on such membranes or equipment. See, for example, Tye, U.S.
Pat. No. 6,894,150. The dilute solution of hemoglobin used for
washing is discarded after each use. Preferably, any ion removal or
buffer equilibration can be performed using counter flow dialysis
so as to prevent accumulation of endotoxin in the subsequent
product. 2. Step 101. Washing of RBCs to remove Plasma Proteins and
endotoxin.
[0091] The RBCs in the blood solution can be washed by any suitable
means, such as by diafiltration or by a combination of discrete
dilution and concentration steps with at least one solution, such
as an isotonic solution, to separate RBCs from extracellular plasma
proteins, such as serum albumins or antibodies (e.g.,
immunoglobulins (IgG)). It is understood that the RBCs can be
washed in a batch or continuous feed mode. Acceptable isotonic
solutions are well known in the art and include solutions, such as,
for example, citrate/saline solution or PBS which have a pH and
osmolarity which does not rupture the cell membranes of RBCs and
displaces the plasma portion of the whole blood. Sources of
purified water which can be used in the method of invention
includes distilled water, deionized water (DI), water-for-injection
(WFI) and/or low pyrogen water (LPW). WFI, which is preferred, is
deionized, distilled water. The specific method of purifying water
is not as important as the requirement that it needs to be low in
endotoxin content.
[0092] The water and the reagents used in the present invention are
substantially free from endotoxin contamination. Preferably, the
water and the reagents used in the present invention are completely
free from endotoxin contamination. One way to reduce the risk of
endotoxin contamination can be to reduce the amount of water and
reagent buffers exposed to the hemoglobin preparation. Therefore,
under some embodiments of the present invention, the hemoglobin
preparations are made using counter-flow or counter-current
dialysis for equilibration of buffers and/or removal of reaction
products. Counter flow dialysis methods are suitable for use in the
present invention are commercially available e.g., VariPerm M,
bitop, Witten (see, e.g., Schwarz, T. et al, Electrophoresis
15:1118-1119 (1994)), Spectrum Laboratories, Inc., Laguna Hills,
Calif., etc. It is estimated that the hollow fiber technique may
yield a hemoglobin preparation of the present invention that has a
100 fold reduction in the amount of endotoxin as compared to
standard synthesis techniques. It is understood that other methods
of removing the endotoxins are known in the art and are within the
scope of the present invention.
[0093] In one method used to collect the erythrocytes, the blood
samples can be washed several times with an isotonic solution and
the plasma can be separated by centrifugation at 3,000 rpm in a 4''
diameter bowl. Preferably, the isotonic solution used is a saline
solution. Preferably, the cells are washed at least three times,
rinsed between each centrifugation, and resuspended in a final
volume of an equal volume of isotonic solution. Alternatively,
concentration of RBCS may be accomplished by filtration over a
tangential flow membrane.
[0094] The use of a sonicator may be discouraged as it makes
membrane spheres (often referred to as "dust"). Agitation methods
suitable for use in the present invention may include a magnetic
stir bar (0.25'' in diameter) and a mechanical rocker or shaker.
(one to two liter container capacity may be used). This exemplary
protocol describes equipment to illustrate the limitation of forces
acting upon the collected cells to prevent undesirable fracturing
of the cell membranes at this point.
[0095] It is understood that methods generally known in the art for
separating RBCs from other blood components can be employed. For
example, sedimentation, wherein the separation method does not
rupture the cell membranes of a significant amount of the RBCs,
such as less than about 30% of the RBCs, prior to RBC separation
from the other blood components such as, white blood cells (WBCs)
and platelets.
[0096] White blood cells can cause febrile reactions in human
recipients when present in transfused packed RBCs. It is desirable
to use a leucoreduction filter which can pass the RBCs but markedly
reduce the number of WBCs. A larger prototype than that used for
single human unit of packed cells is used to evaluate the
leucoreduction. Prechilling washed bovine erythrocytes for about 12
h permits leukoreduction of filtration in about 15 minutes. The
results are shown in Table 1. A 3 log reduction in WBCs, as
quantified by instruments such as a Coulter Counter.RTM. Cell and
Particle Counter is achieved by the passage of the red cell
suspension through a leucocyte reduction filter. This is an
alternative to the method wherein RBCs are selectively lysed in the
presence of WBCs without lysing the WBCs, which are subsequently
removed by filtration. This selective lysing is discussed more
fully below.
TABLE-US-00001 TABLE 1 Leucocyte Reduction Filter Initial Vol
Adjusted % Sample WBC/mm.sup.3 Final WBC/mm.sup.3 Removal
Log.sub.10 Removal 12 h Cold 6.13 .times. 10.sup.3 28 99.6% 3
Bovine
3. Step 102. Lysis of Erythrocytes.
[0097] Various lysis methods can be used, such as mechanical lysis,
chemical lysis, hypotonic lysis or other known lysis methods which
release hemoglobin without significantly damaging the ability of
the Hb to transport and release oxygen. Hemoglobin may be released
from the erythrocyte by hypotonic lysis in deionized water.
Preferably, lysis is accomplished in four to twenty volumes of
deionized water. In one method, plasma free blood cells are
equilibrated with NS, and then diluted into 4 volumes of deionized
water (DI). This can result in the fracturing of the plasma free
blood cells by the hypotonic lysis. The cells are fractured by the
rapid uptake of water. Red blood cells can be lysed in about 30
seconds, while WBCs are more resistant. RBCs are collected in a
flow process after the RBCs are allowed to lyse but just before the
WBCs begin to lyse, an additional volume of a 9% saline solution is
added to arrive at a total concentration of 0.9% saline content
overall. This timed increase in salinity prevents WBCs from lysing.
The stroma and WBCs are removed from the lysed RBCs by filtration.
The hemoglobin can then be removed by a 0.22 .mu.m filter as
filtrate while the retentate would concentrate the stroma, red cell
membranes, and the unlysed WBCs. FIG. 2 depicts the time course of
a lysis experiment showing resistance of WBC lysis for up to 5
minutes. Erythrocyte lysis can be stopped during the two minute
period before appreciable leucocyte lysis occurs.
[0098] Other methods of erythrocyte lysis, such as "slow hypotonic
lysis" or "freeze thaw", may also be employed. See, e.g., Chan et
al., J. Cell Physiol. 85:47-57 (1975), incorporated by reference in
its entirety. In some embodiments of the present invention, the
cells are lysed by flow mixing red blood cells in isotonic saline
with 12 volumes of deionized, endotoxin-free water and subjecting
the cells to gentle agitation. It is understood that other methods
of lysing the RBCs are known in the art and are within the scope of
the present invention.
4. Step 103. Separation of Stroma from Hemoglobin.
[0099] The contents of the erythrocyte are about 98.5% in pure
hemoglobin, with some small amount of other proteins including
carbonic anhydrase. The membranes of red blood cells are referred
to as ghosts or stroma and contain all of the blood type antigens.
Rabiner et al. first demonstrated that some of the toxic properties
of hemolyzed red blood cells were related to the membrane (stroma)
of red blood cells and their related lipids (Rabiner et al., J.
Exp. Med. 126:1127 (1967), incorporated by reference in its
entirety). The membranes can be destroyed by freezing so that
storage requirements for blood may require climate controlled
refrigeration. In addition, many of the human viral diseases
transmitted through blood transfusions may adhere to the stroma of
red blood cells. Thus, stroma-free hemoglobin ("SFH") can be
beneficial in light of the immunogenic properties, such as
inflammation, agglutination, clotting, an immune mediated
complement response, platelet activation, etc, of the cell
membranes of red blood cells, and possibility of viral
contamination.
[0100] An effective stroma-free hemoglobin blood substitute or
oxygen delivery therapy can offer several advantages over
conventional blood based therapies. Significantly, the use of
stroma-free hemoglobin blood substitutes can reduce the extent and
severity of undesired immune responses, and the risk of
transmission of viral diseases, including hepatitis and HIV.
Moreover, in contrast to the limited storage capacity of
erythrocytes, a stroma-free hemoglobin blood substitute or oxygen
delivery therapeutic can exhibit an extended shelf life, and
require less rigorous environmentally controlled storage
facilities.
[0101] The stroma may be removed by ultrafiltration of the
hemolysate over a 0.65 micron filter which retains the cellular
components and passes the hemoglobin. Alternatively, the cellular
debris may be removed by subsequent filtration through a 0.22
micron filter or a 300,000 Dalton molecular weight filter.
Ultrafiltration membranes suitable for use in the present invention
are commercially available from, for example, Millipore
Corporation. Other methods for separating Hb from the lysed RBC
phase can be employed, including sedimentation, precipitation (Tye,
U.S. Pat. No. 4,529,719), centrifugation or microfiltration It is
understood that other methods of removing stroma are known in the
art and are within the scope of the present invention.
[0102] Carbonic Anhydrase.
[0103] Carbonic anhydrase will be removed through diafiltration
once the red cell membrane has been lysed, which is employed at
several points in this method. For example, diafiltration and
buffer exchanges occur before, during and after cross-linking. The
presence of carbonic anhydrase may be quantified by ELISA.
[0104] Microscopic analysis of 10 ml spun samples does not reveal
any cellular debris.
[0105] Phospholipid Level Reduction.
[0106] Another key element to the stable NO-blocked tetrameric
hemoglobins of the invention is the low level of phospholipids
present. Phospholipids derive from the surface lipid layer of the
red cells, the source of the hemoglobin. The steps of processing
given above remove these unwanted lipids, thus eliminating the
problems associated with their presence. Phospholipid assays can be
measured by HPLC and/or ELISA as is well known to one skilled in
the art. Phosphatidylcholine is found to be below the limit of
detection.
[0107] Concentration of Hemoglobin to 14% Solution.
[0108] After such treatment, the stroma-free hemolysate is
concentrated by a membrane that does not allow for the passage of
hemoglobin. Preferably, the stroma-free hemolysate is concentrated
using a filter having a 10,000 MW cut-off. Preferably, the
stroma-free hemolysate is concentrated to a 1%-25% (g/1) solution.
More preferably, the stroma-free hemolysate is concentrated to
about 5% to about 20%. Most preferably, the stroma-free hemolysate
is concentrated to about 6% to about 10%. The concentrated solution
can be equilibrated with buffer and the pH is adjusted. Preferably,
the pH is adjusted to a pH of 7.40. However, a pH of between about
6.5 and about 8.5 can be used in the present invention.
[0109] Optionally, the concentrated Hb solution can then be
directed into one or more parallel chromatographic columns to
further separate the hemoglobin by high performance liquid
chromatography from other contaminants such as antibodies,
endotoxins, phospholipids and enzymes and viruses. Examples of
suitable media include anion exchange media, cation exchange media,
hydrophobic interaction media and affinity media. The
chromatographic columns may contain an anion exchange medium
suitable to separate Hb from non-hemoglobin proteins. Suitable
anion exchange mediums include, for example, silica, alumina,
titanium gel, cross-linked dextran, agarose or a derivatized
moiety, such as a polyacrylamide, a polyhydroxyethyl-methacrylate
or a styrene divinylbenzene, that has been derivatized with a
cationic chemical functionality, such as a diethylaminoethyl or
quaternary aminoethyl group. A suitable anion exchange medium and
corresponding eluents for the selective absorption and desorption
of Hb as compared to other proteins and contaminants, which are
likely to be in a lysed RBC phase, are readily determinable by one
of reasonable skill in the art.
[0110] Removal of Phosphate Ion.
[0111] Bucci et al. (U.S. Pat. No. 5,290,919) have reported that
removal of organic phosphates, e.g., 2,3-diphosphoglycerate, may be
necessary in human hemolysates because the site of the
cross-linking reaction is the same as that occupied by
2,3-diphosphoglycerate in hemoglobin. In some embodiments of the
present invention, the stroma free human Hb solution is
substantially free from inorganic phosphate. Accordingly, in some
embodiments of the present invention, the stroma free human Hb
(before cross linking) that has passed through the filter may be
then treated to exchange phosphate for chloride. For this purpose,
the stroma free human Hb can be passed in the absence or presence
of oxygen, through an ion exchange column that has been previously
prepared and equilibrated with chloride. Efficacy of this step may
be measured by total inorganic phosphate analysis. Suitable ionic
resins are commercially available and are within the scope of the
present invention. The ionic resin removes phosphate that may
compete for the site to which aspirin binds during the reaction
with DBSF. The solution can then be concentrated to the desired
range. This operation is not necessary when using bovine Hb.
5. Step 104. Removal of Oxygen.
[0112] The thiol blocked stroma free Hb or more specifically, the
CMSFH can be treated under conditions sufficient to remove oxygen
present in the preparation. One aspect of the present invention
concerns an improved process for removing oxygen from CMSFH
preparations. Without limiting the scope of the present invention,
such deoxygenation can be carried out before or after any of the
steps disclosed herein. For example, the deoxygenation step can be
performed prior to or after the step of removing stroma, the step
of removing the endotoxins, the step of thiol protection, the step
of phosphate removal, the step of lysis of RBCs, or the step of
cross-linking of the hemoglobin. In one embodiment, such
deoxygenation is performed prior to the protection of the thiol
group in the cysteine moiety of the hemoglobin of the present
invention. In another embodiment of the invention, deoxygenation is
performed prior to cross linking the hemoglobin. In some
embodiments of the invention, the steps of the method may require
more time to be completed. In such cases, deoxygenated conditions
may be preferred.
[0113] The extent of deoxygenation can be measured by gas
chromatograph, zirconium-based detector (e.g., a "MOCON" analyzer
(Mocon, Minneapolis, Minn.), by measuring pO.sub.2 or by measuring
the spectral shift that is characteristic of deoxyhemoglobin
formation.
[0114] Oxygen in the hemoglobin can be removed by vacuum, or by
vacuum centrifugation. The CMSFH used may be an ultrafiltrate
obtained from the removal of stroma (dilute) or a retentate from
the ultrafiltration of the second stage ultrafiltration conducted
to concentrate the hemoglobin to approximately 10% (w/v). Either of
these solutions of CMSFH obtained can be readily deoxygenated by
applying a vacuum sufficient to equal the partial pressure of water
at the temperature of the solution, while the solution can be
centrifuged at a speed sufficient to produce a force greater than
the surface tension of the solution. These are generally low speeds
and can be met with preparatory centrifuges, or those of a
continuous flow variety. It may be desirable to consider the
geometry of the containers of the CMSFH to insure that there may be
adequate surface area for gas exchange and that the temperature can
be maintained and the solution not allowed to freeze.
[0115] Contactor membrane technology can be used to remove O.sub.2
from Hb solutions. The contactor membrane technology can also be
used for oxygenation where oxygen gas may be used instead of
nitrogen gas. Three or four of such membranes may be attached in
series for higher throughput and can be used for commercial
production of deoxygenated or oxygenated hemoglobin solution. The
Hb concentration affects the rate at which the dissolved O.sub.2 is
removed. As the Hb concentration is lowered the O.sub.2 removal
rate increases. The experiment may not lower O.sub.2 concentration
to <100 ppb. However, the test can be performed in the anaerobic
glove box to make the system gas tight. The glove box can maintain
the environment at very low O.sub.2 levels (<5 ppb). This glove
box environment can provide the O.sub.2 barrier required to ensure
that no O.sub.2 can be re-absorbed by the Hb. Hg vacuum greater
than 28.5'' (<50 mm Hg) can be used for optimum O.sub.2
removal.
[0116] The deoxygenated, endotoxin free, stroma free,
carboxamidomethylated Hb (dCMSFH) prepared in the manner described
above may be preferably maintained in an inert environment and the
pH of the preparation may be preferably adjusted to a range between
6.0 and 9.5, and most preferably about pH 8.3-8.4. The pH of the
solution may be adjusted using 1.0 N acetic acid or 1.0 N NaOH.
Where dilution, suspension, or addition of water (including
buffers, etc.) for other purposes is desired, such water may be
deoxygenated and be free of endotoxin. All subsequent steps may be
carried out in the absence of oxygen, maintained by what ever means
is desired. As indicated above, a preferred method involves the use
of nitrogen positive pressure environmental glove box, however,
other inert gases (e.g., argon) may be equivalently employed in
lieu of nitrogen.
6. Step 105. Protecting the Sulfhydryl of the Cysteine(s) of the
Hemoglobin.
[0117] The step of protecting cysteine of the hemoglobin with thiol
protecting groups may be carried out before the cross linking step
or after the cross linking step. In one embodiment of the present
invention, the step of protecting the thiol group in the cysteine
moiety is carried out before the cross-linking step. In another
embodiment of the present invention, the step of deoxygenating the
hemoglobin is carried out before the step of protecting the thiol
group in the cysteine moiety. In some embodiments of the invention,
deoxygenation is not performed prior to protecting the sulfhydryl
groups in the hemoglobin. All the reagents known in the art for the
protection of a functional group such as, but not limited to,
hydroxyl, thiol, or carboxyl, are included in the present
invention.
[0118] Some of the examples of the reagents include, but are not
limited to, 4-pyridylmethyl chloride, alkoxyalkylchloride,
dimethoxymethane, N-(hydroxymethyl)acetamide, triphenylmethyl
chloride, acetyl chloride, 2-chloroacetic acid, acetic anhydride,
haloacetamide such as, iodoacetamide, bromoacetamide,
chloroacetamide, or fluoroacetamide, haloacetate such as
iodoacetate, bromoacetate, chloroacetate, or fluoroacetate, benzyl
chloride, benzoyl chloride, di-tert-butyl dicarbonate,
p-hydroxyphenacyl bromide, p-acetoxybenzyl chloride,
p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride,
tetrahydropyran, acetamidohydroxymethane, acetone,
bis-carboethoxyethene, 2,2,2-trichloroethoxycarbonyl chloride,
tert-butoxycarbonyl chloride, alkyl isocyanate, and alkoxyalkyl
isocyanate. In a specific example of the sulfhydryl protected
hemoglobins of the invention, the reagent is iodoacetamide. It is
understood that any reagent known in the art that can be used for
carboxamidomethylation.
[0119] Optimization of the Reaction with Iodoacetamide (IAM):
[0120] The iodoacetamide reaction is followed with an iodide
specific electrode, since one of the byproducts is iodide ion. A
two molar excess per equivalent of sulfhydryl group can be used.
Table 2 shows a grid of time course of the iodoacetamide reaction
for bovine Hb vs. the moles of IAM reagent used in the IAM
reaction. The results show the amount of free sulfhydryl per mole
of Hb and are given in units of molar equivalents relative to
bovine Hb.
TABLE-US-00002 TABLE 2 Reaction of Bovine Hb with IAM. Results
given in equivalents of free sulfhydryl remaining. Moles Time IAM 0
15 30 45 60 75 90 120 2 2 1.0 0.5 0.5 4 0.5 0.2 0.1 <0.1
7. Step 106. Separating Thiol-Protected Hemoglobin from
Reactants.
[0121] After the reaction is complete, as determined by the rate of
iodide evolution observed, excess IAM is removed by equilibration
with Ringer's Acetate and diafiltration.
8. Step 107. Cross-Linking with DBSF and Reaction with PLP.
[0122] Stroma-free Hb can be prevented from dissociation into
.alpha., .beta. dimers by cross-linking intramolecularly to prevent
dissociation of the tetramer into .alpha., .beta. dimers and thus
increase its circulatory half-life. This restricts Hb into the T
state and resultantly can modify the affinity for oxygen and
therefore modifies the oxygen transport properties of the Hb.
[0123] Examples of suitable cross-linking agents include
polyfunctional agents that will cross-link Hb proteins, such as
glutaraldehyde, succindialdehyde, activated forms of
polyoxyethylene and dextran, .alpha.-hydroxy aldehydes, such as
glycolaldehyde, N-maleimido-6-aminocaproyl-(2'-nitro,4'-sulfonic
acid)-phenyl ester, m-maleimidobenzoic acid-N-hydroxysuccinimide
ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
m-maleimidobenzoyl-N-hydroxysuccinimide ester,
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester,
N-succinimidyl(4-iodoacetyl)aminobenzoate,
sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl
4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl
4-(p-maleimidophenyl)butyrate,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,
N,N-phenylene dimaleimide, and compounds belonging to the
bis-imidate class, the acyl diazide class or the aryl dihalide
class, among others. The saccharides can be used as cross-linking
agents. The examples of saccharides include, but are not limited
to, monosaccharides (galactose, glucose, methylglucopyranoside, and
mannitol), disaccharides (lactose, maltose, cellobiose, sucrose,
and trehalose), a trisaccharide (raffinose) and polysaccharides
(dextrans with molecular weights of 15,000 and 71,000 Da).
[0124] The cross-linking of hemoglobin may be conducted in the
absence of oxygen. Inorganic phosphate, which binds tightly to the
hemoglobin molecule and interferes with the cross-linking reaction,
may be removed to increase yield. Endotoxins, which bind tightly to
the hemoglobin molecule and become a hepatic toxin when the
hemoglobin is metabolized, may not be allowed to contact the
hemoglobin.
[0125] A suitable amount of a cross-linking agent may be that
amount which may permit intramolecular cross-linking to stabilize
the Hb and also intermolecular cross-linking to form polymers of
Hb, to thereby increase intravascular retention. Without limiting
the scope of the present invention, various strategies can be
employed to cross-link Hb with desirable molecular weight
distributions and oxygen binding properties. The type and
concentration of both cross linking and quenching agents, the
duration of the cross-linking/polymerization reaction, and
utilization of reducing agents are all possible variables that can
be modified in these reactions to engineer the molecular weight
distribution, oxygen binding properties, and metHb levels of
cross-linked Hb dispersions.
[0126] Optimization of Cross Linking Using pH Control and Excess
Cross Linking Agent:
[0127] Table 3 shows a grid of increasing pH vs. mole ratio of
cross linking agent (DBSF) over 2 h. The results show the
percentage of alpha chain left unreacted at different pH and
equivalents (in moles) DBSF at the end of the 2 h period. The pH is
maintained constant with titration of the acid produced by reaction
with NaOH. After 2 h, the production of acid has long since ceased.
Results of cross linking are determined using gel electrophoretic
separations, looking for residual uncross linked alpha chains. In
the standard method of production of the stable NO blocked
tetrameric hemoglobins of the present invention, using 2
equivalents of DBSF, better than 98% cross-linking is achieved.
TABLE-US-00003 TABLE 3 pH vs. Molar Ratio DBSF. Percentage of
Uncross-Linked Hemoglobin Remaining. Equivalents pH DBSF 7 7.5 8
8.2 8.4 1 21% 1.2 19% 1.5 3% 2.0 38% 11% 2.5% 1.7% 1.5% 2.5
1.7%
[0128] In some embodiments of the present invention, the dCMSFH is
cross-linked with bis 3',5' dibromo salicyl fumarate (DBSF) (Tye,
U.S. Pat. No. 4,529,719, hereby incorporated by reference in its
entirety). DBSF cross-linker may be added with stirring to the
dCMSFH preparation at a molar ratio of DBSF cross-linker:dCMSFH of
greater than 1:1. Preferably, the molar ratio of DBSF
cross-linker:dCMSFH is 2:1. Prior to such addition, the pH of the
dCMSFH preparation is adjusted to 8.4 and maintained at 8.4
throughout the reaction. The pH of the reaction mixture is
carefully maintained by the addition of acid or base since the
solution is not buffered. The reaction is permitted to go to
completion.
[0129] Determination of Extent of Cross Linking Using SDS Gel
Electrophoresis:
[0130] SDS can denature proteins to form long rods covered by
negative charges of the carboxyl group at neutral pH. Proteins can
then be separated by size exclusion using electrophoresis since the
manifold excess of negative charge by the SDS can dwarf the charge
heterogeneity of the native proteins. The Beckman Coulter
PA-800.RTM. provides rapid record of the gel electrophoresis by
using the absorption at 216 nm for the peptide bond. Standard
protein mixtures can be used to calibrate the column for the range
of molecular weights of interest, between 10 KDa and 100 KDa in the
present case, as shown in FIG. 3.
[0131] Native hemoglobin run on SDS gel electrophoresis can give
two peaks with almost baseline separation between them. The first
peak has been shown to be the alpha chain and the second the beta
chain of hemoglobin. They can occur in almost equal amounts as
there are equal numbers of chains in the molecule. This is
illustrated in FIG. 4A, which is an expanded region of the overlaid
electropherograms shown in FIG. 4B. Native hemoglobin appears as
the green trace (rt13.98 and rt14.14), cross linked hemoglobin
according to the present invention (dXCMSFH) appears as the red
trace, and size standards are blue. The overlaid electropherograms
shown in FIG. 4A are slightly offset for ease of viewing. The
expanded region illustrated is clustered about the 20 kDa size
standard, which is the region of interest for the two alpha and
beta subunits of native hemoglobin. The experiment shown here for
the cross-linked material is taken from a midpoint in the process,
when most of the alpha chains have already cross linked, but
significant amounts of beta chain still remain unreacted.
[0132] In FIG. 4B, the overlay of the full width electropherograms
are shown, again with native hemoglobin in green, the cross linking
experiment in red, and the size standards in blue. The fumaryl
cross linking of the two alpha chains yields a pair of peptides
tethered together and thus will appear at later elution times than
the unreacted beta chains. The series of three new major product
peaks at a higher molecular weight of about 36,000-45,000 KDA are
seen in FIG. 4B at rt of 16 to 17 min. The product peaks may not be
quantified as the size standards are all single peptide chains and
cannot be extended for quantification for the dimerized products of
this reaction. However, it can be seen that three higher molecular
weight products are being formed.
[0133] There is a formation of .beta.-.beta. crosslink's in
addition to the .alpha.-.alpha. crosslink's. There is a small
amount of material at 90,000 KDa which can indicate the formation
of a small number of inter molecular crosslink's.
[0134] Reaction of Human Hb with PLP.
[0135] Pyridoxal-5-phosphate (PLP) has the ability to modify many
hemoglobins. Although the properties of dXCMSFH from human
hemoglobin benefit from the pyridoxal-5-phosphate reaction, dXCMSFH
from bovine hemoglobin does not require this step. PLP modifies
human hemoglobin by introducing a negative charge near a
penultimate .beta. chain histidine residue and by removing a
positive charge at the amino terminal end of the same chain. These
charge changes stabilize a new molecular configuration that is
similar to the hemoglobin-DPG (diphosphoglycerate) complex.
Significantly, the hemoglobin of this new configuration has an
oxygen affinity resembling that of native hemoglobin within the red
cell. The product may have one or two PLP molecules attached per
tetramer. In prior PLP-hemoglobin preparations the intravascular
retention time was too short to permit such preparations to be
acceptable as a resuscitation fluid. Additionally, they were found
to cause osmotic diuresis.
[0136] Accordingly, after the cross-linking reaction has been
completed, where using human Hb, pyridoxal-5-phosphate (PLP) is
added to the deoxygenated, endotoxin free, stroma free,
carboxamidomethylated cross-linked human Hb (dXCMSFH) preparation.
The PLP is reacted with the dXCMSFH and then reduced with sodium
borohydride to form dXCMSFH-pyridoxal-5'-phosphate (dXCMSFH-PLP)
using the methods described by Benesch et al. (Benesch et al.,
Biochemistry 11:3576 (1972) and references therein; Benesch et al.
Proc. Natl. Acad Sci. 70 (9): 2595-9 (1974); Benesch et al.,
Biochem. Biophys. Res. Commun. 63(4): 1123-9 (1975); Benesch et
al., Methods Enzymol. 76:147-59 (1981); Benesch et al., J. Biol.
Chem. 257(3):13204 (1982); Schnackerz et al.; and, J. Biol. Chem.
258(2):872-5 (1983), all of which references are incorporated
herein by reference in their entirety) with the change that all
reagents are free of endotoxin and oxygen and the reaction occurs
in the absence of oxygen. This treatment is not necessary when
using bovine Hb
[0137] Determination of Residual Uncross-Linked Hemoglobin.
[0138] High performance liquid chromatography (HPLC) Size Exclusion
Chromatography (SEC) can be used to determine the percent of total
cross-linked Hb, percent of cross-linked tetramer, or percent of
cross linked higher order species of Hb/polyHb dispersions. A salt
such as, MgCl.sub.2 can serve to dissociate any non-cross-linked
tetrameric Hb into .alpha.-.beta. dimers, while cross-linked
tetrameric Hb may remain intact. Hence, non-cross-linked Hb may
elute in a separate peak away from intramolecularly cross-linked
Hb.
[0139] Size exclusion HPLC on a Biorad Bio-Sil.sup.R SEC-12.5-5
column of the reaction mixture using 0.5M MgCl.sub.2 solution as a
buffer under conditions that would otherwise not denature the
hemoglobin secondary structure, may be used to examine the amount
of unreacted material, since under these conditions the equilibrium
would favor the alpha beta dimer with a molecular weight of 32 KDa,
which would be expected at greater retention times than seen for
any peak in this experiment. As seen in FIG. 5, the HPLC trace
shows the major peak at 64 KDa, with a minor peak at 128 KDa. There
is no material at later elution times, and hence no materials with
lower molecular weight. This experiment demonstrates the complete
absence of unreacted hemoglobin and illustrates that most of the
material is the stabilized tetramer of Hb with a molecular weight
of 64 KDa.
9. Step 108. Preparing Hemoglobin Solutions by Equilibration.
[0140] The deoxygenated stable NO blocked tetrameric Hb and, in
particular, dXCMSFH can be equilibrated with Ringer's lactate or
Ringer's acetate solution, which under conditions of diafiltration,
removes excess DBSF and byproducts of the reaction, for example,
dibromosalicylic acid. Preferably, any ion removal or buffer
equilibration can be performed using counter flow dialysis so as to
prevent accumulation of endotoxin in the subsequent product. After
equilibration, the solution can be sterile filtered into suitable
infusion containers. Infusion containers suitable for use in the
present invention may include, but are not limited to, sterile IV
bags. Preferred infusion containers may prevent gas exchange (i.e.,
impermeable to oxygen) and the dXCMSFH can be stored in the absence
of oxygen. This is expected to prevent heme oxidation which forms
methemoglobin.
[0141] Determination of the Affinity for Oxygen by Modified
Hemoglobin.
[0142] Hemoglobin has an ability to bind and release oxygen under
physiological conditions as a function of the partial pressure of
oxygen in the system. Oxygen affinity of the hemoglobin derivative
of the present invention can be measured using the Hemox-Analyzer
(made by TCS Corporation or the gill cell described by Dolman et
al., Anal. Biochem. 87:127 (1978), incorporated by reference in its
entirety.
[0143] Hemox-Analyzer (made by TCS Corporation) allows the
determination of the hemoglobin oxygen dissociation curve. Other
methods to obtain a hemoglobin oxygen dissociation curve may not be
as reproducible, accurate and easy to perform. The hemoglobin
oxygen dissociation curves can be altered by changes in pH,
temperature, CO.sub.2 concentration, species of hemoglobin, variant
of human hemoglobin, hemolyzed hemoglobin, and the like. The shape
of the curve and the shift of the curve along the X axis can
describe the ability of the hemoglobin to load and unload oxygen.
The information can be useful in research for blood and modified
hemoglobin as it is an in vitro test of in vivo function. It can
measure the ability of hemoglobin to load and unload oxygen. A
shorthand description of the entire hemoglobin dissociation curve
can be given by the p50 for O.sub.2, the partial pressure of oxygen
in mm of Hg, which can cause the hemoglobin to be half saturated
with oxygen.
[0144] Chemical modifications to hemoglobin or genetic variants to
hemoglobin can cause the p50 to decrease, i.e. bind oxygen more
tightly at any given oxygen pressure. In vivo this can mean less
oxygen to perfused tissue.
[0145] The Hemox-Analyzer relies upon the change in color of blood
(hemoglobin) that is arterial (oxygenated, red) and venous (less
oxygenated, blue), and an oxygen electrode. A small dilute sample
is prepared in a special spectrophotometric cell that has a small
orifice in the bottom that allows a purified gas to be slowly
bubbled through a stirred solution and also fitted with an oxygen
electrode. The entire cell is precisely temperature controlled at
37.degree. C., to equilibrate to body temperature. The outputs of
the spectrophotometer and the oxygen electrode are analyzed and
plotted. At the beginning of a plot the sample is fully oxygenated
by bubbling pure oxygen through the sample until the oxygen
saturation is greater than the fraction of oxygen in air (21%),
then the gas bubbler is switched to nitrogen and the removal of
oxygen begins. Oxygen equilibrium curves can thus be generated.
[0146] The p50 for human hemoglobin in RBCs can be about 28. The
p50 falls to about 14 when RBCs are separated from the 2,3DPG which
forms a salt bridge in the red cell to decrease the oxygen
affinity. The p50 for bovine hemoglobin whether in the red cell or
in free solution is about 25 to about 40 depending on the pH and
the concentration of CO.sub.2. FIG. 6 shows oxygen affinity curves
for bovine whole blood, stroma free Hb, cross linked dXCMSFH of the
invention, and fresh human blood. In the cross linked hemoglobin,
the cross linking locks the hemoglobin in the tense state and
therefore loses the sigmoidal curve. At lower pO.sub.2, when
O.sub.2 is delivered to the tissues, the cross linked hemoglobin
delivers more oxygen as compared to bovine whole blood, stroma free
Hb, and fresh human blood. The p50 of cross linked hemoglobin is
higher than that of human hemoglobin, and bovine hemoglobin. The
p50 value for bovine whole blood is 24.73 mm Hg, for stroma free Hb
is 21.20 mm Hg, for fresh human blood is 23.72 mm Hg and for
dXCMSFH is 32.43 mm Hg, which is significantly higher than that of
human RBCs. Therefore, the stable NO-blocked tetrameric Hb of
present invention, and in particular, XCMSFH, may demonstrate
greater efficiency to the delivery of oxygen per gram of
hemoglobin. Although these numbers, as measured herein, are not
identical to literature values (i.e., human blood p50 literature
value is 27 mm Hg compared to 23.72 mm Hg reported here), the
present values are a good relative measure of oxygen offloading
performance.
[0147] The deoxygenated, endotoxin free, stroma free,
carboxamidomethylated cross-linked Hb (dXCMSFH) of the present
invention is a stabilized tetramer of bovine hemoglobin that is
locked in the tense or T state, and has a p50 similar to hemoglobin
within normal human red blood cells or, as shown in FIG. 6, higher.
Thus, an equal amount of hemoglobin from a human red blood cell and
hemoglobin from dXCMSFH, can carry the same amount of oxygen
leaving the lung. However, dXCMSFH can deliver slightly more oxygen
before its venous return, based upon the data shown in FIG. 6.
III. ANALYSIS
1. Physical Characteristics of the Stable NO-Blocked Tetrameric
Hemoglobins of the Invention
[0148] A stable NO-blocked tetrameric hemoglobin of the present
invention has a molecular weight distribution of about 65 kDa, a
p50 of 20-45 mm Hg, an osmolality of 290-310 mOsm/Kg, with a pH of
6.0 to 7.9 at 10-22.degree. C. The modified hemoglobins of the
invention have a total hemoglobin of 6.0-20 g/dL, with
methemoglobin levels of less than or equal to 5%, oxyhemoglobin
levels of less than or equal to 10%. The modified hemoglobins of
the invention have endotoxin levels of less than or equal to 0.02
EU/ml, phosphatidylcholine levels below detection limits, meet test
for sterility, and a low level of extraneous organics. The modified
hemoglobins of the present invention have a sodium ion level of
125-160 mmol/l, a potassium ion level of 3.5-5.5 mmol/l, a chloride
ion level of 105-120 mmol/l, and a calcium level of 0.5-1.5 mmol/l.
The modified hemoglobins of the invention have levels of N-acetyl
cysteine of less than or equal to 0.22%.
2. Analytical Methods
Physical Chemical Analysis
[0149] The deoxygenated stable NO blocked tetrameric Hb and
dXCMSFH, in particular, as disclosed herein can be analyzed at any
step of the process of making it. Hemoglobin can be analyzed, for
example, but not limited to, after removing stroma, after removing
endotoxin, after lysis, after removing oxygen, after protection of
thiol group in the cysteine moiety, or after cross-linking etc. The
hemoglobin can be analyzed for purity, absorbance, structure, p50,
nitric oxide binding capacity, white blood cell (WBC) count,
microorganism growth in the hemoglobin solution, cross-linking,
amino acid analysis, protein analysis, or effect of refrigeration
or storage. Various analytical techniques are known in the art and
are all within the scope of the present invention. Some of the
examples of the analytical techniques are provided herein but they
are not limiting to the scope of the present invention.
[0150] A. Mass Spectrometry (MS).
[0151] There are many types of mass spectrometers and sample
introduction techniques which allow a wide range of analyses and
they are all included herein. In some preferred embodiments of the
present invention, the technique used is mass spectrometry. Mass
spectrometers may consist of three distinct regions: Ionizer, Ion
Analyzer, and Detector. Ionization methods include, but are not
limited to, electron impact (EI), chemical ionization (CI),
electrospray (ESI), fast atom bombardment (FAB), and matrix
assisted laser desorption (MALDI). Analyzers include but are not
limited to, quadrupole, sector (magnetic and/or electrostatic),
time-of-flight (TOF), and ion cyclotron resonance (ICR). Other
related techniques are, for example, ion mobility spectrometry/mass
spectrometry (IMS/MS), Tandem mass spectrometry (MS/MS), Orbitrap
mass spectrometry. FTICR mass spectrometry, single-stage or a
dual-stage reflectron (RETOF-MS, ladder sequencing with TOF-MS),
Post-source decay with RETOF-MS MALDI, In-source decay with linear
TOF-MS, and surface-enhanced laser desorption ionization-time of
flight (SELDI-TOF). The mass spectrometer may be coupled with LC or
GC.
[0152] B. UV-Vis.
[0153] In some embodiments of the present invention, optical
absorption spectroscopy (UV/VIS) has been used to determine the
absorbance range for the hemoglobin. UV/VIS plays a role for the
determination of concentrations of macromolecules such as proteins.
Organic dyes can be used to enhance the absorption and to shift it
into the visible range (e.g. Coomassie blue reagents).
Understanding the forces that govern the interaction of proteins
with one another assists in the understanding of such processes as
macromolecular assembly, chaperone-assisted protein folding and
protein translocation. Resonance Raman spectroscopy (RRS) is a tool
which can be used to study molecular structure and dynamics.
Resonance Raman scattering requires excitation within an electronic
absorption band and results in a large increase of scattering. This
approach may help to investigate specific parts of macromolecules
by using different excitation wavelengths.
[0154] C. Liquid Chromatography (LC).
[0155] Liquid chromatography is a tool for isolating proteins,
peptides, and other molecules from complex mixtures. In some
embodiments of the present invention, LC has been used for
separation, purification and analysis of the hemoglobin and
excipients used in the formulations of the invention. Examples of
LC include affinity chromatography, gel filtration chromatography,
anion exchange chromatography, cation exchange chromatography,
diode array--LC and high performance liquid chromatography (HPLC)
and affinity and size exclusion chromatography HPSEC.
[0156] Gel filtration chromatography and HPSEC chromatography
separates proteins, and peptides on the basis of size. Gel
Filtration Chromatography may be used for analysis of molecular
size, for separations of components in a mixture, or for salt
removal or buffer exchange from a preparation of
macromolecules.
[0157] Affinity chromatography is the process of bioselective
adsorption and subsequent recovery of a compound from an
immobilized ligand.
[0158] Ion exchange chromatography separates molecules based on
differences between the overall charges of the proteins. It is
usually used for protein purification but may be used for
purification of peptides, or other charged molecules. Elution can
be achieved by increasing the ionic strength to break up the ionic
interaction, or by changing the pH of the protein.
[0159] HPLC can be used in the separation, purification and
detection of hemoglobin of the present invention. Use of
reversed-phased chromatography (RPC) can be utilized in the process
of protein structure determination. The normal procedure of this
process can be 1) fragmentation by proteolysis or chemical
cleavage; 2) purification; and 3) sequencing. A common mobile phase
for RPC of peptides can be, for example, a gradient of 0.1%
trifluoroacetic acid (TFA) in water to 0.1% TFA in a suitable
organic solvent, such as acetonitrile, which provides for the
solubilization of the proteins/peptides, permits detection at
approximately 230-240 nmm, and is easily removable, i.e by
evaporation, from the proteins/peptides.
[0160] The use of size-exclusion chromatography (SEC) and
ion-exchange chromatography (IEC) can be used in determining the
structure of the hemoglobin of the present invention. Full recovery
of activity after exposure to the chromatography may be achieved,
and SEC columns can allow fractionation from 10 to 1000
kilodaltons. The use of gradient elution with the IEC column may be
favorable because of equivalent resolution as polyacrylamide gel
electrophoresis (PAGE) and increased loading capability when
compared to SEC. In liquid affinity chromatography (LAC)
interaction may be based on binding of the protein due to mimicry
of substrate, receptor, etc. The protein may be eluted by
introducing a competitive binding agent or altering the protein
configuration which may facilitate dissociation. HPLC may be
coupled with MS.
[0161] D. Electrophoresis.
[0162] Electrophoresis can be used for the analysis of the
hemoglobin of the present invention. Electrophoresis can be gel
electrophoresis or capillary electrophoresis.
[0163] Gel Electrophoresis:
[0164] Gel electrophoresis is a technique that can be used for the
separation of proteins. Separation of large (macro) molecules may
depend upon two forces: charge and mass. During electrophoresis,
macromolecules are forced to move through the pores when the
electrical current is applied. Their rate of migration through the
electric field depends on the strength of the field, size and shape
of the molecules, relative hydrophobicity of the samples, and on
the ionic strength and temperature of the buffer in which the
molecules are moving. Using this technology it is possible to
separate and identify protein molecules that differ by as little as
a single amino acid. Also, gel electrophoresis allows determination
of crucial properties of a protein such as its isoelectric point
and approximate molecular weight. Electrofocusing or isoelectric
focusing is a technique for separating different molecules by their
electric charge differences, taking advantage of the fact that a
molecule's charge changes as the pH of its surroundings
changes.
[0165] Capillary Electrophoresis:
[0166] Capillary electrophoresis is a collection of a range of
separation techniques which may involve the application of high
voltages across buffer filled capillaries to achieve separations.
The variations include separation based on size and charge
differences between analytes (termed capillary zone electrophoresis
(CZE) or free solution CE (FSCE)), separation of neutral compounds
using surfactant micelles (micellar electrokinetic capillary
chromatography (MECC) or sometimes referred to as MEKC) sieving of
solutes through a gel network (capillary gel electrophoresis, GCE),
separation of cations (or anions) based on electrophoretic mobility
(capillary isotachophoresis, CITP), and separation of zwitterionic
solutes within a pH gradient (capillary isoelectric focusing,
CIEF). Capillary electrochromatography (CEC) can be an associated
electrokinetic separation technique which involves applying
voltages across capillaries filled with silica gel stationary
phases. Separation selectivity in CEC can be a combination of both
electrophoretic and chromatographic processes. Many of the CE
separation techniques rely on the presence of an electrically
induced flow of solution (electroosmotic flow, EOF) within the
capillary to pump solutes towards the detector. GCE and CIEF are of
importance for the separation of biomolecules such as proteins.
[0167] E. Nuclear Magnetic Resonance (NMR).
[0168] NMR can be used for the analysis of the hemoglobin of the
present invention. NMR spectroscopy is capable of determining the
structures of hemoglobin at atomic resolution. In addition, it is
possible to study time dependent phenomena with NMR, such as
intramolecular dynamics in macromolecules, reaction kinetics,
molecular recognition or protein folding. Heteronuclei like
.sup.15N, .sup.13C and .sup.2H, can be incorporated in proteins by
uniformly or selective isotopic labeling. Spectra from these
samples can be drastically simplified. Additionally, some new
information about structure and dynamics of macromolecules can be
determined with these methods.
[0169] F. X-Ray Crystallography.
[0170] X-ray crystallography can be used for the analysis of the
hemoglobin of the present invention. X-ray crystallography is a
technique in which the pattern produced by the diffraction of
X-rays through the closely spaced lattice of atoms in a crystal is
recorded and then analyzed to reveal the nature of that lattice.
This generally leads to an understanding of the material and
molecular structure of a substance. The spacings in the crystal
lattice can be determined by using Bragg's law. The electrons that
surround the atoms, rather than the atomic nuclei themselves, are
the entities which physically interact with the incoming X-ray
photons. This technique can be used to determine the structure of
the hemoglobin of the present invention. X-ray diffraction is
commonly carried out using single crystals of a material, but if
these are not available, microcrystalline powdered samples may also
be used which may require different equipment.
[0171] G. Arrays.
[0172] Arrays can be used for the analysis of the hemoglobin of the
present invention. Arrays involve performing parallel analysis of
multiple samples against known protein targets. The development of
various microarray platforms can enable and accelerate the
determination of protein abundance, localization, and interactions
in a cell or tissue. Microarrays provide a platform that allows
identification of protein interaction or function against a
characterized set of proteins, antibodies, or peptides.
Protein-based chips array proteins on a small surface and can
directly measure the levels of proteins in tissues using
fluorescence-based imaging. Proteins can be arrayed on either flat
solid phases or in capillary systems (microfluidic arrays), and
several different proteins can be applied to these arrays.
Nonspecific protein stains can be then used to detect bound
proteins.
[0173] H. Amino Acid Analysis.
[0174] In some embodiments, amino acid analysis (AAA) is a
technique used in the analysis of the hemoglobin of the present
invention. AAA is a process to determine the quantities of each
individual amino acid in a protein. There can be four steps in
amino acid analysis: hydrolysis, derivatization, separation of
derivatized amino acids, and data interpretation and
calculations.
[0175] In the hydrolysis step, a known amount of internal standard
(norleucine) may be added to the sample. The sample, containing at
least 5 nmoles of each amino acid (i.e. 10 .mu.g of protein) can be
then transferred to a hydrolysis tube and dried under vacuum. The
tube can be placed in a vial containing HCl and a small amount of
phenol and the protein is hydrolyzed by the HCl vapors under
vacuum. The hydrolysis is carried out for about 24 h at about
110.degree. C. Following hydrolysis, the sample can be dried.
[0176] Derivatization can be performed automatically on the amino
acid analyzer by reacting the free amino acids, under basic
conditions, for example, with phenylisothiocyanate (PITC) to
produce phenylthiocarbamyl (PTC) amino acid derivatives. A standard
solution containing a known amount (500 pmol) of 17 common free
amino acids can also be loaded on a separate amino acid analyzer
sample spot and derivatized. This can be used to generate a
calibration file that can be used to determine amino acid content
of the sample. Following derivatization, a methanol solution
containing the PTC-amino acids can be transferred to a narrow bore
HPLC system using a reverse phase C18 silica column for separation.
The buffer system used for separation can be for example, 50 mM
sodium acetate at pH 5.45 as buffer A and 70% acetonitrile/32 mM
sodium phosphate at pH 6.1 as buffer B. The program can be run
using a gradient of buffer A and buffer B. Chromatographic peak
areas can be identified and quantitated using a data analysis
system that can be attached to the amino acid analyzer system.
[0177] Alternatively, the classical method of amino acid analysis
of Moore and Stein using ninhydrin may be used.
Clinical Chemistry Analysis Methods
[0178] A. Oxygen Transport.
[0179] A CO-oximeter is used for comprehensive hemoglobin analysis
to establish saturation, desaturation and methemoglobin levels.
Ultraviolet illumination is used to for oxygen transport tests
including levels of deoxyhemoglobin (HHb), oxyhemoglobin
(O.sub.2Hb), methemoglobin (MetHb), carboxyhemoglobin (COHb), total
hemoglobin (tHb), oxygen saturation (SO.sub.2%), oxygen content
(O.sub.2Ct), and oxygen capacity (O.sub.2Cap) in the hemoglobins of
the invention. One suitable instrument is manufactured by Nova
Biomedical Instrumentation.
[0180] B. Electrolytes.
[0181] Electrolytes such as potassium, calcium, sodium chloride,
and others are measured using standard electrolyte/chemistry
analyzers. Suitable instrumentation is produced by Nova Biomedical,
Hitachi, Roche, among others.
[0182] C. Osmolality.
[0183] The osmolality of the hemoglobins of the invention is also
measured. Freezing point depression is the methodology used to
perform this analysis, in order to produce biocompatible volume
expansion and oxygen delivery agents of the invention. Suitable
instrumentation is available from Advanced Instruments Inc., and
can measure all osmoticially active solutes within the range of 0.0
to 4000 mOsmol/kgH.sub.2O.
[0184] D. Carbonic Anhydrase.
[0185] Carbonic anhydrase may be detected by a double sandwich
ELISA, wherein a polystryene support is coated with rabbit
anti-bovine CA, to which CA in the samples will bind. The enzyme
substrate reaction is quantified by visible absorbance of the
products of the reaction.
[0186] E. Phospholipid Level Reduction.
[0187] Phospholipid assays can be measured by HPLC and/or ELISA.
The ELISA validation protocol is designed according to current USP
guidelines for a Category II, quantitative assay to determine the
presence of phosphatidylcholines. The protocol includes validation
of linearity/range, accuracy and precision.
[0188] F. Assay for Endotoxin.
[0189] The final product, ready for infusion, must be endotoxin
free. Endotoxin is actually material from bacterial cell walls, and
is responsible for initiation of a fever in the recipient, in low
doses; while higher levels will initiate a more serious
constellation of symptoms. The LAL (Limulus Ameobocyte Lysate)
kinetic-turbidometric assay was chosen over other assays, such as
the chromogenic and the gel-clot, because of its reproducible
results and high degree of sensitivity.
[0190] The potency of Control Standard Endotoxin (CSE) used for
routine testing is determined by comparison with Reference Standard
Endotoxin (RSE), EC5, Lot F manufactured by the USPC. It is
necessary to perform this comparison whenever an endotoxin other
than the Referenced endotoxin is to be used for creating spikes and
curves in routine testing. This is a consequence of the fact that
different lots of CSE have markedly different potencies. The
RSE/CSE comparison is performed by comparing one vial of RSE to
four vials of the same lot of CSE and calculating an average
potency. A standard curve is assayed in triplicate, with a
coefficient of correlation of -0.98 or less required for
qualification. Numerous CSE standard curves are run and one
standard curve is archived for future testing.
Inhibition/enhancement studies are performed on all products to be
tested with the LAL assay. The LAL assay is performed using the
protocol of Associates of Cape Cod, Falmouth Technology Park, East
Falmouth Mass. 02536-4445, using a Pyros Kinetix.RTM. Incubating
Tube Reader as manufactured by Associates of Cape Cod.
i. Reagent and Equipment Preparation
[0191] All reagents used for the kinetic-turbidometric assay are
used according to the specific manufacturers' instructions with the
following two exceptions: 1) the LAL is reconstituted with 5 ml of
Pyrosol.TM. reconstitution buffer instead of LAL Reagent Water and
2) the CSE is not reconstituted with exactly 5 ml of LAL Reagent
Water. The reconstitution of LAL with buffer is performed to
overcome extreme enhancement of the LAL assay by pure hemoglobin
solutions. The CSE is reconstituted with an amount of water which
will yield a final solution concentration of 1000 EU/ml. The amount
to be added is determined by standardizing the CSE against the USPC
RSE and may be more or less than the 5 ml recommended by Associates
of Cape Cod. This yields a constant CSE solution concentration and
prevents recalculation of endotoxin spikes every time the CSE lot
changes. All other reagents are used as directed.
[0192] All glassware is depyrogenated by heating to 180.degree. C.
for 4 h. All dilutions and solution transfers are performed under a
class 100 laminar flow hood. All pipette tips used are sterile and
pyrogen-free.
ii. Determination of CSE Potency
[0193] One vial of RSE (10,000 EU/vial by definition) was
reconstituted with 5 ml of LAL Reagent Water to yield a 2,000 EU/ml
solution. Two RSE curves were run to cover full range of current
Q.C. testing. The mid-range curve contained the following
concentrations (EU/ml): 1.0, 0.5, 0.25, 0.125, 0.0625, and 0.03125.
The low range curve consisted of the following concentrations
(EU/ml): 0.004, 0.002, 0.001, 0.0005, 0.00025, and 0.0001. These
curves were run in duplicate, linear regression was performed to
determine the slope and Y-intercept of the curves, and the curves
were archived for the purpose of comparison with the CSE
curves.
[0194] After the RSE curves had been run, 4 vials of CSE (500
ng/vial) were reconstituted with an amount of water which will
yield a final solution concentration of 1000 EU/each. Dilutions of
each vial were prepared in each of the two ranges so that at least
three concentrations of the CSE curve would fall directly on the
RSE curve. The mid-range curve contained the following
concentrations (ng/ml): 0.1, 0.05, 0.025, 0.0125, 0.00625 and
0.003125. The low-range curve consisted of the following
concentrations (ng/ml): 0.004, 0.002, 0.001, 0.0005, 0.00025, and
0.0001. These curves were run in duplicate and the onset times were
interpolated off the corresponding RSE curve.
[0195] The endotoxin concentration in EU/ml for each CSE standard
was divided by the corresponding concentration in ng/ml. Any onset
times which did not fall directly on the RSE curve, indicated on
the raw data by an asterisk, were not included in the calculations.
The resulting EU/ng potencies for each standard were averaged to
determine the CSE potency in each range. The two range potencies
were then averaged to determine the potency of the CSE through the
entire range of 1.0 to 0.001 EU/ml. The overall CSE potency was
then used to calculate the amount of LAL Reagent Water to be added
to each CSE Vial in the lot to yield a 1000 EU/ml solution
according to the following calculations:
500 ng vial 1000 EU ml .times. potency EU ng = water ml vial
##EQU00001##
iii. Test Method
[0196] 100 .mu.l of LAL is added to a depyrogenated 10.times.75 mm
culture tube containing 400 .mu.l of sample. The tube is vortexed
gently for approximately 2 seconds and placed in the incubation
module of the LAL device. Each tube is added individually in this
manner. Timing is initiated for each tube as the bottom of the tube
actuates a mechanical switch. Tubes are incubated at
37.0.+-.0.5.degree. C. throughout the test. No readings are taken
for the first 60 seconds; this allows time for the contents of the
tube to come to temperature and for air bubbles to disperse. For 60
to 120 seconds after tube insertion, photodetectors in each well
take 7 readings 10 seconds apart. The readings are then averaged
and taken to represent 100% transmittance. This zeroing period
eliminates data errors due to tube imperfections and sample color
or endogenous turbidity. Subsequent readings are taken every ten
seconds, converted to transmittance, and then to optical density
(OD). From 400 to 550 seconds, the OD values collected are averaged
and then subtracted from all subsequent OD values for the test.
(This baseline correction compensates for the OD of the
background.)
[0197] Onset time, T.sub.o, is defined as the number of seconds
between placement of a sample in and incubating well and the
development of an optical density of 20 mAU. The endotoxin level is
determined by comparing the onset times to an archived standard
curve of log.sub.10(T.sub.o) versus log.sub.10 (known endotoxin
concentration).
[0198] A small-programmed-computer is used to collect data from the
LAL device. The LAL device software stores the OD values in data
files and performs data analysis upon command such as onset time
correction, linear regression on standards, and endotoxin
concentration determination.
All in-process samples are tested in duplicate, unspiked and
spiked, with a four-lambda spike, where lambda equals the lowest
standard on the standard curve. In addition, a four-lambda spike in
LAL Reagent Water and unspiked LAL Reagent Water are tested. All
final product samples are tested unspiked and spiked, in
triplicate.
Results.
[0199] The levels of endotoxin in dXCMSFH are below 1 EU per/ml. In
preferred embodiments of the process, the elimination of endotoxins
to a level below 0.01 EU are achieved and allow for complex usage
and for larger volumes. This results in readings of 0.1 EU per ml
to below 0.02 EU per ml.
V. FORMULATIONS
[0200] 1. Pharmaceutical Compositions Including Excipients, Routes
of Administration and Dosages.
[0201] The deoxygenated stable NO blocked tetrameric Hb, and
dXCMSFH, in particular, of the present invention may be
incorporated in conventional pharmaceutical formulations (e.g.
injectable solutions) for use in treating mammals in need thereof.
Pharmaceutical compositions can be administered by subcutaneous,
intravenous, or intramuscular injection, or as large volume
parenteral solutions and the like. In some embodiments, dXCMSFH of
the present invention may be formulated by encapsulating Hb within
liposomes. Liposome encapsulation is often used in drug delivery to
reduce the toxicity of encapsulated therapeutic agents, as well as
to increase drug half-life. The liposome-encapsulated hemoglobin
(LEHb) encases Hb in a structure physiologically similar to RBCs,
thus preventing Hb dissociation and its rapid clearance in the
blood stream. The half-life of LEHb dispersions is dependent on the
surface chemistry of the bilayer, as well as the bilayer surface
charge and the vesicle size distribution. Hence, decreasing vesicle
size and modifying the vesicle surface can significantly increase
the circulatory lifetime. Surface conjugation of liposomes with
polyethylene-glycol (PEG) can extend the half-life. The uptake of
liposomes by the reticuloendothelial system (RES) affects the LEHb
concentration that can be safely administered, since overloading
the RES would impair the immune system.
[0202] The deoxygenated stable NO-blocked tetrameric Hb and, in
particular, dXCMSFH of the present invention can also be formulated
into other artificial blood and oxygen delivery therapeutic
formulations. Such formulations can include other components in
addition to the dXCMSFH. For example, a parenteral therapeutic
composition can comprise a sterile isotonic saline solution. The
formulations can be either in a form suitable for direct
administration, or in a concentrated form requiring dilution prior
to administration. The formulations of the present invention can
thus contain between 0.001% and 90% (w/v) dXCMSFH. In some
embodiments of the present invention, the extracellular hemoglobin
solution of dXCMSFH of the present invention may contain from about
5 percent to about 20 percent, from about 5 percent to about 17
percent, from about 8 to about 14 percent, and about 10 percent
hemoglobin in solution (% weight per volume). In some embodiments
of the invention, the extracellular hemoglobin solution of dCMSFH
may contain from about 5% to about 7% hemoglobin in solution (%
w/v). In some embodiments of the invention the solution containing
dCMSFH contains about 6.4% hemoglobin. The selection of percent
hemoglobin depends on the oncotic properties of the chosen
hemoglobin product. The hemoglobin solutions formulated for use in
the present invention may be normo-oncontic to hyperoncotic. The
percent hemoglobin may be adjusted to obtain the desired oncotic
pressure for each indication.
[0203] dXCMSFH of the present invention can be used in compositions
useful as blood substitutes and oxygen delivery therapeutics in any
mammal that uses red blood cells for oxygen transport. The mammals
include but are not limited to, human, livestock such as cattle,
cat, horse, dog, sheep, goat, pig etc. In some embodiments of the
invention, the mammal is human.
[0204] A dose of the dXCMSFH of the present invention can be from
about 1 to about 15,000 milligrams of hemoglobin per kilogram of
patient body weight over the appropriate time period either from
initial dose or repeat dose. When used as an oxygen delivery
composition, or as a blood volume supplement, the dosage may range
between 100 to 7500 mg/kg patient body weight, 500 to 5000 mg/kg
body weight, or 700 to 3000 mg/kg body weight. Thus, a dose for a
human patient might be from a gram to over 1000 grams. It will be
appreciated that the unit content of active ingredients contained
in an individual dose of each dosage form need not in itself
constitute an effective amount, as the necessary effective amount
could be reached by administration of a number of individual doses.
The selection of dosage depends upon the dosage form utilized, the
condition being treated, and the particular purpose to be achieved
according to the determination of those skilled in the art.
[0205] For use in the present invention, the deoxygenated stable NO
blocked tetrameric HB and, in particular, the dXCMSFH of the
present invention can be dialyzed or exchanged by ultrafiltration
into a physiologically acceptable solution. The dXCMSFH of the
present invention may be formulated at a concentration of 50-150
g/l. The solution may comprise a physiologically compatible
electrolyte vehicle isosmotic with whole blood and which may
maintain the reversible oxygen-carrying and delivery properties of
the hemoglobin. The physiologically acceptable solution can be, for
example, physiological saline, a saline-glucose mixture, Ringer's
acetate, Ringer's solution, lactated Ringer's solution,
Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's
balanced saline, heparinized sodium citrate-citric acid-dextrose
solution, and polymeric plasma substitutes, such as polyethylene
oxide, polyvinyl pyrrolidone, polyvinyl alcohol and ethylene
oxide-propylene glycol condensates.
[0206] Each formulation according to the present invention may
additionally comprise inert constituents including
pharmaceutically-acceptable carriers, diluents, fillers, salts, and
other materials well-known in the art, the selection of which
depends on the dosage form utilized, the condition being treated,
the particular purpose to be achieved according to the
determination of the ordinarily skilled artisan in the field and
the properties of such additives. For example, the hemoglobin
solution of the present invention in addition to dXCMSFH may
include 0-200 mM of one or more physiological buffers, 0-200 mM of
one or more carbohydrates, 0-200 mM of one or more alcohols or poly
alcohols, 0-200 mM of one or more physiologically acceptable salts,
and 0-1% of one or more surfactants, 0-20 mM of a reducing agent.
The hemoglobin solution of the present invention in addition to
dXCMSFH may include, 0-50 mM sodium gluconate, 0-50 mM of one or
more carbohydrates (e.g. glucose, mannitol, sorbitol or others
known to the art), 0-300 mM of one or more chloride salts and,
optionally, 0-0.5% surfactant, e.g. Tween.TM. [polysorbate 80],
and/or 0-20 mM N-acetyl cysteine.
[0207] Administration of the dXCMSFH of the present invention can
occur for a period of seconds to hours depending on the purpose of
the hemoglobin usage. For example, when used as an oxygen carrier
for the treatment of severe hemorrhage, the usual time course of
administration is as rapidly as possible. Typical infusion rates
for hemoglobin solutions as volume enhancer or oxygen therapeutics
can be, for example, from about 100 ml/h to about 3000 ml/h, from
about 1 ml/kg/h to about 300 ml/kg/h, or from about 1 ml/kg/h to
about 25 ml/kg/h. In some embodiments of the invention, the rates
of administration may be higher.
[0208] Suitable compositions can also include 0-200 mM of one or
more buffers (for example, acetate, phosphate, citrate,
bicarbonate, or Goode's buffer). Salts such as sodium chloride,
potassium chloride, sodium acetate, calcium chloride, magnesium
chloride can also be included in the compositions of the invention.
The salt can be in concentrations of 0-2M.
[0209] In addition, the compositions of the invention can include
one or more carbohydrate (for example, reducing carbohydrates such
as glucose, maltose, lactose or non-reducing carbohydrates such as
sucrose, trehalose, raffinose, mannitol, isosucrose or stachyose)
and one or more alcohol or poly alcohol (such as polyethylene
glycols, propylene glycols, dextrans, or polyols). The
concentration of carbohydrate or alcohol can be 0-2 M.
[0210] The dXCMSFH of the present invention can also contain one or
more surfactant and 0-200 mM of one or more chelating agent (for
example, ethylenediamine tetraacetic acid (EDTA), ethylene
glycol-bis(beta-aminoethyl ether) N,N,N,N'-tetraacetic acid (EGTA),
ophenanthroline, diethylamine triamine pentaacetic acid (DTPA also
known as pentaacetic acid) and the like). The surfactant can be
0.005-1% of the composition. The compositions of the invention can
be at pH of about 6.0-9.5. In some embodiments, the composition may
contain 0-150 mM NaCl, 0-10 mM sodium phosphate, 0.01-0.1%
surfactant, and/or 0-50 .mu.M of one or more chelating agents at pH
6.0-9.5. The formulation may contain 5 mM sodium phosphate, 150 mM
NaCl, 0.025% to 0.08% polysorbate 80, and/or 25 .mu.M EDTA at pH
6.0-9.5.
[0211] Additional additives to the formulation can include
anti-bacterial agent, oncotic pressure agent (e.g. albumin or
polyethylene glycols) and other formulation acceptable salt, sugar
and other excipients known in the art. Each formulation according
to the present invention can additionally comprise constituents
including carriers, diluents, fillers, salts, and other materials
well-known in the art, the selection of which depends upon the
particular purpose to be achieved and the properties of such
additives which can be readily determined by one skilled in the
art. The compositions of the present invention can be formulated by
any method known in the art. Such formulation methods include, for
example, simple mixing, sequential addition, emulsification,
diafiltration and the like.
[0212] 2. Packaging and Storage of the NO-Blocked Tetrameric Hb of
the Invention, Including Both Stable (Cross Linked) and
Unstabilized (Uncross Linked) Hb.
[0213] Various embodiments of the NO-blocked tetrameric Hb of the
invention, including dXCMSFH, dCMSFH, and dTBSFH may be stored in
conventional, and preferably oxygen impermeable containers (for
example, stainless steel tanks, glass containers, oxygen
impermeable plastic bags, or plastic bags overwrapped with low
oxygen permeable plastic bags wherein an oxygen scavenger is placed
between the internal plastic bag and the overwrapped plastic bag).
In some preferred embodiments, the dXCMSFH, dCMSFH, or dTBSFH of
the present invention is stored in the absence of oxygen. The
dXCMSFH, dCMSFH, or dTBSFH may be oxygenated prior to use such as,
by way of example only, oxygenating before using in the catheter
for cardiac therapy. In some embodiments, the dXCMSFH, dCMSFH, or
dTBSFH can be stored in oxygen permeable or oxygen impermeable
("anoxic") containers in an oxygen controlled environment. Such
oxygen controlled environments can include, for example, glove
boxes, glove bags, incubators and the like. Preferably the oxygen
content of the oxygen controlled environment is low relative to
atmospheric oxygen concentrations (see, Kandler, R. L. et al., U.S.
Pat. No. 5,352,773; herein incorporated by reference). In some
embodiments of the present invention, the dXCMSFH, dCMSFH, or
dTBSFH can be packaged in sealed Tyvek or Mylar (polyethylene
terephthalate) bags or pouches. In some embodiments, the dXCMSFH,
dCMSFH, or dTBSFH of the present invention can be lyophilized and
stored as a powder. The preparations may be stored at room or
elevated temperature (Kandler et al., PCT Publication No. WO
92/02239; Nho, PCT Publication No. WO 92/08478, both herein
incorporated by reference), or more preferably under refrigeration.
In some embodiments, the dCMSFH or dTBSFH may be stored in HyClone
BioProcess Containers.TM. for ease of shipping and further
handling.
[0214] Where the package is an oxygen impermeable film, the
container can be manufactured from a variety of materials,
including polymer films, (e.g., an essentially oxygen-impermeable
polyester, ethylene vinyl alcohol (EVOH), or nylon), and laminates
thereof. Where the container is an oxygen impermeable overwrap, the
container can be manufactured from a variety of materials,
including polymer films, (e.g., an essentially oxygen-impermeable
polyester, ethylene vinyl alcohol (EVOH), or nylon) and laminates,
such as a transparent laminate (e.g. a silicon oxide or EVOH
containing-laminate) or a metal foil laminate (e.g., a silver or
aluminum foil laminate). The polymer can be a variety of polymeric
materials including, for example, a polyester layer (e.g., a 48
gauge polyester), nylon or a polyolefin layer, such as
polyethylene, ethylene vinyl acetate, or polypropylene or
copolymers thereof.
[0215] The containers can be of a variety of constructions,
including vials, cylinders, boxes, etc. In a preferred embodiment,
the container is in the form of a bag. A suitable bag can be formed
by continuously bonding one or more (e.g., two) sheets at the
perimeter(s) thereof to form a tightly closed, oxygen impermeable,
construction having a finable center. In the case of laminates
comprising polyolefins, such as linear low density, low density,
medium or high density polyethylene or polypropylene and copolymers
thereof, the perimeter of the bag may be bonded or sealed using
heat. It is well within the skill of the art to determine the shape
of the bag and the appropriate temperature to generate a tightly
closed, oxygen and/or moisture impermeable construction. Where the
container is a film, such as a polyester film, the film can be
rendered essentially oxygen-impermeable by a variety of suitable
methods. The film can be laminated or otherwise treated to reduce
or eliminate the oxygen permeability.
[0216] In some embodiments, one or more antioxidants, such as
ascorbate (Wiesehahn, G. P. et al., U.S. Pat. No. 4,727,027; and,
Kerwin, B. D. et al., U.S. Pat. No. 5,929,031), glutathione,
N-acetylcysteine, methionine, tocopherol, butyl hydroxy toluene,
butyl hydroxy anisole, or phenolic compounds (Osterber et al., PCT
Publication No. WO 94/26286; and, Kerwin, B. D. et al., U.S. Pat.
No. 5,929,031) may be added to further stabilize the dXCMSFH,
dCMSFH, and dTBSFH (all references herein incorporated by
reference). Alternatively, and more preferably, the dXCMSFH of the
present invention can be lyophilized and stored as a powder, or can
be packaged in sealed Tyvek, or Mylar (polyethylene terephthalate)
bags or pouches. Packaging such as, Kerwin, B. D. et al., U.S. Pat.
No. 5,929,031, is herein incorporated by reference. In some
embodiments, the dXCMSFH, dCMSFH, and dTBSFH in such storage
containers may be subjected to irradiation or other sterilization
treatment sufficient to extend the shelf-life of the compositions.
An oxygen scavenger such as n-acetyl-cysteine may be included in
the formulation.
[0217] The dXCMSFH, dCMSFH, and TBSFH of the present invention may
be stored at suitable storage temperatures for periods of two years
or more, and preferably for periods of two years or more, when
stored in a low oxygen environment. Suitable storage temperatures
for storage of one year or more are between about 0.degree. C. and
about 40.degree. C. The preferred storage temperature range is
between about 0.degree. C. and about 25.degree. C. The process of
making dXCMSFH, dCMSFH, and dTBSFH of the present invention
includes maintaining the steps of the process under conditions
sufficient to minimize microbial growth, or bioburden, such as
maintaining temperature at less than about 20.degree. C. and above
0.degree. C.
VI. METHODS OF USE
[0218] The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, the dXCMSFH may be used to
form pharmaceutical compositions that may be administered to
recipients, for example, by infusion, by intravenous or
intra-arterial injection, or by other means. The dXCMSFH
formulations of the present invention can be used in compositions
useful as blood substitutes, volume expanders within the blood
volume, and oxygen perfusion agents in any application where red
blood cells are used. One application uses compositions of the
present invention for the treatment of hemorrhage where blood
volume is lost and both fluid volume and oxygen delivery capacity
must be replaced. Moreover, because the deoxygenated stable
NO-blocked tetrameric Hb of the present invention, and, in
particular, dXCMSFH, can be made pharmaceutically acceptable, the
formulations of the present invention can not only deliver oxygen
but also act as simple volume expanders that provide oncotic
pressure due to the presence of the large hemoglobin protein
molecule. The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, dXCMSFH, can thus be used as
replacement for blood that is removed during surgical procedures
where the patient's blood is removed and saved for reinfusion at
the end of surgery or during recovery (e.g., acute normovolemic
hemodilution or hemoaugmentation, etc.).
[0219] A typical dose of the deoxygenated stable NO-blocked
tetrameric Hb of the present invention, and, in particular, dXCMSFH
as a blood substitute is from 10 mg to 7 grams or more of
extracellular hemoglobin per kilogram of patient body weight. Thus,
a typical dose for a human patient might be from a few grams to
over 350 grams. It will be appreciated that the unit content of
active ingredients contained in an individual dose of each dosage
form need not in itself constitute an effective amount since the
necessary effective amount could be reached by administration of a
plurality of administrations as injections, etc. The selection of
dosage depends upon the dosage form utilized, the condition being
treated, and the particular purpose to be achieved according to the
determination of the ordinarily skilled artisan in the field.
[0220] In some embodiments of the invention, a solution of a
deoxygenated stable NO-blocked tetrameric Hb, for example, dXCMSFH,
will contain about 5% to about 25% dXCMSFH by weight for
administration to a mammal. In some preferred embodiments of the
invention a solution of dXCMSFH will contain about 7% to about 15%
dXCMSFH by weight for administration to a mammal. In some other
preferred embodiments of the invention, a solution of dXCMSFH will
be 10% by weight of dXCMSFH for administration to a mammal. In some
embodiments of the invention, a dose to be administered to a mammal
contains about 7 g of dXCMSFH. In some embodiments of the invention
a dose to be administered to a mammal contains about 1 g of a
deoxygenated stable NO-blocked tetrameric Hb, and in particular,
dXCMSFH. In some embodiments of the invention, an exemplary unit of
production for use in a therapeutic setting is a container with 500
ml of a 0.5 mmol solution of dXCMSFH (about 64 g/L, or about 6.4%
by weight in solution). The larger unit solutions may be used for
replacement of blood or for augmenting oxygen delivery for a number
of therapeutic interventions. The smaller unit solutions may be
used for labeling and diagnostic purposes, as well as therapeutic
interventions. The smaller unit solutions may, in a preferred
embodiment, contain a solution of dXCMSFH of up to 4%, 5%, 6%, 7%,
8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21,
22%, 23%, 24%, or up to 25% by weight of dXCMSFH. In another
embodiment, a solution may contain dXCMSFH with a concentration as
low as 2%, 3%, 4%, 5%, 6%, 7%, 8% 9%, 10%, 11%, 12%, 13% or 14%, by
weight of dXCMSFH.
[0221] Administration of the deoxygenated stable NO-blocked
tetrameric Hb of the present invention, and, in particular,
dXCMSFH, can occur for a period of seconds to hours depending on
the purpose of the hemoglobin usage. For example, as a volume
augmentation therapy, the usual time course of administration is as
rapid as possible. Typical infusion rates for hemoglobin solutions
as oxygen delivery/perfusion agents or volume enhancers can be from
about 100 ml to 3000 ml/h. However, when used to stimulate
hematopoiesis, administration can be made more slowly and therefore
administration rates can be slower because the dosage of the
deoxygenated stable NO-blocked tetrameric Hb of the present
invention, and, in particular, dXCMSFH may be much less than
dosages that may be required to treat hemorrhage.
[0222] In some embodiments, the deoxygenated stable NO-blocked
tetrameric Hb of the present invention, and, in particular,
dXCMSFH, can be used to treat anemia as caused by renal failure,
diabetes, AIDS, chemotherapy, radiation therapy, hepatitis, G.I.
blood loss, iron deficiency, menorrhagia, and the like, by
providing additional oxygen delivery capacity in a mammal that is
suffering from anemia, as well as by stimulating hematopoiesis,
providing an effective iron supplement to support RBC production,
and by serving as an adjuvant to erythropoietin therapy.
[0223] Likewise, the deoxygenated stable NO-blocked tetrameric Hb
of the present invention, and, in particular, dXCMSFH, can be used
to provide additional oxygen delivery capacity to a mammal (such as
an athlete, soldier, mountaineer, aviator, smoke victim, etc.)
desiring such additional oxygen delivery capacity. Such additional
oxygen delivery capacity can be used to overcome environmental
(i.e, for example, high altitudes and smoke inhalation) and
physical (i.e., for example, acute performance demands) stresses.
The stable NO-blocked tetrameric Hbs of the present invention, and
in particular, dXCMSFH, thus are useful in treating carbon monoxide
poisoning and its concurrent hypoxia and ischemia, as the compounds
and compositions of the present invention can supply oxygen to
tissue while the carbon monoxide bound cellular hemoglobin is being
eliminated, thus bridging the oxygen needs of the patient until new
RBCs are produced.
[0224] The deoxygenated stable NO-blocked tetrameric Hbs of the
present invention, and, in particular, dXCMSFH, can be used for
applications requiring administration to a mammal of high volumes
of hemoglobin as well as in situations where only a small volume of
the hemoglobin of the present invention is administered. The
deoxygenated stable NO-blocked tetrameric Hb of the present
invention, and, in particular, dXCMSFH can be used in applications
during surgery where large volumes of blood are normally lost, or
in treatment of trauma victims who have lost large volumes of
blood. This can include both civilian accidents and military
situations.
[0225] The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, dXCMSFH may be used as a
blood substitute in veterinary clinical applications.
[0226] In addition, because the distribution throughout the
vasculature of the deoxygenated stable NO-blocked tetrameric Hb of
the present invention, and, in particular, dXCMSFH, is not limited
by viscosity or by the size of red blood cells, the compositions of
the present invention can be used to deliver oxygen to areas that
red blood cells cannot penetrate. These areas can include any
tissue areas that are located downstream of obstructions to red
blood cell flow, such as areas downstream of thrombi, sickle cell
occlusions, arterial occlusions, angioplasty balloons, surgical
instrumentation, tissues that are suffering from oxygen starvation
or are hypoxic, and the like.
[0227] Additionally, all types of tissue ischemia, including
ischemic events in the brain, can be treated using the methods of
the instant invention. Such tissue ischemias include, for example,
stroke, emerging stroke, transient ischemic attacks, myocardial
stunning and hibernation, acute or unstable angina, emerging
angina, infarct, and the like. The recovery of tissues from
physical damage such as burns can also be accelerated by
pretreatment with the hemoglobin of the present invention, which
allows increased perfusion and oxygenation of the tissues which may
also reduce infection risk. The use of the stable NO-blocked
tetrameric Hbs of the present invention also will allow for better
oxygen uptake in the lungs due to better distribution of these
smaller molecules within small capillaries. In and after cosmetic
surgery, fine tissue beds suffer microcirculatory disruption,
thereby losing flow of RBCs. Use of the stable NO-blocked
tetrameric Hbs of the present invention provide better oxygenation
for tissue metabolism and regrowth, to decrease scarring with its
loss of vascularization.
[0228] The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, dXCMSFH, can be used for the
treatment of sickle cell anemia patients. Sickle cell anemia
patients in vasoocclusive crisis are currently treated by
transfusion of red blood cells in conjunction with dilution and
pain management. The deoxygenated stable NO-blocked tetrameric Hb
of the present invention, and, in particular, dXCMSFH, may not only
deliver oxygen thereby preventing further sickling (as do red blood
cells), they may also penetrate vessels already occluded with
deformed red cells to better alleviate pain and minimize tissue
damage. Also, frequent transfusions in the sickle cell anemia
population may result in alloimmunization to red cells and to
platelets, an adverse effect that would be avoided by use of
hemoglobin of the present invention. The deoxygenated stable
NO-blocked tetrameric Hb of the present invention, and, in
particular, dXCMSFH, n offer a significant therapeutic advantage in
treatment of sickle cell anemia patients, since they elicit a
lesser degree of vasoconstriction or none at all. This is an
advantage in the treatment of vasoocclusive crisis, and is also an
advantage in other treatments of sickle cell anemia patients in
situations where there is a risk of sudden onset of vasoocclusive
crisis. For example, dXCMSFH of the present invention may be used
in place of packed red cells for preoperative transfusion of sickle
cell anemia patients to minimize risk of anesthesia. The
deoxygenated stable NO-blocked tetrameric Hb of the present
invention, and, in particular, dXCMSFH may also be administered
periodically to minimize risk of stroke.
[0229] The stable NO-blocked Hb of the present invention, i.e,
re-oxygenated XCMSFH can perfuse because of its size, and deliver
oxygen to tissue beds that would normally be dependent upon
diffusion alone due to the poor perfusion of bulky large red blood
cells. For example, the compounds and compositions of the present
invention can be used as a tissue protectant in acute coronary
syndrome (ACS) and in transplantation, where the area of insult or
harvested organ is perfused during stopped flow situations. This
may prevent reperfusion injury and allow for the salvage and
preservation of tissues that have been perfused, with subsequent
normal circulation. Additionally, in transplantation procedures,
organs may be prepared for harvesting by flushing with the
compounds and compositions of the present invention to remove
native blood agents and components prior to removal and continue to
support tissue viability as discussed above.
[0230] The compounds and compositions of the present invention may
also be utilized as a wound healing reagent where the molecular
size and oxygen delivery capabilities may yield superior perfusion
in poorly vascularized regions such as, for example, diabetic foot
injuries, recovery from cardiac revascularization and post surgical
recovery, i.e. for example, cosmetic surgery or cancer resection
breast reconstruction, where RBCs may not perfuse well due to size
or rigidity. Another application of the stable NO-blocked
tetrameric Hbs of the present invention may be in poorly
vascularized tumor tissue beds of cancer cells where appropriate
use of the invention can allow for an increase in oxygen tension
and allow for more effective use of radiation therapy and for the
enhancement of oxygen dependent pharmaceutical agents.
[0231] The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, dXCMSFH, may be used as a
small molecule inhibitor of nitric oxide for cardiogenic shock.
Cardiogenic shock afflicts a significant number of patients
presenting with acute myocardial infarction, whereby circulatory
shutdown occurs after the infarct. Despite intervention with
catheters or bypassgrafting, the mortality rate is about 50%. The
use of the compounds and compositions of the present invention may
provide the level of oxygenation to heart and blood vessels to
forestall excessive production of nitric oxide and support survival
past the critical initial thirty day post infarct time period.
Survival is greatly enhanced after this timepoint.
[0232] The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, dXCMSFH contains iron, and
as such, may be detected via MRI (magnetic resonance imaging).
Thus, in some embodiments, the present invention contemplates the
use of deoxygenated stable NO-blocked tetrameric Hb of the present
invention, and, in particular, dXCMSFH as an imaging agent.
[0233] The present invention also concerns implantable delivery
devices (such as cartridges, implants, etc.) that contain
deoxygenated stable NO-blocked tetrameric Hb of the present
invention, and, in particular, dXCMSFH, and that are capable of
releasing dXCMSFH, for example, into the circulation in response to
a sensed need for increased oxygen delivery capacity. In some
embodiments, such devices can deliver dXCMSFH, for example, at a
constant rate, so as to facilitate erythropoiesis (either alone, or
in combination with erythropoietin). In some embodiments, the
devices can be controlled by sensing means (such as electronic
probes of hemoglobin, O.sub.2 level, CO.sub.2 level, etc.) so as to
deliver the deoxygenated stable NO-blocked tetrameric Hb of the
present invention at a rate commensurate with the patient's oxygen
delivery capacity needs. Such sensing means may themselves be
implantable, or part of the implanted device, or may be located
extracorporeally. In some embodiments, such devices may be used to
accomplish or facilitate the hemo-diagnosis of individuals.
[0234] The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, dXCMSFH may also be used to
form non-pharmaceutical compositions that can be used, for example,
as reference standards for analytical instrumentation needing such
reference standards, reagent solutions, control of gas content of
cell cultures; for example by in vitro delivery of oxygen to a cell
culture, and removal of oxygen from solutions. Additionally, the
dXCMSFH of the present invention may be used to oxygenate donated
tissues and organs during transport.
[0235] The deoxygenated stable NO-blocked tetrameric Hb of the
present invention, and, in particular, dXCMSFH may be used to
scavenge endotoxin from surfaces or liquids. The invention thus
contemplates devices, such as cartridges, filters, beads, columns,
tubing, and the like that contain the deoxygenated stable
NO-blocked tetrameric Hb of the present invention. Liquids, such as
water, saline, culture medium, albumin solutions, etc., may be
treated by passage over or through such devices in order to remove
endotoxin that may be present in such liquids, or to lessen the
concentration of endotoxin present in such liquids. The
deoxygenated stable NO-blocked tetrameric Hb of such devices is
preferably immobilized (as by affinity, ionic, or covalent bonding,
etc.) to solid supports present in such devices. In some
embodiments, the deoxygenated stable NO-blocked tetrameric Hb is
bound to beads that may be added to the liquids being treated, and
then subsequently removed (as by filtration, or affinity
immobilization). In some embodiments, the beads may be of
ferromagnetic or paramagnetic metal, or may be themselves magnetic,
such that they may be readily separated from the treated liquid by
magnetic means.
[0236] The deoxygenated thiol blocked Hb, both cross linked and
uncross linked, i.e., dCMSFH, dTBSFH, and dXCMSFH, can be used to
remove oxygen from solutions requiring the removal of oxygen, and
as reference standards for analytical assays and instrumentation.
The deoxygenated thiol blocked Hb, both cross linked and uncross
linked, i.e., CMSFH, TBSFH, and XCMSFH, can also be used in vitro
to enhance cell growth in cell culture by maintaining oxygen
levels.
[0237] The re-oxygenated stable NO-blocked tetrameric Hb of the
present invention, and in particular, XCMSFH, can be used for the
use in visualizing intravascular space. Present optical techniques
for the observation of vascular walls are relegated to non-optical
techniques due to the opaque effects of the transfusion of red
blood cells. The stable NO-blocked Hb of the present invention,
i.e., XCMSFH may not only deliver oxygen thereby preventing
ischemia, but they may also present an optically translucent field
of view to allow for visualization of tissue beds in situ for the
determination of pathology; cancer, vulnerable plaques, lipid
damage, stent placement, etc, using visible light in the red
wavelength band, for example, to illuminate the targeted feature.
The use of Optical Coherence Tomography may be expanded by
employing the stable NO-blocked tetrameric Hbs of the present
invention. Intermittent saline flushes are currently employed to
create transient visual fields in-vivo, but superior visualization
and sustained examinations may be possible with use of the present
stable NO-blocked tetrameric Hb, which can continue to oxygenate
the local area.
VII. EXAMPLES
Example 1
Comparison of Two Methods of Initial Processing of Whole Blood
[0238] Materials: Bovine Blood Collection:
[0239] Bovine blood is collected in a 1 gallon collection container
which may hold 100 ml of 6% sodium ethylenediaminetetraacetic acid
(EDTA) solution and is cooled in ice.
[0240] The whole bovine arterial blood is divided into Batch A and
Batch B. Batch A consists of 2200 ml of whole blood and is washed
in the haemonetics Cell Saver 5 to obtain concentrated red blood
cells free of platelets, clotting factors, extra cellular
potassium, anticoagulants, and cell stroma (Method A). Batch B
consists of 1800 ml of whole blood and is washed on a Millipore
0.65 .mu.m filter (Method B).
[0241] Method A. Cell Saver 5 Removal of Plasma Proteins:
[0242] Cell Saver 5 from Haemonetics is used to concentrate
erythrocytes from other components in freshly collected
anticoagulated bovine blood. It may be desirable not to fracture
either leucocytes or erythrocytes at this point.
[0243] After being passed through a coarse filter of 150 .mu.m, the
cells are washed in a spinning bowl holding 225 ml of packed red
blood cells and washed with 3 liters of saline in a reverse flow
from the outside edge of the bowl towards the center. The
centrifugation is gentle, and some of the WBCs are eluted in the
wash, which may be desirable. Table 4 shows the progress of serum
protein removal at 500 ml increments. It is a continuous flow
technique. Sample in Table 4 is the sample volume applied to the
bowl. Progress is followed by reading the protein concentration at
280 nm spectrophotometrically, with the values given in A.sub.280
units (absorbance units at 290 nm). The data points in Table 4 are
taken at the indicated points during the washing process. The
results indicate that when a full bowl of red cells is washed with
3 liters of NS, there is a greater than 3 log reduction in serum
proteins. This also infers a greater than 3 log reduction in
viruses, prions, etc that are not bound to red cell membranes. All
values in Table 4 are corrected for dilution.
TABLE-US-00004 TABLE 4 Cell Saver 5 removal of plasma proteins
Sample status A.sub.280 Absorbance Units A.sub.280 of initial crude
plasma containing sample 277.35 A.sub.280 of filtrate after 500 cc
NS 43.20 A.sub.280 of filtrate after 1000 cc NS 7.69 A.sub.280 of
filtrate after 1500 cc NS 0.86 A.sub.280 of filtrate after 2000 cc
NS 0.05
[0244] Method B. Millipore 0.65 .mu.m Filter Removal of Plasma
Proteins:
[0245] Batch B is passed through a 150 .mu.m filter. The material
is then filtered with a tangential flow membrane that will pass
plasma proteins but retain cellular components such as leucocytes
and erythrocytes. The tangential flow membrane filtration can be
slower but it may require less labor as it can run unattended. It
can be more suitable for scaleup. Other types of large scale
centrifuges may be used. The results of this continuous
diafiltration are shown in Table 5, where all results are corrected
for dilution. This method reveals a greater than 3 log reduction in
plasma proteins, which also implies a similar log reduction in
viruses, prions, etc.
TABLE-US-00005 TABLE 5 Millipore 0.65 .mu.m filter removal of
plasma proteins Sample status A.sub.280 Absorbance Units A.sub.280
of plasma in 1000 cc blood 221.6 A.sub.280 of filtrate after 1000
cc NS 82.1 A.sub.280 of filtrate after 2000 cc NS 26.5 A.sub.280 of
filtrate after 3000 cc NS 8.07 A.sub.280 of filtrate after 4000 cc
NS 2.76 A.sub.280 of filtrate after 5000 cc NS 0.81 A.sub.280 of
filtrate after 6000 cc NS 0.29 A.sub.280 of filtrate after 7000 cc
NS 0.102
[0246] Evaluation of Leucocyte Loss/Removal.
[0247] It is desirable during the preparation of hemoglobin to
remove any WBCs to remove granolocyte proteolytic enzymes from the
hemoglobin solution. Thus at the stage of removing plasma proteins
it is desirable to remove the WBCs without causing their lysis. The
Cell Saver 5 technology can remove some of the WBCs in the floating
buffy coat during centrifugation. However, the tangential flow
membrane may retain all of the WBCs so an observed loss of WBCs may
mean that cell lysis of the WBCs had occurred. Table 6 shows
evaluation of the conservation of WBCs after filtration with Cell
Saver 5 or Millipore 0.65 .mu.m filter. When appropriately
corrected for volume, both methods provide adequate protection from
leucocyte lysis in the presence of RBCs.
TABLE-US-00006 TABLE 6 Evaluation of leucocyte loss/removal Sample
Initial WBC Final WBC (Vol Adj) % Recovery Cell Saver 5 5.79
.times. 10.sup.3 6.10 .times. 10.sup.3 100% Millipore 0.65 .mu.m
5.79 .times. 10.sup.3 5.63 .times. 10.sup.3 100%
[0248] Batches A and B are then refrigerated for 8 h, whereupon
both batches are passed through a Baxter leuko-reducing filter,
which also removes viral materials. Batch A yields 1500 ml of RBCs
while Batch B yields 1200 ml of RBCs. Samples are extracted from
each batch throughout the course of cleaning.
[0249] Lysing Cells and Removing Stroma.
[0250] The 1500 ml of RBCs from Batch A are diluted with 6000 ml of
DI water. After allowing the cells 45 seconds to lyse, 750 ml of 9%
N saline (NS) is added to the solution to minimize the lysing of
any leukocytes present. The 1200 ml of RBCs from Batch B are lysed
with 4800 ml of DI water. No saline is added to Batch B at this
point. Next, both batches are passed over a 0.22 micron Pellicon
filter. Once the hemoglobin has been filtered out and collected in
a separate flask, it is passed over a second 10K Dalton Pellicon
filter. This filters out any saline present which is discarded. The
pure hemoglobin is recirculated into the original flask and is
concentrated to a desired percentage, such as 13.5% (w/v).
[0251] Blocking Sulfhydryls of Hemoglobin with Iodoacetamide
(IAM).
[0252] After the samples are concentrated to 13.5% Hb, oxygen is
removed as previously described, and the pH is adjusted to 7.4 with
0.1M sodium phosphate buffer. Oxygen remaining is <10 ppb. Two
molar equivalents of IAM per mole of dSFH are added and the
reaction is allowed to proceed for 1 h. Progress of the reaction is
monitored by an iodide electrode. Unreacted IAM by ultrafiltration
using PBS. Once iodide is removed the intermediate dCMSFH is stable
and may be is packaged in oxygen barrier containers and can be
safely stored at room temperature.
[0253] Deoxygenation and Cross-Linking:
[0254] The stable intermediate product, dCMSFH, is again placed in
an oxygen free (<10 ppm) environment and dissolved oxygen
removed to a level of less than 0.010 ppm. Membrane contactor
technology can be the method utilized to deoxygenate the hemoglobin
in a controlled atmosphere with an oxygen level of less than 10
ppm. An initial oxygen saturation reading is taken for both batches
using a polorgraphic dissolved oxygen probe. Batch A has an initial
reading at 4 mg/L. Batch B has an initial reading at 7 mg/L. The
hemoglobin is pumped and recirculated through the membrane
contactor using a peristaltic pump at a flow rate of 600 ml/min
with applied vacuum pressure of >28.5 in/Hg. The final oxygen
saturation level for both batches is <0.01 ppm. The batches are
then pH adjusted to 8.4 with 0.5M NaOH for cross-linking. Once the
pH is adjusted, 2.94 g of bis-3,5-dibromosalicyl fumarate (DBSF) is
added to Batch A (2 molar equivalents per sulfhydryl) while 1.47 g
is added to Batch B (2 molar equivalents per sulfhydryl). Once
cross-linking is complete, pH is adjusted with 0.5M citric acid
back to 7.4 and the batches are stored in air-tight containers in
the refrigerator.
[0255] The reaction is monitored by Capillary gel electrophoretic
analysis is performed using a Beckman CoulterPA-800.RTM. Proteomics
instrument with standard sample preparation, to determine the
extent of cross-linking to monitor the reaction time course. The
reaction is complete when 95% or more of the tetramer are
cross-linked (data not shown). The samples are also evaluated in a
Hemox Analyzer for the recording of blood oxygen equilibrium curves
based on dual wavelength spectrophotometry.
[0256] Comparison of Method A and Method B Overall:
[0257] The Method A and Method B purifications are run side by side
to compare efficiency in releasing Hb while removing stroma and
preventing leucocyte lysis. Either method will provide purified
materials of acceptable quality, and both methods will provide
purified materials of acceptable quality, and a combination of both
methods may also be envisioned to be used in the methods of the
invention.
Example 2
Lysing White Blood Cells
[0258] Determination of the relative time of lysing of WBCs is
demonstrated. Preferential lysing of RBCs relative to WBCs allows
optimization of red blood cell lysis to obtain the maximum amount
of hemoglobin, without also introducing proteases from lysed
WBCs.
[0259] Procedure:
[0260] 2000 mls of whole blood is filtered through only the 100 g
reservoir filter of the Cell Saver 5. Seven beakers are then filled
with 200 mls of blood. One beaker is designated as the control. The
other six beakers are then assigned a specific time for lysing at
times of 30 seconds and then 1, 2, 3, 4, and 5 minutes. The control
beaker is started and 910 mls of 0.9% saline solution is added. At
time increments of 30 seconds, 1, 2, 3, 4, and 5 minutes a 10 ml
sample is taken for white blood cell analysis.
[0261] For the remaining six beakers, 800 ml of DI water is added.
After 30 seconds 110 ml of 9% saline is added to the first beaker
to stop lysing. After being allowed to stir for approximately 30
seconds, a 10 ml sample is taken for white blood cell analysis.
After 1 minute, 110 ml of 9% saline is then added to the second
beaker. Again after being allowed to stir for 30 seconds, a 10 ml
sample is taken. For the remaining four beakers, 110 ml of 9%
saline is added at 2, 3, 4 and 5 minutes to stop lysing. After each
time increment, a 10 ml sample is taken from each for white blood
cell analysis via standard WBC quantitation.
Conclusion:
[0262] As seen in FIG. 2, the lysing of the white blood cells can
take place between 2 and 3 minutes. So in order to optimize red
blood cell lysis, lysing can be stopped at two minutes. Due to the
addition of DI water and saline, the volume is increased. The
results shown in Table 7 are corrected for dilution.
TABLE-US-00007 TABLE 7 Determination of Relative Lysing Time of
WBCs Control Experimental (K/mm.sup.3) (K/mm.sup.3) Time (Control
Beaker) (Beakers 1-6) 30 seconds 5.9 5.2 1 minute 5.4 4.9 2 minutes
5.8 5.0 3 minutes 7.4 4.5 4 minutes 6.8 4.0 5 minutes 5.7 3.4
Example 3
Rabbit Safety Trial
[0263] Materials:
[0264] Domestic rabbits are raised and treated by standard animal
husbandry. IV access is established with a 22 or 24 gauge catheter
into a shaven topically anesthetized ear vein for dXCMSFH infusion.
IV infusion is metered with a syringe pump; the total volume
usually given over 45-60 minutes. If blood is removed from rabbits,
it is performed by inserting a 20-22 gauge catheter into the artery
of the other ear. Procedures and infusions are done using a Velcro
cloth wrap type restraint.
[0265] Methods: Protocol A:
[0266] Rabbits are prepared for infusion as above dXCMSFH to be
administered has a p50 for O.sub.2 of 28-32 as determined on a TCS
Hemox-Analyzer at 37.degree. C. in pH 7.40 buffered NS, and is 12%
w/v for the modified hemoglobin of the invention. The amount of
dXCMSFH to be infused is based upon 10% of the estimated blood
volume of a rabbit (56 ml/kg). This amount is placed into the
syringe, and infused by the pump over 45-60 minutes. The IV
catheter is then removed from the rabbit's ear and the rabbit
returned to its cage for observation. The control rabbits receive
Hb without the NO blockage chemical modification.
[0267] Protocol B:
[0268] Venous and arterial access is prepared as above. A
significant amount of rabbit blood, 54-75 cc, is removed from the
arterial catheter while simultaneously an identical volume of
dXCMSFH is infused through the venous access. This procedure is
accomplished in 12-20 minutes total time. On subsequent days 2 and
3, 15 cc of dXCMSFH is infused through the heparin locked venous
catheter over a 30 minute interval, twice each day with 4 h between
infusions.
Results:
[0269] Twenty-five (25) rabbits are infused according to protocol
A, over an approximately 1 h period as shown in Table 8. All of
these rabbits live beyond 72 h up to 75 days, without any deaths.
Three (3) rabbits are infused with Hb without the NO blockage
chemical modification. All of these rabbits die within 7-12 minutes
of starting the infusion, as shown in Table 9. Four (4) rabbits
receive large amounts of dXCMSFH, according to protocol B, as shown
in Table 10. All of these rabbits are alive and well for more than
two weeks, without any subsequent deaths. The rabbits treated with
dXCMSFH in either protocol A or B show 100% survival and no obvious
signs of morbidity. Therefore, experimental subjects receiving
650-7500 mg/kg of XCMSFH tolerate the experimental protocols A and
B well, while the treatment group receiving cell free Hb, with no
further modifications, at levels of 125-160 mg/kg, expire upon
first administration.
TABLE-US-00008 TABLE 8 Dose Rabbit Weight dXCMSFH ID (Kg) (mg/Kg)
Outcome 11 2.47 567 survive 15 2.4 583 survive 13 2.62 534 survive
B7 2.36 636 survive CO 2.52 476 survive B3 2.24 670 survive B1 2.8
714 survive C02 2.65 566 survive E03 2.38 630 survive 04 2.1 1143
survive 07 2.26 1062 survive C8 2.39 1004 survive MH2 3.12 808
survive LHD 2.78 647 survive D03 2.3 783 survive D01 2.06 874
survive 05 2.29 629 survive 03 2.02 713 survive 01 2.12 679 survive
JSD 2.13 676 survive D04 2.26 637 survive MH4 2.51 574 survive K03
2.27 634 survive D08 2.28 632 survive D12 2.42 595 survive
TABLE-US-00009 TABLE 9 Dose Weight Elapsed dSFH Rabbit ID (Kg) time
(min) (mg/Kg) Outcome 06 2.04 7 98 expired 10 2.39 12 126 expired
09 2.42 8 83 expired
TABLE-US-00010 TABLE 10 Dose dXCMSFH dXCMSFH Rabbit ID Weight (Kg)
(mg) (mg/Kg) Outcome B03 3.1 12000 3871 survive F02 2.79 11500 4122
survive F01 3.02 13800 4570 survive F05 2.65 12700 4792 survive
Example 4
Pie Safety Trial
[0270] Materials and Methods:
[0271] Twelve piglets weighing 10-16 kilogram were studied. Prior
to inclusion in the study, noninvasive screening by 2D
echocardiogram for cardiac anomalies and aortic valve diameter
measurements were performed. 5 mg of Lasix 40 mg/ml concentration
was administered to each piglet immediately after establishing an
IV, prior to initiating the top loading of the XCMSFH solution The
XCMSFH solution had a p50 of 32 for Oxygen and a concentration of
12 gram percent (120 mg XCMSFH/ml). Each piglet received 1200 mg
XCMSFH/kg body weight.
[0272] Non invasive cardiac data was obtained using a blood
pressure cuff on a hind limb. Doppler ultrasound (USCOM) was used
to measure the beat to beat cardiac output at various times
throughout the infusion. The data selected for analysis represented
the best ultrasound wave form obtained at any time point. Top
loading the addition of study material was limited by the upper
boundary of fluids producing congestive heart failure.
[0273] An amount equal to 14.3 percent of the calculated blood
volume (70 ml blood/kg body weight) was infused through a
peripheral IV over the course of one hour. Subject pigs were not
anesthetized, sedated, or invasively monitored.
[0274] Results:
[0275] FIGS. 7A-D represent, in order, cardiac output, systemic
vascular resistance, systolic blood pressure and diastolic blood
pressure as a function of XCMSFH infused, corrected for body
weight. All of the piglets tolerated the infusion well; there were
no subject deaths. A least squares method of correlating variables
was used to evaluate the data, providing slope and intercept. It is
readily apparent that there is great variability in the data of any
parameter, regardless of the amount of XCMSFH infused. This relates
to the fact that the subjects were not sedated, nor restrained.
Arousal, toileting, and handling seemed to account for most of the
variation, though there was significant variation between subjects'
parameters even before the initiation of any of the experiment.
[0276] The results of this study showed that the average (max, min)
for these indices were: Cardiac Output, 1.12 L/min (2.7, 0.45);
Systemic Vascular Resistance, 7008 dynessec/cm.sup.5 (21590-3364);
Systolic Blood Pressure, 156.69 mmHg (193-130); and Diastolic Blood
Pressure, 95.75 mmHg (113-72); respectively. The baseline values
for Cardiac Output were 1.26 L/min (1.62, 0.72); Systemic Vascular
Resistance 9588 dynessec/cm.sup.5 (12662-4991); Systolic Blood
Pressure 140 mmHg (159-123); and Diastolic Blood Pressure, 84.25
mmHg (111-72); respectively. Cardiac output was observed to
decrease slightly (<5%), while the systemic resistance increased
by approximately 30%. Both systolic and diastolic blood pressure
increased slightly (12 & 14%, respectively) during the
infusion. The relationship between the varied dose overload of
XCMSFH and the hemodynamic indices are represented in FIGS. 7A-D.
Although, the effect of XCMSFH on these indices is minimal, this
effect seems to be dependent on the % volume overload
delivered.
[0277] In this study, significant variability was found for all
parameters apparently independent of the amount of XCMSFH infused.
The amount of minimal change in these cardiac parameters can be
attributed to the increase in blood volume, essentially fluid
overloaded subjects. Results of this study indicate that all
subjects survived the experiment, and there were minimal changes in
the cardiac parameters observed relating to vasoactivity.
[0278] Prion Safety:
[0279] A number of measures are taken to ensure that the Hb of the
invention are prion free. Selection of suitable animals is an
initial step, choosing only animals from a closed herd, which have
been fed no animal protein, given no antibiotics, and which are
less than 30 months old. A second point for prevention of
contamination is scrupulous attention to avoidance of mixing brain
matter into blood. The sacrificial method of the "mushroom stunner"
approach is chosen to eliminate the possibility of brain matter
contamination, and thus eliminate potential introduction of prion
containing materials into the collected blood. Further, when the Hb
is processed, the washing procedure to remove plasma proteins will
also remove prions. Additionally, when the Hb is filtered through
the 300,00 Da molecular weight filter, any prions can be
eliminated. Lastly, the Hb of the invention is processed through
the Pall filter to remove leucocytes. At this point, small formed
bodies such as prions and viruses can be removed. All of these
precautions operate to secure the safety of the Hb of the invention
for use in human therapeutics and emergency procedures.
[0280] Deoxygenated and Oxygenated States.
[0281] The NO-blocked and stable NO-blocked tetrameric hemoglobins
of the invention are packaged for storage and transport as
deoxygenated species. For many therapeutic applications, the
modified hemoglobins are used in the deoxygenated state. For
applications where perfusion is required, for example, clearing a
field of living tissue for observation, perfusing an ischemic
region, or maintaining an organ ex-vivo prior to transplantation,
the modified hemoglobins may be used in their re-oxygenated states
to support tissue function.
[0282] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *