U.S. patent application number 11/034457 was filed with the patent office on 2005-10-27 for methods and compositions for optimization of oxygen transport by cell-free systems.
Invention is credited to Winslow, Robert M..
Application Number | 20050239686 11/034457 |
Document ID | / |
Family ID | 26708291 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239686 |
Kind Code |
A1 |
Winslow, Robert M. |
October 27, 2005 |
Methods and compositions for optimization of oxygen transport by
cell-free systems
Abstract
Compositions, and methods of use thereof, for use as blood
substitute products comprise aqueous mixtures of oxygen-carrying
and non-oxygen carrying plasma expanders and methods for the use
thereof. The oxygen-carrying component may consist of any
hemoglobin-based oxygen carrier, while the non-oxygen carrying
plasma expander my consist of any suitable diluent.
Inventors: |
Winslow, Robert M.; (La
Jolla, CA) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Family ID: |
26708291 |
Appl. No.: |
11/034457 |
Filed: |
January 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11034457 |
Jan 11, 2005 |
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10199269 |
Jul 18, 2002 |
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10199269 |
Jul 18, 2002 |
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09544656 |
Apr 5, 2000 |
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6432918 |
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09544656 |
Apr 5, 2000 |
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09032342 |
Feb 27, 1998 |
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6054427 |
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09032342 |
Feb 27, 1998 |
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08810694 |
Feb 28, 1997 |
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5814601 |
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Current U.S.
Class: |
514/13.4 ;
424/78.3 |
Current CPC
Class: |
Y10S 514/832 20130101;
Y10S 514/833 20130101; Y10S 530/815 20130101; A61K 2300/00
20130101; A61K 38/42 20130101; A61K 38/42 20130101; Y10S 530/829
20130101; Y10S 530/813 20130101; A61P 7/08 20180101 |
Class at
Publication: |
514/006 ;
424/078.3 |
International
Class: |
A61K 038/42 |
Goverment Interests
[0002] This invention was made with Government support under the
National Institutes of Health (NIH) awarded by contract P01
HL48018. The Government has certain rights in this invention.
Claims
We claim:
1-44. (canceled)
45. An aqueous cell-free composition for administration to a
subject, comprising polyalkylene oxide modified hemoglobin in an
aqueous solution, wherein said modified hemoglobin has a molecular
radius larger than native hemoglobin, wherein said composition has
a viscosity at least half that of blood, and an oncotic pressure
higher than that of plasma, and wherein said polyalkylene oxide is
covalently attached to cysteine residues on the hemoglobin.
46. The composition according to claim 45, wherein P50 of said
composition is equal to or lower than that of blood.
47. The composition according to claim 45, wherein the modified
hemoglobin is human hemoglobin.
48. The composition according to claim 45, wherein the viscosity is
from 2 to 4.5 cPs.
49. The composition according to claim 48, wherein the viscosity is
at least 2 cPs.
50. The composition according to claim 49, wherein the viscosity is
2.5 cPs.
51. The composition according to claim 45, wherein the modified
hemoglobin concentration is greater than 4.0 g/dl.
52. The composition according to claim 45, wherein P50 of said
composition is less than 28 mm Hg.
53. The composition according to claim 45, wherein the polyalkylene
oxide is polyethylene glycol.
54. The composition according to claim 45, wherein the oncotic
pressure is greater than 25 mm Hg.
55. The composition according to claim 45, wherein the oncotic
pressure is from 20 to 60 mm Hg.
56. The composition according to claim 45, wherein said
polyalkylene oxide is covalently attached to sulfhydryl groups of
said cysteine residues.
57. An aqueous cell-free composition for administration to a
subject, comprising polyethylene glycol conjugated to sulfhydryl
groups on human hemoglobin to form a modified hemoglobin in an
aqueous solution, wherein said modified hemoglobin has a molecular
radius larger than native hemoglobin, wherein said composition has
a viscosity from 2 to 4.5 cPs, an oncotic pressure from 20 to 60 mm
Hg, and a modified hemoglobin concentration greater than 4.0
g/dl.
58. The composition according to claim 57, wherein said sulfhydryl
groups are on cysteine residues.
Description
[0001] The present application is a Continuation-in-Part of U.S.
patent application Ser. No. 08/810,694, filed Feb. 28, 1997.
FIELD OF THE INVENTION
[0003] The present invention relates generally to blood products,
and more particularly to compositions comprising mixtures of
oxygen-carrying and non-oxygen carrying plasma expanders and
methods for their use.
BACKGROUND OF THE INVENTION
[0004] A. The Circulatory System and the Nature of Hemoglobin
[0005] The blood is the means for delivering nutrients to the
tissues and removing waste products from the tissues for excretion.
The blood is composed of plasma in which red blood cells (RBCs or
erythrocytes), white blood cells (WBCs), and platelets are
suspended. Red blood cells comprise approximately 99% of the cells
in blood, and their principal function is the transport of oxygen
to the tissues and the removal of carbon dioxide therefrom. The
left ventricle of the heart pumps the blood through the arteries
and the smaller arterioles of the circulatory system. The blood
then enters the capillaries, where the majority of the exchange of
nutrients and cellular waste products occurs. (See, e.g., A. C.
Guyton, Human Physiology And Mechanisms Of Disease (3rd. ed.; W.B.
Saunders Co., Philadelphia, Pa.), pp. 228-229 [1982]). Thereafter,
the blood travels through the venules and veins in its return to
the right atrium of the heart. Though the blood that returns to the
heart is oxygen-poor compared to that which is pumped from the
heart, in resting man the returning blood still contains about 75%
of the original oxygen content.
[0006] The reversible oxygenation function (i.e., the delivery of
oxygen and the removal of carbon dioxide) of RBCs is carried out by
the protein hemoglobin. In mammals, hemoglobin has a molecular
weight of approximately 68,000 and is composed of about 6% heme and
94% globin. In its native form, it contains two pairs of subunits
(i.e., it is a tetramer), each containing a heme group and a globin
polypeptide chain. In aqueous solution, hemoglobin is present in
equilibrium between the tetrameric (MW 68,000) and dimeric forms
(MW 34,000); outside of the RBC, the dimers are prematurely
excreted by the kidney (plasma half-life of approximately two to
four hours). Along with hemoglobin, RBCs contain stroma (the RBC
membrane), which comprises proteins, cholesterol, and
phospholipids.
[0007] B. Exogenous Blood Products
[0008] Due to the demand for blood products in hospitals and other
settings, extensive research has been directed at the development
of blood substitutes and plasma expanders. A blood substitute is a
blood product that is capable of carrying and supplying oxygen to
the tissues. Blood substitutes have a number of uses, including
replacing blood lost during surgical procedures and following acute
hemorrhage, and for resuscitation procedures following traumatic
injury. Plasma expanders are blood products that are administered
into the vascular system but are typically not capable of carrying
oxygen. Plasma expanders can be used, for example, for replacing
plasma lost from burns, to treat volume deficiency shock, and to
effect hemodilution (for, e.g., the maintenance of normovolemia and
to lower blood viscosity). Essentially, blood products can be used
for these purposes or any purpose in which banked blood is
currently administered to patients. (See, e.g., U.S. Pat. No.
4,001,401 to Bonson et al. and U.S. Pat. No. 4,061,736 to Morris et
al., hereby incorporated by reference).
[0009] The current human blood supply is associated with several
limitations that can be alleviated through the use of an exogenous
blood product. To illustrate, the widespread availability of safe
and effective blood substitutes would reduce the need for banked
(allogeneic) blood. Moreover, such blood substitutes would allow
the immediate infusion of a resuscitation solution following
traumatic injury without regard to cross-matching (as is required
for blood), thereby saving valuable time in resupplying oxygen to
ischemic tissue. Likewise, blood substitutes can be administered to
patients prior to surgery, allowing removal of autologous blood
from the patients which could be returned later in the procedure,
if needed, or after surgery. Thus, the use of exogenous blood
products not only protects patients from exposure to non-autologous
(allogeneic) blood, it conserves either autologous or allogeneic
(banked, crossmatched) blood for its optimal use.
[0010] C. Limitations of Current Blood Substitutes
[0011] Attempts to produce blood substitutes (sometimes referred to
as "oxygen-carrying plasma expanders") have thus far produced
products with marginal efficacy or whose manufacture is tedious and
expensive, or both. Frequently, the cost of manufacturing such
products is so high that it effectively precludes the widespread
use of the products, particularly in those markets where the
greatest need exists (e.g., emerging third-world economies).
[0012] The blood substitutes that have been developed previously
are reviewed in various references (See e.g., Winslow, Robert M.,
"Hemoglobin-based Red Cell Substitutes," Johns Hopkins University
Press, Baltimore [1992]). They can be grouped into the following
three categories: i) perfluorocarbon-based emulsions, ii)
liposome--encapsulated hemoglobin, and iii) modified cell-free
hemoglobin. As discussed below, none has been entirely successful,
though products comprising modified cell-free hemoglobin are
thought to be the most promising. Perfluorochemical-based
compositions dissolve oxygen as opposed to binding it as a chelate.
In order to be used in biological systems, the perfluorochemical
must be emulsified with a lipid, typically egg-yolk phospholipid.
Though the perfluorocarbon emulsions are inexpensive to
manufacture, they do not carry sufficient oxygen at clinically
tolerated doses to be effective. Conversely, while
liposome-encapsulated hemoglobin has been shown to be effective, it
is far too costly for widespread use (See e.g., Winslow,
supra).
[0013] Most of the blood substitute products in clinical trials
today are based on modified hemoglobin. These products, frequently
referred to as hemoglobin-based oxygen carriers (HBOCs), generally
comprise a homogeneous aqueous solution of a chemically-modified
hemoglobin, essentially free from other red cell residues (stroma).
Although stroma-free human hemoglobin is the most common raw
material for preparing a HBOC, other sources of hemoglobin have
also been used. For example, hemoglobin can be obtained or derived
from animal blood (e.g., bovine hemoglobin) or from bacteria or
yeast or transgenic animals molecularly altered to produce a
desired hemoglobin product. (See generally, Winslow, supra).
[0014] The chemical modification is generally one of intramolecular
crosslinking and/or oligomerization to modify the hemoglobin such
that its persistence in the circulation is prolonged relative to
that of unmodified hemoglobin, and its oxygen binding properties
are similar to those of blood. Intramolecular crosslinking
chemically binds together subunits of the tetrameric hemoglobin
unit to prevent the formation of dimers which, as previously
indicated, are prematurely excreted. (See, e.g., U.S. Pat. No.
5,296,465 to Rausch et al., hereby incorporated by reference).
[0015] The high costs of manufacturing HBOC products have greatly
limited their commercial viability. In addition, the present
inventors have found that known HBOCs have a tendency to release
excessive amounts of oxygen to the tissues at the arteriole walls
rather than the capillaries; this can result in insufficient oxygen
available for delivery by the HBOC to the tissues surrounding the
capillaries. This is despite the fact that the initial loading of
the HBOC with oxygen may be relatively high, even higher than that
normally achieved with natural red blood cells.
[0016] What is needed is a blood product that is relatively
inexpensive to manufacture and that delivers adequate amounts of
oxygen to the tissues.
SUMMARY OF THE INVENTION
[0017] The present invention is directed at compositions comprising
mixtures of an oxygen-carrying component and a non-oxygen carrying
component and methods for their use. The compositions overcome the
limited oxygen delivery characteristics of previous blood
substitutes, and therefore lower doses may be used. They are a
safer and more effective alternative to currently available blood
substitutes.
[0018] The present invention contemplates a means of improving the
oxygen delivering capacity of an oxygen carrier by combining that
carrier with a non-oxygen-carrying component like a conventional
plasma expander. In preferred embodiments, the oxygen carrier
(i.e., the oxygen-carrying component) is a hemoglobin-based oxygen
carrier. The hemoglobin may be either native (unmodified);
subsequently modified by a chemical reaction such as cross-linking,
polymerization, or the addition of chemical groups (i.e.,
polyethyleneglycol, polyoxyethylene, or other adducts); or it may
be recombinant or encapsulated in a liposome. A non-oxygen-carrying
plasma expander is any substance used for temporary replacement of
red cells which has oncotic pressure (e.g., starches such as
hetastarch or pentastarch, dextran such as dextran-70 or
dextran-90, albumin, or any other colloidal intravenous
solution).
[0019] More specifically, it is contemplated that the compositions
of the present invention will contain one or more of the following
properties: i) viscosity at least half that of blood, ii) oncotic
pressure higher than that of plasma; iii) hemoglobin oxygen
affinity higher than or equal to (i.e., P50 equal to or lower than)
that of blood; and iv) oxygen capacity less than that of blood. It
is not intended that the invention be limited to how the
compositions are used. A variety of uses are contemplated for the
compositions of the present invention, including, but not limited
to, the treatment of hemorrhage or use in hemodilution.
[0020] Particular non-oxygen carrying plasma expanders have been
used (e.g., for hemodilution) for a number of years, and their
physiological effects following administration are well
characterized. Previously, researchers have assumed that
administration of an oxygen-carrying blood product (e.g., a blood
substitute like an HBOC), should result in physiological
cardiovascular responses similar to those observed following
administration of non-oxygen carrying diluent materials of similar
molecular weight (e.g., dextran 70,000 MW, albumins and starches).
Furthermore, researchers in the field of blood substitutes have
been working under several other key assumptions. More
specifically, prior to the present invention, it has been thought
that blood substitutes should have viscosity less than that of
blood, oxygen affinity similar to or equal to or lower than that of
red cells, minimal colloidal osmotic (oncotic) pressure, and
hemoglobin concentration as high as possible. As described in
detail below, the compositions and methods of the present invention
are counter-intuitive to some of these assumptions.
[0021] The present invention contemplates a blood product solution,
comprising an oxygen-carrying component and a non-oxygen carrying
component, the blood product solution having oncotic pressure
higher than that of plasma and viscosity at least half that of
blood. In some embodiments, the blood product solution further
comprises oxygen affinity equal to or greater than that of blood.
In other embodiments, the blood product solution further comprises
oxygen capacity less than that of blood. In particular embodiments,
the oxygen-carrying component is a polyethylene glycol-modified
hemoglobin. Furthermore, in certain embodiments the
non-oxygen-carrying component is a colloid starch. When the
non-oxygen-carrying component is a colloid starch, it has an
average molecular weight of from approximately 200,000 daltons to
approximately 400,000 daltons is some embodiments. In particular
embodiments, the colloid starch is pentastarch.
[0022] The present invention also contemplates a blood product
solution, comprising a) an oxygen-carrying component, the
oxygen-carrying component comprising a polyethylene glycol-modified
hemoglobin; and b) a non-oxygen carrying component, the
non-oxygen-carrying component comprising a colloid starch having an
average molecular weight of from approximately 200,000 daltons to
approximately 400,000 daltons. In some embodiments, the
polyethylene glycol-modified hemoglobin comprises hemoglobin
selected from the group consisting of animal hemoglobin, human
hemoglobin, and recombinant hemoglobin. In particular embodiments,
the colloid starch has an average molecular weight of from
approximately 225,000 daltons to approximately 300,000 daltons, and
in other embodiments the colloid starch is pentastarch. In still
other embodiments, the pentastarch comprises from approximately 20
percent to approximately 80 percent by volume of the blood product
solution, whereas the pentastarch comprises from approximately 40
percent to approximately 60 percent by volume of the blood product
in other embodiments. Moreover, the blood product solution has a
viscosity from approximately 2 centipoise to approximately 4.5
centipoise in particular embodiments.
[0023] The present invention also contemplates a method of
enhancing oxygen delivery to the tissues of a mammal, comprising a)
providing a blood product solution, comprising an oxygen-carrying
component and a non-oxygen carrying component, the blood product
solution having oncotic pressure higher than that of plasma and
viscosity at least half that of blood; and b) administering the
blood product solution to the mammal, thereby enhancing oxygen
delivery to the tissues of the mammal. In some embodiments, the
blood product solution further comprises oxygen affinity equal to
or greater than that of blood, while in other embodiments the blood
product solution further comprises oxygen capacity less than that
of blood. In some embodiments, the oxygen-carrying component is a
polyethylene glycol-modified hemoglobin. The non-oxygen-carrying
component is a colloid starch in particular embodiments; in some
embodiments, the colloid starch has an average molecular weight of
from approximately 200,000 daltons to approximately 400,000
daltons. The colloid starch is pentastarch in still further
embodiments.
[0024] In addition, the present invention contemplates a method of
enhancing oxygen delivery to the tissues of a mammal, comprising a)
providing a blood product solution, comprising i) an
oxygen-carrying component, the oxygen-carrying component comprising
a polyethylene glycol-modified hemoglobin, and ii) a non-oxygen
carrying component, the non-oxygen carrying component comprising a
colloid starch having an average molecular weight of from
approximately 200,000 daltons to approximately 350,000 daltons; and
b) administering the blood product solution to the mammal, thereby
enhancing oxygen delivery to the tissues of the mammal.
[0025] In some embodiments, the polyethylene glycol-modified
hemoglobin comprises hemoglobin selected from the group consisting
of animal hemoglobin, human hemoglobin, and recombinant hemoglobin.
In other embodiments, the colloid starch has an average molecular
weight of from approximately 200,000 daltons to approximately
400,000 daltons. In still other embodiments, the colloid starch is
pentastarch. In particular embodiments, the pentastarch comprises
from approximately 20 percent to approximately 80 percent by volume
of the blood product.
[0026] In certain embodiments, the blood product solution has a
viscosity of from approximately 2 centipoise to approximately 4.5
centipoise. Finally, in other embodiments, the mammal is a
human.
[0027] The present invention also provides an aqueous cell-free
composition comprising hemoglobin, in which the hemoglobin is
present in a concentration of between 0.1 and 4.0 g/dl, and the
aqueous composition has a viscosity that is greater than 2.5 cP. In
some preferred embodiments, the viscosity of the aqueous
composition is between 2.5 and 4 cP. Thus, it is not intended that
the present invention be limited to any viscosity that is greater
than approximately 2.5 cP. Indeed, it is contemplated that the
present invention encompass compositions in which the viscosity is
6 cP or greater. In addition, the present invention encompasses
compositions in which the hemoglobin concentration is less than 0.1
or greater than 4 g/dl, although in particularly preferred
embodiments, the hemoglobin concentration is between 0.1 and 4
g/dl. Furthermore, in some embodiments, the K* of the composition
is approximately equal or similar to that of a red blood cell
suspension when measured at the same hemoglobin concentration.
[0028] In other embodiments of the composition, the hemoglobin has
an increased affinity for molecular oxygen as compared to red blood
cells. The present invention provides compositions that are
suitable for use in any animal, including humans. Thus, in some
embodiments, the hemoglobin of the composition has an increased
affinity as compared to mammalian red blood cells, although in
other embodiments, it is contemplated that the red blood cells are
from reptiles, avians, or any other animal. In most preferred
embodiments, the red blood cells used in this comparison are human
red blood cells. In preferred embodiments, the composition has a
P50 of less than 28 mm Hg. However, it is not intended that the
present invention be limited to this P50 value, as in some
embodiments, the P50 is higher than 28 mm Hg.
[0029] In other embodiments, the composition further comprises a
diluent selected from the group consisting of proteins,
glycoproteins, polysaccharides, and other colloids. It is not
intended that these embodiments be limited to any particular
diluent. Thus, it is intended that the diluent encompass solutions
of albumin, other colloids, or other non-oxygen carrying
components. In preferred embodiments, the diluent comprises
polysaccharide. In other preferred embodiments, the polysaccharide
comprises starch. In particularly preferred embodiments, the starch
comprises pentastarch.
[0030] In other embodiments, the hemoglobin within the composition
is surface-modified. It not intended that these embodiments be
limited to any particular type of surface modification. In
preferred embodiments, the surface modification includes the use of
polyalkylene oxide groups of varying chain lengths and charges. In
preferred embodiments, the hemoglobin is surface-modified with
polyethylene glycol of varying chain lengths and charges. It is not
intended that the surface modification be limited to any particular
type or a single type of modification. It is contemplated, that
multiple types of surface-modifications will be made to hemoglobin
of the composition.
[0031] The present invention also provides an aqueous cell-free
composition comprising surfaced-modified hemoglobin, wherein the
surface-modified hemoglobin is present in a concentration of
between 0.1 and 4.0 g/dl, and the aqueous composition has a
viscosity that is greater than 2.5 cP. As discussed above, in some
preferred embodiments, the viscosity of the aqueous composition is
between 2.5 and 4 cP. Thus, it is not intended that the present
invention be limited to any viscosity that is greater than
approximately 2.5 cP. Indeed, it is contemplated that the present
invention encompass compositions in which the viscosity is 6 cP or
greater. In further embodiments, the hemoglobin concentration is
less than 0.1 or greater than 4 g/dl, although in particularly
preferred embodiments, the hemoglobin concentration is between 0.1
and 4 g/dl. In some embodiments, the K* of the composition is
approximately equal or similar to that of a red blood cell
suspension when measured at the same hemoglobin concentration.
[0032] In preferred embodiments of this composition, the hemoglobin
has an increased affinity for molecular oxygen as compared to red
blood cells. As above, these embodiments are suitable for use in
any animal, including humans. Thus, in some embodiments, the
hemoglobin has an increased affinity as compared to mammalian red
blood cells, although in other embodiments, it is contemplated that
the red blood cells are from reptiles, avians, or any other animal.
In most preferred embodiments, the red blood cells used in this
comparison are human red blood cells. In other preferred
embodiments, the composition has a P50 of less than 28 mm Hg.
However, it is not intended that the present invention be limited
to this P50 value, as in some embodiments, the P50 is higher than
28 mm Hg.
[0033] Furthermore, in other embodiments, the present invention
provides compositions which further comprise a diluent selected
from the group consisting of proteins, glycoproteins,
polysaccharides, and other colloids. It is not intended that the
these embodiments be limited to any particular diluent. Thus, it is
intended that the diluent encompass solutions of albumin, other
colloids, or other non-oxygen carrying components. In preferred
embodiments, the diluent comprises polysaccharide. In other
preferred embodiments, the polysaccharide comprises starch. In
particularly preferred embodiments, the starch comprises
pentastarch. In these embodiments, it not intended that the present
invention be limited to any particular type of surface
modification. In preferred embodiments, the surface modification
includes the use of polyalkylene oxide groups of varying chain
lengths and charge. In preferred embodiments, the hemoglobin is
surface-modified with polyethylene glycol of varying chain lengths
and charges.
[0034] The present invention further provides an aqueous cell-free
composition comprising a mixture of hemoglobin and a diluent,
wherein the hemoglobin is present in a concentration between 0.1
and 4 g/dl, and wherein the diluent is selected from the group
consisting of proteins, glycoproteins, polysaccharides, and other
colloids, and wherein the aqueous composition has a viscosity of at
least 2.5 cP. As discussed above, in some preferred embodiments,
the viscosity of the aqueous composition is between 2.5 and 4 cP.
Thus, it is not intended that these embodiments be limited to any
viscosity that is greater than approximately 2.5 cP. Indeed, it is
contemplated that the present invention encompass compositions in
which the viscosity is 6 cP or greater. Furthermore, in some
embodiments, the diluent comprises a polysaccharide, while in
preferred embodiments, the diluent comprises starch, and in
particularly preferred embodiments, the diluent comprises
pentastarch. In addition, the present invention encompasses
compositions in which the hemoglobin concentration is less than 0.1
or greater than 4 g/dl, although in particularly preferred
embodiments, the hemoglobin concentration is between 0.1 and 4
g/dl. In some embodiments, the K* of the composition is
approximately equal or similar to that of a red blood cell
suspension when measured at the same hemoglobin concentration.
[0035] In some embodiments, the compositions comprise hemoglobin
with an increased affinity for molecular oxygen as compared to red
blood cells. The present invention provides compositions that are
suitable for use in any animal, including humans. Thus, in some
embodiments, hemoglobin has an increased affinity as compared to
mammalian red blood cells, although in other embodiments, it is
contemplated that the red blood cells are from reptiles, avians, or
any other animal. In most preferred embodiments, the red blood
cells used in this comparison are human red blood cells. In
preferred embodiments, the composition has a P50 of less than 28 mm
Hg. However, it is not intended that the present invention be
limited to this P50 value, as in some embodiments, the P50 is
higher.
[0036] As indicated above, these embodiments may also comprise
hemoglobin that is surface-modified. It not intended that the
present invention be limited to any particular type of surface
modification. In preferred embodiments, the surface modification
includes the use of polyalkylene oxide groups of varying chain
lengths and charge. In preferred embodiments, the hemoglobin is
surface-modified with polyethylene glycol of varying chain lengths
and charges.
[0037] The present invention also provides methods comprising
providing an animal and an aqueous cell-free composition comprising
hemoglobin, wherein the hemoglobin is present in a concentration of
between 0.1 and 4.0 g/dl, and the aqueous composition has a
viscosity that is greater than 2.5 cP; and administering the
aqueous composition to the animal. In preferred embodiments, the
animal is a mammal, while in particularly preferred embodiments,
the animal is human. In some embodiments, the human is suffering
from the symptoms of disease, pathology, insufficiency, or
abnormality. In some embodiments, the human has symptoms of
disease, wherein the disease is selected from the group consisting
of hypovolemic shock symptoms, hypoxia, chronic lung disease,
ischemia, stroke, trauma, hemodilution, cardioplegia, cancer,
anemia, sickle-cell anemia, septic shock, or disseminated
intravascular coagulation. However, it is not intended that the
methods of the present invention be limited to the administration
of the aqueous composition to alleviate any particular disease,
condition, pathology, insufficiency, or abnormality. Rather, it is
intended that the methods encompass any and all applications for
which the methods are suitable.
[0038] As above, the methods of present invention encompass an
aqueous cell-free composition comprising hemoglobin, wherein the
hemoglobin is present in a concentration of between 0.1 and 4.0
g/dl, and the aqueous composition has a viscosity that is greater
than 2.5 cP. In some preferred embodiments, the viscosity of the
aqueous composition is between 2.5 and 4 cP. Thus, it is not
intended that the present invention be limited to any viscosity
that is greater than approximately 2.5 cP. Indeed, it is
contemplated that the present invention encompass compositions in
which the viscosity is 6 cP or greater. In addition, the present
invention encompasses compositions in which the hemoglobin
concentration is less than 0.1 or greater than 4 g/dl, although in
particularly preferred embodiments, the hemoglobin concentration is
between 0.1 and 4 g/dl. In some embodiments, the K* of the
composition is approximately equal or similar to that of a red
blood cell suspension when measured at the same hemoglobin
concentration.
[0039] In alternative embodiments, the compositions comprise
hemoglobin with an increased affinity for molecular oxygen as
compared to red blood cells. In addition, these embodiments are
suitable for use with any animal, including humans. Thus, in some
embodiments, hemoglobin has an increased affinity as compared to
mammalian red blood cells, although in other embodiments, it is
contemplated that the red blood cells are from reptiles, avians, or
any other animal. In most preferred embodiments, the red blood
cells used in this comparison are human red blood cells. In
preferred embodiments, the composition has a P50 of less than 28 mm
Hg. However, it is not intended that the present invention be
limited to this P50 value, as in some embodiments, the P50 is
higher than 28 mm Hg.
[0040] The other embodiments, the compositions which further
comprise a diluent selected from the group consisting of proteins,
glycoproteins, polysaccharides, and other colloids. It is not
intended that the present invention be limited to any particular
diluent. Thus, it is intended that the diluent encompass solutions
of albumin, other colloids, or other non-oxygen carrying
components. In preferred embodiments, the diluent comprises
polysaccharide. In other preferred embodiments, the polysaccharide
comprises starch. In particularly preferred embodiments, the starch
comprises pentastarch.
[0041] In yet other embodiments, the hemoglobin within the
composition is surface-modified. It not intended that the present
invention be limited to any particular type of surface
modification. In preferred embodiments, the surface modification
includes the use of polyalkylene oxide groups of varying chain
lengths and charge. In preferred embodiments, the hemoglobin is
surface-modified with polyethylene glycol of varying chain lengths
and charges.
[0042] The present invention also provides methods comprising the
steps of providing: an organ from an animal, and an aqueous
cell-free composition comprising hemoglobin, wherein the hemoglobin
is present in a concentration of between 0.1 and 4.0 g/dl, and the
aqueous composition has a viscosity that is greater than 2.5 cP;
and perfusing the organ with said aqueous composition. In preferred
embodiments, the animal is a mammal, while in particularly
preferred embodiments, the animal is a human. However, it is not
intended that the methods be limited to humans or mammals. In
preferred embodiments, the organ is selected from the group
consisting of kidneys, liver, spleen, heart, pancreas, lung, and
muscle, although it is not intended that the methods of the present
be limited to these organs, as any organ may be perfused with the
aqueous solution of the present invention.
[0043] In some preferred embodiments of the methods, the viscosity
of the aqueous composition is between 2.5 and 4 cP. Thus, it is not
intended that the present invention be limited to any viscosity
that is greater than approximately 2.5 cP. Indeed, it is
contemplated that the present invention encompass compositions in
which the viscosity is 6 cP or greater. In addition, the present
invention encompasses compositions in which the hemoglobin
concentration is less than 0.1 or greater than 4 g/dl, although in
particularly preferred embodiments, the hemoglobin concentration is
between 0.1 and 4 g/dl. In some embodiments, the K* of the
composition is approximately equal or similar to that of a red
blood cell suspension when measured at the same hemoglobin
concentration.
[0044] These embodiments also provide compositions comprising
hemoglobin with an increased affinity for molecular oxygen as
compared to red blood cells. As above, these embodiments are
suitable for use in any animal, including humans. Thus, in some
embodiments, hemoglobin has an increased affinity as compared to
mammalian red blood cells, although in other embodiments, it is
contemplated that the red blood cells are from reptiles, avians, or
any other animal. In most preferred embodiments, the red blood
cells used in this comparison are human red blood cells. In
preferred embodiments, the composition has a P50 of less than 28 mm
Hg. However, it is not intended that the present invention be
limited to this P50 value, as in some embodiments, the P50 is
higher than 28 mm Hg.
[0045] The other embodiments, the compositions further comprise a
diluent selected from the group consisting of proteins,
glycoproteins, polysaccharides, and other colloids. It is not
intended that the present invention be limited to any particular
diluent. Thus, it is intended that the diluent encompass solutions
of albumin, other colloids, or other non-oxygen carrying
components. In preferred embodiments, the diluent comprises
polysaccharide. In other preferred embodiments, the polysaccharide
comprises starch. In particularly preferred embodiments, the starch
comprises pentastarch.
[0046] In yet other embodiments, the hemoglobin within the
composition is surface-modified. It not intended that the present
invention be limited to any particular type of surface
modification. In preferred embodiments, the surface modification
includes the use of polyalkylene oxide groups of varying chain
lengths and charge. In preferred embodiments, the hemoglobin is
surface-modified with polyethylene glycol of varying chain lengths
and charges.
[0047] It is not intended that the present invention be limited to
any particular oncotic pressure. Indeed, it is intended that the
compositions of the present invention encompass a range of oncotic
pressure. In some embodiments, the oncotic pressure ranges from 70
to 80 mm Hg, while in the most preferred embodiments, the oncotic
pressure is approximately 90 mm Hg. However, in other embodiments,
the oncotic pressure can be as low as 60 mm Hg. Furthermore, it is
intended that the present invention encompass hypooncotic,
hyperoncotic, and isooncotic pressures. As used herein, the term
"hyperoncotic" encompasses any oncotic pressure that is greater
than 25 mm Hg, although in preferred embodiments, solutions with
oncotic pressures of 20-60 mm Hg are provided. In some embodiments
of the methods of the present invention, it is contemplated that
the composition chosen for administration will be customized to the
particular needs of the animal. The present invention provides the
means to customize the composition to meet the needs of various
clinical and veterinary uses.
[0048] FIG. 19 provides a graph showing the hemoglobin
concentration and viscosity of various hemoglobin preparations. The
square positioned within this graph (i.e., at approximately 2.5-4
cP and 0.1 to 4 g/dl hemoglobin) indicates the properties of the
most preferred compositions of the present invention. As indicated,
the only hemoglobin solution that meets the criteria is the
"Hemospan" solution which was made according to the methods of the
present invention. The other samples in this graph include blood,
PEG-Hb (Enzon), PHP (Apex), and .alpha..alpha.-hemoglobin (US
Army). As discussed in more detail below, the characteristics of
the compositions of the present invention provide many heretofore
unknown and unexpected advantages.
[0049] The present invention further provides a method comprising:
providing i) liganded hemoglobin, ii) means for treating
hemoglobin, and iii) means for surface decorating hemoglobin;
treating the liganded hemoglobin with the treating means under
conditions such that a treated hemoglobin is produced having
greater affinity for molecular oxygen than unliganded hemoglobin;
and surface decorating the treated hemoglobin with the surface
decorating means.
[0050] In some embodiments of the method, the means for treating is
selected from the group consisting of crosslinking means and
polymerizing means. In alternative embodiments, the surface
decoration of step (c) comprises reacting said treated hemoglobin
with a polyalkylene oxide.
[0051] The present invention also provides a method comprising:
providing i) liganded hemoglobin, ii) means for treating hemoglobin
selected from the group consisting of crosslinking means and
polymerizing means, and iii) means for surface decorating
hemoglobin; treating the liganded hemoglobin with the treating
means under conditions such that a treated hemoglobin is produced
having greater affinity for molecular oxygen than unliganded
hemoglobin; and surface decorating the treated hemoglobin with the
surface decorating means. In some embodiments of the method, the
surface decoration of step (c) comprises reacting the treated
hemoglobin with a polyalkylene oxide.
[0052] The present invention further provides a method comprising:
providing i) hemoglobin, ii) means for enzymatically treating
hemoglobin (e.g., with enzymes such as carboxy peptidase), and iii)
means for surface decorating hemoglobin; treating the liganded
hemoglobin with the enzymatic treating means under conditions such
that an enzymatically treated hemoglobin is produced having greater
affinity for molecular oxygen than hemoglobin in red blood cells;
and surface decorating the enzymatically treated hemoglobin with
the surface decorating means.
[0053] Definitions
[0054] To facilitate understanding of the invention set forth in
the disclosure that follows, a number of terms are defined
below.
[0055] The phrase "oxygen capacity less than that of blood" means
that when the oxygen capacity of the blood product solutions of the
present invention is compared with that of blood, the oxygen
capacity of the blood product solutions is less. The oxygen
capacity of the blood product solutions of the present invention is
not required to be less than that of blood by any particular
amount. Oxygen capacity is generally calculated from hemoglobin
concentration, since it is known that each gram of hemoglobin binds
1.34 mL of oxygen. Thus, the hemoglobin concentration in g/dL
multiplied by the factor 1.34 yields the oxygen capacity in mL/dL.
The present invention contemplated the use of a suitable
commercially available instruments to measure hemoglobin
concentration, including the B-Hemoglobin Photometer (Hemocue,
Inc.). Similarly, oxygen capacity can be measured by the amount of
oxygen released from a sample of hemoglobin or blood by using, for
example, a fuel-cell instrument (e.g., Lex-O.sub.2-Con; Lexington
Instruments).
[0056] The phrase "oxygen affinity equal to or greater than that of
blood" means that when the oxygen affinity of the blood product
solutions of the present invention is compared with that of blood,
the oxygen affinity of the blood product solutions is greater. The
oxygen capacity of the blood product solutions of the present
invention is not required to be greater than that of blood by any
particular amount. The oxygen affinity of whole blood (and
components of whole blood such as red blood cells and hemoglobin)
can be measured by a variety of methods known in the art. (See,
e.g., Vandegriff and Shrager in Methods in Enzymology (Everse et
al., eds.) 232:460 [1994]). In preferred embodiments, oxygen
affinity may be determined using a commercially available
HEMOX.RTM. Analyzer (TCS Medical Products). (See, e.g., Winslow et
al., J. Biol. Chem., 252(7):2331-37 [1977]).
[0057] The phrase "oncotic pressure higher than that of plasma"
means that when the oncotic pressure of the blood product solutions
of the present invention is compared with that of plasma, the
oxygen affinity of the blood product solutions is greater. The
oncotic pressure of the blood product solutions of the present
invention is not required to be greater than that of blood by any
particular amount. Oncotic pressure may be measured by any suitable
technique; in preferred embodiments, oncotic pressure is measured
using a Colloid Osmometer (Wesco model 4420).
[0058] The phrase "viscosity at least half of that of blood" means
that when the viscosity of the blood product solutions of the
present invention is compared with that of blood, the oxygen
affinity of the blood product solutions is at least 50% of that of
blood; in addition, the viscosity may be greater than that of
blood. Preferably, viscosity is measured at 37.degree. C. in a
capillary viscometer using standard techniques. (See Reinhart et
al., J. Lab. Clin. Med. 104:921-31 [1984]). Moreover, viscosity can
be measured using other methods, including a rotating
cone-and-plate viscometer such as those commercially available from
Brookfield. The viscosity of blood is approximately 4 centipoise.
Thus, at least half of the blood value corresponds to at least
approximately 2 centipoise.
[0059] The term "blood product" refers broadly to formulations
capable of being introduced into the circulatory system of the body
and carrying and supplying oxygen to the tissues. While the term
"blood products" includes conventional formulations (e.g.,
formulations containing the fluid and/or associated cellular
elements and the like that normally pass through the body's
circulatory system, including, but not limited to, platelet
mixtures, serum, and plasma), the preferred blood products of the
present invention are "blood product mixtures." As used herein,
blood product mixtures comprise a non-oxygen-carrying component and
an oxygen-carrying component.
[0060] The term "oxygen-carrying component" refers broadly to a
substance capable of carrying oxygen in the body's circulatory
system and delivering at least a portion of that oxygen to the
tissues. In preferred embodiments, the oxygen-carrying component is
native or modified hemoglobin. As used herein, the term
"hemoglobin" refers to the respiratory protein generally found in
erythrocytes that is capable of carrying oxygen. Modified
hemoglobin includes, but is not limited to, hemoglobin altered by a
chemical reaction such as cross-linking, polymerization, or the
addition of chemical groups (e.g., polyethyleneglycol,
polyoxyethylene, or other adducts). Similarly, modified hemoglobin
includes hemoglobin that is encapsulated in a liposome.
[0061] The present invention is not limited by the source of the
hemoglobin. For example, the hemoglobin may be derived from animals
and humans; preferred sources of hemoglobin are cows and humans. In
addition, hemoglobin may be produced by other methods, including
recombinant techniques. A most preferred oxygen-carrying-component
of the present invention is "polyethylene glycol-modified
hemoglobin."
[0062] The term "polyethylene glycol-modified hemoglobin" refers to
hemoglobin that has been modified such that it is associated with
polyethylene glycol
(.alpha.-Hydro-.omega.-hydroxypoly-(oxy-1,2-ethanediy- l);
generally speaking, the modification entails covalent binding of
polyethylene glycol (PEG) to the hemoglobin. PEGs are liquid and
solid polymers of the general chemical formula
H(OCH.sub.2CH.sub.2).sub.nOH, where n is greater than or equal to
4. PEG formulations are usually followed by a number that
corresponds to its average molecular weight; for example, PEG-200
has a molecular weight of 200 and a molecular weight range of
190-210. PEGs are commercially available in a number of
formulations (e.g., Carbowax, Poly-G, and Solbase).
[0063] The term "non-oxygen-carrying component" refers broadly to
substances like plasma expanders that can be administered, e.g.,
for temporary replacement of red blood cell loss. In preferred
embodiments of the invention, the non-oxygen-carrying component is
a colloid (i.e., a substance containing molecules in a finely
divided state dispersed in a gaseous, liquid, or solid medium)
which has oncotic pressure (colloid osmotic pressure prevents,
e.g., the fluid of the plasma from leaking out of the capillaries
into the interstitial fluid). Examples of colloids include
hetastarch, pentastarch, dextran-70, dextran-90, and albumin.
[0064] Preferred colloids of the present invention include starches
like hetastarch and pentastarch. Pentastarch (hydroxyethyl starch)
is the preferred colloid starch of the present invention.
Pentastarch is an artificial colloid derived from a starch composed
almost entirely of amylopectin. Its molar substitution is 0.45
(i.e., there are 45 hydroxyethyl groups for every 100 glucose
units); hydroxyethyl groups are attached by an ether linkage
primarily at C-2 of the glucose unit (and less frequently at C-3
and C-6). The polymerized glucose units of pentastarch are
generally connected by 1-4 linkages (and less frequently by 1-6
linkages), while the degree of branching is approximately 1:20
(i.e., there is one branch for every 20 glucose monomer units). The
weight average molecular weight of pentastarch is about 250,000
with a range of about 150,000 to 350,000. Unless otherwise
indicated, reference to the "average molecular weight" of a
substance refers to the weight average molecular weight.
Pentastarch is commercially available (e.g., DuPont Merck) as a 10%
solution (i.e., 10 g/100 mL); unless otherwise indicated, reference
to blood product solutions comprising pentastarch (and other
non-oxygen-carrying components as well as oxygen-carrying
components) is on a volume basis.
[0065] The phrase "enhancing oxygen delivery to the tissues of a
mammal" refers to the ability of a fluid (e.g., a blood product)
introduced into the circulatory system to deliver more oxygen to
the tissues than would be delivered without introduction of the
fluid. To illustrate, a patient may experience substantial blood
loss following acute hemorrhage, resulting in decreased transport
of oxygen to the tissues via the blood. The administration of a
blood product to the patient can supplement the ability of the
patient's own blood to deliver oxygen.
[0066] The term "mixture" refers to a mingling together of two or
more substances without the occurrence of a reaction by which they
would lose their individual properties. The term "solution" refers
to a liquid mixture. The term "aqueous solution" refers to a
solution that contains some water. In many instances, water serves
as the diluent for solid substances to create a solution containing
those substances. In other instances, solid substances are merely
carried in the aqueous solution (i.e., they are not dissolved
therein). The term aqueous solution also refers to the combination
of one or more other liquid substances with water to form a
multi-component solution.
[0067] The term "approximately" refers to the actual value being
within a range of the indicated value. In general, the actual value
will be between 10% (plus or minus) of the indicated value.
DESCRIPTION OF THE DRAWINGS
[0068] FIGS. 1A-B are a diagrammatic cross-sectional illustration
of the flow of whole blood (FIG. 1A) and a hemoglobin-based oxygen
carrier (FIG. 1B) through an arterial vessel.
[0069] FIG. 2 depicts a plot of flow velocity in the
microcirculation as a function of hematocrit reductions with
dextran hemodilution and saline hemodilution.
[0070] FIG. 3 graphically presents mean arterial blood pressure in
rats prior to and during an exchange transfusion (arrow) with
Hemolink.RTM. (.tangle-soliddn.), pentastarch (.tangle-solidup.)
and a 50/50 (volume/volume) mixture of Hemolink.RTM.+pentastarch
(.gradient.).
[0071] FIG. 4 graphically presents mean arterial blood pressure in
rats following exchange transfusion with Hemolink.RTM.
(.tangle-soliddn.), pentastarch (.tangle-solidup.) and a 50/50
(volume/volume) mixture of Hemolink.RTM.+pentastarch (.gradient.),
during a 60% blood volume hemorrhage.
[0072] FIG. 5 depicts rat survival following exchange transfusion
with pentastarch (.tangle-solidup.), .alpha..alpha.-Hb
(.box-solid.), PEG-Hb (.circle-solid.),
pentastarch+.alpha..alpha.-Hb (.quadrature.), and
pentastarch+PEG-Hb (.smallcircle.) and after the initiation of a
60% hemorrhage.
[0073] FIG. 6A-D graphically depict the acid-base status of control
rats (.diamond-solid.) and of rats following exchange transfusion
with pentastarch (.tangle-solidup.), .alpha..alpha.-Hb
(.box-solid.), PEG-Hb (.circle-solid.),
pentastarch+.alpha..alpha.-Hb (.quadrature.), and
pentastarch+PEG-Hb (.smallcircle.) and after the initiation of a
60% hemorrhage. FIG. 6A depicts PaO.sub.2, FIG. 6B depicts
PaCO.sub.2, FIG. 6C depicts arterial pH, and FIG. 6D depicts base
excess.
[0074] FIG. 7 graphically depicts the production of lactic acid in
control rats (.diamond-solid.) and of rats following exchange
transfusion with pentastarch (.tangle-solidup.), .alpha..alpha.-Hb
(.box-solid.), PEG-Hb (.circle-solid.),
pentastarch+.alpha..alpha.-Hb (.quadrature.), and
pentastarch+PEG-Hb (.smallcircle.) and after the initiation of a
60% hemorrhage.
[0075] FIG. 8A depicts mean arterial blood pressure in control rats
(.diamond-solid.) and of rats following exchange transfusion with
pentastarch (.tangle-solidup.), PEG-Hb (.circle-solid.), and
Pentaspan+PEG-Hb (.smallcircle.) at time -30 minutes, and after the
initiation of a 60% hemorrhage at time 0 minutes.
[0076] FIG. 8B depicts mean arterial blood pressure in control rats
(.diamond-solid.), and rats following exchange transfusion with
pentastarch (.tangle-solidup., point B), .alpha..alpha.-Hb
(.box-solid., point B), and pentastarch+.alpha..alpha.-Hb
(.quadrature., point A), and after the initiation of a 60%
hemorrhage (point C).
[0077] FIG. 9 depicts cardiac output in control rats
(.diamond-solid.) and in rats following exchange transfusion with
pentastarch (.tangle-solidup.), .alpha..alpha.-Hb (.box-solid.),
PEG-Hb (.circle-solid.), and pentastarch+PEG-Hb (.smallcircle.) and
after the initiation of a 60% hemorrhage at 0 minutes.
[0078] FIG. 10 depicts systemic vascular resistance in control rats
(.diamond-solid.) and of rats following exchange transfusion with
pentastarch (.tangle-solidup.), .alpha..alpha.-Hb (.box-solid.),
PEG-Hb (.circle-solid.), and pentastarch+PEG-Hb (.smallcircle.) and
after the initiation of a 60% hemorrhage at 0 minutes.
[0079] FIG. 11 depicts animal survival following exchange
transfusion with pentastarch (.tangle-solidup.), .alpha..alpha.-Hb
(.box-solid.), and pentastarch+.alpha..alpha.-Hb (.quadrature.)
after the initiation of a 60% hemorrhage.
[0080] FIG. 12 depicts animal survival following exchange
transfusion with hetastarch (x), Hemolink.RTM. (.tangle-soliddn.),
Hemolink.RTM.+pentastar- ch (.gradient.), and
hetastarch+Hemolink.RTM. (.diamond.) and after the initiation of a
60% hemorrhage.
[0081] FIG. 13 provides an illustration of a Krogh cylinder.
[0082] FIG. 14 provides a schematic of a capillary system.
[0083] FIG. 15. is a graph showing the exit PO.sub.2 compared to
the residence time of red blood cells, A.sub.0 hemoglobin,
.alpha..alpha.-hemoglobin, and PEG-Hb.
[0084] FIG. 16. is a graph showing the saturation compared with the
residence time of red blood cells, A.sub.0 hemoglobin,
.alpha..alpha.-hemoglobin, and PEG-Hb.
[0085] FIG. 17 is a graph showing the K* compared to the residence
time of red blood cells, A.sub.0 hemoglobin,
.alpha..alpha.-hemoglobin, and PEG-Hb.
[0086] FIG. 18 is a graph showing the MAP over time for .sub.0
hemoglobin, .alpha..alpha.-hemoglobin, and PEG-Hb.
[0087] FIG. 19 is a graph showing the hemoglobin concentration and
viscosity of various hemoglobin solutions.
DESCRIPTION OF THE INVENTION
[0088] The present invention relates generally to blood products,
and more particularly to compositions comprising a mixture of an
oxygen-carrying component and a non-oxygen-carrying component and
methods for the use thereof. The compositions and methods of the
present invention result in improved oxygen delivering capacity of
hemoglobin-based oxygen carriers. Generally speaking, the
compositions of the present invention will exhibit one or more of
the following properties: i) viscosity at least half that of blood;
ii) oncotic pressure higher than that of plasma; iii) hemoglobin
oxygen affinity higher than or equal to (i.e., P50 equal to or
lower than) that of blood; and iv) oxygen capacity less than that
of blood. Because of the more efficient utilization of the oxygen
carried by the HBOC in terms of tissue oxygenation, the
compositions of the present invention comprise a substantially
reduced hemoglobin content and are generally less expensive to
formulate.
[0089] The description of the invention is divided into: I) The
Nature of Oxygen Delivery and Consumption; II) Facilitated
Diffusion and The Design of Hemoglobin-Based Oxygen Carriers; III)
Clinical and Other Applications of the Present Invention; IV) The
Oxygen-carrying Component of the Blood Products of the Present
Invention; V) The Non-oxygen Carrying Component of the Blood
Products of the Present Invention; and VI) Blood Product
Compositions. Each section will be discussed in turn below.
[0090] I. The Nature of Oxygen Delivery and Consumption
[0091] Although the successful use of the compositions and methods
of the present invention do not require comprehension of the
underlying mechanisms of oxygen delivery and consumption, basic
knowledge regarding some of these putative mechanisms may assist in
understanding the discussion that follows. As previously indicated,
it has generally been assumed that the capillaries are the primary
conveyors of oxygen to the tissue; however, regarding tissue at
rest, current findings indicate that there is approximately an
equipartition between arteriolar and capillary oxygen release. That
is, hemoglobin in the arterial system is believed to deliver
approximately one-third of its oxygen content in the arteriolar
network and one-third in the capillaries, while the remainder exits
the microcirculation via the venous system. The arteries themselves
comprise a site of oxygen utilization (e.g., the artery wall
requires energy to effect regulation of blood flow through
contraction against vascular resistance). Thus, the arterial wall
is normally a significant site for the diffusion of oxygen out of
the blood. However, current oxygen-delivering compositions (e.g.,
HBOCs) may release too much of their oxygen content in the arterial
system, and thereby induce an autoregulatory reduction in capillary
perfusion.
[0092] The rate of oxygen consumption by the vascular wall, i.e.,
the combination of oxygen required for mechanical work and oxygen
required for biochemical synthesis, can be determined by measuring
the gradient at the vessel wall. Present technology allows accurate
oxygen partial pressure measurements in vessels on the order of 50
microns diameter. The measured gradient is directly proportional to
the rate of oxygen utilization by the tissue in the region of the
measurement. Such measurements show that the vessel wall has a
baseline oxygen utilization which increases in inflammation and
constriction, and is lowered by relaxation.
[0093] The vessel wall gradient is inversely proportional to tissue
oxygenation. Vasoconstriction increases the oxygen gradient (tissue
metabolism), while vasodilation lowers the gradient. Higher
gradients are indicative of the fact that more oxygen is used by
the vessel wall, while less oxygen is available for the tissue. The
same phenomenon is believed to be present throughout the
microcirculation.
[0094] The present invention demonstrates that increased blood
PO.sub.2 (which can be obtained, e.g., by hemodilution) through
administration of a conventional oxygen-carrying solution (e.g., a
HBOC), though superficially a beneficial outcome of the altered
blood flow characteristics and blood oxygen carrying capacity of
the resulting circulatory fluid, carries with it significant
disadvantages. That is, when the hemoglobin carrying the oxygen is
evenly distributed in the vessel as opposed to being contained in
RBCs, a different set of factors influencing oxygen delivery
apparently come into play. The present invention provides a means
of alleviating these disadvantages, namely by providing and using
an aqueous solution of an oxygen-carrying component (e.g., modified
hemoglobin) and a non-oxygen-carrying component (e.g., a
non-proteinaceous colloid such as dextran or pentastarch). Among
other attributes, the compositions of the present invention can be
manufactured at a much lower cost than that of normal HBOCs and
provide a blood substitute of increased viscosity.
[0095] FIG. 1A diagrammatically illustrates, in cross section, an
arteriole having a wall (2) surrounding the flow passage
therethrough. The wall in turn, is surrounded by muscle (1). As
previously indicated, normal whole blood consists essentially of
red blood cells (3) and plasma (4). Substantially all
(approximately 97%) of oxygen carried by the blood is associated
with the hemoglobin and is inside the red blood cells (3); only
about 3% of the oxygen is in the plasma component.
[0096] Accordingly, the oxygen availability to the artery wall (2)
is limited by the surface area of the RBCs and the rate of
diffusion of oxygen through the RBC membrane and surrounding
unstirred plasma. The artery walls receive an amount of oxygen
proportional to the spacing between RBCs and the mean distance for
diffusion from RBCs to the wall.
[0097] For comparison purposes, FIG. 1B diagrammatically
illustrates oxygen delivery when an artery is perfused with a HBOC
(5) mixed with whole blood. In this situation, the component of the
HBOC that directly binds oxygen is homogeneously distributed
throughout the HBOC (5) and the oxygen is available for diffusion
to all parts of the surface of the artery wall (2). Thus, oxygen
availability to the artery wall (2) is greatly increased,
effectively causing an increase of PO.sub.2 in the arterial system.
Though the present invention does not require an understanding of
the precise mechanisms, it is believed that arterial wall and
muscle reactions (e.g., increased metabolism of the cellular
components of the vessel wall as a consequence of energy-consuming
vasoconstrictor effects) take place in an attempt to maintain the
PO.sub.2 of the tissue; this is evidenced by the establishment of a
large gradient of oxygen partial pressure across the arterial wall
aimed at maintaining arteriolar partial oxygen pressure constant.
As a result, there is excessive loss of oxygen from the blood-HBOC
mixture at the arterial walls, and, concomitantly, insufficient
oxygen is available for capillary delivery to the tissues.
[0098] Though a precise understanding of the underlying mechanism
is not required in order to practice the present invention, the
present invention is based upon the discovery that a HBOC tends to
release too much of the oxygen it carries at the artery walls,
resulting in reaction of the arterial walls to the excess oxygen
and oxygen deficiency at the capillaries. As alluded to above,
researchers have previously assumed that administration of a blood
substitute (e.g., a HBOC) should result in physiological
cardiovascular responses similar to those observed upon
administration of non-oxygen carrying diluent materials of similar
molecular weight. However, it has been observed that HBOCs cause
physiological reactions that differ from those found with
non-oxygen-carrying plasma expanders. The dilution of RBCs,
accompanied by the maintenance of intrinsic oxygen delivering
capacity of the composition (i.e., because the blood substitute
composition is itself an oxygen carrier), changes the distribution
of oxygen in the circulatory system, increasing the PO.sub.2 in the
arteriolar segment. As discussed further below, this in turn
appears to lead to the reaction of the muscles lining the arterial
walls to the excess oxygen availability. In contrast, the
compositions of the present invention result in increased oxygen
delivery to the tissues surrounding the capillaries.
[0099] As set forth in the preceding discussion, the suitability of
a blood product should be determined by analysis of its systemic
effects, and how such effects, in conjunction with the altered
transport properties of the circulating fluid, influence transport
microcirculatory function.
[0100] II. Facilitated Diffusion and the Design of Hemoglobin-Based
Oxygen Carriers
[0101] Vasoconstriction is one of the most perplexing problems in
the development of a safe and efficacious red cell substitute. When
infused into animals and humans, many hemoglobin-based solutions
produce significant hypertension, increased vascular resistance and
decreased O.sub.2 transport. This phenomenon has been observed in
both the systemic and pulmonary circulations in models of clinical
use (Hess et al., J. Appl. Physiol., 74: 1769-78 [1993], Keipert et
al., Transfusion 33: 701-8 [1993]) and in humans (Kasper et al.,
Biochem., 31: 7551-9 [1992]).
[0102] Vasoactivity is usually attributed to the avidity with which
hemoglobin combines with nitric oxide, the endothelium-derived
relaxing factor. The NO affinity of model hemoglobins however does
not correlate with the effect on mean arterial blood pressure in
rats (Rohlfs et al., In R. M. Winslow et al., (eds.), Advances in
Blood Substitutes. Industrial Opportunities and Medical Challenges,
Birkhauser, Boston [1997], pp. 298-327 [1997]), and it is possible
that oversupply of O.sub.2 due to diffusion of HbO.sub.2 or removal
of NO due to diffusion of HbNO also plays an additional, if not
exclusive, role.
[0103] Increased rates of O.sub.2 uptake and release by cell-free
hemoglobin compared to red blood cells have been predicted (See
e.g., Homer, Microvasc. Res., 22: 308-23 [1981]; Federspiel and
Popel, Microvasc. Res., 32: 164-189 [1986]) and shown in vitro
(Page et al., In R. M. Winslow et al., (eds.), Blood Substitutes.
New Challenges, Birkhauser, Boston [1996], pp. 132-145). However,
attempts to demonstrate augmented transport by O.sub.2 diffusion in
vivo by cell-free hemoglobin have been unsuccessful (See, Biro,
Can. J. Physiol. Pharmacol., 69: 1656-1662 [1991]; Hogan et al.,
Adv. Exp. Med. Biol., 361: 375-378 [1994]; and Hogan et al., J.
Appl. Physiol., 361: 2470-5 [1992]). Although an understanding of
the mechanism is not necessary in order to make and use the present
invention, during the development of the present invention, it was
determined, shown, by measurements in artificial capillaries, that
cell-free hemoglobin does, indeed, increase the availability of
O.sub.2 to the surrounding medium.
[0104] In normal blood, O.sub.2 moves from the red blood cell to
the vessel wall by simple diffusion. When hemoglobin is present in
the plasma space, O.sub.2 can also move bound to hemoglobin as
HbO.sub.2. This second process is called "facilitated diffusion."
During the development of the present invention, properties of
cell-free hemoglobin that modulate this facilitated diffusion were
identified. Using this knowledge, hemoglobins that demonstrate
diffusive O.sub.2 transport similar to that of red blood cells by
reduced facilitated diffusion were prepared. It was also confirmed
that these example molecules do not produce vasoconstriction in
animals. Surprisingly, it was found that increased viscosity,
increased O.sub.2 affinity (reduced P50), and increased molecular
size are the key properties required for a cell-free hemoglobin to
avoid vasoactivity and to enable success as a red cell
substitute.
[0105] In addition, the present invention provides teachings
regarding the optimal properties of hemoglobin-based blood
substitutes in regard to oxygen affinity, viscosity and molecular
size and a method to evaluate such products by an instrument based
on an artificial capillary. This method enables the quantitative
determination of the ability of a blood substitute to transfer
O.sub.2 (or any other gas such as CO.sub.2, NO, or CO) across a
capillary membrane as a model of in vivo gas transfer.
[0106] A. Facilitated Diffusion
[0107] During the development of the present invention, it was
shown that unexpectedly, arterioles, particularly at the A2/A3
level consume large amounts of O.sub.2. This was determined by a
technique for measuring O.sub.2 concentration in localized areas of
the microcirculation (Torres and Intaglietta, Am. J. Physiol., 265:
H1434-H1438 [1993]). These results indicate that these arterioles
are capable of prodigious metabolic activity. Innervation of these
arterioles is particularly dense (Saltzman et al., Microvasc. Res.,
44: 263-273 [1992]), suggesting that they regulate downstream
capillary blood flow. Based on these results, increasing the
O.sub.2 available to these arterioles would be expected to provide
a potent stimulus to engage mechanisms that regulate the delivery
of O.sub.2 to capillary beds (autoregulation). Although an
understanding of the exact biochemical mechanism(s) which underlie
these events is not necessary in order to use the present
invention, it is contemplated that they could be mediated by
O.sub.2-- or NO-- sensitive pathways; the presence of hemoglobin,
free in the plasma space, as in a "blood substitute" is likely to
engage these mechanisms because of its capacity for facilitated
diffusion.
[0108] The transport of O.sub.2 in the blood by two pathways
(O.sub.2 and HbO.sub.2 diffusion) can be expressed mathematically.
The transport (flux, -J) of O.sub.2 to the vessel wall is the sum
of the diffusion of free (O.sub.2) and chemically bound oxygen
(HbO.sub.2): 1 - J = D O 2 P O 2 X + D HbO 2 [ Hb ] T Y X ( 1 )
[0109] where D.sub.02 and D.sub.HbO2 are the diffusion constants
for O.sub.2 and cell-free HbO.sub.2, respectively, .alpha. is the
solubility of O.sub.2 in plasma, .DELTA.PO.sub.2 is the difference
in partial pressure of O.sub.2 inside and outside the vessel,
.DELTA.Y is the gradient of hemoglobin saturation from the center
of the vessel to its wall, and [Hb].sub.T is the total cell-free
hemoglobin concentration. D.sub.02 and D.sub.Hbo2 have been
measured experimentally in static solution (Table 1). The distance
for diffusion, .DELTA.X, is considered to be the same for the two
molecules, O.sub.2 and HbO.sub.2. The references cited in Table 1
are: Wittenberg, Physiol. Rev., 50(4): 559-636 [1970], and Bouwer,
Biochim. Biophys. Acta 1338: 127-136 [1977]).
1TABLE 1 Values For Diffusion Constants From The Literature
D.sub.O2 (cm.sup.2/sec) D.sub.HbO2 (cm.sup.2/sec) Wittenberg 2.13
.times. 10.sup.-5 11.3 .times. 10.sup.-7 Bouwer 1.40 .times.
10.sup.-5 7.0 .times. 10.sup.-7 Mean 1.76 .times. 10.sup.-5 9.15
.times. 10.sup.-7
[0110] Table 1 shows that D.sub.HbO2 is about {fraction
(1/20)}.sup.th of D.sub.02. However because the solubility of
O.sub.2 in plasma is low (.alpha.=1.2074 .mu.M/Torr), and D.sub.02
is relatively high, when plasma hemoglobin concentration is only 3
mM (4.83 g/dl) at PO.sub.2 of 100 Torr, the product of diffusion
and concentration (the numerators in equation 1) for free O.sub.2
and HbO.sub.2 are nearly equal. Thus plasma hemoglobin contributes
as much O.sub.2 as dissolved O.sub.2, effectively doubling the
amount of O.sub.2 available from red blood cells. These
relationships are shown quantitatively in Table 2.
2TABLE 2 The Product Of Diffusion And Concentration For Dissolved
O.sub.2 vs. HbO.sub.2 Concentration at Diffusion constant
Concentration .times. 100 Torr, mM (see table 3) Diffusion O.sub.2
0.1207 176 .times. 10.sup.-7 2.48 .times. 10.sup.-6 HbO.sub.2 3.0
9.15 .times. 10.sup.-7 2.74 .times. 10.sup.-6
[0111] In order to develop a strategy to minimize the facilitated
diffusion of O.sub.2 by plasma HbO.sub.2, it was necessary to
analyze the biophysical properties which contribute to it. Because
water is much smaller than HbO.sub.2, D.sub.HBO2 is a function of
viscosity and molecular radius, as defined by the Stokes-Einstein
equation: 2 D HbO 2 = k T 6 solution r HbO 2 ( 2 )
[0112] where k is Boltzman's constant, .eta..sub.solution is the
viscosity of the solution, and r.sub.HbO2 is the radius of the
hemoglobin molecule (HbO.sub.2). For molecular oxygen, where the
molecular radius (r.sub.O2) is approximately the same as that of
water, the Stokes-Einstein equation becomes: 3 D O 2 = k T 4
solution r O 2 ( 3 )
[0113] Thus, for both HbO.sub.2 and dissolved O.sub.2 their
diffusivities are inversely related to the viscosity of the
macromolecular solutions. For cell-free hemoglobin, hemoglobin
molecular size is an additional factor in that D.sub.HbO2 is
inversely proportional to the molecular size of hemoglobin
(r.sub.HbO2 in Equation 2). Thus this analysis predicts that two
potential strategies to reduce or eliminate facilitated diffusion
by cell-free hemoglobin is increasing the molecular radius of the
molecule and increasing solution viscosity.
[0114] Further analysis of the equation 1 leads to an understanding
of an additional strategy to defeat this mechanism. The gradient
along which HbO.sub.2 diffuses is [Hb].sub.T.DELTA.Y and the
distance through which HbO.sub.2 must diffuse (.DELTA.X.sub.HbO2).
The quantity .DELTA.Y at a given PO.sub.2 is the slope of the
oxygen equilibrium curve at that PO.sub.2 and is dependent on the
shape of the curve (a property of the hemoglobin molecule) and its
position (i.e., P50).
[0115] To summarize, the total O.sub.2 transferred in a cylindrical
section of the Krogh cylinder (see FIG. 1) can be described as
follows: 4 O 2 r = ( r 2 R ) [ ( D O 2 P O 2 X O 2 ) + ( D HbO 2 [
Hb ] T Y X HbO 2 ) ] ( 4 )
[0116] In this equation, r is the radius of the capillary, and R is
the flow rate. The equation shows the contribution of HbO.sub.2
diffusion to total O.sub.2 transport. This form of the O.sub.2
transfer equation has the interesting property in that it shows
that the contribution of the HbO.sub.2 diffusion is dependent on 4
variables: the diffusion constant (D.sub.HbO2), hemoglobin
concentration ([Hb].sub.T), the difference in saturation between
the center and the edge of the capillary (.DELTA.Y) and the
distance for diffusion of HbO.sub.2 (.DELTA.X.sub.HBO2).
[0117] Equation 4 reveals a number of strategies that can be
employed independently or in combination to modulate O.sub.2 flux
(.DELTA.O.sub.2T). The strategies are defined by the relationship
of .DELTA.O.sub.2T to the alterable solution properties such that
.DELTA.O.sub.2T is:
[0118] (1) inversely proportional to solution viscosity (.eta.),
according to Eqs. 2 and 3, through changes in both DO.sub.2 and
DHbO.sub.2;
[0119] (2) inversely proportional molecular size (r.sub.HbO2),
according to Eq. 2, through a change D.sub.HbO2;
[0120] (3) directly proportional to [Hb]T; and
[0121] (4) directly proportional to .DELTA.Y (.DELTA.Y can be
altered by changing O.sub.2 affinity and/or cooperativity of
O.sub.2 binding).
[0122] Thus, to minimize effects of facilitated diffusion on
.DELTA.O.sub.2T from cell-free hemoglobin-based oxygen carriers, a
given .DELTA.O.sub.2T based on the value for red blood cells can be
achieved using the above strategies independently or in
combination. For the purpose of example, .DELTA.O.sub.2T can be
decreased to within a desired range by:
[0123] (1) altering a single parameter independently through:
[0124] increasing .eta.;
[0125] increasing r.sub.HbO2;
[0126] decreasing [Hb]T or
[0127] adjusting .DELTA.Y through its O.sub.2 affinity and/or
cooperativity;
[0128] (2) altering any combination of the above properties such
that, quantitatively, .DELTA.O.sub.2T is within the desired
range.
[0129] B. Evaluation of Cell-Free Hemoglobins
[0130] Evaluation of cell-free hemoglobins with regard to their
facilitated diffusion of oxygen and hence their potential to
produce autoregulatory vasoactivity in arterioles is based on the
Krogh cylinder, an idealized segment of vessel (See FIG. 13).
Through a detailed analysis of the shape and position of the oxygen
equilibrium curve, the amount of O.sub.2 delivered to this
sensitive region is analyzed as a function of diffusion, hemoglobin
concentration, and P50.
[0131] 1. Artificial Capillary System
[0132] The artificial capillary system is shown diagrammatically in
FIG. 14. The capillary is polydimethlysiloxane (e.g., Silastic,
Point Medical Corporation, Crown Point, Ind.) with a wall thickness
approximately the same size as the diameter (57 .mu.m). The glass
capillary is a 2 .mu.l pipette (e.g., Drummond Scientific,
Broomall, Pa.). The glass and silicone junction is sealed with a
silicone sealant (e.g., RTV 60, General Electric). The typical
length of a capillary, 100 mm, produces residence times similar to
in vivo times (i.e., 0.37 sec-1.5).
[0133] The infusion syringe pump (e.g., KD Scientific, Boston,
Mass.) is connected to the entry oxygen flow cell by a short length
of low-permeable Tygon tubing. The Clark-type oxygen electrodes
(e.g., Instech, Plymouth Meeting, Pa.) are used to monitor the
system. Data collection is accomplished by analysis of the effluent
fluid with a blood-gas analyzer (ABL-5, Radiometer). The exit from
the flow cell is connected to another short length of Tygon tubing,
which in turn is tightened to the glass capillary of the silicone
capillary unit by the use of a micro-tube connector (e.g.,
Cole-Palmer, Niles, Ill.). The artificial capillary is encased in a
gas-tight exchange chamber made of clear acrylic plastic.
Bimetallic temperature probes (e.g., YSI 700, Yellow Springs, Ohio)
are attached near the entry and exit points of the fluid flow to
ensure proper temperature control and held constant at 37.degree.
C.
[0134] The collection cell is mated directly to the end of the
artificial capillary unit by use of a silicone sealant and a
polypropylene microfitting (Cole-Palmer). The collection cell is
solid acrylic with a T shaped channel (diameter of 0.75 mm) drilled
through it. The first channel is shunted through a calibrated
measuring tube that serves as a flow meter. Periodically the flow
meter can be replaced with an oxygen electrode to monitor system
conditions. The second flow channel is directed toward the back of
the collection cell, where a gas-tight septum seals the exit. A
Hamilton gas-tight syringe (Hamilton Co., Reno, Nev.) pierces this
septum and collects the sample as a syringe pump slowly withdraws
fluid at a rate lower than the flow rate in the capillary. This
entire apparatus is enclosed within an acrylic container which
maintains the temperature 37.degree. C. through the use of a fin
heater.
[0135] 2. Artificial Capillary Experimental Protocol
[0136] The equilibrated samples are aspirated from the tonometer
into a Hamilton gas-tight syringe which is mounted onto the
infusion pump. Constant flow is established throughout the system
to achieve the desired residence time. The test solutions are
equilibrated with 20% O.sub.2, balance N.sub.2, to simulate air.
The chamber outside of the capillary is filled with 100% N.sub.2.
The inlet gas is routed through a 37.degree. C. water bath and a
flow meter to maintain constant flow rate, so that the volume of
gas in the chamber is exchanged every 10 seconds. Oxygen electrodes
monitor the extracapillary gas compartment.
[0137] The effluent from the capillary is collected in a second
Hamilton gas-tight syringe and is injected into the blood gas
analyzer (e.g., ABL-5, Radiometer, West lake, OH). A minimum of
three samples are taken at each residence time. Flow conditions are
changed, and a new set of samples is tested. Three flow rates, 10,
20 and 40 .mu.l/min, give residence times in the capillary of 1.56,
0.75 and 0.39 seconds, respectively.
[0138] 3. Mathematical Analysis Of Artificial Capillary Data
[0139] For each segment (dx, FIG. 13) of distance along the
capillary, the total O.sub.2 present in the solution is:
O.sub.2.sub..sub.r=.alpha.PO.sub.2+Y[Hb].sub.T
[0140] where .alpha. is the solubility coefficient of O.sub.2 in
plasma (1.2074 .mu.M/Torr) (Winslow et al, J. Biol. Chem.,
252(7):2331-2337 [1977]), Y is hemoglobin saturation, and
[Hb].sub.T is total hemoglobin concentration. The amount of O.sub.2
transferred out of the capillary in the segment dx is 5 O 2 = K * (
P O 2 ) ( r 2 ) d x R ( 6 )
[0141] where K* is a lumped diffusion parameter, consisting of the
diffusion constants given in equation 1 and the length of the
diffusion gradient for 2. .DELTA.PO.sub.2 is the PO.sub.2 gradient
(essentially the interior PO.sub.2 when N.sub.2 is the outside
gas), r is the radius of the capillary, and R is the flow rate.
Total O.sub.2 is now decremented by .DELTA.O.sub.2. At this point,
the Adair equation, using the known parameters for the hemoglobin
in question, is used to empirically find the PO.sub.2 and Y
combination that provide the new O.sub.2T according to equation
(5). The process is repeated until the end of the capillary is
reached, and the final PO.sub.2 is matched with the value actually
measured in the experiment. A FORTRAN program was used to perform
this analysis in finite elements of dx. Experiments were conducted
using these methods and devices, as described in the Experimental
section below (See Example 16).
[0142] C. Possible Modifications of Hemoglobin
[0143] No product currently under development can replace all the
functions of blood. Instead, these blood product solutions are
distinguished from other plasma expanders by their ability to
increase the total oxygen that can be delivered. Of these, there
are two general types: those that increase dissolved oxygen (i.e.,
perfluorocarbons) and those that carry oxygen chemically bound to
hemoglobin (hemoglobin-based O.sub.2 carriers). There are
significant differences between the two types and they transport
O.sub.2 in fundamentally different ways.
[0144] Hemoglobin is a protein made up of 4 polypeptide subunits, 2
.alpha. and 2 .beta. chains. One of each, tightly bound together,
make up a half molecule (.alpha..beta. dimer) and two dimers are
more loosely bound to form the fully functional molecule
(.alpha..sub.2.beta..sub.2 tetramer). The interface between the
.alpha..beta. dimers slides apart as O.sub.2 is reversibly bound,
forming two structures, one each corresponding to the fully
deoxygenated (T, tense) and one to the fully oxygenated (R,
relaxed) structure. These two conformers have vastly different
affinities for O.sub.2 so that as O.sub.2 molecules are
sequentially bound and the transition from deoxy to oxy occurs, the
affinity for O.sub.2 increases. This change in affinity is called
"cooperativity" and is represented by the Hill coefficient, n (FIG.
13).
[0145] The loose interface between .alpha..beta. dimers is of
critical importance for hemoglobin-based blood substitutes. The
equilibrium constant for this dissociation reaction is 10.sup.-6 M
for HbO.sub.2 which means that as hemoglobin concentration falls,
the relative proportion of dimeric molecules increases. These
dimers are very quickly and efficiently filtered in the glomerulus
of the kidney. Mechanisms to remove dimers which are present when
mild hemolysis occurs include haptoglobin binding which can remove
free hemoglobin in concentrations up to 200 mg/dl. When this
threshold is exceeded renal clearance of hemoglobin is very high,
and renal toxicity may result.
[0146] Many chemical modifications of hemoglobin have been devised
(See, Table 3). The purposes of these modifications are to prevent
tetramer-dimer dissociation, modulate oxygen affinity, and prolong
vascular retention. They take advantage of several reactive sites
on the surface of hemoglobin, in its internal cavity and at the
amino terminus. One of the most useful modifications for
researchers (.alpha..alpha.-hemoglobin, DCLHb.TM., HemAssist.TM.)
incorporates a single cross-link between a deoxyhemoglobin Lysine
99 residues with the reagent DBBF (Walder et al., J. Mol. Biol.,
141: 195-216 [1980]. This single modification at once binds
.alpha..beta. dimers together and reduces the O.sub.2 affinity of
cell-free molecules to approximately that of intact human red blood
cells. When crosslinking is carried out with oxygenated hemoglobin,
the dimensions of the internal cavity change enough so that the
reaction occurs between .beta.82 Lysines. In this case, the final
crosslinked product has a much higher O.sub.2 affinity than that of
the deoxy cross-linked product. This material can also be produced
easily, but has been less well studied because its O.sub.2 affinity
has been traditionally thought to be too high to be physiologically
or clinically useful.
3TABLE 3 Examples Of Hemoglobin Modifications Useful In Preparation
Of Blood Substitutes Reagent/Modification Name Reference Amino acid
modification: N-carboxymethylation DiDonato (1983) J. Biol. Chem.,
258: 4 amino termini 11890-11895 monoisothiocyanate 2-, 3-, 4-ICBS
Currell (1994) Meth. Enzymol., 231: 281 4 amino termini pyridoxal
phosphate PLP Benesch (1982) J. Biol. Chem., 257: Val-1(.beta.)
1320-1324 Cross-linked tetramers: mono-(3,5-dibromosalicyl) FMDA
Bucci (1989) J. Biol. Chem., 264: 6191-6195 fumarate
mono-(3,5-dibromosalicyl) Rayzynska (1996) Arch. Biochem. muconate
Biophys., 325: 119-125 bis(2,3-dibromo-salycyl) Bucci (1986)
Biochim. Biophys. Acta fumarate 874: 76-81 bis(3,5-dibromosalicyl)
DBBF Walder (1979) Biochem., 18: 4265-4270; fumarate
.beta..beta.-Hb .alpha..alpha.- and Chaterjee (1986) J. Biol.
Chem., Lys-82(.beta..sub.1)-Lys-82(.- beta..sub.2) Hb, 261:
9929-9937 Lys-99(.alpha..sub.1)-Lys-99(.alpha- ..sub.2) DCLHb
(HemAssist) bis-(3,5-dibromosalicyl) Bucci (1996) J. Lab. Clin.
Med., 128: sebacate 146-153 2-nor-2-formylpyridoxal 5'- NFPLP
Benesch (1981) Meth. Enzymol., phosphate 76: 147-158
Lys-82(.beta..sub.1)-Val-1(.beta..sub.2) bis(pyridoxal) diphosphate
(bisPL)P2 Benesch (1988) BBRC 156: 9-14
Lys-82(.beta..sub.1)-Val-1(.beta..sub.2) bis(pyridoxal)
tetraphosphate (bisPL)P4 Benesch (1994) Meth. Enzymol.,
Lys-82(.beta..sub.1)-Val-1(.beta..sub.2) 231: 267 Diisothiocyanato
benzene DIBS Manning (1991) PNAS 88: 3329 sulfonate
(.alpha.-DIBS-.alpha.).beta.2 Val-1(.alpha..sub.1)-Val-1-
(.alpha..sub.2) diisothiocyanate Kavanaugh (1988) Biochem., 27: 804
Trimesoyl tris(methyl Tm-Hb Kluger (1992) Biochem., 31: 7551-7559
phosphate) .beta.82-Hb Val-1(.beta..sub.1)-Lys-82-
(.beta..sub.1)-Lys- 82(.beta..sub.2) Lys-82(.beta..sub.1)-Ly-
s-82(.beta..sub.2) Recombinant dialpha fusion rHb 0.1 Looker (1992)
Nature 356: 258-260 wild type (Optro) .beta.N108K (Presbyterian)
Polymers: glycolaldehyde & Fantl (1987) Biochem., 26: 5755-5761
carboxymethylation glycolaldehyde & PLP MacDonald (1991) Eur
Pat 9,104,011.3 glycolaldehyde & NFPLP MacDonald (1991) BACIB.
19: A424 glycolaldehyde & DBBF MacDonald (1994) Meth. Enzymol.,
231: 287-308 glutaraldehyde (Hemopure), Lysines, N-term Valines
(Oxypure) glutaraldehyde & PLP PolyHeme DeVenuto (1982) Surg.
Gyn. Obst., Lysines, N-term Valines SFH 155: 342-346 glutaraldehyde
& NFPLP polyHbNFPLP Berbers (1991) J. Lab. Clin. Med., Lysines,
N-term Valines 117: 157-65 glutaraldehyde & DBBF Nelson (1992)
BACIB 20: 253-258 Oxidative ring-opened (Hemolink) Hsia (1989) US
Pat 4,857,636 raffinose Lysines, N-term Valines Surface Conjugates:
cellulose Flemming (1973) Acta Biol. Med. Ger., 30: 177-182 dextran
dialdehyde Tam (1976) Proc. Natl. Acad. Sci., 73: 2118-2121
dextran-alkylation Dx-Hb Chang (1977) Can. J. Biochem.,
Cys-93(.beta.) 55: 398-403 dextran sulfate SF-Dx Barberousse (1986)
J. Chromatogr., 369: 244-247 dextran phosphate P-Dx Sacco (1990)
Biochim. Biophys. Acta 1041: 279-284 dextran benzene Dx-BHC
Prouchayret (1992) BACIB 20: 319-322 hexacarboxylate hydroxyethyl
starch Cerny (1984) Appl. Biochem. Biotech., 10: 151-153 inulin
Iwasaki (1983) BBRC 113: 513-518 polyvinylpyrrolidone Schmidt
(1979) Klin. Wochenschr. 57: 1169-1175 polyethylene glycol Ajisaka
(1980) BBRC 97: 1076-1081 methoxy-polyoxyethylene (PEG-Hb) Zalipsky
(1991) Polymeric Drugs, pp 91-100 (mPEG) 10-12 Lysines
.alpha.-carboxymethyl, .omega.- (PHP) Iwashita (1995) Artificial
Red Cells, pp carboxymethoxypolyoxyethylene 151-176 (dicarboxyPEG)
8-10 Lysine & PLP
[0147] Another unique class of crosslinkers, trimesic acid
derivatives, result in 2- or 3-point reactions (Kluger et al.,
Biochem., 31:7551-7559 [1992]). In early reports, the resulting
modified hemoglobins produced with these appeared to be stable and
the reaction seemed to have a high degree of specificity.
[0148] A variation on this 64,000 kD molecular weight hemoglobin is
the genetically produced "rHb1.1" (Looker et al., Nature
356:258-260 [1992]) in which crosslinking is done genetically. In
this case, 2 .alpha. chain genes are introduced into the E. coli
genome such that when they are transcribed, a single gene product
results in which one .alpha. chain is contiguous with the other
(dialpha peptide). Thus, the product has a molecular weight of
64,000 kD and does not dissociate in to dimers. Its physiological
properties are similar to .alpha..alpha.-hemoglobin.
[0149] Other crosslinking agents are analogs of 2,3-DPG. NFPLP, a
prototype of such a crosslinker, binds in the 2,3-DPG "pocket"
between .beta. chains and has the dual effects of preventing
dimerization and reducing O.sub.2 affinity. This product has been
extensively studied (Bleeker et al., Biomater. Artific. Cells
Immobil. Biotechnol., 20:747-750 [1992]) but unfortunately the
crosslinker itself is difficult to synthesize, and scaleup has not
been achieved practically.
[0150] Conjugated hemoglobins are those to which some modifying
molecule has been attached to the surface (See e.g., Nho et al.,
Biomat. Artif. Cells Immobil. Biotechnol., 20:511-524 [1992]).
Modifying groups include polyethylene glycol (PEG), polyoxyethylene
(POE), or dextran. These products have increased molecular weights,
depending on the number and size of the modifying groups, but are
relatively easy to produce. Increasing the molecular size may also
increase the hydration shell around the protein molecule, in the
case of POE and PEG, and may thereby restrict the reaction of
hemoglobin with other molecules in the cell-free environment.
[0151] Finally, nonspecific reagents can react with any of the 44
the .epsilon.-amino lysine groups on the surface of hemoglobin or
the 4 amino-terminal groups. Such bifunctional reactants include
glutaraldehyde and o-raffinose and have been used in at least three
of the products presently in clinical trials. While the
modification reactions are clearly understood chemically, the
extent of reaction can sometimes be difficult to control, and a
range of molecular weights of product may result (Marini et al.,
Biopolymers 29:871-882 [1990]).
[0152] The present invention provides methods to improve the
current hemoglobin-based red cell substitutes which have serious
problems. In general, molecular size can be increased by
polymerization of hemoglobin with polyfunctional cross-linkers or
by surface conjugation to polymers such as PEG, dextran, or other
starches, carbohydrates, or proteins. Viscosity can be increased by
conjugation to PEG or its analogues. The viscosity of the solution
can be increased by formulation with a high viscosity material such
as pentastarch, dextrans, carbohydrates or proteins which are,
themselves, viscous. Finally, oxygen affinity can be increased by
intramolecular crosslinking of hemoglobin in the R conformational
state. This can be achieved by placing the hemoglobin in an
environment such as O.sub.2, CO or other ligand which favors the R
conformation. Examples of specific changes to the production of
modified hemoglobins to be used as cell-free oxygen carriers
include the following.
[0153] 1. .alpha..alpha.-Hemoglobin
[0154] This hemoglobin, initially designed as a model compound for
study by the U.S. Army, has been produced by Baxter Healthcare and
is being tested as a replacement for human blood in the immediate
postoperative period and in selected trauma patients. Both the Army
and Baxter have reported that this product produces significant
elevations of blood pressure and vascular resistance, and
preclinical animal studies have shown that these undesirable
properties eliminate any advantage to be derived from
administration of hemoglobin solution (See e.g., Hess et al., J.
Appl. Physiol., 74:1769-1778 [1993]).
[0155] As presently formulated, .alpha..alpha.-Hb has low viscosity
(approximately 1 cP, shear rate of 160 s.sup.-1, 37.degree. C.),
high [Hb] at approximately 10 g/dl, a molecular size that is the
same as that of tetrameric hemoglobin, and it has oxygen affinity
similar to or lower than that of blood. The present invention
provides .alpha..alpha.-hemoglobin for which the viscosity has been
increased by formulation in pentastarch or any high viscosity
colloid. Indeed, the viscosity and molecular size can be increased
by surface conjugation with PEG or any other suitable methods of
surface decoration. In addition, the composition can be formulated
with a lower [Hb]. In addition, its oxygen affinity can be
increased by carrying out crosslinking chemistry using DBBF with
the starting hemoglobin material in a (e.g., CO or O.sub.2
liganded) high-affinity conformational state.
[0156] Thus, the present invention provides methods to improve this
composition by reducing its P50, for example by crosslinking the
hemoglobin in a liganded (e.g., CO or O.sub.2) state, and by
increasing its molecular size by surface decoration with PEG or
other materials that increase its molecular radius and viscosity.
Diffusion of O.sub.2 in a solution of .alpha..alpha.-hemoglobin
could be reduced by formulation in pentastarch.
[0157] 2. rHb1.1
[0158] Recombinant hemoglobin (e.g., Optro.TM., Somatogen) may be
produced using various hosts (e.g., bacteria). Currently available
recombinant hemoglobin consists, primarily, of fused .alpha. chains
and the introduction of a mutation (Presbyterian) which reduces its
oxygen affinity. The present invention provides methods to improve
this product by reducing its P50, for example, by eliminating the
Presbyterian mutation or by introducing other mutations that
increase its oxygen affinity or reduce cooperativity, and by
increasing its molecular size by surface decoration with PEG or
other materials that increase its molecular radius and viscosity.
Diffusion of O.sub.2 in a solution of Optro.TM. could be reduced by
formulation in pentastarch.
[0159] As presently formulated, rHb1.1 has low viscosity, its
molecular size is that of tetrameric hemoglobin, and it has an
oxygen affinity similar to or lower than that of blood. The present
invention provides methods to increase the viscosity of rHb1.1, by
formulation in pentastarch or any high viscosity colloid. In
addition, its viscosity and molecular size can be increased by
surface conjugation using surface conjugation with PEG or any other
suitable methods of surface decoration. Furthermore, it can be
formulated with a low [Hb].
[0160] 3. PHP (Pyridoxylated Hemoglobin Polyoxyethylene)
[0161] PHP (pyridoxylated hemoglobin polyoxyethylene; e.g., Apex
Bioscience). This hemoglobin is from a human source, reacted with
pyridoxal phosphate (PLP) to increase its P50, and then surface
modified with a form of polyethylene glycol. The present invention
provides methods to improve this product by eliminating the PLP
reaction, crosslinking the liganded (e.g., CO or O.sub.2) state,
and by more extensive surface decoration with PEG, either by
increasing the number of PEG strands per molecule or by increasing
the length of individual PEG strands. Diffusion of O.sub.2 in a
solution of PHP could be reduced by formulation in pentastarch.
[0162] As presently formulated, PHP has an intermediate viscosity
of approximately 2 cP (under conditions described herein), high
[Hb] at approximately 8 g/dl, its molecular size is slightly more
than 2-fold larger than a hemoglobin tetramer, and an oxygen
affinity that is slightly greater than that of blood. The present
invention provides methods to increase the viscosity of this
product by formulation in pentastarch or any high viscosity
colloid. Its viscosity and molecular size can be increased by more
extensive surface decoration with PEG, either by increasing the
number of PEG strands per molecule or by increasing the length of
individual PEG strands. It can also be formulated at lower [Hb],
and/or its oxygen affinity increased, by eliminating PLP during
hemoglobin modification reaction.
[0163] 4. Hemolink.TM.
[0164] Hemolink.TM. (Hemosol, Ltd.) is a human-derived hemoglobin
product with a very high P50, and is polymerized with o-raffinose,
a multifunctional crosslinking reagent. The present invention
provides methods to improve the product by crosslinking the
liganded (e.g., CO or O.sub.2) protein and by increasing its
molecular radius and viscosity. This could be accomplished by
surface decoration with any PEG derivative or conjugation to a
polysaccharide or other polymer that would increase its molecular
size. Diffusion of O.sub.2 in a solution of Hemolink.TM. could be
reduced by formulation in pentastarch.
[0165] As presently formulated, Hemolink has low viscosity (ca. 1.4
cP under conditions of our measurement), high [Hb] at approximately
10 g/dl, its molecular size is about 1.7-fold greater than
tetrameric hemoglobin, and it has low oxygen affinity compared to
blood. The present invention provides methods to increase the
viscosity by formulation in pentastarch or any high viscosity
colloid. Its viscosity and molecular size can be increased by
surface conjugation using surface conjugation with PEG or other
methods of surface decoration. In addition, it can be formulated at
lower [Hb]. Its oxygen affinity can be increased by carrying out
its polymerization chemistry using o-raffinose with the starting
hemoglobin material in a (e.g., CO or O.sub.2 liganded)
high-affinity conformational state.
[0166] 5. HemoPure.TM.
[0167] HemoPure.TM. (Bio-Pure) is a bovine-derived hemoglobin
product with a moderately high P50, and is polymerized with
glutaraldehyde, a bifunctional crosslinking reagent. The present
invention provides methods to improve the product by crosslinking
the liganded (e.g., CO or O.sub.2) protein and by increasing its
molecular radius and viscosity. This could be accomplished by
surface decoration with any PEG derivative or conjugation to a
polysaccharide or other polymer that would increase its molecular
size. Diffusion of O.sub.2 in a solution of HemoPure.TM. could be
reduced by formulation in pentastarch.
[0168] 6. Polyheme.TM.
[0169] Polyheme.TM. (Northfield Laboratories) is a human-derived
hemoglobin product with a moderately high P50 due to reaction with
PLP, and is polymerized with glutaraldehyde, a bifunctional
crosslinking reagent. The present invention provides methods to
improve the product by crosslinking the liganded (e.g., CO or
O.sub.2) protein, eliminating the PLP and by increasing its
molecular radius and viscosity. This could be accomplished by
surface decoration with any PEG derivative or conjugation to a
polysaccharide or other polymer that would increase its molecular
size. Diffusion of O.sub.2 in a solution of Polyheme.TM. could be
reduced by formulation in pentastarch.
[0170] As presently formulated, Polyheme has high [Hb] at
approximately 10 g/dl, its molecular size is larger than that of
tetrameric hemoglobin by being polymerized, and its oxygen affinity
is lowered by reaction with PLP. The present invention provides
methods to increase the viscosity by formulation in pentastarch or
any high viscosity colloid. Its viscosity and molecular size can be
increased by surface conjugation using surface conjugation with PEG
or other methods of surface decoration. It can also be formulated
at lower [Hb]. Its oxygen affinity can be increased by carrying out
its polymerization chemistry using glutaraldehyde in the absence of
PLP and with the starting hemoglobin material in a (e.g., CO or
O.sub.2 liganded) high-affinity conformational state.
[0171] 7. PEG-Hb
[0172] Commercially available PEG-Hb compositions (e.g., Enzon) may
be improved using the present invention by decreasing its
concentration and by formulation in pentastarch. Enzon's hemoglobin
consists of a bovine hemoglobin modified by conjugation to linear
5000 MW polyethylene glycol (PEG) chains. Polyalkylene oxide (PAO)
is a generic term for a group of molecules that includes PEG.
Attachment of PAO to hemoglobin is achieved by formation of a
covalent bond between the PAO and the .epsilon.-amino groups of
lysine residues. Enzon's hemoglobin is conjugated to 10-12 PAO
chains per hemoglobin tetramer. When measured at a shear rate of
160 s.sup.-1, 37.degree. C., a 5 g/dl solution of Enzon's PEG-Hb
exhibits a viscosity of 3.39 cP.
[0173] As presently formulated, PEG-Hb has a viscosity of
approximately 3.5 cp (under conditions of described herein) at a
[Hb] of 5.5 g/dl, its molecular size is 4-fold greater than that of
tetrameric hemoglobin, and it has high oxygen affinity relative to
blood. The present invention provides methods to increase the
viscosity of this product at lower [Hb] by formulation in
pentastarch or any high viscosity colloid. Furthermore, its
viscosity and molecular size can be increased by more extensive
surface decoration with PEG, either by increasing the number of PEG
strands per molecule or by increasing the length of individual PEG
strands. It can also be formulated at lower [Hb].
[0174] Additional methods to increase the viscosity (.eta., cP)
unit per unit concentration ([Hb], g/dl) of a hemoglobin solution
include, but are not limited to the following:
[0175] 1. Increase the Number of Sites Conjugated to 5000 MW PAO
Per Hemoglobin Tetramer
[0176] Human hemoglobin contains 44 lysine residues (11 on each
chain). In combination with the 4 N-terminal amino groups, this
gives 48 theoretically possible sites for covalent attachment of
PAO using the chemistry described for modification of amino groups.
Additional chemistry has been described (See e.g., Acharya's U.S.
Pat. No. 5,585,484; herein incorporated by reference) that allows
covalent attachment of PAO to the sulfhydryl group of a cysteine
residue. There are 6 cysteine residues per Hb tetramer (i.e., one
on each .alpha. chain, and two on each .beta. chain) increasing the
number of theoretically possible attachments to 54. Further PAO
modifications are contemplated, including the use of suitable
conjugation chemistry lead to attachment to serine, threonine,
tyrosine, asparagine, glutamine, arginine, and histidine residues.
It is also contemplated that chemistry that allows conjugation to
carboxylic acid groups may allow PAO conjugation to aspartic acid
and glutamic acid residues as well as the C-terminal carboxy groups
of hemoglobin.
[0177] If the number of conjugation sites per tetramer is
sufficiently large, it is contemplated that PAO molecules of lower
molecular mass (i.e., MW<5000) will still achieve an increased
viscosity per unit concentration over Enzon's product without the
modifications described herein.
[0178] 2. Maintain the Number of Covalent PAO Attachments and
Increase the Size of Each PAO Moiety
[0179] An increased viscosity per unit hemoglobin concentration is
contemplated in situations in which the PAO groups attached to the
10-12 sites per tetramer are of greater molecular mass (i.e.,
MW>5000). This can be achieved by using PAO starting material
consisting of molecules containing longer and/or branched PAO
chains.
[0180] If the molecular size of the PAO units is sufficiently
large, it may be possible to modify a fewer number of sites on the
tetramer (i.e., <10) and still achieve an increased viscosity
per unit concentration over Enzon's product.
[0181] IV. The Oxygen-Carrying Component of the Blood Products of
the Present Invention
[0182] In preferred embodiments of the present invention, the
oxygen-carrying component is native or modified hemoglobin (e.g., a
HBOC). Modified hemoglobin is altered by chemical reaction (e.g.,
cross-linking or polymerization) or through the addition of adducts
(e.g., polyethyleneglycol, polyoxyethylene). Furthermore, the
oxygen-carrying component of the present invention may be
recombinantly-produced hemoglobin or a hemoglobin product
encapsulated in a liposome. The present invention also contemplates
the use of other means for oxygen delivery that do not entail
hemoglobin or modified hemoglobin.
[0183] Though the present invention contemplates the use of any
oxygen-carrying component, preferred oxygen-carrying components
entail solutions of human or animal (e.g., bovine) hemoglobin,
intramolecularly crosslinked to prevent dissociation into dimeric
form. Optionally, the preferred oxygen-carrying components of the
present invention may be oligomerized to oligomers of molecular
weight up to about 750,000 daltons, preferably up to about 500,000
daltons. Hemoglobin preparations prepared by genetic engineering
and recombinant processes are also among the preferred
oxygen-carrying components.
[0184] The preferred oxygen-carrying components of the present
invention should be stroma free and endotoxin free. Representative
examples of preferred oxygen-carrying components are disclosed in a
number of issued United States Patents, including U.S. Pat. No.
4,857,636 to Hsia; U.S. Pat. No. 4,600,531 to Walder, U.S. Pat. No.
4,061,736 to Morris et al.; U.S. Pat. No. 3,925,344 to Mazur; U.S.
Pat. No. 4,529,719 to Tye; U.S. Pat. No. 4,473,496 to Scannon; U.S.
Pat. No. 4,584,130 to Bocci et al.; U.S. Pat. No. 5,250,665 to
Kluger et al.; U.S. Pat. No. 5,028,588 to Hoffman et al.; and U.S.
Pat. No. 4,826,811 and U.S. Pat. No. 5,194,590 to Sehgal et al.;
the contents of each are hereby incorporated by reference. In a
more preferred embodiment, the oxygen-carrying components comprise
human, recombinant, or animal hemoglobin, either cross-linked or
not, modified by reaction with polyethyleneglycol (PEG) or
polyoxyethylene (POE).
[0185] The capacity of a solution to deliver oxygen to tissues can
be determined in a number of ways routinely used by researchers,
including direct measurement of oxygen tension in tissues,
increased mixed venous oxygen tension, and reduced oxygen
extraction ratio.
[0186] V. The Non-Oxygen-Carrying Component of the Blood Products
of the Present Invention
[0187] As noted above, the present invention contemplates a mixture
comprising an oxygen-carrying component and a non-oxygen-carrying
component. The non-oxygen-carrying component of the present
invention is any substance used for temporary replacement of RBCs
which has oncotic pressure (e.g., dextran-70, dextran-90, hespan,
pentastarch, hetastarch, albumin, or any other colloidal
intravenous solution).
[0188] Non-oxygen-carrying plasma expander products for the
treatment of hypovolemia and other conditions are commercially
available; representative products include, but are not limited to,
Pentaspan.RTM. (DuPont Merck, Fresenius), Hespan.RTM. (6%
hetastarch in 0.9% sodium chloride for injection; DuPont Merck),
and Macrodex.RTM. (6% Dextran 70 in 5% dextrose in water for
injection, or 6% Dextran 70 in 0.9% sodium chloride for injection;
Pharmacia). Non-oxygen-carrying fluids available for clinical use
(e.g., hemodilution or resuscitation) can be broadly classified as
crystalloid solutions (i.e., salt solutions) and colloid solutions.
In preferred embodiments of the present invention, colloid
solutions comprise the non-oxygen-carrying component of the
mixture.
[0189] In one embodiment of the present invention, the problems of
the prior art products are alleviated by the formulation and use of
a composition (an aqueous solution) that contains both an
oxygen-carrying component (e.g., a HBOC) and a non-oxygen-carrying
component comprising an inert, non-proteinaceous colloid. Such
compositions result in two effects, either alone or in combination.
First, the oxygen carrying capacity of the composition is
decreased, while colloid osmotic (oncotic) pressure and plasma
retention are maintained. The resulting colloid-diluted
oxygen-carrying component has fewer oxygen-delivering colloidal
particles per unit volume than the oxygen-carrying component alone,
and hence there is less oxygen presented to the arterial walls.
That is, the oxygen delivery more closely approximates that of
whole blood, so that the combination according to the invention is
able to deliver and distribute its oxygen loading in a manner more
closely resembling that achieved by RBCs.
[0190] Second, by proper choice of type and amount of
non-proteinaceous colloid (discussed below), the viscosity of an
oxygen-carrying component-colloid composition can be increased,
preferably close to that of whole blood. This also appears to
reduce or counteract arterial wall reaction. Though an
understanding of the mechanism of this effect is not required in
order to practice the present invention, it is believed to be due
to i) reduced oxygen delivery as a result of decreased hemoglobin
and ii) increased shear stress at the vessel wall (which results in
the increased release of endogenous vasodilators such as
prostacyclin).
[0191] Suitable examples of non-proteinaceous colloids for use in
the compositions of the present invention include dextran and
pharmaceutically-acceptable derivatives thereof, starch and
pharmaceutically acceptable derivatives thereof, and
polyvinylpyrrolidone (PVP). Particularly preferred among suitable
non-proteinaceous colloids is pentastarch. Indeed, suitable
non-proteinaceous colloids include substantially all
non-proteinaceous colloidal substances which have previously been
successfully used as hemodiluents. Acceptable candidates should be
water soluble, exhibit oncotic pressure, and be biologically inert
and otherwise pharmaceutically acceptable. The cost of these
materials (e.g., oncotic non-proteinaceous colloids like dextran
and hetastarch), on a weight for weight basis, is much lower than
that of hemoglobin and HBOCs.
[0192] III. Clinical and Other Applications of the Present
Invention
[0193] The present invention finds use in many settings, ranging
from the emergency room to the operating table, as well as military
conflicts, and veterinary applications. This versatility is due to
the optimized formulations of the present invention, which may be
stored as desired, and avoid the necessity for cross-matching or
other laboratory tests to determine compatibility with the patient
to be treated. Extensive research on chemical and genetic
modifications of hemoglobin, in conjunction with the present
invention now permit the design of molecules with nearly any
desired combination of physical and physiological properties in a
heretofore unexpected and highly efficient manner.
[0194] A. Clinical Applications
[0195] Various clinical applications are matched with properties of
the proposed red cell substitutes in Table 4, below. In this Table,
T.sub.1/2 refers to the half-life.
4TABLE 4 Potential Clinical Applications For Red Cell Substitutes
And Optimal Properties Appilcation COP P50 Viscosity T.sub.1/2
Hemodilution .Arrow-up bold. .dwnarw. .Arrow-up bold. .Arrow-up
bold. Trauma .Arrow-up bold. .dwnarw. .Arrow-up bold. .Arrow-up
bold. Septic Shock .Arrow-up bold. .dwnarw. .Arrow-up bold.
.Arrow-up bold. Ischemia (e.g., stroke) .Arrow-up bold. .dwnarw. ?
.dwnarw. Cancer -- .dwnarw. .dwnarw. .Arrow-up bold. Chronic Anemia
-- .dwnarw. .Arrow-up bold. .Arrow-up bold. Sickle Cell Anemia
.Arrow-up bold. .Arrow-up bold. .dwnarw. .Arrow-up bold.
Cardioplegia .Arrow-up bold. .Arrow-up bold. .dwnarw. -- Hypoxia --
.dwnarw. .Arrow-up bold. .Arrow-up bold. Organ Perfusion --
.Arrow-up bold. ? -- Cell Culture -- -- -- -- Hematopoiesis
.dwnarw. .Arrow-up bold. .dwnarw. .dwnarw.
[0196] It is contemplated that high oncotic activity (COP) will
find use in the short term, immediate, resuscitation from
hypovolemic shock. The utility of hypertonic saline/dextran (HSD)
has been shown in animal studies (Kramer et al., Surgery,
100(2):239-47 [1986]). Oncotic activity (COP) expands the vascular
volume very quickly and it is contemplated that perhaps this,
combined with the rapid restoration of O.sub.2 capacity, might lead
to significantly better salvage of patients and tissues after acute
blood loss. However, there numerous settings in which the
compositions and methods of the present invention find use
including the following:
[0197] Hemodilution. In this clinical application, the patient
comes to surgery and some volume of blood is removed, to be
replaced with the substitute. The goal is preventative, not to
correct some imbalance. A solution that performs very close to
blood is needed. A slightly increased COP is desired because it
increases blood volume and cardiac output, in anticipation of
surgical blood loss. Since the replacement fluid is
hemoglobin-based, a reduced P50 is preferred, in order to overcome
facilitated diffusion. Viscosity should be increased for the same
reason, and the T.sub.1/2 should be prolonged to eliminate or
reduce the need for postoperative transfusion with allogeneic blood
units, should the ones collected prior to surgery (autologous) not
be sufficient. The solution for hemodilution would have the same
properties as one used in cardiopulmonary bypass.
[0198] Trauma. In trauma, the patient has lost whole blood. In
response to this blood loss, fluid shifts from the interstitial and
intracellular spaces to attempt to replace lost volume. In the
process, hematocrit and viscosity fall and vasoconstriction occurs
to shunt blood from organs that have low priority. These include
the skin and gut, for example, while blood flow to the kidneys,
heart and brain are preserved for as long as possible. The goal of
a therapeutic blood replacement here would be to first replace lost
volume as fast as possible. Hence, increased COP are desired. Since
the replacement fluid is hemoglobin-based, a reduced P50 is
preferred, in order to overcome facilitated diffusion. The
viscosity should be increased for the same reason.
[0199] Septic Shock. In overwhelming sepsis, some patients may
become hypertensive in spite of massive fluid therapy and treatment
with vasoconstrictor drugs. The mechanism of lowered blood pressure
in this instance is overproduction of nitric oxide (NO). Therefore
hemoglobin is close to an ideal agent to treat these patients with
because of the avidity with which hemoglobin binds NO. In general,
NO binding affinity parallels O.sub.2 binding affinity, so an agent
for use in this application should have very high O.sub.2 affinity
(low P50). Since the patients are often fluid overloaded, increased
COP would be desired, but not essential, and increased viscosity
would reduce autoregulatory vasoconstriction. The T.sub.1/2 should
be moderately long, but it is not necessary to be markedly
prolonged, since continuous infusions can be used in these
patients.
[0200] Ischemia (e.g., stroke). Ischemia refers to the condition
where tissue is "starved" for oxygen. This usually results from
limitation of blood flow as in, for example, a heart attack or
cerebrovascular accident. The tissue, starved of O.sub.2 dies in
small patches, called "infarcts." The goal of blood replacement
therapy here would be to increase blood flow and to promote O.sub.2
delivery into capillary beds. Hence, a solution of lower viscosity
may be preferred, in order to better perfuse capillary beds. This
can be done only if the blood volume is maintained or expanded, and
therefore an increased COP would be desirable. In most situations
of heart attack and stroke, the tissue damage is acute, so therapy
is only necessary for a few hours. Thus, the T.sub.1/2 is less
important than in other applications.
[0201] Cancer. To increase the radiosensitivity (or sensitivity to
chemotherapy), the goal is to deliver as much O.sub.2 to the
hypoxic core of the tumor as possible. The microcirculation of
tumors is unlike that of other tissues, because it lacks
endothelial lining of capillaries, and normal vasoactivity does not
occur. Thus, it should be possible to provide solutions of low
viscosity. The P50 should be very low so that little, if any,
O.sub.2 is unloaded in tissues before it reaches the hypoxic core
of the tumor. In other words, we would like O.sub.2 to be unloaded
at very low PO.sub.2, if possible Plasma T.sub.1/2 can be as long
as possible, so that repeated doses of irradiation or chemotherapy
can be administered.
[0202] Chronic anemia. These patients are unable to regenerate lost
red cells or they are not able to keep production up with normal
(or accelerated) destruction. In this situation, it is desired that
the transfusion substitute to behave as much as possible like
native red cells. Thus, facilitated diffusion should be overcome by
increasing oxygen affinity and viscosity. In this application, more
than any other, the T.sub.1/2 is very important because patients
will be unable to replace lost or metabolized hemoglobin on their
own.
[0203] Sickle cell anemia. This is a unique clinical condition in
that red cell turnover is very high, and the sickling process in
the affected person's red cells is a function of PO.sub.2. That is,
the lower the PO.sub.2, the greater the sickling rate. Sickling is
also a function of red cell density and viscosity, which, in turn,
is strongly dependent on hematocrit. The ideal solution in a sickle
cell crisis would be one that delivers O.sub.2 to sickled red
cells. Thus, it may be preferable to use a high, rather than low,
P50 so there is a net transfer of O.sub.2 in favor of the sickled
red cells. In order to do this, it would be necessary to decease
diffusion in any way possible, to reduce vasoactivity which could
offset any potential benefit of oxygenating the red cells. At the
same time, it is preferred that the solution to have good flow
properties. Thus, a balance between P50 and viscosity would have to
be struck such that red cells are oxygenated while vasoconstriction
is blocked or, at least, not induced.
[0204] Cardioplegia. In certain cardiac surgical procedures, the
heart is stopped by appropriate electrolyte solutions and reducing
the temperature of the patient. Reduction of the temperature will
reduce P50 drastically, possibly to the point where O.sub.2 may not
be unloaded under any ordinary physiological conditions. Thus, the
P50 of a solution for this purpose might be higher than for other
applications. The viscosity is also temperature-dependent and
appropriate adjustments would be made such that the in vivo
viscosity is close to that of blood under the specific conditions
of the patient.
[0205] Hypoxia. In altitude dwellers and world-class athletes and
soldiers under extreme conditions, extraction of O.sub.2 from air
in the lung may become limiting to overall O.sub.2 transport. This
aspect of O.sub.2 transport would probably be more important than
the ability of the solution to unload O.sub.2 in tissues. In this
case, lower P50 would be advantageous, and cooperativity should be
maximal. Vasoactivity would not be desired, so viscosity would be
elevated. The COP of such solutions would not need to be elevated,
and the plasma T.sub.1/2 should be as long as possible.
[0206] Organ Perfusion. Here, the main goal is to increase O.sub.2
content of the perfusate. The parameters of O.sub.2 loading and
unloading are less important than in other conditions, since the
fluid is not flowing. Therefore, nearly complete extraction is
possible. P50 can be relatively normal or even elevated, since the
solutions can be oxygenated with external oxygenators.
[0207] Cell Culture. This requirement is almost identical to that
of organ perfusion, except that the rate of O.sub.2 consumption may
be higher, depending on the cells and their concentration.
[0208] Hematopoiesis. Here, the hemoglobin is serving as a source
of heme and iron, to be resynthesized into new hemoglobin. Thus,
the hemoglobin should be taken up into the monocyte-macrophage
system and broken down in such a way as to make its components
available for red cell metabolism and maturation. The properties of
COP, P50 and viscosity can be the same as the hemodilution
solution. The T.sub.1/2 can be relatively short, as long as
metabolism is efficient.
[0209] Many workers in the field of oxygen transport have assumed
that oxygen affinity of modified hemoglobin should be low, or at
least not significantly different from that of red cells, in order
to maximize tissue oxygenation. During the development of the
present invention, it was found that this concept is invalid. In
severe hypoxia, pulmonary O.sub.2 diffusion may become limiting to
O.sub.2 uptake in the alveolus, as demonstrated in mountaineers at
extreme altitude (Winslow et al., [1984]). In this instance,
increased, rather than decreased O.sub.2 affinity is beneficial
because it increases arterial O.sub.2 saturation. Based on the high
altitude data, this point is reached at approximately 6,000 meters
altitude, or at a PaO.sub.2 of about 40 Torr. By extrapolation, one
might conclude that sea level patients with severe restrictions in
diffusive pulmonary O.sub.2 uptake might also benefit from
increased hemoglobin O.sub.2 affinity. If the pulmonary capillary
PO.sub.2 reaches a maximal value of 40 Torr (or less), then
shifting the oxygen equilibrium curve to the left will increase
saturation, in effect providing the same increase in O.sub.2
content as a transfusion, without adding the burden of increased
red cell mass and, hence, viscosity.
[0210] In general, plasma retention times should be as long as
possible. However, it is also contemplated that perhaps for O.sub.2
delivery to specific tissues (e.g., tumors, myocardium, ulcers,
sickle cell disease) this property might not be so important.
Furthermore, if the reason to give a hemoglobin solution is to
stimulate erythropoiesis, it is contemplated that a short retention
time is desired.
[0211] The present invention provides data that show if the
properties of viscosity, oncotic pressure, oxygen affinity and
hemoglobin concentration are optimized as described, the hemoglobin
can be formulated with additional components to serve additional
functions of blood. For example, coagulation factors (e.g., Factors
VIII, IX, and/or II), immunoglobulins, antioxidants, iron
chelators, peroxidases, catalase, superoxide dismutase, carbonic
anhydrase, and other enzymes may be mixed with the hemoglobin
solution in order to provide benefit to patients in need of such
compositions. Similarly, drugs such as cytotoxins, antibiotics or
other agents may be mixed with the solution or chemically
conjugated to other components, such as hemoglobin or other
polymers.
[0212] In addition, the final product can be formulated at any
desired electrolyte and salt composition. It can be stored in the
liquid state, frozen or lyophilized as the final product or the
hemoglobin component itself can reconstituted with any solution
subsequently. Such reconstitution medium could be, but need not be
limited to, saline, Ringer's lactate, albumin solution, or
PlasmaLyte, for example. The final product can be stored in any
biocompatible container such as glass or plastic.
[0213] B. Veterinary Applications
[0214] The present invention is not limited to use in humans. In
addition to the clinical applications briefly described above, the
present invention finds utility in the veterinary arena. The
compositions of the present invention may be used with domestic
animals such as livestock and companion animals (e.g., dogs, cats,
birds, reptiles), as well as animals in aquaria, zoos, oceanaria,
and other facilities that house animals. For example, as with
humans, the compositions of the present invention may be used for
emergency treatment of domestic and wild animals traumatized by
blood loss due to injury, hemolytic anemias, etc. For example, it
is contemplated that embodiments of the present invention in such
as equine infectious anemia, feline infectious anemia, hemolytic
anemias due to chemicals and other physical agents, bacterial
infection, Factor IV fragmentation, hypersplenation and
splenomegaly, hemorrhagic syndrome in poultry, hypoplastic anemia,
aplastic anemia, idiopathic immune hemolytic conditions, iron
deficiency, isoimmune hemolytic anemia, microangiopathic hemolytic,
parasitism, etc.). In particular, the present invention finds use
in areas where blood donors for animals of rare and/or exotic
species are difficult to find.
[0215] VI. Blood Product Compositions
[0216] The relative proportions of the oxygen-carrying component
and the non-oxygen-carrying component (e.g., a colloid plasma
expander) included in the compositions of the present invention can
vary over wide ranges. Of course, the relative proportions are, to
some extent, dependent upon the nature of the particular
components, such as the molecular weight of the colloid used as a
non-oxygen-carrying plasma expander. However, the present invention
is not limited to the use of colloids as the non-oxygen-carrying
component.
[0217] In preferred embodiments of the present invention, the
hemodilution effect of the non-oxygen-carrying component (e.g., a
non-proteinaceous colloid) predominates, i.e., the overall
oxygen-carrying capacity of the oxygen-carrying component is
reduced by dilution so that the adverse effects of excessive oxygen
release at the arterial walls are alleviated. In such embodiments,
substantial economic benefits are derived from a composition that
preferably contains at least 20% by weight of each of the
components, and more preferably at least 25% by weight of each
component. Most preferable compositions comprise from approximately
30 to approximately 70 parts of the oxygen-carrying component
(e.g., HBOC), correspondingly, from approximately 70 to
approximately 30 parts of the non-oxygen carrying component (e.g.,
inert colloid) (per 100 parts by weight of the combination of the
two).
[0218] In preferred embodiments, the viscosity of the blood
substitute compositions of the present invention is preferably
close to that of normal blood. Thus, when it is desirable to
utilize a composition whose primary purpose is to increase
viscosity, high molecular weight colloids in amounts of from
approximately 1 to approximately 20 parts by weight per 100 parts
by weight of oxygen-carrying component are preferred.
[0219] In other preferred embodiments of the present invention,
increased viscosity (i.e., to a value approaching that of whole
blood) of the composition is the predominant effect. In these
compositions, the viscosity of the composition is high enough so
that shear stresses at the arterial walls are sufficient to release
endogenous vasodilators to counteract the effects of the plentiful
oxygen availability at the arterial walls. In such embodiments, the
non-oxygen carrying component (e.g., non-proteinaceous colloid)
should have a substantially higher molecular weight than the oxygen
carrying-component, but should be used in smaller amounts to avoid
excessive viscosities. Polyvinylpyrrolidone (PVP) of molecular
weight 300,000-750,000 used in amounts from about 1 to about 20
parts by weight per 100 parts by weight of oxygen-carrying
component is particularly suitable in these embodiments. Similarly,
high molecular weight starches (e.g., approximately 200,000-750,000
molecular weight) are also preferred in these embodiments. The
amounts are chosen so as to result in an oxygen-carrying
component--colloid solution having a viscosity, relative to whole
blood (assigned a value of 1), of from about 0.5 to about 1.2.
[0220] In certain embodiments of the present invention, advantage
is taken of both of the above-mentioned effects. That is, an amount
and type of the non-oxygen-carrying component (e.g.,
non-proteinaceous inert colloid plasma expander) is chosen which
both reduces the amount of oxygen carried by a unit volume of the
solution, and increases its viscosity to a level approximating that
of normal whole blood. For this purpose, PVP and starches of
molecular weights higher than that of the oxygen-carrying component
are used, and in amounts sufficient to increase the viscosity, to
reduce the amount of oxygen carried, and to reduce the cost of the
solution. Specifically, PVP and starches possessing molecular
weights from about 200,000 to about 600,000 used in amounts from
about 5 to about 50 parts by weight of inert colloid per 100 parts
by weight of the oxygen-carrying component are contemplated for use
with the present invention.
[0221] In some embodiments, the present invention contemplates that
the concentration of the combined oxygen-carrying component and
non-oxygen-carrying component (e.g., inert colloid plasma expander)
in the aqueous solution compositions will generally be in the same
range as that usually employed when one of the ingredients is used
alone for the same purpose (i.e., from about 5 to about 15 grams of
the combination per decaliter of solution).
[0222] The compositions of the present invention provide the
following improvements over current blood substitutes: i) decreased
concentration of hemoglobin to which the patient is exposed,
thereby reducing the toxicity and cost of the blood product; ii)
oncotic pressure, which more effectively expands the vascular
volume than the currently used blood substitutes; iii) optimal
viscosity which maintains capillary blood flow; iv) optimal oxygen
affinity which reduces oversupply of oxygen to arteriolar walls;
and v) optimal oxygen carrying capacity. All of these improvements
increase the effectiveness of the blood products as a cell-free
oxygen carrier.
[0223] Several prior art references discuss the possibility of
mixing hemoglobin solutions with non-oxygen carrying plasma
expanders. For example, U.S. Pat. No. 4,061,736 to Morris et al.
and U.S. Pat. No. 4,001,401 to Bonson et al. describe
pharmaceutical compositions comprising an analog of hemoglobin and
a pharmaceutically acceptable carrier; the carrier may comprise,
for example, polymeric plasma substitutes (e.g., polyethylene
oxide). Similarly, U.S. Pat. No. 5,349,054 to Bonaventura et al.
describes a pharmaceutical composition comprising a hemoglobin
analog which can be mixed with a polymeric plasma substitute (e.g.,
polyvinylpyrrolidone). However, the prior art does not describe the
specific compositions nor the techniques of the present invention
for improving the effectiveness of a blood substitute and reducing
the toxicity of those solutions.
DESCRIPTION OF SOME PREFERRED EMBODIMENTS
[0224] Generally speaking, compositions comprising i) an
oxygen-carrying component (e.g., a HBOC) with high oncotic
pressure, oxygen affinity and viscosity and ii) a
non-oxygen-carrying component with similar oncotic pressure and
viscosity provide an optimal blood product. In the most preferred
embodiments of the present invention, the oxygen-carrying component
of the mixture comprises a polyethylene glycol-modified hemoglobin
and the non-oxygen-carrying component comprises pentastarch.
[0225] As described in more detail in the Experimental section,
there are currently two commercially available hemoglobin products
modified with polyethylene glycol. The first product, Pyridoxal
Hemoglobin Polyoxyethylene (PHP), is a human-derived product from
Apex Bioscience. The second product, PEG-Hb, is a bovine-based
product obtained from Enzon, Inc. Though most of the experimental
work was performed using PEG-Hb, the two PEG-modified hemoglobin
products gave qualitatively the same results. It is to be
understood that the preferred oxygen-carrying components of the
present invention are not limited to PEG-Hb and PHP; indeed, any
hemoglobin products associated with polyethylene glycol are
contemplated for use with the most preferred mixtures of the
present invention.
[0226] Pentastarch, the most preferred non-oxygen-carrying
component of the present invention, is commercially available from
DuPont Merck (Pentaspan.RTM.) as well as from other sources. It
comprises hydroxethyl starch and has a molecular weight of
approximately 250,000 Daltons. Because of its lower molecular
weight and lower degree of hydroxyethyl substitution compared to
other starches (e.g., hetastarch), it exhibits higher oncotic
pressure and faster enzymatic degradation in the circulation. As
described in detail in the Experimental section, dilution of PEG-Hb
with a different non-oxygen-carrying component like hetastarch
reduces the resulting blood product's viscosity and oncotic
pressure, and reduces the oxygen capacity of the resulting mixture.
In contrast, the mixtures resulting from combination of
PEG-modified hemoglobin with pentastarch have viscosity and oncotic
pressure values very close to that of PEG-Hb alone, and have been
shown to lead to enhanced animal survival and physiological
parameters compared to other mixtures (see Experimental
section).
[0227] Preferred mixtures of polyethylene glycol-modified
hemoglobin and pentastarch contain at least 20% by weight of each
of the components, and more preferably at least 25% by weight of
each component. Most preferable compositions comprise from
approximately 30 to approximately 70 parts of the oxygen-carrying
component PEG-modified hemoglobin, and, correspondingly, from
approximately 70 to approximately 30 parts of the non-oxygen
carrying component pentastarch (per 100 parts by weight of the
combination of the two).
[0228] The experimental results presented below indicate that a
mixture of PEG-Hb and pentastarch performed similarly to a solution
of PEG-Hb alone. This was true even though the hemoglobin
concentration to which the animals were exposed and the amount of
hemoglobin product used were less by half with the mixture,
offering the advantage of reducing the concentration of hemoglobin
given to patients, thereby reducing both cost and potential adverse
effects.
[0229] As previously indicated, the compositions and methods of the
present invention can be used in any situation in which banked
blood is currently administered to patients. For example, the
compositions can be administered to patients who have lost blood
during surgery or due to traumatic injury. The compositions of the
present invention are advantageous in that they save the patient
exposure to possible infectious agents, such as human
immunodeficiency virus and hepatitis virus.
EXPERIMENTAL
[0230] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0231] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); mM (millimolar);
.mu.M (micromolar); g (grams); mg (milligrams); .mu.g (micrograms);
kg (kilograms); L (liters); mL (milliliters); dL (deciliters);
.mu.L (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); min. (minutes); s and sec.
(seconds); b.w. (body weight); i.p. (intraperitoneal or
intraperitoneally); Da (Daltons); dP/dt (change in pressure over
time); IU (international units); Hg (mercury); Hz (hertz); MHz
(mega hertz); COP (colloid osmotic pressure); CRBCv (Capillary red
blood cell velocity); FCD (functional capillary density); FDA
(United States Food and Drug Administration); Hb (hemoglobin); MAP
(mean arterial pressure); Pd (palladium); PEG (polyethylene
glycol); PEGHb (bovine hemoglobin modified by conjugation with
polyethylene glycol); sat. (saturation); sem and s.e.m. (standard
error of the mean); TM (trimesic acid); Abbott (Abbott
Laboratories, Chicago, Ill.); Beckman (Beckman Instruments,
Fullerton, Calif.); Bectron (NJ); Dupont (Dupont Pharmaceuticals,
Wilmington, Del.); EG&G Electro Optics (Salem, Mass.); Enzon,
Inc., (Piscataway, N.J.); Fresenius (Walnut Creek, Calif.);
Hemocue, Inc. (Mission Viejo, Calif.); Hemosol Inc. (Etobicoke, ON,
Canada); IPM (IPM, Inc., San Diego, Calif.); Lexington Instruments
(Waltham, Mass.); Pharmacia (Pharmacia, Inc., Piscataway, N.J.);
Porphyrin Products, Inc. (Logan, Utah); Sharp (Japan); Sony
(Japan); TCS Medical Products (Hintingdon Valley, Pa.); Tektronix
(Tektronix Inc., Beaverton, Oreg.); Wescor (Logan, Utah).
[0232] The following general methods were used in the examples that
follow unless otherwise indicated.
[0233] Animal Model And Preparation
[0234] Experiments (except those described in Example 16) were
carried out with 10 Syrian golden hamsters of 40-50 g body weight.
A "hamster window preparation" was then generated in each animal
using a described surgical technique. (See, e.g., H. D. Papenfuss
et al., "A transparent access chamber for the rat dorsal skin
fold," Microvasc. Res. 18:311-318 [1979]; H. Kerger et al.,
"Systemic and subcutaneous microvascular oxygen tension in
conscious Syrian golden hamsters," Am. J. Physiol., 267 (Heart.
Circ. Physiol. 37):H802-810 [1995]). Briefly, each animal's dorsal
skinfold, consisting of 2 layers of skin and muscle tissue, was
fitted with two titanium frames with a 15 mm circular opening and
surgically installed under pentobarbital anesthesia (50 mg/kg b.w.,
i.p., Membutal.RTM., Abbott). Layers of skin muscle were carefully
separated from the subcutaneous tissue and removed until a thin
monolayer of muscle and one layer of intact skin remained.
[0235] Thereafter, a cover glass held by one frame was placed on
the exposed tissue, allowing intravital observation of the
microvasculature. The second frame was open, exposing the intact
skin. PE10 catheters were implanted in the jugular vein and the
carotid artery. The catheters were passed subcutaneously from the
ventral to the dorsal side of the neck, and exteriorized through
the skin at the base of the chamber. The patency of the catheters
was ensured by daily flushing of the implanted tip with 0.005-0.01
mL of heparinized-saline (40 IU/mL). Microvascular observations of
the awake and unanesthetized hamster were performed at least two
days after chamber implantation, thus mitigating post-surgical
trauma. During these investigations, the animals were placed in a
tube from which the window chamber protrudes to minimize animal
movement without impeding respiration.
[0236] A preparation was considered suitable for experimentation if
microscopic examination of the window chamber met the following
criteria: i) no signs of bleeding and/or edema; ii) systemic mean
blood pressure above 80 mm Hg; iii) heart rate above 320
beats/minute (Beckman recorder, R611, Spectramed transducer P23XL);
iv) systemic hematocrit above 45% (Readacrit.RTM. centrifuge,
Bectron); and v) number of immobilized leukocytes and leukocytes
flowing with venular endothelial contact less than 10% of all
passing leukocytes at time point control.
[0237] Unless otherwise indicated, the experiments described
hereafter were carried out exclusively in the hamster window
preparation. This model was selected because it allows observation
of the microcirculation for prolonged periods (i.e., several days)
in the absence of anesthesia; previously performed microvascular
studies indicated that data obtained from anesthetized animals is
not representative of the awake condition. The hamster window
preparation also presents the tissue being observed in a state that
is isolated from the environment in order to obtain representative
data.
[0238] Intravital Microscopy
[0239] Microscopical observations were performed using an
intravital microscope (Leitz, Ortholux II) with a 25.times.SW 0.60
n.a. water immersion objective. The preparation was observed
visually with a 10.times. ocular at a total optical magnification
of 250.times.. Contrast enhancement for the transilluminated image
was accomplished by using a blue filter (420 nm), which selectively
passes light in the maximum absorption band of hemoglobin, causing
the red blood cells to appear as dark objects in an otherwise gray
background. A heat filter was placed in the light path prior to the
condenser.
[0240] The microscopic images were viewed by a closed circuit video
system consisting of two different cameras, a video cassette
recorder (Sharp XA-2500S) and a monitor (Sony, PVM 1271Q), where
total final magnification at the monitor was 650.times..
[0241] Capillary Red Blood Cell Velocity
[0242] Capillary red blood cell velocity (CRBCv) was measured using
the video dual window technique with a velocity tracing correlator
(IPM, model 102B). CRBCv for each capillary was measured for a
period of 20 seconds in order to obtain an average velocity over
the period of observation. All measurements were performed in the
same capillaries. Those capillaries that had blood flow and which
stopped at subsequent time points were not included in the
statistics with a zero value at the time point in which there was
no flow; this is because their effect on tissue perfusion index is
accounted for by their effect on the functional capillary density
(FCD), i.e., the number of capillaries in a unit area observed to
be passing RBCs. CRBCv was measured in one-to-two vessels per field
of observation (10-12 per animal), since not all capillaries in a
field are in the same focal plane.
[0243] Arteriolar and Venular Diameters
[0244] Arteriolar and venular diameters were measured at each time
point using an image shearing monitor (IPM, model 907) during video
playback.
[0245] Measurement of pO.sub.2 in Microcirculation
[0246] Before collection of data, each animal received a slow
intravenous injection of palladium (Pd)-coproporphyrin (Porphyrin
Products, Inc.) previously bound to albumin. The concentration used
was 30 mg/kg body weight. During pO.sub.2 measurements, a xenon
strobe arc (EG&G Electro Optics) with a decay constant of 10
microseconds was flashed at 30 Hz over a selected area.
Epi-illumination was only used during pO.sub.2 measurements, in
order to avoid possible tissue damage which may be caused by the
intense illumination. The phosphorescence emission from the
epi-illuminated area passes through an adjustable slit and a long
band pass filter (cut off at 630 mm) before being captured by a
photomultiplier (EMI, 9855B). Slit size was usually kept at
15.times.100 .mu.m (relative to the actual microscopic field), and
it was always positioned along the length of the vessel.
[0247] When interstitial measurements were performed, the slit was
positioned parallel to the nearest vessel, at various distances.
The signals from the photomultiplier were sent to a digital
oscilloscope (Tektronix, 2430). The oscilloscope averages 200-500
curves, and a single smoothed curve was then digitized (10 bit
resolution) at a rate of 0.5 MHz and stored for later analysis.
Each curve was also processed by a specialized analog processor for
the calculation of pO.sub.2.
[0248] General Experimental Protocol
[0249] Unless otherwise indicated below, the following general
exchange transfusion procedure was utilized in the examples that
follow. The chamber window of the window preparation was implanted
at day one. The chamber was inspected for compliance with inclusion
criteria at day 3, and, if satisfactory, carotid artery and jugular
vein catheters were implanted. The animal was investigated at day 5
for compliance with systemic and microvascular inclusion criteria,
and, if satisfactory, an exchange experiment was started.
[0250] Each experiment served as its own control, and all data were
relative to the conditions of the animal at the start of the
experiment. Video microscopic measurements, systemic hematocrit,
heart rate, blood gasses (pO.sub.2, pH, pCO.sub.2) and blood
hemoglobin content (this measurement was initiated with the
experiments involving Hemolink.RTM./dextran and continued with the
experiments conducted thereafter) were taken at control prior to
exchange of blood. Microscopic measurements at control included
capillary flow velocity and arteriolar and venular diameters.
Microvascular pO.sub.2 measurements were not taken at control,
since this measurement can only be carried out at one time point
due to toxicity. Macro and micro data collection at control lasted
one hour.
[0251] After control measurements were collected, the first
exchange was initiated. The target was 40% of the original blood
mass to be withdrawn and replaced with a blood substitute at the
rate of 100 .mu.L/min (the duration of this procedure was 10-20
minutes). At the end of this procedure and after an equilibration
and stabilization period of ten minutes, micro and macro
measurements, described above, were taken (the duration of this
procedure was one hour).
[0252] A second exchange targeted at extracting 30% of the original
volume was then instituted, using the procedure described above.
Micro and macro measurements were taken, and, if this was the final
exchange target, the animal was transferred to the pO.sub.2
measurement microscope. The animal was injected with the porphyrin
compound and intravascular and extravascular pO.sub.2 measurements
were made in arterioles, venules and the tissue (the duration of
this procedure was one hour).
[0253] If the final hematocrit target was in the range of 20%, then
a third exchange was performed, and microvascular pO.sub.2 was not
measured during the second exchange. After the third exchange,
micro and macro measurements were made, and the animal was
transferred to the pO.sub.2 measurement microscope.
[0254] Statistical Analysis
[0255] Data obtained for each group were analyzed to determine if
the changes observed within groups were statistically significant.
The results of each group are presented by treating each data point
as resulting from an independent experiment. The Mann-Whitney
non-parametric test was used on the normalized means to assess if
the changes in the parameters were significantly different from
control. Results are given in terms of median and interquartile
ranges. Changes were deemed statistically significant for
p<0.05.
[0256] The examples that follow are divided into the following
sections:
[0257] I) Microcirculation Experiments; and II) Clinical Model
Experiments.
[0258] 1. Microcirculation Experiments
EXAMPLE 1
Blood Flow and Hematocrit During Colloid and Saline
Hemodilution
[0259] The experiments of this example were directed at determining
the effect of decreasing hematocrit, as a result of hemodilution,
on blood flow velocity. The experiments of this example were
conducted on hamsters using dextran 70 and saline.
[0260] The general experimental procedures (e.g., General
Experimental Protocol and Capillary Red Blood Cell Velocity)
described above were performed. FIG. 2 depicts a plot of flow
velocity in the microcirculation as a function of hematocrit
reductions with dextran hemodilution and saline hemodilution. The
following designations are used in FIG. 2: i) dextran hemodilution:
small circle=mesentery; square=skin; plus sign=muscle; and ii)
saline hemodilution: large circle=skin fold. The results indicate
that blood flow, as evidenced by the velocity of blood in the
vessel of the microcirculation, increases as blood is diluted. The
increase is linearly related to the decrease of hematocrit,
reflecting the fact that most of the viscous losses in the
circulation occur in the microcirculation where the relationship
between blood viscosity and hematocrit is linear.
[0261] The majority of previous studies have shown that the number
of RBCs can be reduced to 25% of the original amount, i.e., a loss
of 75% of the original RBC mass, while maintaining circulatory
function and flow. Most free hemoglobin solutions (e.g., HBOCs) do
not show the linear increase in blood flow with the reduction in
hematocrit for very low hematocrits, which is evidenced by
non-oxygen carrying diluents. These results indicate the presence
of additional processes in the case of free hemoglobin solutions,
such as the arterial wall reactions previously alluded to and
described in further detail below.
EXAMPLE 2
pO.sub.2 Distribution During Dextran 70 and Hemolink.RTM.
Hemodilution
[0262] The experiments of this example were directed at determining
the effect of hemodilution on pO.sub.2 in the microcirculation by
the phosphorescence decay method described above.
Dextran 70 Hemodilution
[0263] Measurements of pO.sub.2 were made in 50 .mu.m arterioles
and the tissue surrounding those arterioles. The results were as
follows: arteriole pO.sub.2 (pO.sub.2.A)=53 mm Hg; tissue pO.sub.2
(pO.sub.2.T)=21 mm Hg. The following equation may then be utilized
to calculate K.sub.A*, the constant representing the difference in
the decrease in the oxygen partial pressure between i) the
arterioles and the tissues and ii) the central arteries and the
tissues:
K.sub.A*=In[(pO.sub.2.A-pO.sub.2.T)/(pO.sub.2.a-pO.sub.2.T)]
[0264] where pO.sub.2.a is the oxygen tension in a central artery.
If one assumes a pO.sub.2.a=100 mm Hg, then KA*=In
[(53-21)/(100-21)]=-0.90.
[0265] Table 5 sets forth previously obtained (by the present
inventors) pO.sub.2 values for various hematocrit (.alpha.) levels
with dextran 70 hemodilution. The convection diffusion model allows
comparison of measured values to theoretical values. Changes in
blood viscosity (.gamma.) were not measured directly, but were
inferred from the change in blood flow velocity in the
microcirculation; the relative viscosity y relates to the viscosity
of whole blood (.gamma.=1.0). The oxygen carrying capacity was
assumed to be directly proportional to hematocrit (i.e., ignoring
oxygen carried by plasma). Table 5 summarizes measured and
theoretical pO.sub.2.A values following dextran 70 hemodilution.
Predicted values for each level of hemodilution were obtained by
using model results where K.sub.A* was multiplied by the
corresponding .gamma./.alpha. ratio.
5TABLE 5 pO.sub.2.A pO.sub.2.A Theor. mm Meas. Wall Grad.
pO.sub.2.T .alpha. .gamma. .gamma./.alpha. Hg mm Hg mm Hg mm Hg 1.0
1.00 1.0 53 55 21 0.8 0.80 1.0 53 0.6 0.67 1.12 56 55 21 21 0.4
0.57 1.42 42 54 22 20 0.2* 0.50 2.50 29 37 17 8 *Animals do not
tolerate this low hematocrit. The viscosity factor .gamma. is
deduced from the effect on velocity.
[0266] The results presented in Table 5 indicate that a reduction
of hematocrit to 60% of the original amount, i.e., a loss of 40% of
the original RBC mass, or a hemoglobin concentration (in RBCs) of
9%, does not normally change tissue oxygenation. This is true in
terms of autoregulatory responses and in terms of tissue
oxygenation. The model predicts that blood pO.sub.2 in the
arterioles would be significantly lower as hematocrit is reduced to
40% and 20% of the normal value. However, as the data exhibit, this
does not take place for reductions of 40%, indicating that the
arterioles elicit a sufficiently strong autoregulatory response
aimed at sustaining pO.sub.2. Further reductions of hematocrit
cause an important decline in tissue pO.sub.2. Moreover, the wall
gradient at extreme hemodilution is low, reflecting vasodilation
needed to respond to lower arteriolar oxygen tension.
Hemolink.RTM. Hemodilution
[0267] Hemodilution with Hemolink.RTM. was carried out in an
analogous manner to that described above for dextran 70. The
results are set forth in Table 6.
6TABLE 6 pO.sub.2.A Theor. pO.sub.2.A Meas. Wall Grad. pO.sub.2.T
(Htc).alpha.* .gamma. .gamma./.alpha. mm Hg mm Hg mm Hg mm Hg
(0.6)0.86 0.65 0.97 61 (0.4)0.80 0.66 0.89 59 55 23 17 (0.2)0.73
0.54 0.91 62 53 28 5 *.alpha. shows the oxygen carrying capacity of
the mixture of HemoLink .RTM. (concentration: 10 g/100 mL) and
RBCs. The numbers are normalized relative to the oxygen carrying
capacity of normal blood.
[0268] The results in Table 6 indicate that Hemolink.RTM.
maintained arteriolar pO.sub.2 for all levels of hemodilution.
Animals tolerated hemodilution to 20% of the original RBC mass,
which is not the case with dextran hemodilution. Though an
understanding of the mechanism is not required in order to practice
the present invention, the maintenance of arteriolar pO.sub.2
appears to be due to a vasoconstrictor effect that reduces blood
flow by about 25%. This is evidenced by: i) increased vessel wall
gradient (a sign of vasoconstriction); ii) arteriolar
vasoconstriction; and iii) a flow increase due to viscosity effects
that is lower than that obtained with dextran 70 hemodilution, as
evidenced by higher .gamma. values at any given level of RBC mass
dilution with Hemolink.RTM..
[0269] If dilution with Hemolink.RTM. were to increase blood flow
only according to the viscosity effect resulting from colloids, one
would expect to obtain pO.sub.2 values at the level of 50 .mu.m
arterioles that, when calculated according to theoretical
predictions, would be approximately 60 mm Hg (for hematocrit=0.4).
Though the practice of the present invention does not require an
understanding of why the values are approximately the same, the
differences between the theoretical figures and the measured
figures indicate the existence of some sort of arterial wall
reaction. The results suggest that there is a vasoconstrictor
effect accounting for decreased blood flow on the order of 25%,
since this would be due to a decrease in vessel diameter on the
order of 6%. The data obtained shows that arteriolar diameters
decrease to 93% of control for hematocrit 0.4 and to 88% of control
for hematocrit 0.2. This level of vasoconstriction is also evident
from the increase in pressure for hematocrit 0.4 (but not different
from control for the greater exchange level).
[0270] The results obtained with Hemolink.RTM. indicate that,
following an isovolemic reduction of hematocrit from 10% to 40%,
tissue oxygenation (in terms of the pO.sub.2 of 50 .mu.m arterioles
and tissue to the same level) is sustained at those levels present
in normal conditions. Though a precise understanding of the
methodology of this effect is not necessary in order to practice
the present invention, the observed slight increase in blood
pressure and vessel wall gradient and decrease in functional
capillary density may be the direct consequence of autoregulatory
phenomena, i.e., phenomena aimed at maintaining pO.sub.2 in 50
.mu.m arterioles constant in the presence of potentially excess
oxygen carrying capacity due to lowered blood viscosity.
Effect of the Results on Blood Substitute Formulations of the
Present Invention
[0271] The results or this example indicate that Hemolink.RTM., in
its present formulation, provides too much oxygen and that the
viscosity of the resulting blood mixture is too low. While
hemodilution with inert colloids depends on low blood viscosity to
maintain oxygen carrying capacity, the resulting increase in
cardiac output may not be a desirable effect in all cases.
Therefore, in some embodiments of the present invention,
Hemolink.RTM. and other oxygen-carrying components, especially
HBOCs, are formulated in a solution that contains an inert colloid.
In this way, either an increase in viscosity is achieved and/or the
oxygen carrying capacity is decreased, while colloid osmotic
pressure and plasma retention are maintained.
EXAMPLE 3
Tissue Oxygenation Resulting from Hemodilution with 50%
Hemolink.RTM./50% Dextran 70
[0272] The experiments of this example are directed at determining
the adequacy of tissue oxygenation following administration of a
mixture of Hemolink.RTM. and dextran 70.
[0273] A mixture of 50% Hemolink.RTM. and 50% dextran 70 was
prepared, and tissue oxygenation was determined at hematocrit
levels of 60% and 40% of baseline levels. Hemoglobin concentration
in the resulting mixture was measured directly by
spectrophotometry. In addition, the number of RBCs and the amount
of Hemolink.RTM. were measured directly in blood samples. Though
testing was initiated using four animals, only two animals
satisfied all criteria for inclusion in an experimental run; the
results for the two animals are set forth in Table 7.
7TABLE 7 pO.sub.2.A Meas. Wall Grad. pO.sub.2.T Htc/.alpha. .gamma.
.gamma./.alpha. pO.sub.2.A mm Hg mm Hg mm Hg 0.6/0.68 0.64 0.86 55
0.4/0.54 0.76 1.41 43 51 27 15
[0274] When the data in Table 7 is compared with that derived from
use of Hemolink.RTM. alone (see Table 6), it is observed that the
values of pO.sub.2.T (17 mm Hg v. 15 mm Hg, respectively, for
hematocrit=0.4) are very similar; these values are acceptable in
practice. Therefore, both the diluted mixture and Hemolink.RTM.
itself provide adequate tissue oxygenation, despite the fact that
the mixture carries only half as much oxygen per unit weight as is
carried by Hemolink.RTM. alone.
EXAMPLE 4
Tissue Oxygenation with Hemolink.RTM., Dextran 70 and
Hemolink.RTM./Dextran 70 at Hematocrit 0.4
[0275] The experiments of this example are directed at determining
and comparing the tissue oxygenation of Hemolink.RTM., Dextran 70,
and Hemolink.RTM./Dextran 70 (50%/50%) at hematocrit 0.4. These
experiments build upon those set forth in the preceding
example.
[0276] The efficacy of tissue oxygenation following administration
of the above-mentioned compositions was evaluated from information
of arteriolar and venular pO.sub.2, the percent oxygen saturation
of hemoglobin, capillary flow velocity (1/.gamma.), and intrinsic
oxygen carrying capacity (.alpha.). These parameters were
determined as previously described, and oxygen extraction by the
microcirculation was determined by the method discussed hereafter.
The results are set forth below in Table 8 (relative numbers are
indicated where applicable).
8 TABLE 8 Normal Dextran HemoLink .RTM./ Blood 70 HemoLink .RTM.
Dextran Arteriolar pO.sub.2 53 54 55 51 Arteriolar O.sub.2 % sat.
0.84 0.85 0.85 0.81 Venular pO.sub.2 33 30 20 22 Venular O.sub.2 %
sat. 0.52 0.50 0.30 0.32 Cap. Velocity 1.0 1.75 1.51 1.32 O.sub.2
carrying capacity 1.0 0.40 0.80 .54 Extraction 0.32 0.22 0.50
0.26
[0277] The data in Table 8 for oxygen extraction are derived from
measurements of the pO.sub.2 gradients at the vessel wall. This
value, in combination with the value for oxygen carrying capacity
normalized to blood=1, gives an indication of the relative amount
of oxygen which is lost between the arterial vessel and the tissue
for a given level of tissue oxygenation. In the case of normal
(i.e., undiluted blood), the figure is 32%. When blood is diluted
with dextran 70, the figure is 9% (i.e., 22% of 40%); when blood is
diluted with Hemolink.RTM., the figure is 40% (50% of 80%); and
when blood is diluted with a dextran/Hemolink.RTM. mixture, the
figure is 14% (26% of 54%).
[0278] The results indicate that the dextran/Hemolink.RTM. mixture
is considerably more efficient in delivering oxygen to the tissues
than is Hemolink.RTM. alone. Because the mixture loses much less of
its oxygen in moving from the arteries to the capillaries than does
Hemolink.RTM. alone, the mixture has greater reserves of oxygen
available to the tissue for oxygenation purposes. Therefore, the
compositions of the present invention comprising a non-oxygen
carrying component and an oxygen carrying component provide greater
reserves of oxygen for the tissues; this result represents an
additional, unexpected advantage of the compositions.
EXAMPLE 5
Wall Gradients with Hemolink.RTM. and Hemolink.RTM./Dextran 70 at
Hematocrit 0.4
[0279] Several of the previous examples were directed at the use of
the "awake hamster" model to determine i) partial oxygen pressures
in arteries, veins and tissue, and ii) blood pressure in normal
blood (control) with Hemolink.RTM. at hematocrit 0.4, and 50:50
dextran:Hemolink.RTM. at hematocrit 0.4. This example is directed
at the determination of wall gradients using each of those
compositions.
[0280] As previously indicated, the vessel wall gradient is
inversely proportional to tissue oxygenation. In this example, wall
gradients were derived from the pO.sub.2 measurements in previous
studies. The blood pressure data represents mean arterial blood
pressure relative to the control. The results are shown in Table
9.
9TABLE 9 HemoLink .RTM./ Parameter Control HemoLink .RTM. Dextran
Wall Gradient - Arteriole 17.8 24.3 26.8 (mm Hg) Wall Gradient -
Venular 10.1 10.8 7.6 (mm Hg) Tissue pO.sub.2 21.4 17.0 19.2 Blood
Pressure 100% 112% 109%
[0281] The data in Table 9 indicate that the Hemolink.RTM./dextran
composition is effectively equivalent to Hemolink.RTM. alone when
compared for the measured parameters. Moreover, the results of this
example, in conjunction with the examples set forth above, indicate
that the desirable properties of a blood substitute obtainable by
using Hemolink.RTM. (and, by extrapolation, other HBOCs) alone are
also obtainable with the compositions of the present invention
(i.e., compositions comprising solutions of an oxygen carrying
component in combination with a non-oxygen carrying component).
EXAMPLE 6
Microcirculatory Parameters at Hematocrit of 12-13%
[0282] The experiments of this example utilized the previously
described procedures to assess various microcirculatory parameters
following administration of several different compositions.
[0283] Six different compositions were administered to hamsters in
separate experiments: 1) control (i.e., normal blood); 2) dextran
70 alone; 3) Hemolink.RTM. alone; 4) Hemolink.RTM. 33%/dextran 66%
(by volume); 5) Hemolink.RTM. 50%/dextran 50%; and 6) L-Name
(L-nitrosyl-arginine-monomethyl-ether; commercially available from,
e.g., Sigma). A hematocrit of approximately 12% of the control was
achieved in experiments 3)-5) following three exchange perfusions.
Only two hemodilutions (i.e., two exchange perfusions) were
performed for the experiment with dextran alone (experiment number
3) because the animals do not tolerate three dilutions with this
composition. The L-name composition was injected into animals
(i.e., it was not administered to effect hemodilution).
[0284] The resulting data is set forth in Table 10. Referring to
Table 10, PaO.sub.2=arterial PO.sub.2; Grad(A)=arteriolar/tissue
gradient; and Grad(V)=venular/tissue PO.sub.2 gradient. The data
regarding vasoconstriction is relative to the control (experiment
number 1).
[0285] The data in Table 10 indicate that hemodilution with the
hemoglobin-based oxygen carrier (HBOC) Hemolink.RTM. decreased
tissue PO.sub.2 from approximately 20 to 5 mm Hg. This was
accompanied by an increase of the arteriolar/tissue PO.sub.2
gradient from about 17 to 28 mm Hg, consistent with the
vasoconstriction previously determined to be caused by this
product. When the Hemolink.RTM. was mixed with the
non-oxygen-carrying plasma expander dextran, tissue PO.sub.2
increased to 13 and 17 mm Hg, respectively, with 33% and 50%
mixtures of Hemolink.RTM./dextran. However, in the experiments with
the Hemolink.RTM./dextran compositions, the arteriolar/tissue
PO.sub.2 gradient remained high, a consequence of vasoconstriction
still being produced by the hemoglobin.
[0286] These experiments, in conjunction with some of the results
from the previous examples indicate that if the O.sub.2
availability is increased by the extracellular location of
hemoglobin, then, in order to prevent autoregulatory
vasoconstriction at the arteriolar level, one or more of the
following compensations must take place: i) increased viscosity,
ii) decreased O.sub.2 carrying capacity, or iii) increased O.sub.2
affinity.
10TABLE 10 PaO.sub.2 Grad(A) Tissue Venular Grad(V) Arteriolar Exp.
n Material Hct % BP Torr Torr pO.sub.2Torr pO.sub.2Torr Torr
Vasoconstriction Velocity 1 Control stable 53 17.8 20 33 10 2
Dextran 19 unstable 54 22 20 30 11 none 1.4 3 many HemoLink 12
stable 53 28 5 10 4 not done 1.2 4 1 HemoLink 33% 12 stable 69 34
13 25 18 0.2 Dextran 66% 5 2 HemoLink 50% 12 stable 73 41 17 21 8
0.3 2.6 Dextran 66% 6 many L-Name stable 57 26 21 28 9 not done
1.8
EXAMPLE 7
Use of a Composition Comprising Hemolink.RTM. and
Polyvinylpyrrolidone
[0287] The experiments of this example provide evidence that
increased viscosity prevents autoregulatory vasoconstriction at the
arteriolar level. The microvasculature experiments of this example
were performed utilizing a composition comprising Hemolink.RTM. and
polyvinylpyrrolidone (PVP), 750,000 dalton molecular weight.
[0288] Aqueous solutions of i) Hemolink.RTM., ii) 50:50
Hemolink.RTM.:dextran molecular weight 70,000 (by volume), and iii)
100:4 Hemolink.RTM.:PVP molecular weight 750,000 (by volume) were
prepared at a total solute concentration, in each case, of 10 g/100
mL. The compositions were tested in the "awake hamster" model
described above. PVP is used experimentally as a plasma expander
and has also been used in humans for the same purpose; its
principal property is that of increasing plasma blood viscosity.
The use of PVP substantially increases the viscosity of the
solution, to a value estimated at about 15 centipoise
(substantially equivalent to that of whole blood).
[0289] The animals were subjected to an isovolemic exchange of
blood with each of the compositions to achieve a final hematocrit
of 0.20 of control (i.e., 20% of original RBC mass) or an effective
hematocrit of about 10%. By the procedures previously described,
measurements were taken of the arterial pressure, wall gradient,
blood pressure and tissue oxygen. The results are set forth below
in Table 11.
[0290] The results in Table 11 indicate that the increased
viscosity of the Hemolink.RTM.:PVP composition significantly lowers
the vessel wall gradient, making more oxygen available to the
tissue, compared to the other two compositions. This increased
viscosity causes dilation of the vasculature and normalizes the
distribution of oxygen in the microcirculation. Though an
understanding of the underlying mechanism is not required in order
to practice the present invention, the mechanism for vasodilation
with compositions of increased viscosity is believed to be
two-fold. First, decreased oxygen delivery of blood due to lower
hemoglobin causes autoregulatory effects analogous to those
observed with the previously described oxygen-carrying compositions
comprising other inert, non-proteinaceous colloids.
11 TABLE 11 Relative pO.sub.2 Arterioles Wall Gradient Mean
Arterial Blood pO.sub.2 Tissue Hb Content .alpha. Viscosity .gamma.
mm Hg mm Hg FCD Pressure % Normal mm Hg HemoLink .RTM. 0.73 0.54 53
28 0.64 -14% 5 HemoLink .RTM. & 0.54 0.76 51 27 0.78 +9% 13
Dextran HemoLink .RTM. & 0.51 1.00 46 15 0.45 -3% 16 PVP
[0291] Second, increased shear stress at the vessel wall increases
release of endogenous vasodilators such as prostacyclin.
[0292] In addition, even though the O.sub.2 capacity of the
Hemolink.RTM./PVP mixture is lower than that of Hemolink.RTM. alone
and its viscosity is higher, the arteriolar/tissue PO.sub.2
gradient is reduced, and tissue PO.sub.2 is increased from 5 to 16
mm Hg. These results are consistent with the theoretical
formulation alluded to previously. However, it is believed that the
mixture of Hemolink.RTM. and PVP is not suited to development as a
blood substitute, and the functional capillary density is lower
than desired.
[0293] II. Clinical Model Experiments
EXAMPLE 8
Use of Pentastarch, Hemolink.RTM., and a Mixture Thereof Under
Clinical Conditions
[0294] This example relates to experiments conducted in vivo using
male Sprague-Dawley rats under severe stress. The experiments of
this example provide information relevant to the clinical use
(e.g., in an operating theater environment) of the compositions of
the present invention.
Exchange Transfusion
[0295] The animals were instrumented 24 hours prior to initiation
of experiments, and all experiments were conducted in the awake
state. A catheter was placed in the femoral artery and another in
the femoral vein. The animal was restrained in an experimental
cage. First, an exchange transfusion was performed in which about
50% of the blood of the animal was removed and replaced with a test
composition; the test compositions assessed were pentastarch,
Hemolink.RTM. and a Hemolink.RTM./pentastarch mixture (50:50 by
volume). A peristaltic pump was used to simultaneously withdraw
blood and infuse one of the test compositions at a rate of 0.5
mL/min. The duration of the exchange was calculated to achieve
exchange of 50% of the estimated total blood volume, based on 65 mL
of blood per kg body weight as the standard blood volume of the
rat.
Mean Arterial Blood Pressure During Exchange
[0296] As the exchange transfusions proceeded, mean arterial
pressures were measured through the catheter, by standard
procedures in the art. FIG. 3 graphically presents arterial blood
pressure prior to and during the exchange transfusion (indicated by
the arrow in FIG. 3). Referring to FIG. 3, (.tangle-soliddn.)
represents Hemolink.RTM., (.tangle-solidup.) represents pentastarch
and (.gradient.) represents the mixture of Hemolink.RTM.
pentastarch. Using the statistical analyses described above, there
are no significant differences between Hemolink.RTM. alone and the
composition of Hemolink.RTM./pentastarch.
Physiological Status During Hemorrhage
[0297] Animals were subjected to a 60% hemorrhage procedure
analogous to that described in the preceding example. More
specifically, 60% of the total blood volume was calculated, using
the aforementioned 65 mL/kg estimate. The calculated amount of
blood was then removed using a simplified exponential protocol
similar to that developed by Hannon et al. ("Blood and Acid-base
Status of Conscious Pigs subjected to Fixed-volume Hemorrhage and
Resuscitated with Hypertonic Saline Dextran," Circulatory Shock
32:19-29 [1990]). At the beginning of each 10 minute period of the
hemorrhage, blood was removed from an arterial site using a syringe
pump running at a rate of 0.5 mL/min. The duration of each
withdrawal was calculated so that 60% of the total blood volume was
removed over 60 minutes.
[0298] Mean arterial blood pressure was measured through the
catheter, and data are presented graphically in FIG. 4; in FIG. 4,
the symbols depicting each composition are the same as set forth in
FIG. 3. Of note, the animals transfused with the
Hemolink.RTM./pentastarch composition start the bleed with a higher
blood pressure, which initially falls quite steeply. Both the
Hemolink.RTM./pentastarch composition and Hemolink.RTM. alone
preserve the blood pressure well during the first 50 minutes.
[0299] The hemorrhage test described above represents a relatively
severe test model. Only about 50% of the animals, even without an
exchange transfusion, survive beyond 120 minutes from the onset of
the 60% hemorrhage, and even fewer of those transfused with a test
solution survive (data not shown).
[0300] Other measurements were also determined during the
hemorrhaging, including heart rate (measured from the pressure
trace of the mean arterial pressure measurements), and pH,
pCO.sub.2, pO.sub.2, lactate accumulation, and base excess
(measured by standard analysis of the blood). The results (not
shown) from animals transfused with Hemolink.RTM. and those
transfused with the Hemolink.RTM./pentastarch composition were
substantially equivalent with the following exception. The
Hemolink.RTM./pentastarch composition resulted in more lactate
accumulation, reflecting the fact that this composition carries
less oxygen. Lactate accumulation is a direct reflection of the
status of tissue oxygenation; that is, lactate accumulates when
tissue is not supplied with sufficient oxygen.
[0301] The findings of the experiments of this example indicate
that a mixture of an oxygen-carrying component and a non-oxygen
carrying component provides similar, if not superior, results to
that achieved with an oxygen-carrying component alone.
EXAMPLE 9
Use of Pentastarch, Modified Hemoglobins, and Mixtures Thereof
Under Clinical Conditions
[0302] The experiments of this example evaluate two oxygen-carrying
components, bovine hemoglobin modified by conjugation with
polyethylene glycol (PEGHb or PEG) and .alpha..alpha.-Hb, alone and
in combination with a the non-oxygen-carrying component, the plasma
expander pentastarch (Pentaspan.RTM.; DuPont).
Nature of the Compositions
[0303] The properties of several of the compositions used in this
example are compared in Table 12. The PEGHb+pentastarch composition
and the .alpha..alpha.-Hb+pentastarch composition comprised 50% of
each composition by volume. As indicated in Table 12, both PEGHb
and pentastarch have high colloid osmotic pressure (COP) values,
and both have a viscosity that approximates that of blood (in the
measuring system used, water and purified hemoglobin have
viscosities of 1 centipoise).
12TABLE 12 Hemoglobin Viscosity Solution COP (mm Hg) (g/dL)
(centipoise) Blood 26.0 15.0 4.0 Pentaspan .RTM. 85.0 0.0 4.0 PEGHb
81.3 6.0 3.4 PEGHb + Pentaspan 98.0 3.0 3.2
Exchange Transfusion
[0304] A 50% isovolemic exchange transfusion was performed in awake
rats using the procedure described in the preceding example. Table
13 indicates the effect of the exchange transfusion (.+-.sem) on
blood volume, hematocrit, total hemoglobin, and plasma hemoglobin
for several of the compositions.
13TABLE 13 Blood Volume Plasma Hb Solution (mL/kg) Hct (%) Total Hb
(g/dL) (g/dL) Controls 56.3 .+-. 2.5 38.6 .+-. 0.9 13.8 .+-. 0.3
0.0 .+-. 0.0 Pentastarch 71.1 .+-. 5.7 18.4 .+-. 1.0 6.8 .+-. 0.4
0.0 .+-. 0.0 PEGHb 74.0 .+-. 1.6 15.8 .+-. 0.4 7.6 .+-. 0.1 2.0
.+-. 0.1 PEGHb + 91.0 .+-. 3.0 14.8 .+-. 0.3 5.6 .+-. 0.2 1.0 .+-.
0.1 Pentaspan
[0305] Referring to Table 13, the decreases in hematocrit and
hemoglobin concentration for the experimental groups indicate that
the exchange procedure led to significant expansion of the plasma
volume in the PEGHb, Pentaspan.RTM. and PEGHb+Pentaspan.RTM.
animals.
Physiological Status During Hemorrhage
[0306] Next, the rats were subjected to a 60% hemorrhage over 1
hour; this protocol, known to be lethal in approximately 50% of
animals, was performed as described in Example 8.
[0307] In FIGS. 5-10, the following designations apply: pentastarch
(.tangle-solidup.), .alpha..alpha.-Hb (.box-solid.), PEG-Hb
(.circle-solid.), pentastarch+.alpha..alpha.-Hb (.quadrature.),
pentastarch+PEG-Hb (.smallcircle.), and control animals
(.diamond-solid.). FIG. 5 depicts animal survival over a 2 hour
period beginning with the start of hemorrhage. As indicated by the
data in FIG. 5, hemodilution with pentastarch alone led to
significantly reduced survival, while hemodilution with either
PEGHb alone or PEGHb+pentastarch led to complete survival; survival
following hemodilution with the compositions comprising
.alpha..alpha.-Hb was much lower than with the compositions
containing PEGHb.
[0308] FIG. 6A-D graphically depict the acid-base status of control
rats (.diamond-solid.) and of rats following exchange transfusion
with pentastarch (.tangle-solidup.), .alpha..alpha.-Hb
(.box-solid.), PEG-Hb (.circle-solid.),
pentastarch+.alpha..alpha.-Hb (.gradient.), and pentastarch+PEG-Hb
(.smallcircle.) and after the initiation of a 60% hemorrhage. FIG.
6A depicts PaO.sub.2, FIG. 6B depicts PaCO.sub.2, FIG. 6C depicts
arterial pH, and FIG. 6D depicts base excess.
[0309] FIGS. 6A-D are directed at the animals' acid-base status
determined over a 2 hour period from the start of hemorrhage. More
specifically, FIG. 6A depicts PaO.sub.2, FIG. 6B depicts
PaCO.sub.2, FIG. 6C depicts arterial pH, and FIG. 6D depicts base
excess. As indicated in FIGS. 6A-C, neither the PEGHb nor the
PEGHb+pentastarch animals had significant respiratory alkalosis
compared to the pentastarch animals. Moreover, neither the PEGHb
nor the PEGHb+pentastarch animals developed significant acidosis,
even at the end of the hemorrhage period. Acid base status was well
preserved in the PEGHb and PEGHb+pentastarch animals (FIG. 6D).
Again, neither of the compositions comprising .alpha..alpha.-Hb
performed as well as PEGHb+pentastarch animals or the pentastarch
animals.
[0310] FIG. 7 shows the production of lactic acid following
administration of each of the compositions. As depicted in FIG. 7,
generation of lactic acid during the hemorrhage was significantly
greater in the .alpha..alpha.-Hb animals (alone and in combination
with pentastarch) and the pentastarch animals than in the other
groups. Notably, the controls animals (no prior exchange
transfusion) and the PEGHb+pentastarch animals had approximately
equal minimal rises in lactic acid, even though the total
hemoglobin concentration and hematocrit were significantly less in
the PEGHb+pentastarch group. (See Table 13).
[0311] FIG. 8A depicts mean arterial blood pressure of control rats
(.diamond-solid.) and of rats following exchange transfusion with
pentastarch (.tangle-solidup.), PEG-Hb (.circle-solid.), and
Pentaspan+PEG-Hb (.smallcircle.) at time -60 minutes, and after the
initiation of a 60% hemorrhage at time 0 minutes. As indicated by
the data in FIG. 8A, blood pressure did not rise in any of the
groups during the exchange transfusion (i.e., from -60 to 0
minutes), but fell significantly in the controls and in the
pentastarch animals during hemorrhage (i.e., from 0 to 120
minutes). Both the PEGHb and the PEGHb+pentastarch compositions
"protected" the animals from hypotension.
[0312] FIG. 8B depicts mean arterial blood pressure in control rats
(.diamond-solid.), and rats following exchange transfusion with
pentastarch (.tangle-solidup., point B), .alpha..alpha.-Hb
(.box-solid., point B), and pentastarch+.alpha..alpha.-Hb
(.quadrature., point A), and after the initiation of a 60%
hemorrhage (point C). As set forth in FIG. 8B, the control animals
and the pentastarch animals maintained mean arterial pressure to a
greater extent than the pentastarch+.alpha..alpha.- -Hb
animals.
[0313] FIG. 9 and FIG. 10 depict relative cardiac output and
systemic vascular resistance, respectively. Cardiac output refers
to the amount of blood pumped by the heart in a unit period of time
(e.g., liters per minute); relative cardiac output refers to the
cardiac output of the three experimental groups relative to the
control period (-30 minutes). As depicted in FIG. 9, cardiac output
was higher in PEGHb and PEGHb+pentastarch compared to the other
groups. FIG. 10 indicates that systemic vascular resistance
remained low in both PEGHb and PEGHb+pentastarch animals relative
to the other animals.
[0314] The results presented above indicate that the
PEGHb+pentastarch mixture was superior to compositions comprising
.alpha..alpha.-Hb. In addition, the PEGHb+pentastarch mixture
performed similarly to the PEGHb composition alone. This was true
even though the hemoglobin concentration to which the animals were
exposed and the amount of hemoglobin product used were less by half
with the mixture, offering the advantage of reducing the
concentration of hemoglobin given to patients, thereby reducing
both cost and potential side effects. Though a precise
understanding of why the mixture is effective is not required in
order to practice the present invention, the effectiveness of
PEGHb+pentastarch is thought to result from its preservation of all
four of the previously discussed properties, namely oncotic
pressure, viscosity, oxygen affinity, and low oxygen capacity.
Indeed, the results indicate that compositions comprising i) an
oxygen-carrying component (e.g., a HBOC) with high oncotic
pressure, oxygen affinity and viscosity and ii) a
non-oxygen-carrying plasma expander with similar oncotic pressure
and viscosity provide an optimal blood product.
EXAMPLE 10
Survival Data with Modified Hemoglobins, Non-Oxygen-Carrying
Components, and Compositions Thereof
[0315] This example is directed at animal survival using several
modified hemoglobin products (i.e., oxygen-carrying components),
non-oxygen-carrying components, and several mixtures comprising an
oxygen-carrying component and a non-oxygen-carrying component.
Experimental Protocol
[0316] Generally speaking, the experiments of Examples 10-15 were
carried out as described in Example 8. Briefly, male Sprague-Dawley
rats were instrumented, under anesthesia, 24 hours prior to
hemodilution. Instrumentation consisted of cannulation of the
femoral artery and vein and exteriorizing the catheters so that the
animals had free range in their cages in the following 24 hours.
The experiments were all carried out in awake animals, loosely
constrained to restrict gross movements. Arterial pressure was
continuously monitored at one femoral artery. Thereafter, 50% of
the estimated blood volume (60 mL/kg) was exchanged with test
material at a rate of 0.5 mL/min. This was performed with a
peristaltic pump so that withdrawal and infusion were done
simultaneously at the same rate.
[0317] Hemorrhage was initiated after the exchange transfusion; the
hemorrhage volume was calculated to be 60% of the original blood
volume. Blood was removed using a simple exponential protocol so
that the hemorrhage was complete after 60 minutes. Specifically,
the withdrawal pump was driven at 0.5 mL/min for decreasing periods
of time at the start of each 10 minute period for a total of 60
minutes.
Animal Survival
[0318] Table 14 summarizes all the materials used in the
experiments. Referring to Table 14, it should be noted that the
designation "DBBF" refers to human hemoglobin crosslinked between
the alpha chains (".alpha..alpha.-Hb"); this was produced by the
United States Army and provided as a gift. Two hemoglobin products
modified with polyethylene glycol were tested. PHP Hemoglobin is a
human-derived product from Apex Bioscience, and PEGHb is a
bovine-based product obtained from Enzon, Inc. The two PEG-modified
hemoglobin products (PHP and PEGHb) gave qualitatively the same
results. Though the experiments described hereafter utilize PEGHb,
other products comprising PEG-modified hemoglobin and a
non-oxygen-carrying component, including, but not limited to,
products comprising PHP, are contemplated by the present
invention.
14TABLE 14 Materials Raw Oxygen Abbr. Name Material Source Mol Wt.
COP Viscosity Affinity PS Pentaspan Corn DuPont Merck *250,000 High
High None HS Hetastarch Corn Fresenius *480,000 Low undetermined
None BOV Bovine Hemoglobin Cow Blood Enzon 64,000 Low Low High DBBF
.alpha..alpha.-Hemoglobin Human Blood U.S. Army 64,000 Low Low
Normal .beta.82 .beta.82 Hemoglobin Human Blood Hemosol 64,000 Low
Low High TM TM Hemoglobin Human Blood Hemosol 64,000 Low Low Low HL
HemoLink .TM. Human Blood Hemosol 128,000 Low Low High PHP PHP
Hemoglobin Human Blood Apex Bioscience 105,000 Moderate Moderate
Mod High PEG PEG Hb Cow Blood Enzon 118,000 High High High
*weight-average molecular weight.
[0319] One of the major criteria for an effective blood substitute
product is enhanced survival, and Table 15 provides several indices
of animal survival. Specifically, Table 15 sets forth the mean
times to death; the column indicating "initial death" refers to the
number of minutes that elapsed following the initiation of
hemorrhage before the death of the first animal, and the column
indicating "% survival" refers to the number of minutes that have
elapsed when 50% of the animals have expired.
[0320] Referring to Table 15, all of the mixture blood products
(i.e., Pentaspan.RTM.+Hemolink.RTM.; hetastarch+Hemolink.RTM.;
Pentaspan.RTM.+PEGHb; and Pentaspan.RTM.+DBBF) in Table 15
contained 50% (by volume) oxygen-carrying component and 50%
non-oxygen-carrying component. These data show that all of the
modified hemoglobins (regardless of their properties), with the
single exception of hemoglobin modified by conjugation with
polyethylene glycol (PEG), show a diminished survival compared to
controls or Pentaspan.RTM.. Indeed, in studies with a mixture of
PEGHb and a non-oxygen-carrying component, most of the animals were
still alive after the one-hour observation period following
hemorrhage.
[0321] As indicated in Table 15, of the mixture blood products,
only Pentaspan.RTM.+PEGHb performed as well as or better than the
controls (control animals underwent no exchange transfusion).
Moreover, Pentaspan.RTM.+PEGHb was nearly as effective in survival
as PEGHb, which is surprising given the fact that the total
hemoglobin is less in the Pentaspan"+PEGHb animals, and the plasma
hemoglobin is only approximately 1 g/dL. The animal survival data
with the other mixture blood products was much less than the
control animals.
15TABLE 15 Survival Initial 50% Survival Sample Death (Min) Slope
(minutes) Controls 110 -0.0247 130.2 PS 96 -0.0325 111.4 HS 38
-0.0237 59.1 DBBF 46 -0.0175 74.6 TM 41 -0.0559 49.9 B82 40 -0.0383
53.1 HL 39 -0.0289 56.3 PEG Hb >120 >120 PS/HL 33 -0.0182
60.5 HS/HL 40 -0.0204 64.5 PS/DBBF 33 -0.0491 43.2 PS/PEGHb >120
>120
[0322] As previously indicated, blood products comprising
pentastarch (e.g., Pentaspan.RTM.) and PEGHb optimize viscosity,
oncotic pressure, oxygen affinity and oxygen capacity. Of the
products listed in Table 14, only PEGHb has all of these
properties. Diluting PEGHb with a different non-oxygen-carrying
component (e.g., the plasma expander hetastarch) would reduce the
resulting blood product's viscosity and oncotic pressure, not
change the oxygen affinity, but reduce the oxygen capacity. In
contrast, the mixtures resulting from combination of PEG-modified
hemoglobin with pentastarch have viscosity and oncotic pressure
values very close to that of PEGHb alone.
[0323] The examples that follow compare several different blood
product mixtures and solutions and summarize the physiological data
generated from each set of experiments. The data indicate that
preferred substitute blood products incorporate most, if not all,
of the above-mentioned properties (i.e., oncotic pressure,
viscosity, oxygen affinity and oxygen content).
EXAMPLE 11
Conventional Plasma Expanders
[0324] This example specifically compares animal survival and
physiological data following exchange transfusions and hemorrhage
with two conventional plasma expanders (i.e., non-oxygen-carrying
components, hetastarch (HS) and pentastarch (PS) (see Table 14)).
The experiments were performed as described in Example 10.
Product Characteristics
[0325] Hetastarch is commercially available from Fresenius, and
pentastarch was commercially obtained from DuPont Merck. Both
products comprise hydroxyethyl starch, but pentastarch's low
molecular weight (250,000 Da vs 480,000 Da) is a result of a lower
degree of hydroxyethyl substitution (0.45 compared to 0.70). This
difference results in higher oncotic pressure for pentastarch and
its faster enzymatic degradation in the circulation. Because of its
higher oncotic pressure, pentastarch has a greater plasma expanding
capability.
Animal Survival
[0326] Overall animal survival for the two groups of test animals
(pentastarch and hetastarch) and control animals are set forth in
Table 15, supra. The data are consistent with the hemodynamic,
oxygen transport, and acid-base data. That is, survival in the
pentastarch animals is significantly longer than that of the
hetastarch animals, but both are shorter than the controls.
Hematocrit and Hemoglobin
[0327] Tables 16, 17, and 18 indicate hematocrit, total hemoglobin,
and plasma hemoglobin, respectively. In Tables 16-18, "n"=the
number of animals in the experiment, "ND"=not determined, "post ET"
immediately following the exchange transfusion, and "60
min"=following the 60 minute hemorrhage.
16TABLE 16 Hematocrit Solution n Baseline Post ET 60 Min. Control 7
38.6 .+-. 0.9 24.8 .+-. 0.9 PS 4 42.6 .+-. 1.3 18.4 1.0 15.0 .+-.
0.7 LHS 2 4.0 .+-. 2.0 18.3 1.8 12.7 .+-. DBBF 6 39.5 .+-. 0.7 18.5
0.4 13.4 .+-. 0.6 TM 6 42.4 .+-. 0.9 21.8 0.5 *13.9 .+-. 0.2 B82 4
2.7 .+-. 1.3 18.3 1.0 14.4 .+-. 0.6 HL 4 40.7 .+-. 1.2 18.1 1.3
12.2 .+-. 0.8 PEG 5 40.5 .+-. 1.2 15.8 0.4 12.9 .+-. 0.2 Bovine 1
40.0 22.0 #18.2 PS/DBBF 5 40.3 .+-. 1.1 22.2 2.3 *17.1 .+-. 2.0
PS/HL 5 43.3 .+-. 0.9 20.2 0.6 15.0 .+-. 1.0 HS/HL 4 40.5 .+-. 0.4
19.0 0.1 13.1 .+-. 0.3 PS/PEG 2 40.2 .+-. 0.8 14.8 0.4 12.6 .+-.
0.4 *50 minute sample. #30 minute sample.
[0328]
17TABLE 17 Total Hemoglobin Solution n Baseline Post ET 60 Min.
Control 7 13.8 .+-. 0.3 8.8 .+-. 0.3 PS 4 15.2 .+-. 0.4 6.8 .+-.
0.4 5.4 .+-. 0.3 HS 2 14.1 .+-. 0.9 6.6 .+-. 0.5 4.2 .+-. DBBF 6
14.0 .+-. 0.3 10.2 .+-. 0.2 7.2 .+-. 0.4 TM 6 14.8 .+-. 0.3 10.9
.+-. 0.3 *7.4 0.4 B82 4 14.7 0.4 9.2 0.3 7.5 0.4 HL 5 14.2 0.2 10.8
0.2 7.8 0.1 PEG 5 14.5 .+-. 0.6 7.6 0.1 6.4 0.1 Bovine 1 14.0 9.9
#8 PS/DBBF 5 13.9 .+-. 0.4 9.1 0.7 *7.1 .+-. 0.3 PS/HL 5 13.9 .+-.
1.5 8.8 .+-. 0.4 6.0 .+-. 0.1 HS/HL 4 14.4 .+-. 0.2 8.6 .+-. 0.1
6.0 .+-. 0.1 PS/PEG 2 14.0 .+-. 0.2 5.6 0.2 5.0 0.2 *50 minute
sample. #30 minute sample.
[0329]
18TABLE 18 Plasma Hemoglobin Solution n Baseline Post ET 60 Min.
Control 6 No 3.9 .+-. 0.1 2.3 .+-. 0.1 PS 6 No 3.7 0.2 *2.2 0.1 HS
4 No 3.6 0.2 2.4 0.1 DBBF 4 No 4.8 .+-. 0.1 2.6 .+-. 0.3 TM 5 No
1.9 0.1 1.5 0.1 B82 5 No 1.6 .+-. 0.4 *1.3 .+-. 0.2 PS/HL 5 No 2.1
.+-. 0.4 1.1 .+-. 0.3 PS/PEG 2 No 1.0 0.0 0.8 0.0 Bovine 1 No 2.5
#1.9
[0330] The data in Table 16 indicate that both pentastarch and
hetastarch hemodilute to a similar degree, as measured by
post-exchange hematocrit. However, the hematocrit in the hetastarch
animals was significantly lower than in the pentastarch animals
after the 60% hemorrhage. Similarly, Table 17 shows that the total
hemoglobins were similar in both groups of animals after
hemodilution, but the hetastarch animals had significantly lower
hemoglobin after the hemorrhage.
Hemodynamics
[0331] Compared to controls, both pentastarch- and
hetastarch-hemodiluted groups dropped their blood pressure very
rapidly after start of the hemorrhage (data not shown). Recovery
was faster in the pentastarch animals and was sustained better than
in the hetastarch group, but both have significantly lower blood
pressure than the controls after the first 40 minutes of
hemorrhage.
[0332] Both hetastarch and pentastarch groups increased their heart
rates in response to the volume loss, but the rise in the
hetastarch group was more abrupt than in the pentastarch or control
groups (data not shown). Though the practice of the present
invention does not require an understanding of this effect, it is
most likely due to the more significant plasma volume expansion
expected after exchanging with the hyper-oncotic pentastarch. Both
test groups raised their blood pressure sooner than the controls
during the hemorrhage, probably because of the significantly lower
hemoglobin and hematocrit in the exchanged animals.
[0333] The parameter dP/dt represents the maximum positive slope of
the pulse pressure contour. This parameter is proportional to the
onset of the systolic contraction, and is therefore a reflection of
the strength, or inotropic action of the heart. In the control
animals, dP/dt rose after onset of hemorrhage, as the heart
attempts to increase its output. The dP/dt value rose in all three
groups, but sooner in the hetastarch group compared to pentastarch
group and controls (data not shown). The increase in dP/dt in the
pentastarch group was actually very similar to that seen in the
controls, suggesting that the plasma volume expansion of the
pentastarch animals was beneficial.
Ventilation
[0334] Ventilation is reflected by PO.sub.2 and PCO.sub.2
measurements. The rise in PO.sub.2 and fall in PCO.sub.2 (data not
shown) was more pronounced in the hetastarch animals compared to
the pentastarch animals, but both were more significant than in the
controls. This is a reflection of compromise in oxygen delivery
during hemorrhage in the rank hetastarch>pentastarch>Control.
Though the practice of the present invention does not require an
understanding of the mechanism, it is probable that both starch
products reduce the hemoglobin significantly compared to the
control, explaining why both seem to stress the animals more than
the controls. Of pentastarch and hetastarch, however, pentastarch
affords better compensation to hemorrhage, most likely because of
its better plasma expanding ability.
Acid-Base Balance and Lactic Acid
[0335] Regarding pH and base excess, the most significant
compromise during hemorrhage was seen in the hetastarch animals,
which exhibited dramatic drops in pH and base excess (data not
shown). The pentastarch animals were slightly more compromised
compared to controls. It is noteworthy that the controls actually
seemed to compensate fairly adequately to the 60% hemorrhage;
specifically, although PCO.sub.2 fell and base excess became more
negative, the animals were able to maintain their pH essentially
constant.
[0336] Lactic acid is an accurate indicator of tissue oxygenation.
The lactic acid accumulation in the hetastarch animals was
significantly greater than in the pentastarch animals, and both
groups accumulated more lactic acid than the controls (data now
shown). Of note, the lactic acid level plateaued in the controls,
suggesting that the rate of production and clearance is equal,
another indication of adequate compensation to the hemorrhage.
[0337] The results of this example show that in the
exchange-transfusion/hemorrhage model utilized, all of the control
animals were dead by approximately 130 minutes after start of the
hemorrhage. Thus, any perturbation in the oxygen transport system
was reflected in a number of measured variables. The results
indicate that neither pentastarch or hetastarch was able to
compensate for loss of half of the circulating blood volume.
However, comparison of the two plasma expanders reveals that
pentastarch is clearly superior to hetastarch. Though the rationale
for this finding is not required in order to practice the
invention, it is believed to be due to the higher oncotic pressure
of pentastarch, which thus affords more significant plasma volume
expansion in the pentastarch animals compared to the hetastarch
group.
EXAMPLE 12
Blood Product Mixtures of Pentastarch and DBBF
[0338] This example specifically compares animal survival and
physiological data following exchange transfusions and hemorrhage
with a blood product mixture (50:50) of pentastarch and DBBF
(.alpha..alpha.-Hb). The experiments were performed as described in
Example 10.
Animal Survival
[0339] Animal survival of the control animals, pentastarch (PS)
alone animals, DBBF (.alpha..alpha. hemoglobin) alone animals, and
animals administered a pentastarch+.alpha..alpha.-Hb mixture is
shown in FIG. 11. Referring to FIG. 11, (.tangle-solidup.)
represents pentastarch, (.box-solid.) represents .alpha..alpha.-Hb,
and (.quadrature.) represents pentastarch+.alpha..alpha.-Hb. As
indicated in FIG. 11, survival of the .alpha..alpha.-Hb animals is
significantly worse than either the controls or the pentastarch
animals, and a mixture of 50/50 .alpha..alpha.-Hb and pentastarch
is even worse. It should also be noted that there was no obvious
relationship between survival and hematocrit (see Table 16, supra)
or hemoglobin (see Table 17, supra), so survival does not appear to
be a linear function of the oxygen carried in the blood.
Mean Arterial Pressure
[0340] Mean arterial pressure rose in the PS/.alpha..alpha.-Hb
animals and the .alpha..alpha.-Hb animals (data not shown).
Moreover, even though hemoglobin dose was half in the
PS/.alpha..alpha.-Hb animals, the magnitude of the blood pressure
rise was the same. Thus, the presence of PS did not attenuate the
hemoglobin-induced hypertension of approximately 20 mm Hg. The fall
in blood pressure, however, after starting the hemorrhage, was more
abrupt in the PS/.alpha..alpha.-Hb animals than in any of the other
3 groups. The recovery was somewhat faster, possibly due to the
plasma expansion afforded by the presence of pentastarch.
Nevertheless, when blood pressure began to fall terminally, it fell
very fast, and animals rapidly died. Thus, the rise in blood
pressure resulting from the presence of .alpha..alpha.-Hb
hemoglobin does not appear to confer any advantage, and the
presence of PS does not attenuate this effect.
Heart Rate
[0341] In control animals, heart rate gradually increased after
start of the hemorrhage (data not shown). Though the present
invention does not require an understanding of the underlying
mechanism of this effect, it is most likely due to loss of
intravascular volume. This interpretation is supported by the
somewhat lower heart rate response seen in the pentastarch animals
who, in spite of a lower hemoglobin concentration, did not raise
their heart rate to the same degree (data not shown). A different
pattern of heart rate response was seen in the .alpha..alpha.-Hb
animals; more specifically, there was an immediate drop in heart
rate after starting the exchange transfusion, followed by a gradual
rise after starting the hemorrhage (data not shown). The drop
cannot be explained by volume changes, since a contraction of the
plasma volume would be expected to raise the heart rate, not lower
it. More likely, this a direct chronotropic effect on the
myocardium. Of note, this depressant effect is lessened when the
.alpha..alpha.-Hb is diluted with pentastarch (data not shown). The
PS/.alpha..alpha.-Hb animals exhibited a brisk rise in heart rate
after starting hemorrhage, rapidly reaching approximately 500/min,
a rate not reached in the other groups until a later time. Thus,
the PS/.alpha..alpha.-Hb mixture did not seem to offer any
advantage over .alpha..alpha.-Hb alone.
dP/dt
[0342] As previously set forth, the dP/dt is the maximum positive
slope of the pulse pressure contour. This parameter is proportional
to the onset of the systolic contraction, and is therefore a
reflection of the strength, or inotropic action of the heart. In
the control animals, dP/dt rose after onset of hemorrhage (data not
shown). The pentastarch animals showed the same pattern, although
the magnitude of the response was less, presumably because these
animals had a somewhat increased vascular volume compared to the
controls at the onset of hemorrhage. The .alpha..alpha.-Hb animals
never increased their dP/dt (data not shown); in fact, the value
dropped rapidly after the onset of hemorrhage, suggesting that one
of the normal compensatory mechanisms is disordered. The same
observation was made in the PS/.alpha..alpha.-Hb animals, even
though they were expected to have a somewhat greater vascular
volume than the .alpha..alpha.-Hb animals by virtue of the presence
of oncotically-active pentastarch.
Ventilation
[0343] When oxygen transport is diminished, either because of
anemia or hypoxia, a normal physiologic response is to
hyperventilate. The result of hyperventilation is a drop in
PCO.sub.2, since the elimination of CO.sub.2 by the lung is a
direct function of ventilation. A reciprocal effect is increased
PO.sub.2, again, because of the greater minute volume of gas being
exchanged by the lung. In the control animals, PCO.sub.2 dropped
after the onset of hemorrhage (data not shown); by comparison, the
pentastarch animals also lowered their PCO.sub.2 (data not shown),
but the effect persisted for a longer period of time and appeared
to be more pronounced, probably as a result of the lower hemoglobin
concentration in the pentastarch animals compared to the controls.
(See Table 17). The PCO.sub.2 drop was significantly greater in the
.alpha..alpha.-Hb animals, and still greater in the
PS/.alpha..alpha.-Hb animals. Comparison of the .alpha..alpha.-Hb
and PS/.alpha..alpha.-Hb animals is interesting, since the former
has a higher total hemoglobin concentration, but a lower blood
volume. Thus, the addition of PS to the .alpha..alpha.-Hb did not
confer any advantage on the animals and, in fact, appears to have
induced greater hyperventilation.
[0344] The PO.sub.2 changes are the mirror image of the PCO.sub.2
response; the greatest rise in PO.sub.2 (and drop in PCO.sub.2) was
seen in the .alpha..alpha.-Hb and PS/.alpha..alpha.-Hb animals,
while the controls and pentastarch animals had the smallest
increase in PO.sub.2 (data not shown). Thus, the data are
consistent with the belief that reduced oxygen delivery leads to
hyperventilation, and the degree of hyperventilation correlates
with overall survival.
Acid-Base Status
[0345] As hemorrhage progresses and the delivery of oxygen to
tissues becomes compromised, lactic acid is produced and pH drops.
For the control animals, pH was maintained nearly constant as
hemorrhage progressed. Another index of the degree of compensation
is the base excess, which is defined as the amount of base that
would be required to return plasma pH to 7.4 in the presence of a
PCO.sub.2 of 40 Torr. In the case of both the controls and PS
animals, base excess was not significantly changed from baseline
(data not shown). In contrast, .alpha..alpha.-Hb and, especially,
PS/.alpha..alpha.-Hb produced a marked drop in pH which is not
compensated by the brisk hyperventilation (data not shown), and the
result was a dramatic drop in base excess (i.e., a "base deficit"
results). By usual clinical standards, a base excess of -10 mEq/L
or less is indicative of poor recovery from hemorrhagic shock.
[0346] Finally, lactic acid is a direct measure of the degree of
insufficient delivery of oxygen to tissues (i.e., the "oxygen
debt"). The accumulation of lactic acid was very significant in
both the .alpha..alpha.-Hb and PS/.alpha..alpha.-Hb animals, the
latter rising even more sharply than the former (data not shown).
It is also of interest that in the controls and pentastarch
animals, there was a rather more modest rise in lactate which then
seemed to plateau, as the animals' compensatory mechanisms
(increased cardiac output, ventilation) seemed to compensate for
the blood loss. However, the continued linear rise of lactic acid
in both the .alpha..alpha.-Hb and PS/.alpha..alpha.-Hb animals
indicated progressive, uncontrolled tissue acidosis.
[0347] The results discussed above indicate that the use of blood
product mixtures comprising .alpha..alpha.-Hb as the
oxygen-carrying component, even though it provides some plasma
hemoglobin, rendered the animals in a more vulnerable position with
regard to hemorrhage than either the controls or the animals
hemodiluted with pentastarch. The addition of pentastarch to
.alpha..alpha.-Hb did not compensate for the detrimental effects of
.alpha..alpha.-Hb and, in fact, worsened oxygen delivery, acidosis
and overall survival.
EXAMPLE 13
Blood Product Mixtures of Hemolink.RTM./Pentastarch and
Hemolink.RTM./Hetastarch
[0348] Example 8 compared the effects of pentastarch,
Hemolink.RTM., and a mixture thereof. This example compares a
mixture of Hemolink.RTM./pentastarch with a mixture of
Hemolink.RTM. and another non-oxygen-carrying component,
hetastarch. The experiments of this example, performed as described
in Example 10, specifically compare animal survival and
physiological data following exchange transfusions and
hemorrhage.
[0349] As previously indicated, Hemolink.RTM. (Hemosol) is a
polymerized human hemoglobin that has a mean molecular weight of
approximately 128,000 Da. Since Hemolink.RTM. is a polymerized
product, an array of molecular sizes is present in the final
product. When tested alone, hemodilution with Hemolink.RTM. did not
perform as well as pentastarch, and animals died sooner than those
in the control or pentastarch groups. (See, e.g., Example 8). In
view of the surprising and positive results with a mixture of
pentastarch and PEGHb, additional experiments involving mixtures
(50/50) of Hemolink.RTM. and a non-oxygen-carrying components
(hetastarch or pentastarch) were performed in this example.
Animal Survival
[0350] As shown in FIG. 12, exchange transfusion with Hemolink.RTM.
alone reduced survival from a 60% hemorrhage. More specifically,
FIG. 12 depicts animal survival following exchange transfusion with
hetastarch (x), Hemolink.RTM. (.tangle-soliddn.),
Hemolink.RTM.+pentastarch (.gradient.), and
hetastarch+Hemolink.RTM. (.diamond.) and after the initiation of a
60% hemorrhage.
[0351] The post-exchange hematocrit in the Hemolink.RTM. animals
(Table 16, supra) was about half that of controls, and slightly
lower than the pentastarch, DBBF (.alpha..alpha.-Hb), or PS/DBBF
animals. However plasma hemoglobins were slightly higher in the
Hemolink.RTM. animals than in these other groups (Table 16, supra).
FIG. 12 shows that no combination of Hemolink.RTM. and either
pentastarch or hetastarch was as effective (as measured by
short-term survival) as the control animals. However, in contrast
to the situation with DBBF and pentastarch described in Example 12,
dilution of Hemolink.RTM. with either pentastarch or hetastarch did
not worsen survival.
Mean Arterial Pressure
[0352] Exchange transfusion with Hemolink.RTM. raised mean arterial
blood pressure slightly (data not shown), but not as significantly
as DBBF (.alpha..alpha.-Hb) (Example 12). When the arterial
hemorrhage was begun, blood pressure in all four groups (i.e.,
Hemolink.RTM. alone; Hemolink.RTM./pentastarch;
Hemolink.RTM./hetastarch; and control) of animals fell abruptly
(data not shown). The degree of initial fall in blood pressure was
greatest in the Hemolink.RTM./hetastarch group (120 to 50 mm Hg)
compared to 110 to 80 mm Hg for the controls, 120 to 90 mm Hg for
the Hemolink.RTM./pentastarch animals, and 120 to 80 mm Hg for the
Hemolink.RTM. alone animals. Thus, as judged by the fall in blood
pressure and overall survival, the Hemolink.RTM./hetastarch
animals, Hemolink.RTM., and Hemolink.RTM./pentaspan animals (in
that order) all seemed to be worse than the controls. Nevertheless,
overall survival for the Hemolink.RTM./hetastarch and
Hemolink.RTM./pentaspan animals was not different (Table 15, supra)
and only marginally better than the Hemolink.RTM. alone
animals.
Heart Rate
[0353] The Hemolink.RTM. and Hemolink.RTM./pentaspan animals both
raised their heart rates in response to the hemorrhage, but the
rise was earlier and steeper than in the controls (data not shown);
this indicates less cardiovascular stability in the
exchange-transfused animals compared to the controls. Surprisingly,
the Hemolink.RTM./hetastarch animals dropped heart rate abruptly
after starting the hemorrhage; this abnormal response might have
indicated severe compromise in these animals compared to the other
groups.
dP/dt
[0354] An increase in dP/dt was observed in the Hemolink.RTM.
animals after exchange transfusion compared to the controls (data
not shown), indicating that the exchange by itself conferred
instability on the cardiovascular system. The
pentastarch/Hemolink.RTM. animals demonstrated little, if any,
increase in dP/dt, whereas the response in the
hetastarch/Hemolink.RTM. animals was striking, increasing abruptly
after initiating the hemorrhage, reaching a peak value of nearly
2000 mm Hg/sec, and then rapidly falling as animals became severely
compromised and died (data not shown).
Ventilation and Acid Base Status
[0355] The rise in PO.sub.2 and fall in PCO.sub.2 observed in each
of the three experimental groups was greater than the control
values, but no distinction can be made between those groups (data
not shown).
[0356] All experimental groups demonstrates lower pH during the
hemorrhage than the control group. The acid-base disturbance was
more clearly shown in the base excess, as all three experimental
groups become severely acidotic (negative base excess) beginning
abruptly after start of the hemorrhage. Finally, lactic acid
increase was very significant in all three experimental groups,
again confirming the presence of severe acidosis (data not
shown).
[0357] Previously it was shown that Hemolink.RTM. did not perform
as well as the controls or as well as pentastarch alone; moreover,
Hemolink.RTM. alone and hetastarch alone performed comparably, but
neither afforded as much protection as pentastarch alone. As set
forth in this example, attempts to improve the performance of
Hemolink.RTM. by mixing it with either pentastarch or hetastarch
did not improve the results.
EXAMPLE 14
Blood Product Mixtures of TM Hemoglobin/Pentastarch
[0358] This example specifically compares animal survival and
physiological data following exchange transfusions and hemorrhage
with a blood product mixture (50:50) of pentastarch and TM
hemoglobin; trimesic acid (TM) is used to crosslink hemoglobin. The
experiments were performed as described in Example 10.
Animal Survival
[0359] TM hemoglobin (Hemosol) is a human-hemoglobin derived
product of molecular weight approximately 64,000 Da. It has a
relatively low oxygen affinity (P.sub.50 about 35 Torr). Studies
using TM hemoglobin alone were not significantly different from
those with DBBF (.alpha..alpha.-Hb) alone. (See Example 12). All
animals that received TM hemoglobin alone or in combination with
pentastarch died within 60 minutes after start of hemorrhage (only
one animal was tested using a mixture of TM hemoglobin and
pentastarch, and it died at 60 minutes following initiation of
hemorrhage).
Mean Arterial Pressure, Heart Rate and dP/dt
[0360] Exchange transfusion resulted in a moderate rise in mean
arterial blood pressure of the single animal tested using TM
hemoglobin/pentastarch. Pressure abruptly fell after start of the
hemorrhage, but then recovered rather quickly; however as the
hemorrhage progressed, when the mean arterial pressure began to
fall again, the animal died very suddenly (data not shown).
[0361] There was a slight fall in heart rate after the exchange
transfusion with either TM hemoglobin or the TM
hemoglobin/pentastarch mixture. After a delay of about 20 minutes,
the heart rate rose during hemorrhage in both groups.
[0362] Regarding the dP/dt, in contrast to many of the other
hemoglobin preparations, TM hemoglobin/pentastarch or TM hemoglobin
alone did not lead to an increase in dP/dt. Rather, a steady fall
occurred starting after the hemorrhage was initiated (data not
shown).
Ventilation and Acid-Base Status
[0363] As noted in previous examples, PO.sub.2 and PCO.sub.2 change
in mirror image, reflecting the hyperventilation that accompanies
diminished oxygen transport as hemorrhage progresses. The rise in
PO.sub.2 and fall in PCO.sub.2 observed in both of the experimental
groups was greater than the control values (no distinction can be
made between those groups; data not shown).
[0364] In regards to arterial pH acid and base, both experimental
groups demonstrated lower pH during the hemorrhage than the control
group; base excess determinations showed that both experimental
groups became severely acidotic (negative base excess) beginning
abruptly after start of the hemorrhage (data not shown). Finally,
lactic acid increase was very significant in both experimental
groups (data not shown), again confirming the presence of severe
acidosis.
[0365] As previously indicated (see Table 15), TM hemoglobin did
not perform as well as the controls or as well as pentastarch. TM
hemoglobin and pentastarch/TM hemoglobin performed comparably, but
neither afforded as much protection as pentastarch alone. The
attempts to improve the performance of TM hemoglobin by mixing it
with pentastarch, reported in this example, did not improve the
results. TM hemoglobin has a low O.sub.2 affinity compared to other
hemoglobin derivatives studied, and the results reported above
indicate that this low affinity did not confer advantage over other
cross-linked hemoglobins whose other physical properties are the
same (e.g., DBBF).
EXAMPLE 15
Modified Hemoglobins
[0366] As set forth above, mixtures of polyethylene glycol-modified
bovine hemoglobin and pentastarch lead to increased animal survival
when compared to mixtures comprising other non-oxygen-carrying
components. In order to determine whether these results were due to
the mixture or to the bovine hemoglobin itself, an experiment was
performed evaluating purified bovine hemoglobin. In addition,
experiments were performed with .beta.82 Hemoglobin, a product
which has a high oxygen affinity, to determine whether this product
alone might be superior to the mixtures of an oxygen-carrying
component and a non-oxygen-carrying component contemplated for use
with the present invention. As with the previous examples, the
experiments of this example specifically compare animal survival
and physiological data following exchange transfusions and
hemorrhage using the experimental protocol described in Example
10.
Bovine Hemoglobin
[0367] Briefly, when the animal was exchange-transfused with bovine
hemoglobin, there was only a transient rise in mean arterial blood
pressure, followed by a steady fall (data not shown). When the
hemorrhage started, mean arterial pressure fell precipitously, and
the animal died approximately 30 minutes after start of the
hemorrhage (data not shown).
[0368] The heart rate in this animal did not rise significantly
when hemorrhage started but there was a modest rise terminally, a
few minutes before the animal died. The dP/dt remained constant, in
contrast to controls in which this parameter always rose in
response to hemorrhage. Finally, regarding pH and acid-base status,
the animal severely hyperventilated, as indicated by a rise in
PO.sub.2 and a drop in PCO.sub.2. Accordingly, there was a very
precipitous fall in pH and base excess and a sharp rise in lactic
acid (data not shown).
.beta.82 Hemoglobin
[0369] .beta.82 Hemoglobin (Hemosol) is a derivative of human
hemoglobin that is crosslinked between the .beta. chains (in
contrast to DBBF [.alpha..alpha.-Hb]). This product has high oxygen
affinity, but low viscosity and oncotic pressure.
[0370] When animals were exchange-transfused with .beta.82
Hemoglobin, there was a very transient, but pronounced, rise in
blood pressure; the magnitude of the rise was approximately the
same as that seen with .alpha..alpha.-Hb, but the mean arterial
pressure rapidly returned to the pre-infusion level (data not
shown). When hemorrhage began, blood pressure rapidly fell, and
animals died by approximately 70 minutes. Thus, overall survival
was not better than .alpha..alpha.-Hb hemoglobin, and less than
either the controls or the pentastarch animals.
[0371] Heart rate did not rise significantly either after exchange
or after hemorrhage, nor did dP/dt. The animals did have pronounced
hyperventilation (increase in PO.sub.2 and fall in PCO.sub.2).
Severe acidosis was shown by a dramatic drop in pH, base excess,
and rise in lactic acid (data not shown).
[0372] Thus, the experiments with the modified hemoglobin products
of this example did not lead to superior results than those
obtained when mixtures of pentaspan and PEGHb were employed.
EXAMPLE 16
Evaluation of Various Hemoglobin Solutions
[0373] In this Example, three hemoglobin solutions were evaluated
(See, Table 19), including: 1) Purified human hemoglobin A.sub.0
(Hb-A.sub.0); 2) .alpha..alpha.-hemoglobin, human hemoglobin
cross-linked with bis(3,5-dibromo salicyl)fumarate; 3)
PEG-hemoglobin, bovine hemoglobin surface-modified with
polyethylene glycol. The PEG units have a molecular weight of 5,000
Da.
[0374] Human red blood cells were drawn from healthy volunteers
into heparin anticoagulant, washed 3 times in 0.9% NaCl by gentle
centrifugation, and resuspended in 0.1 M Bis-tris Cl buffer, pH
7.4. The hemoglobin concentration of all solutions and red cell
suspensions was approximately 3 mM (heme). The methemoglobin was
always less than 2-4% of total hemoglobin. The test solutions were
equilibrated to the appropriate gas concentrations and 37.degree.
C. using a tonometer (e.g., model 2000, Instrumentation
Laboratories, Lexington, Mass.). Human serum albumin (HSA) was
purchased commercially.
[0375] The test methods used included the following protocols, the
results of which are shown in Table 19. While this Example provides
methods to determine various characteristics of a test preparation,
it is not intended that the present invention be limited to these
particular protocols. Indeed, those of skill in the art know
additional methods that would be suitable for making these
determinations.
[0376] Oxygen Equilibrium Binding Curves:
[0377] Cell-free hemoglobin-oxygen equilibrium curves were measured
by coupling diode array spectrophotometry with enzymatic
deoxygenation of oxyhemoglobin solutions (Vandegriff et al., Anal.
Biochem., 256:107-116 [1998]). The protocatechuic acid
(PCA)/protocatechuic acid 3,4-dioxygenase (PCD) enzyme system
consumes one mole of O.sub.2 for each mole of PCA converted to
product.
[0378] Reactions were carried out 0.1 M bis-Tris propane (Sigma),
0.1 M Cl.sup.-, and 1 mM EDTA at pH 7.4 and 37.degree. C.
Hemoglobin samples were diluted to a concentration of approximately
60 .mu.M (in heme) in air-equilibrated, temperature-equilibrated
buffer containing a small amount of catalase (e.g., 0.2 to 0.5
.mu.M). The final hemoglobin concentration was determined by the
extinction coefficient at 523 nm (.epsilon..sub.523=7.12.sup.-1
mM). Substrate (PCA) was added at a concentration of 1 mM. A volume
of this reaction solution was used to completely fill the reaction
cell to eliminate any gas phase present prior to addition of
enzyme. The cuvette was sealed using a gas-tight teflon stopper
fitted with a micro-oxygen electrode (Microelectrodes, Inc.,
Londonderry, N.H.) inserted through an o-ring imbedded in the
stopper. The electrode was immersed in the solution to a position
just above the light path of a Milton Roy 3000 diode array
spectrophotometer (SLM Instruments, Inc., Urbana, Ill.). The
temperature was controlled using a Peltier controller in the
reaction cell holder, and the solution was mixed using a micro-stir
bar spun by a stirring motor in the reaction cell holder. The
deoxygenation reaction was initiated by addition of enzyme (PCD)
(0.05 to 0.1 units/ml).
[0379] The spectral change of hemoglobin during enzymatic
deoxygenation was measured in the visible range from 480 to 650 nm
at every .about.0.35 nm. Polarographic determination of PO.sub.2
was measured using a Clark-type oxygen electrode, giving a voltage
change in proportion to the change in oxygen concentration. The
electrode was calibrated each day by immersing the electrode in
water bubbled either with air to determine the 100%-air voltage or
with pure N.sub.2 to set the zero point. During the hemoglobin
desaturation reaction, voltages from the O.sub.2 electrode were
collected at a sampling rate of 10 Hz.
[0380] The spectral and O.sub.2-electrode-voltage data were
converted into files for analysis using the MATLAB
technical-computing program (The Mathworks, Natick, Mass.). The
voltage output from the O.sub.2 electrode was converted to mm Hg
based on the barometric pressure and the water vapor pressure at
the temperature of the experiment. An average PO.sub.2 value was
calculated from 50 data points during each 5-second interval that
corresponds in time to the collection of each spectrum.
[0381] The spectral matrix was analyzed using a multicomponent
decomposition algorithm. The program returns the fractions of each
base spectrum (oxy-, deoxy-, and methemoglobin) which combined from
the measured spectrum being evaluated. Fractional saturation was
calculated as the ratio of oxyhemoglobin to the total of oxy-plus
deoxyhemoglobin. Fitted values for the Adair constants
a.sub.1-a.sub.4) were determined by least-squares analysis with
uniform weighting. Values for P50 and the Hill coefficient(n) were
calculated from the fitted Adair constants (i.e., the values shown
in Table 19).
[0382] Oxygen equilibrium curves for red blood cell suspensions
were measured at pH 7.4 and 37.degree. C. by the gas exchange
method using a Hemox-Analyzer.RTM. (TCS Medical Products,
Huntingdon Valley, Pa.).
[0383] COP
[0384] COP was measured using a Wescor 4420 colloid osmometer
(Logan, Utah) with a 30,000 molecular weight cut-off membrane. The
osmometer was calibrated prior to measurement of each hemoglobin
sample with 5% albumin as recommended by the manufacturer.
Measurements were performed at room temperature, which ranged from
20-23.degree. C. Values reported in Table 19 are for hemoglobin
concentrations of 5 g/dl.
[0385] Viscosity
[0386] Viscosity measurements are performed using a capillary
viscometer (Reinhardt, 1984). The device uses the Hagen-Poiseuille
law as its operating principle which defines flow (Q) in terms of
capillary radius (r), pressure change along the capillary (dP/dx)
and viscosity (.eta.).
Q=(.pi.r.sup.4 dP)/(8 .eta.dx)
[0387] This expression can be separated into two components, the
shear stress (L is the capillary length) and the shear rate, where
the shear stress and rate are:
[0388] Shear Stress=(r/2.DELTA.L).DELTA.P
[0389] Shear Rate=(4/.pi.r.sup.3)Q
[0390] Viscosity .eta.=Shear Stress/Shear Rate
[0391] Based on the geometry of the capillary, all parameters were
known, except .DELTA.P and Q. Thus, these were the two variables
measured. Fluid was placed in the syringe pump (Harvard Apparatus,
model 975, S. Natick, Ma.) and flow started. A differential
pressure transducer (Validyne Engineering, model MP-45-14,
Northridge, Calif.) was connected to the ends of a 10 cm glass
capillary tube with an inside diameter of 508 .mu.m (Vitro
Dynamics, Rockway, N.J.) through a T valve. As fluid was driven
through the tube, the transducer sensed the pressure at each T
valve point. The transducer was arranged so that the output is the
.DELTA.P between the two T valve points. The signal was amplified
(Validyne model CD12) and recorded on a strip chart.
[0392] Flow (Q) was measured by use of a calibrated flow tube.
Viscosity was calculated from .DELTA.P and Q. The capillary
viscometer was both statically and dynamically calibrated, while
the pressure transducer was calibrated statically with a head
pressure of saline; a dynamic calibration was accomplished with
water. The solutions were heated to 37.degree. C. and placed in the
viscometer. Measurements in the example used a shear rate of 160
s.sup.-1. The values reported in Table 19 represent the
measurements for hemoglobin concentrations of 5 g/dl.
19TABLE 19 Properties Of The Test Solutions RBC Ao PEG-Hb
.alpha..alpha.-Hb a.sub.1 1.48 .times. 10.sup.-2 4.01 .+-. 0.82
.times. 10.sup.-2 1.47 .+-. 0.39 .times. 10.sup.-1 2.22 .+-. 0.26
.times. 10.sup.-2 a.sub.2 8.53 .times. 10.sup.-4 1.74 .+-. 0.44
.times. 10.sup.-3 4.27 .+-. 0.20 .times. 10.sup.-2 9.51 .+-. 0.19
.times. 10.sup.-4 a.sub.3 4.95 .times. 10.sup.-8 5.95 .+-. 5.95
.times. 10.sup.-13 2.43 .+-. 1.91 .times. 10.sup.-4 1.34 .+-. 0.69
.times. 10.sup.-11 a.sub.4 1.07 .times. 10.sup.-6 2.48 .+-. 0.57
.times. 10.sup.-5 1.48 .+-. 0.13 .times. 10.sup.-4 1.05 .+-. 0.13
.times. 10.sup.-6 P50 (mmHg) 32.8 15.1 10.2 33.8 n 2.59 2.97 1.38
2.43 viscosity 1.4 0.9 3.4 0.9 (cp) (5 g/dl) COP -- 14 79 11 (mm
Hg) (5 g/dl) Radius (nm) -- 2.7.sup.(1) 14.1.sup.(1) 3.1.sup.(1)
.sup.(1)Vandegriff et al., Biophys. Chem., 69: 23-30 [1997].
[0393] Artificial Capillary Experiments
[0394] Exit PO.sub.2 values versus residence times are shown in
FIG. 15. At any given flow rate, the lowest exit PO.sub.2 value is
seen for Hb-A.sub.0 followed by PEG-Hb, .alpha..alpha.-Hb, and RBCs
with the highest exit PO.sub.2 values. The final saturation of
hemoglobin in the artificial capillary (FIG. 6) was calculated from
the Adair constants given in Table 19. PEG-Hb showed the least
desaturation over time at any flow rate. This was closely
paralleled by the RBC profile. Hb-A.sub.0 and .alpha..alpha.-Hb
both showed much greater degrees of desaturation.
[0395] The finite element analysis adjusts values for the lumped
diffusion parameter, K*, until the exit PO.sub.2 equals the
experimental value. The final fitted values for K* as a function of
residence time are shown in FIG. 17. PEG-Hb and RBCs gave similar
values for K* from 900-1200 .mu.M/min/Torr. The K* values for
Hb-A.sub.0 and .alpha..alpha.-Hb are higher than for RBCs because
of the absence of intraluminal resistances for cell-free solutions.
This effect is negated in the cell-free PEG-Hb solution, which has
a K* value equal to that for RBCs at the fastest flow rate and
which is only slightly higher than RBCs at the slowest flow rate.
This is due to at least two physical properties of the PEG-Hb
solutions (See Equation 2, above): (1) its higher viscosity
compared with the tetrameric solutions, due to its larger molecular
size; and (2) its high O.sub.2 affinity.
[0396] Animal Experiments
[0397] Male Sprague-Dawley rats (210-350 g, Charles River Labs)
were anesthetized with 250 .mu.l of a mixture of ketamine (71
mg/ml), acepromazine (2.85 mg/ml), and xylazine (2.85 mg/ml).
Polyethylene catheters (PE-50) were placed into the abdominal aorta
via the femoral artery to allow rapid withdrawal of arterial blood.
A second catheter was placed in the contralateral femoral artery to
monitor blood pressure, and a third catheter was placed in one of
the femoral veins for infusion of test materials. Catheters were
tunneled subcutaneously, exteriorized through the tail, and flushed
with approximately 100 .mu.L of normal saline. Animals were allowed
to recover from the procedure and remained in their cages for 24
hours before being used in experiments. One femoral artery catheter
was connected, through a stopcock, to a pressure transducer (UFI
model 1050, Morro, Calif.), and arterial pressure was sampled
continuously at 100 Hz using a MP100WSW data collection system
(BIOPAC Systems, Inc., Goleta, Calif.). The data were stored in
digital form for subsequent off-line analysis.
[0398] Mean arterial pressures before and during the exchange
transfusion are shown in FIG. 18. All solutions demonstrated
significant vasoactivity except the PEG-hemoglobin, whose K* value
is essentially identical to that of red blood cells (see FIG.
17).
[0399] Based on the data obtained in these experiments, it is
contemplated that autoregulation occurs as a result of oversupply
of oxygen due to facilitated diffusion by cell-free oxygen
carriers. The amount of O.sub.2 delivered should be the greatest
for those solutions that show the greatest vasoactivity. In vivo
experiments of 50% exchange transfusion in a rat are consistent
with this theory in that the increase in mean arterial pressure
corresponds roughly with the estimated diffusion constant, K*. Thus
K* appears to be the key parameter to use to optimize the
characteristics of a potential red cell substitute.
EXAMPLE 17
Other Hemoglobin Preparations
[0400] In this Example, additional hemoglobin preparations are
described. These preparations may be modified to provide blood
substitutes with the desirable properties of high oxygen affinity,
high oncotic pressure, and relatively high viscosity (i.e., at
least half that of blood).
[0401] A. Preparation of Human Hemoglobin A.sub.0
[0402] In this experiment, the human hemoglobin A.sub.0 of
Christensen et al. (Christensen et al., J. Biochem. Biophys. Meth.
17: 143-154 [1988]) is prepared.
[0403] One unit of outdated, packed cells is washed three times in
500 ml plastic centrifuge bottles with sterile 0.9% saline. The
wash solution and the buffy coat are removed with aspiration. The
packed cells are mixed with 2.5 volumes of distilled water and
centrifuged at 20,000.times.g for 1 hour. The supernatant is
removed and passed through a mixed-bed ion-exchange resin (Bio-Rex
RG501-X8, Bio-Rad, Richmond, Calif.) in a column. The iso-ionic
effluent is passed through 0.22 .mu.m filters (Millipore Millistack
40, Bedford, Mass.) into sterile containers.
[0404] For larger quantities of stroma-free hemoglobin: 8 units of
packed cells are washed as above and hemolysis occurs in the cold,
overnight. The lysate is transferred into 600 ml transfer packs
(Fenwal 4B2024, Deerfield, Ill.) and spun for 6 hours at
3500.times.g. Approximately {fraction (1/3)} of the supernatant
hemoglobin solution is then removed with a plasma extractor and
passed through the mixed-bed resin until the conductivity is
.about.15 .mu.mhos. This process requires 1 kg of resin which is
most conveniently packed into three columns. The solutions are
again placed into transfer packs and centrifuged for 4 hours at
3500.times.g. The supernatants are filtered through a 0.22 .mu.m
disposable filter unit (Millipore Millistack, MSG05CHZ), and the
filtrate is collected into sterile transfer packs and stored at 4 C
for chromatography. Long-term storage is best achieved by freezing,
in bulk, at -80.degree. C.
[0405] Stroma-free hemoglobin solutions containing 10-20 g of
hemoglobin are equilibrated with 0.05 M Tris-HCl at pH 8.5. This
can be done as usual by dialysis or by buffer exchange and gel
exclusion columns. However, since these solutions are isoionic, it
is more convenient to merely dilute them with an equal volume of
0.1 M Tris-HCl at pH 8.5. Chromatography is performed with a
preparative HPLC (Waters Delta-Prep 3000). The sample
(.about.250-500 ml, 4-10 g/dl) is applied to a stainless steel
column prepacked with QMA-Acell (Waters), previously equilibrated
with 1-2 liters of buffer A (0.05 M Tris-HCl, pH 8.5) at a flow
rate of 80 ml/min. The chromatogram is developed with a linear
gradient of 0.05 M Tris-HCl, pH 6.5, as the reserve (buffer B) at
the same flow rate. The pH change is linear from 10% to 90% buffer
B, during which all hemoglobin species are eluted. Separations are
complete in 50 minutes at which time the pH of the effluent buffer
is 7.2. Buffer B is the run for an additional 10 minutes to insure
complete elution of the samples, and the column is re-equilibrated
with 1 liter buffer A preparatory to a subsequent separation. It is
possible to process up to 20 g on one column; however, this appears
to be an overload. The column is purged daily with 1 liter of 0.1 M
Tris-HCl, pH 7.4, in 0.1 M NaCl. On standing, it is equilibrated
with 70% ethanol.
[0406] Peak detection using the preparative cell (2.1 mm
pathlength) is at 510 nm and/or 600 nm. The latter wavelength is
necessary for the higher concentrations and to amplify the signal
due to methemoglobin. The major fraction of Hb-A.sub.0 is collected
to avoid the collection of methemoglobin at the leading edge and
the contamination of the minor hemoglobin components at the
trailing edge. The fraction is collected into a 2 liter sterile
transfer pack placed in an ice bucket and transferred aseptically
into a sterile 2 liter Amicon concentrator (Model 2000B, Danvers,
Mass.) equipped with a TMIO membrane (10,000 Da cut-off) filter and
the volume reduced to about 10% of the eluate volume at 4.degree.
C.
[0407] B. Cross-Linking Reactions to Lower P50
[0408] In this experiment, various cross-linking methods are tested
for their ability to lower P50. In one experiment, the method of
Walder et al. (Walder et al. Biochem., 18:4265-4270 [1979]) to
produce bis(3,5-dibromosalicyl) fumarate (DBBF) and
bis(3,5-dibromosalicyl) succinate (DBBS) (.beta.82-.beta.82) is
used. Chemical modifications of human hemoglobin are carried out in
6 g/dl solutions of cell-free oxyhemoglobin in 0.05 M sodium
phosphate, or in 0.05 M Bistris-HCl, pH 7.2. Incubations are for 2
hours at 37.degree. C. in a water bath shaker. Reactions are
terminated by quenching with glycine.
[0409] In another experiment, the method of Manning and Manning
(Manning and Manning Biochem., 27:6640-6644 [1988]), in which
hemoglobin in the R state is cross-linked with glycolaldehyde. In
this experiment, the hemoglobin concentration varied from 45 to 360
.mu.M in 50 mM potassium phosphate buffer, pH 7.3. HbCO is used.
Glycolaldehyde is added to a final concentration of 50 mM. The
cross-linking is performed at room temperature for 4.5 hours, and
the hemoglobin derivative is then dialyzed extensively against 50
mM Tris-acetate, pH 7.3.
[0410] In yet another experiment, diisothiocyanatobenzenesulfonate
(DIBS) is used to cross-link hemoglobin, according to the method of
Manning et al. (Manning et al., PNAS 88:3329-3333 [1991]).
Hemoglobin solutions (200 .mu.M in the deoxygenated state, usually
3-5 .mu.moles) are treated with a 10-fold molar excess of the
crosslinking agent DIBS. The solution is incubated at 25.degree. C.
in 0.1 M potassium phosphate, pH 7.2, for 15 min. The reaction is
terminated by adding glycylglycine in 30-fold molar excess; a
further incubation for 15 min. is then performed. The solution is
dialyzed at 4.degree. C. against the buffer used for the subsequent
chromatographic step. The crosslinked hemoglobin (total 200-250 mg)
is applied to a Whatman DE-52 column (2.times.30 cm) and eluted
with a linear gradient of 50 mM Tris acetate from pH 8.3 to pH 6.3
(500 ml of each). For removal of the most adherent components, the
column is further eluted with 500 ml of the pH 6.3 buffer. Recovery
of hemoglobin from the column is 80-95%. For preparative purposes,
the cross-linked hemoglobin is passed through a mixed bed
resin.
[0411] In another experiment, the method of Kluger et al. (Kluger
et al., Biochem., 31:7551-7559 [1992]) is used to cross-link
hemoglobin with trimesoyl tris(methyl phosphate)
(.beta.82-.beta.82). In this experiment, chemical modifications of
hemoglobin are done using hemolysate diluted with 0.1 M
Bis-Tris-HCl buffer at pH 7.2 to a final concentration of
hemoglobin tetramer of 1 mM Hb. The final concentration of
cross-linking reagent is 2 mM in 0.1 M buffer. During the initial
phases of this study, the reactions are kept at 35.degree. C. for
2-3 hours with hemoglobin in the CO form. To improve yield and to
destroy any viral contaminants, the reactions are carried out at
60.degree. C. Reagent is infused at room temperature into the
60.degree. C. hemoglobin solution over a period of 30-60 min with a
total reaction time of up to 3 hours. Reagents and low molecular
weight byproducts are then removed by gel filtration with Sephadex
G-25 columns.
[0412] In yet another experiment, dicarboxylic acid bis(methyl
phosphates) (fumaryl & isophthalyl) (.beta.82-.beta.82) is used
according to the method of Jones et al. (Jones et al., Biochem.,
32:215-223 [1993]). Chemical modifications of hemoglobin are done
using hemolysate diluted with 0.1 M bis-tris-HCl buffer at pH 7.2
to 1 mM Hb (tetramer) and cross-linking reagent at between 2 mM and
5 mM. The temperature of the reaction is either 35.degree. C. or
60.degree. C., and the duration of the reaction is 2-3 hours. At
the higher temperature, the cross-linking reagent is added slowly
by infusion over 1/2 to 2 hours. The reactions are run with
hemoglobin in the carbon monoxide form (HbCO). The cross-linking
reagents are removed by gel filtration through Sephadex G-25.
[0413] C. Hemoglobin Conjugates
[0414] In this set of experiments, hemoglobin conjugates are
produced using various methods.
[0415] 1. Hemoglobin Conjugated to Polyoxyethylene
[0416] First, 1 ml of a solution containing 1.0 M dibasic phosphate
(Na.sub.2HPO.sub.4) and 1.0 M bicarbonate (NaHCO.sub.3) are added
to 10 ml of a 9 g/dl hemoglobin solution at 4.degree. C. with
gentle stirring. Then, 1 g of N-hydroxysuccinimidyl ester of
methoxypoly(ethylene glycol) propionic acid, molecular weight 5,000
Da (M-SPA-5000, Shearwater Polymers, Huntsville, Ala.) is added to
the solution over a 2 minute period with continued stirring.
Temperature and pH are monitored throughout the reaction. Addition
of the activated polyoxyethylene caused a decrease in solution pH.
Approximately 10 mg amounts of solid sodium carbonate
(Na.sub.2CO.sub.3) are added to the mixture to maintain the pH in
the range 8.5-9.5. After 4 hours, the reaction mixture is
transferred into 30,000 MW dialysis bags (Spectra/Por, Spectrum
Medical Industries, Houston, Tex.) and extensively dialyzed against
0.1 M phosphate buffer, pH 7.4.
[0417] 2. Hemoglobin Conjugated to Polyoxyethylene
[0418] In this experiment, the method of Leonard and Dellacherie
(Leonard and Dellacherie, Biochim. Biophys. Acta 791: 219-225
[1984]) is used.
[0419] Activated polyethylene glycol,
monomethoxypolyoxyethylenesuccinimid- yl ester (MPSE), MW=5,000 Da,
is reacted with stroma-free oxyhemoglobin. 1.5 ml of 10 g/dl
hemoglobin solution are added to 2 ml 0.1 M phosphate buffer,
water, or 0.1 M NaCl solution. When necessary, pH is adjusted to
the desired value (5.7-7.8) by adding small amounts of 0.1 M NaOH
or 0.1 M HCl. Then MPSE is added (20-30 mol MPSE per mol hemoglobin
tetramer). The reaction mixtures are stirred at 6.degree. C. for 2
hours and then analyzed by gel permeation chromatography on AcA 44
Ultrogel (linear fractionation range 10,000-130,000; exclusion
limit 200,000) in 0.05 M phosphate buffer (pH 7.2) at 6.degree. C.
The reactions are considered complete when the free hemoglobin peak
disappeared from the gel permeation chromatograms.
[0420] 3. Hemoglobin-Polyethylene Glycol Conjugate In this
experiment, the method of Zalipsky et al. (Zalipsky et al., In
Polymeric Drugs and Drug Delivery Systems (Dumm, R. L. and
Ottenbrite, R. M., eds) pp. 91-100, American Chemical Society,
Washington, D.C. 91-100 [1991]) is used.
[0421] A. Methoxypoly(ethylene glycol)-N-succinimidyl carbonate
(SC-PEG), MW 2,000-6,000 Da (1 g, .about.0.2 mmol) is added to a
stirred solution of bovine oxyhemoglobin (0.1 g,
.about.1.5.times.10.sup.-6 mol) in 0.1 M sodium phosphate buffer,
pH 7.8 (60 ml). Sodium hydroxide (0.5 N) is used to maintain pH 7.8
for 30 minutes. The excess free PEG is removed by diafiltration
using 50 mM phosphate buffered saline.
[0422] B. Methoxypoly(ethylene glycol)-N-succinimidyl carbonate
(SC-PEG), MW 2,000-6,000 Da (1 g, .about.0.2 mmol) is added to a
stirred solution of bovine oxyhemoglobin (0.1 g,
.about.1.5.times.10.sup.-6 mol) in 0.1 M sodium borate buffer, pH
9.2. Sodium hydroxide (0.5 N) is used to maintain pH 9.2 for 30
minutes. The excess free PEG is removed by diafiltration using 50
mM phosphate buffered saline.
[0423] 4. Hemoglobin-Polyethylene Glycol Conjugate
[0424] In this experiment, the methods of Xue and Wong are used
(Xue and Wong, Meth. Enz., 231: 308-323 [1994]) to produce
hemoglobin-polyethylene glycol conjugates.
[0425] First, activation of PEG: Bis(succinimidyl succinate) is
performed. PEG (200 g; 0.059 mol, average 3400 MW; Nippon Oil and
Fats Co. Ltd., Tokyo, Japan) is dissolved in 200 ml of
dimethylformamide at 100.degree. C., and 15 g of succinic anhydride
(0.15 mol) is added. The mixture is stirred for 3 hours at
100.degree. C. The dimethylformamide solution is cooled to room
temperature and poured into 1 liter of ethyl ether. The resulting
PEG ester of succinic acid is filtered through a glass filter and
washed with ethyl ether. The ester is then dried under vacuum
conditions at 40.degree. C. The weight of the product is about 197
g (93% yield).
[0426] To activate the succinyl groups on PEG, 197 g of the PEG
ester of succinic acid (0.055 mol) is dissolved in 200 ml of
dimethylformamide, after which 13 g of N-hydroxysuccinimide (0.11
mol) and 23 g of dicyclohexylcarbodiimide (0.22 mol) are added. The
solution is stirred vigorously overnight at 30.degree. C. The
precipitate of dicyclohexylurea is filtered out and the filtrate is
poured into 1 liter of ethyl ether. The polyethylene glycol
bis(succinimidyl succinate) formed is isolated, washed with ethyl
ether repeatedly, and dried under vacuum conditions at 40.degree.
C. The weight of the product is about 196 g, representing a yield
from PEG of 87%.
[0427] The purity and the degree of imidylation of polyethylene
glycol bis(succinimidyl succinate) may be estimated by nuclear
magnetic resonance using tetramethylsilane as standard (O ppm) and
chloroform-d, as solvent. Similar procedures may be used for the
electrophilic activation of monomethoxypolyethylene glycol.
[0428] Next, the activated PEG is conjugated to hemoglobin, with
the following procedure being carried out at 4.degree. C. First,
0.95 g (0.25 mmol) of polyethylene glycol bis(succinimidyl
succinate) is added to 100 ml of a 0.25 mM Hb solution in 0.1 M
sodium phosphate, pH 7.4 and the reaction continued for 1 hour. The
solution is concentrated by ultrafiltration on an Amicon XM100
membrane. An electrolyte solution is then added and the
concentration process repeated. By repeating this concentration
procedure three times, unreacted PEG and other low molecular weight
compounds are removed.
[0429] Then, the PEG-Hb is stabilized by taking advantage of the
ester bond between PEG and succinic acid in PEG-Hb, which is labile
to hydrolysis. One approach to increase the stability of the bond
between PEG and Hb is to remove the labile ester linkage between
the polyethylene moiety and the terminal carboxyls by oxidizing
both terminal alcoholic groups of PEG to carboxylic groups through
the use of a metal catalyst, to yield
-carboxymethyl-.omega.-carboxymethoxylpolyoxyethylene, which is
activated and coupled to pyridoxalated Hb as in the case of PEG.
The resultant conjugate is designated "stabilized hemoglobin."
[0430] In addition, monomethoxypolyoxyethlylene-hemoglobin is
produced. PEG has two hydroxyl groups at the two termini. When
these are derivatized into functional groups capable of reacting
with Hb, the presence of two reactive groups on the same polymer
makes possible crosslinking reactions. Such cross-linking is
abolished by blocking one of the two termini, as in the case of
monomethoxypolyoxyethylene (MPOE).
[0431] To produce MPOE, 80 g (4 mmol) of MW 5000 MPOE from Aldrich
(Milwaukee, Wis.) is dissolved in tetrahydrofuran (300 ml) and
treated with naphthalene sodium under nitrogen at room temperature
for 3 hours. Then BrCH.sub.2COOC.sub.2H.sub.5 (1.4 ml; 12 mmol) is
added dropwise with stirring. After 4 hours of reaction, the ethyl
ester obtained is precipitated with ether, dried, dissolved in
water, and saponified with 0.1 N NaOH at 55.degree. C. for 24 hours
to yield MPOE-carboxylic acid (MPOE-O--CH.sub.2COOH). The solution
is then acidified with 1 N HCl down to pH 2.5, and the polymer
taken up with chloroform. After several washings with water, the
organic layer is dried over MgSO.sub.4 and treated with charcoal.
The MPOE-carboxylic acid is precipitated with dry ether, filtered,
and dried under vacuum. This run of operations is repeated until
the potentiometric titration gives a constant value for the
quantity of fixed COOH.
[0432] The MPOE-carboxylic acid (5 g; 1 mmol) is dissolved in dry
ethyl acetate (60 ml) and activated by N-hydroxysuccinimide (0.15
g; 1.25 mmol) and dicyclohexylcarbodiimide (0.26 g; 1.25 mmol) at
30.degree. C. for 15 hours. Dicyclohexylurea is removed by
filtration and the polymer precipitated with dry ether is taken up
with chloroform and crystallized from this solution by dropwise
addition of ether at 0.degree. C. This procedure is repeated
several times until the spectrophotometric analysis of succinimidyl
groups gave a constant value.
[0433] Coupling to hemoglobin is performed at 5.degree. C. by
diluting 1.5 ml of a 10 g/dl hemoglobin solution with 2 ml 0.1 M
phosphate buffer, pH 5.8, and 300 mg of MPOE-carboxylic
succinimidyl ester is added under stirring. The reaction mixture is
stirred at 6.degree. C. for 2 hours and analyzed by gel permeation
chromatography on Ultrogel AcA 34 (linear fractionation range MW
20,000-350,000; exclusion limit 750 000) in 0.05 M phosphate buffer
(pH 7.2) at 6.degree. C.
[0434] 5. Hemoglobin-Dextran Conjugate
[0435] In this experiment, hemoglobin-dextran conjugates are
produced according to the various methods of Kue and Wong (Xue and
Wong, Meth. Enz., 231: 308-323 [1994]).
[0436] Synthesis by Alkylation
[0437] In this method, the dextran (Dx) is first derivatized with
cyanogen bromide and diaminoethane to contain a free amino group,
which is acylated with bromoacetyl bromide. The bromoacetyl
function in turn alkylates the sulfhydryl of the .beta.93 cysteine
on Hb:
[0438] Dx+CNBR+diaminoethane.fwdarw.aminoethyl-Dx
[0439] Aminoethyl-Dx+bromoacetyl
bromide.fwdarw.Dx-NHCOCH.sub.2Br
[0440] Dx-NHCOCH.sub.2Br+HS-Hb.fwdarw.Dx-NHCOCH.sub.2--S-Hb
[0441] In a typical preparation, 1.5 g of cyanogen bromide is
dissolved in 15 ml of acetonitrile and added to 10 g of dextran (MW
20,000) in 375 ml of water. The pH is maintained at 10.8 for 5 min
by the addition of 1 M NaOH; the pH is then lowered to about
2.0-2.5 with concentrated HCl. After stirring for 1 min, 15 ml of
diaminoethane is added along with sufficient HCl to prevent the pH
from exceeding 9.5. The final pH is adjusted to 9.5. After standing
overnight at 4.degree. C., the mixture is thoroughly dialyzed
against distilled water using a Millipore (Marlborough, Mass.)
Pellicon dialyzer and lyophilized. The aminoethyl-Dx so obtained is
dissolved in 250 ml of 0.1 M sodium phosphate, pH 7.0, and 15 ml of
bromoacetyl bromide is added through a Pasteur pipette with a
finely drawn capillary tip, accompanied by vigorous stirring over a
period of 2 hours. Throughout, the pH is maintained at 7.0 with the
use of a pH-stat and addition of 1 M NaOH. Afterward, the mixture
is dialyzed thoroughly against distilled water and is lyophilized
to yield about 7 g of the Dx-NHCOCH.sub.2Br (Br-dextran). The
bromine content of the Br-dextran is in the range of 9-11 glucose
residues per bromine atom.
[0442] To couple hemoglobin to dextran, 3.3 g of Br-dextran is
dissolved in 100 ml of 6 g/dl hemoglobin solution in 0.1 M sodium
bicarbonate, pH 9.5. The coupling reaction is allowed to proceed
with constant mixing at 4.degree. C. To determine the yield of
Dx-NHCOCH.sub.2--S-Hb (Dx-Hb), 0.1 ml of the reaction mixture is
applied to a Sephadex G-75 column equilibrated with 0.05 M
phosphate buffer, pH 7.5, and eluted with the same buffer, at a
flow rate of 40 ml/hr. The hemoglobin content of the eluant
fractions is determined by absorbance at 415 nm, and the
proportions of the faster migrating Dx-Hb peak and the slower
migrating Hb peak were given by the areas under these peaks. After
2 days the formation of the Dx-Hb conjugate is essentially
complete.
[0443] Synthesis by Dialdehyde
[0444] Ten ml of a 12% aqueous solution of sodium periodate is
added to 100 ml of a 10% aqueous solution of dextran, and the
mixture is left overnight in the dark at 4.degree. C. A 3% solution
of sodium bisulfite is added until the mixture turned brown and
then, once again, colorless. The mixture is dialyzed against
distilled water to yield the dextran dialdehyde solution. It is
then added to 2 volumes of 3 g/dl stroma-free hemoglobin in 0.3 M
sodium bicarbonate buffer, pH 9.5; coupling of hemoglobin to
dextran is allowed to proceed overnight at 4.degree. C. The Dx-Hb
complex formed is separated from uncoupled hemoglobin by means of
chromatography on a Sephadex G-75 column.
[0445] Coupling of Hb to Dx-dialdehyde is pH dependent. When
coupling is performed by dissolving 100 mg Dx-dialdehyde in 1 ml of
0.6 M sodium borate buffer and mixing with 1.8 ml of 10 g/dl Hb at
6.degree. C., many labile imine linkages are formed at pH<9.6,
and the conjugates have a high molecular weight, ranging to above
100,000. At higher pH, the major product has a lower molecular
weight range (70,000>MW>100,000) and likely consists of a 1:1
complex between Dx and Hb, which only slowly converts to higher
molecular weight forms. When this conjugate is formed at pH 9.8 and
reduced at pH 7.2 for 30 min with excess NaBH.sub.4 (2 mol per mole
of initial aldehyde) dissolved in 1 mM NaOH, only the .alpha. chain
of hemoglobin is found to be modified by Dx. Coupling of Hb to
Dx-dialdehyde also proceeds much more rapidly at higher pH,
requiring less than 1 hour for completion at pH 10 and only 1.5
hours at pH 9.7, but 6 hours at pH 9.5 and 23 hours at pH 9.1. When
prepared at pH 9.75, the oxygen P50 for Dx-Hb is 10.1 mm Hg when
the conjugate is allowed to form for 1 hour prior to NaBH.sub.4
reduction, 9.5 mm Hg when allowed to form for 4 hours, and 8.1 when
allowed to form for 18 hours.
[0446] 6. Hemoglobin Conjugation to SF-DX and P-Dx
[0447] In this experiment, hemoglobin is conjugated with SF-DX and
P-Dx. Dextran-sulfate (SF-Dx) and dextran-phosphate (P-Dx) (MW
40,000) are treated with sodium periodate to generate the
dialdehydyl derivatives, which are in turn coupled to the amino
groups on hemoglobin and are further stabilized by reduction with
sodium borohydride, as described above in the synthesis of Dx-Hb
from Dx-dialdehyde.
[0448] 7. Hemoglobin Conjugation to Dextran-Benzene Hexacarboxylate
(Dx-BHC)
[0449] In this experiment, hemoglobin is conjugated with
dextran-benzene hexacarboxylate (Dx-BHC). Aminopropyl-Dx is
prepared 35 by dissolving 5 g of dextran in 7.5 ml of 25% aqueous
Zn(BF.sub.4).sub.2 and 5 ml of water. Epichlorohydrin (25 ml) is
added with vigorous stirring; the mixture is allowed to react for 3
hours at 80.degree. C. and subsequently overnight at room
temperature. The polymer is precipitated by pouring the solution
dropwise into acetone, filtered, and dried under reduced pressure.
The resulting dextran derivative has the structure of
Dx-O--CH.sub.2CH(OH)CH.- sub.2Cl. This product (4.1 g containing 3%
Cl) is purified by repeated dissolution in water and precipitation
by acetone and methanol. The chlorine atom is subsequently replaced
by an amino group by dissolving the compound in 60 ml of H.sub.2O
and 20 ml of 14 M aqueous ammonia. The solution is stirred for 20
hours at room temperature and then poured dropwise into 1 liter of
methanol. The resulting precipitate of aminopropyl-Dx
(3-amino-2-hydroxypropyl ether of dextran) is filtered, washed with
acetone, and dried under reduced pressure. The yield at this stage
is about 3.5 g.
[0450] Benzene hexacarboxylic acid is coupled to aminopropyl-Dx to
form Dx-BHC through the use of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
as condensing agent. Because benzene hexacarboxylic acid has six
carboxylic acid groups, reaction with an amino group on
aminopropyl-Dx still leaves it with up to five carboxylic acid
groups. One of these may be linked to an amino group on Hb through
further use of the water-soluble EDCI as condensing agent.
[0451] 8. Hydroxyethyl Starch-Hemoglobin-Conjugate
[0452] In this experiment, the method of Xue and Wong, (Xue and
Wong, Meth. Enz., 231: 308-323 [1994]) is used to produce
hydroxyethyl starch-hemoglobin conjugates.
[0453] To prepare for conjugation to hemoglobin, the hydroxyethyl
starch (Hs) is first converted to aminoethyl-Hs. In a typical
preparation, 1.5 g of cyanogen bromide is dissolved in 15 ml of
acetonitrile and added to 500 ml of 2% Hs solution. The pH of the
solution is maintained at 10.8 for 5-10 min by the addition of 1 M
NaOH solution. The pH is then lowered to 2.0-2.5 with concentrated
HCl, and 10 ml of diaminoethane is added along with additional HCl
to prevent the pH from exceeding 9.5. The final pH is adjusted to
9.5 and the solution is allowed to stand overnight at 4.degree. C.
before being dialyzed against deionized water. The ratio of
cyanogen bromide/diaminoethane to Hs can be varied, allowing the
synthesis of aminoethyl-Hs in which from 7 to 20% of the glucose
residues in the starting polymer are substituted.
[0454] Aldehyde-substituted Hs is prepared by reaction of the
aminoethyl-Hs with glutaraldehyde. In a typical reaction, 500 ml of
dialyzed solution of aminoethyl-Hs is treated with 2 g of sodium
bicarbonate to give a solution 2% in Hs and approximately 0.05 M in
bicarbonate. Then 5 ml of 50% glutaraldehyde solution is added to
the solution, which is stirred at room temperature for 2 hours
before dialysis.
[0455] Hemoglobin is employed as a freeze-dried solid under carbon
monoxide. This is reconstituted under argon using deoxygenated
deionized water at 4.degree. C. to give a solution with
approximately 2.5 g Hb per ml. In a typical reaction, 500 ml of
dialyzed solution of the aldehyde-substituted Hs is treated with
sodium bicarbonate to give 500 ml of solution approximately 2% in
Hs and 0.1 M in bicarbonate, hemoglobin solution (25 ml) is added
and the reaction is stirred at room temperature for 4 hours, after
which time get filtration on Sephadex G-150 indicates that no
unmodified hemoglobin remains. Sodium borohydride (1.0 g) is then
added to the solution, which is stirred for a further 2 hours at
room temperature. The Hs-Hb is dialyzed using an Amicon (Danvers,
Mass.) ultrafiltration unit with a 100,000 molecular weight cutoff
cartridge to enable the removal of any trace of unmodified
hemoglobin. Glucose (10 g) is added to the solution prior to
freeze-drying and storage under carbon monoxide at 4.degree. C.
[0456] 9. An Alternative Method for Producing Hydroxyethyl
Starch-Hemoglobin Conjugates
[0457] In this experiment, another method described by Xue and Wong
(Xue and Wong, Meth. Enz., 231: 308-323 [1994]) was used to produce
hydroxyethyl starch-hemoglobin conjugates.
[0458] Hydroxyethyl starch-hemoglobin-conjugate (Hs-Hb) can be
synthesized from Hs-dialdehyde as follows. 0.03 equivalents of Hs
are dissolved in 250 ml of water and treated with 0.028 mol of
sodium periodate for 12 hours at 5.degree. C. in the dark. The
solution is dialyzed until ion free. The percent oxidation may be
determined using a calorimetric method. The solution is buffered to
pH 8.0 by addition of sodium bicarbonate, cooled to 5.degree. C.,
and treated with 5 g of carbonmonoxyhemoglobin. The reaction is
allowed to proceed for 18 hours at room temperature or until gel
filtration indicates complete modification of hemoglobin. The
solution is dialyzed against 1% ammonium carbonate and freeze-dried
in the presence of glucose.
[0459] 10. Hemoglobin-Inulin Conjugate
[0460] In this experiment, a method described by Xue and Wong (Xue
and Wong, Meth. Enz., 231: 308-323 [1994]) is used to produce
hemoglobin-inulin conjugates.
[0461] To synthesize the inulin-hemoglobin (In-Hb) conjugate,
inulin is first succinylated by reacting with succinic anhydride in
N,N-dimethylformamide at 100.degree. C. for 2 hours. Subsequently,
the succinylated inulin is linked to N-hydroxysuccinimide at room
temperature overnight using dicyclohexylcarbodiimide as
condensation agent in N,N-dimethylformamide. Hemoglobin is allowed
to react with a 10-fold molar excess of the
N-hydroxysuccinimide-activated inulin in 0.1 M Tris buffer, pH 7.0,
at 4.degree. C. for 1 hour to yield In-Hb, which is purified with
an Amicon PM30 membrane filter until the unreacted inulin and other
low molecular weight compounds are removed.
[0462] By controlling the succinic anhydride/inulin ratio, the
number of N-hydroxysuccinimide-activated succinyl groups on the
inulin can be varied. A low density of such groups gives rise to a
82,000 MW In-Hb conjugate, whereas higher densities produce
cross-linked In-Hb ranging up to above 300,000 MW.
[0463] 11. An Alternative Method to Produce Hemoglobin-Inulin
Conjugates
[0464] In this experiment, the method of Iwasaki et al. (Iwasaki et
al., Biochem. Biophys. Res. Comm., 113: 513-518 [1983]) is used to
produce hemoglobin-inulin conjugates.
[0465] The N-hydroxysuccinimidyl ester of inulin was reacted with
oxyhemoglobin in 0.1 M tris buffer (pH 7.0) at 4.degree. C. for one
hour. The reaction mixture was analyzed with a JASCO Trirotor HPLC
apparatus equipped with a TSK G3000 SW column. The modified
hemoglobin solution was purified with an Amicon PM 30 membrane
filter until the unreacted inulin and other low molecular weight
compounds are no longer detected.
[0466] 12. Hemoglobin-Polyvinylpyrrolidone Conjugate
[0467] In this experiment, the method of Xue and Wong (Xue and
Wong, Meth. Enz, 231: 308-323 [1994]) was used to produce
hemoglobin-polyvinylpyrroli- done conjugates.
[0468] Synthesis of Activated PVP
[0469] First, 50 g of polyvinylpyrrolidone (PVP) (MW 25,000-35,000)
is dissolved in 1 liter of 0.25 N NaOH and heated at 140.degree. C.
for 42 hours under nitrogen in an autoclave to bring about partial
hydrolysis. It is then adjusted to pH 5 with concentrated HCl and
ultrafiltered through an Amicon UM10 membrane to remove salts.
Water is removed through azeotropic distillation with benzene, and
the extent of hydrolysis is determined by titration of the
secondary amino groups. To blockade these amino groups, 50 g of the
partially hydrolyzed PVP is dissolved in 300 ml of
dichloromethane/dimethylformamide (1:1) and mixed with 0.5 M of
acetic acid anhydride. It is left at room temperature for 1 hour
and refluxed for 4 hours. Evacuated to about 100 ml, the solution
is added dropwise into ethyl ether under strong stirring. The
acetylated PVP precipitate is filtered, washed with ether, and
dried to constant weight under vacuum over phosphorus
pentoxide.
[0470] To activate its carboxyl groups, 50 g of acetylated PVP
dissolved in 500 ml of dichloromethane/dimethylformamide (1:1) is
mixed at 0.degree. C. with 15.47 g of N-hydroxysuccinimide followed
with a solution of 27.75 g dicyclohexylcarbodiimide in 50 ml of
dichloromethane. The solution is stirred at 0.degree. C. for 14
hours before centrifugation to remove the dicyclohexylurea. The
supernatant solution (about 300 ml) is added dropwise into 5 liters
of cold ether under strong stirring. The white precipitate is
filtered, washed repeatedly with ether, and dried in the cold over
phosphorus pentoxide.
[0471] Binding of Hemoglobin to Activated PVP
[0472] Hemoglobin (27 g) is dissolved in 1 liter of 5% sodium
carbonate and treated at 4.degree. C. with 40 g of activated PVP
for 24 hours with stirring. The preparation is lyophilized and
redissolved in 300 ml of distilled water. After a 20-fold volume
diafiltration, it is again lyophilized.
[0473] From the above, it should be evident that the present
invention provides optimal blood substitute compositions comprising
mixtures of oxygen-carrying and non-oxygen carrying plasma
expanders and methods for the use thereof. These compositions and
methods allow for the production of relatively inexpensive products
that are more effective than currently available compositions.
[0474] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in hematology,
surgical science, transfusion medicine, transplantation, or any
related fields are intended to be within the scope of the following
claims.
* * * * *