U.S. patent application number 09/894237 was filed with the patent office on 2002-01-31 for compositions and methods utilizing nitroxides in combination with biocompatible macromolecules.
Invention is credited to Hsia, Jen-Chang.
Application Number | 20020013263 09/894237 |
Document ID | / |
Family ID | 27537190 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020013263 |
Kind Code |
A1 |
Hsia, Jen-Chang |
January 31, 2002 |
Compositions and methods utilizing nitroxides in combination with
biocompatible macromolecules
Abstract
Compositions and processes to alleviate free radical toxicity
are disclosed based on the use of nitroxides in association with
physiologically compatible macromolecules. In particular,
hemoglo-bin-based red cell substitutes are described featuring
stable nitroxide free radicals for use in cell-free hemoglobin
solutions, encapsulated hemoglobin solutions, stabilized hemoglobin
solutions, polymerized hemoglobin solutions, conjugated hemoglobin
solutions, nitroxide-labelled albumin, and nitroxide-labelled
immunoglobulin. Formulations are described herein that interact
with free radicals, acting as antioxidant enzyme-mimics, which
preserve nitroxides in their active form in vivo. Applications are
described including blood substitutes, radioprotective agents,
imaging agents, agents to protect against ischemia and reperfusion
injury, particularly in cerebral ischemia in stroke, and in vivo
enzyme mimics among others.
Inventors: |
Hsia, Jen-Chang; (Irvine,
CA) |
Correspondence
Address: |
LYON & LYON LLP
633 WEST FIFTH STREET
SUITE 4700
LOS ANGELES
CA
90071
US
|
Family ID: |
27537190 |
Appl. No.: |
09/894237 |
Filed: |
June 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09894237 |
Jun 27, 2001 |
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08824739 |
Mar 26, 1997 |
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08824739 |
Mar 26, 1997 |
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08605531 |
Feb 22, 1996 |
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5840701 |
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08605531 |
Feb 22, 1996 |
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08482952 |
Jun 7, 1995 |
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08482952 |
Jun 7, 1995 |
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08417132 |
Mar 31, 1995 |
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5767089 |
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08417132 |
Mar 31, 1995 |
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08291590 |
Aug 15, 1994 |
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5591710 |
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08291590 |
Aug 15, 1994 |
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08107543 |
Aug 16, 1993 |
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Current U.S.
Class: |
514/21.2 ;
424/450; 514/58; 514/59 |
Current CPC
Class: |
A61K 38/42 20130101;
C07K 14/76 20130101; A61K 47/52 20170801; A61K 49/20 20130101; A61K
49/0002 20130101; C07K 14/805 20130101; C07K 16/00 20130101; B82Y
5/00 20130101; A61K 45/06 20130101; A61K 47/6445 20170801; A61K
47/6895 20170801; A61K 38/42 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/2 ; 514/6;
514/58; 514/59; 424/450 |
International
Class: |
A61K 038/42; A61K
038/38; A61K 031/721; A61K 031/724; A61K 009/127 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 1996 |
US |
PCT/US96/03644 |
Claims
The claims are:
1. A method to promote the in vivo conversion of a hydroxylamine
form of a nitroxide to a paramagnetic form comprising: reacting a
first nitroxide in the hydroxylamine form with a second nitroxide
providing enzyme-mimic activity as a hydroxylamine oxidase, wherein
the enzyme-mimic compound is a nitroxide-containing compound having
a nitroxyl group which is less stable than the first nitroxide in
its free radical form.
2. The method of claim 1 wherein the enzyme mimetic compound is a
biocompatible polynitroxide macromolecule.
3. The method of claim 2 wherein the polynitroxide macromolecule is
a synthetic nitroxide polymer.
4. The method of claim 3 wherein the polynitroxide macromolecule is
selected from the group of dextran, hydroxylethyl starch, liposome,
hemoglobin, or albumin.
5. A method to provide a localized increase in the in vivo
concentration of the free radical form of a nitroxide comprising:
administering a membrane permeable first nitroxide; administering a
localized dose of a second nitroxide having a nitroxyl group
capable of accepting an electron from the hydroxylamine form of the
first nitroxide.
6. The method of claim 5, wherein the second nitroxide is a
compound containing a polynitroxide labelled biocompatible
macromolecule of the group of albumin, hemoglobin, liposome,
dextran, or hydroxylethyl starch.
7. The method of claim 6, wherein the polynitroxide macromolecule
is polynitroxide albumin labelled at a molar ratio of approximately
7 to 95.
8. The method of claim 7, wherein the first nitroxide is selected
from the group consisting of TEMPOL, PROXYL, or DOXYL and the
second nitroxide of the labelled macromolecule is selected from the
same group.
9. A composition comprising a biocompatible polynitroxide
macromolecule in a pharmaceutically acceptable vehicle for
administration.
10. The composition of claim 9 wherein the biocompatible molecule
is selected from the group consisting of hemoglobin, albumin,
hydroxylethyl starch, dextran, or cyclodextran.
11. The-composition of claim 10 wherein the nitroxide is selected
from the group consisting of TEMPOL, PROXYL, or DOXYL.
12. The composition of claim 11 wherein the composition is
polynitroxide albumin having a molar ratio of nitroxide to albumin
of between approximately 7 and 95.
13. A biocompatible composition comprising a membrane permeable
first nitroxide; and a second nitroxide having a nitroxyl group
capable of accepting an electron from the first nitroxide.
14. The composition of claim 13 wherein the second nitroxide is a
substantially membrane impermeable polynitroxide biocompatible
macromolecule.
15. The composition of claim 14 wherein the polynitroxide
biocompatible macromolecule is polynitroxide albumin.
16. The composition of claim 15 wherein the molar ratio of
nitroxide to albumin is between approximately 7 and 95.
17. The composition of claim 14 wherein the first nitroxide is of
the group of TEMPOL, DOXYL, or PROXYL.
18. The composition of claim 17 wherein the nitroxide is the
hydroxylamine derivative thereof.
19. The composition of claim 14 wherein the polynitroxide
biocompatible macromolecule is of the group of native hemoglobin,
cross-linked hemoglobin, polymerized hemoglobin, conjugated
hemoglobin, liposome-encapsulated hemoglobin, hydroxylethyl starch,
dextran, or cyclodextran.
20. A method to enhance the electron paramagnetic resonance or
nuclear magnetic resonance image of a biological structure
comprising: administering a membrane permeable first nitroxide,
administering a second nitroxide having a nitroxyl group capable of
accepting an electron from the first nitroxide, and obtaining an
image of the biological structure.
21. The method of claim 20 wherein the membrane permeable nitroxide
is of the group of TEMPOL, PROXYL, or DOXYL and the second
nitroxide is a biocompatible macromolecule of the group of
hemoglobin, albumin, dextran, cyclodextran, hydroxylethyl starch,
or liposome.
22. The method of claim 21 wherein the polynitroxide macromolecule
is albumin having a molar ratio of nitroxide to albumin of between
approximately 7 to 95.
23. The method of claim 20 wherein the method of administration of
the second nitroxide macromolecule is effective to localize the
image enhancement at a site proximate to the interaction of the
membrane permeable first nitroxide and the second nitroxide.
24. A method to obtain an EPR or MRI image of ischemia in the heart
or brain comprising: intravenous administration of a membrane in
permeable polynitroxide macromolecule.
25. The method of claim 24 wherein the membrane impermeable
nitroxide is a polynitroxide labelled macromolecule selected from
the group consisting of hemoglobin, albumin, hydroxylethyl starch,
liposome, dextran, or cyclodextran and the nitroxide is of the
group of TEMPOL, PROXYL, or DOXYL.
26. The method of claim 25 wherein the polynitroxide membrane
impermeable macromolecule is a polynitroxide hemoglobin-based
oxygen carrier.
27. A method to alleviate ischemic reperfusion injury comprising:
administering a therapeutic dose of a polynitroxide
macromolecule.
28. The method of claim 27 wherein the polynitroxide macromolecule
is of the group of albumin, hemoglobin, dextran, cyclodextran,
hydroxylethyl starch, or liposome, and the nitroxide is of the
group of TEMPOL, DOXYL, OR PROXYL.
29. The method of claim 28 wherein the polynitroxide macromolecule
is polynitroxide albumin having a molar ratio of nitroxide to
albumin of approximately 7 to 95.
30. The method of claim 27 further comprising administration of a
membrane permeable nitroxide.
31. The method of claim 27 wherein the polynitroxide macromolecule
administered intravenously for the treatment of ischemia or
reperfusion injury in the cerebrovascular or cardiovascular
system.
32. The method of claim 27 wherein at least one of the first and
second nitroxide are administered topically to treat ischemia or
reperfusion injury of the dermal layers.
33. A method to protect an organism from ionizing radiation
comprising: administering a membrane permeable first nitroxide, and
administering a membrane impermeable second nitroxide having a
nitroxyl group capable of accepting an electron from the first
nitroxide.
34. The method of claim 33 wherein the second nitroxide is a
nitroxide-labelled macromolecule selected from the group consisting
of albumin, hemoglobin, dextran, hydroxylethyl starch, liposome, or
cyclodextran.
35. The method of claim 34 wherein the polynitroxide macromolecule
is albumin labelled with a nitroxide selected from the group
consisting of TEMPOL, DOXYL, or PROXYL at a molar ratio of
approximately 7 to 95.
36. A method to treat an organism with a physiological condition
using a therapeutic dose of ionizing radiation comprising:
administering a membrane permeable nitroxide, administering a
second nitroxide having a nitroxyl group capable of accepting an
electron from the first nitroxide, and exposing the organism to
ionizing radiation.
37. The method of claim 36 wherein the membrane permeable nitroxide
is selected from the group consisting of TEMPOL, DOXYL, or PROXYL
and the second nitroxide is a nitroxide-labelled macromolecule
selected from the group consisting of hydroxylethyl starch,
albumin, hemoglobin, liposome, dextran, or cyclodextran.
38. The method of claim 37 wherein the nitroxide-labelled
macromolecule is polynitroxide albumin wherein the molar ratio of
nitroxide to albumin is between approximately 7 to 95.
39. The method of claim 36 wherein a dose of ionizing radiation is
delivered to a site coincident to the concentration of the second
nitroxide.
40. The method of claim 39 wherein the polynitroxide albumin is in
a carrier suitable for topical application and further comprising
the topically applying the polynitroxide at the site of radiation
exposure.
41. The method of claim 40 wherein at least one of the membrane
permeable nitroxide or the polynitroxide albumin is administered
intravenously prior to the invitation of radiation therapy.
42. A method to reduce the effect of free radical toxicity in a
biological system comprising: administering a therapeutically
active amount of a membrane permeable first nitroxide, and
administering a catalytically active amount of a second nitroxide
wherein the second nitroxide has a nitroxyl group capable of
accepting an electron from the first nitroxide.
43. The method of claim 42 wherein the first nitroxide is selected
from the group consisting of TEMPOL, DOXYL, or PROXYL and the
second nitroxide is a polynitroxide macromolecule selected from the
group consisting of albumin hydroxylethyl starch, dextran,
liposome, hemoglobin, or immunoglobulin.
44. The method of claim 43 wherein the polynitroxide macromolecule
is polynitroxide albumin molar ratio of nitroxide to albumin of
between approximately 7 and 95.
45. The method of claim 42 wherein the effect is associated with a
disease state selected from the group consisting of sepsis, ulcers,
cataracts, radiation exposure, inflammation alopecia,
ischemia/reperfusion injury, closed head injury, trauma, burns,
psoriasis, aging, stroke, or renal failure.
46. The method of claim 42 including administering a
hemoglobin-based oxygen carrier.
Description
[0001] This application is a continuation-in-part of application
No. 08/605,531 which is a continuation-in-part of application No.
08/482,952 which is a continuation-in-part of application No.
08/417,132 which is a continuation-in-part of application No.
08/291,590 which is a continuation-in-part of application No.
08/107,543 and incorporates each by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the therapeutic and diagnostic use
of nitroxides, including the combined use of a membrane permeable
nitroxide with a membrane impermeable nitroxide including
nitroxide-labelled macromolecules, including polypeptides e.g.,
hemoglobin, albumin, immunoglobulins, and polysaccharides, e.g.r,
dextran, hydroxylethyl starch, and artificial membranes, e.g.,
liquid bilayer, hemoglobin and albumin microbubbles.
Nitroxide-containing formulations are disclosed which alleviate the
toxic effects of oxygen-related species in a living organism and
provide ability to diagnose and treat a wide variety of
pathological physiological conditions. This invention also relates
to nitroxides, synthetic nitroxide polymers and copolymers and
nitroxide-labelled macromolecules used in combination with low
molecular weight and membrane-permeable nitroxides to sustain the
in vivo effect of nitroxides. This invention also discloses novel
compounds and methods featuring nitroxides used in combination with
physiologically compatible cell-free and encapsulated hemoglobin
solutions for use as a red cell substitute. Furthermore, this
invention describes the methods and novel compounds for the topical
delivery of cell membrane impermeable nitroxides in its membrane
permeable form leading to intracellular accumulation of therapeutic
concentrations of the said nitroxide for treatment of skin photo
aging and as an anti-skin wrinkle agent. Additionally, this
invention describes the above nitroxides in combination with other
physiologically active compounds, including other nitroxides, to
protect from pathological damage and oxidative stress caused by
free radicals and describes their use in diagnosis and in the
treatment of disease.
BACKGROUND OF THE INVENTION
[0003] Although the physiological mechanisms of oxygen metabolism
have been known for many years, the role played by oxidative stress
in physiology and medicine is not completely understood. The impact
of oxygen-derived free radicals on physiology and disease is a
topic of increasing importance in medicine and biology. It is known
that disease and injury can lead to levels of free radicals which
far exceed the body's natural antioxidant capacity--the result is
oxidative stress. Oxidative stress is the physiological
manifestation of uncontrolled free radical toxicity, most notably
that which results from toxic oxygen-related species. Toxic free
radicals are implicated as a causative factor in many pathologic
states, including ischemia-reperfusion injury resulting from heart
attack or stroke, shock, alopecia, sepsis, apoptosis, certain drug
toxicities, toxicities resulting from oxygen therapy in the
treatment of pulmonary disease, clinical or accidental exposure to
ionizing radiation, trauma, closed head injury, burns, psoriasis,
in the aging process, and many others.
[0004] Therefore, a need exists for compositions and methods which
detoxify free radicals and related toxic species and which are
sufficiently active and persistent in the body to avoid being
rapidly consumed when increases in free radical concentrations are
encountered.
[0005] Furthermore, evidence has been developed which demonstrates
that free radicals aggravate a number of other disease states
including cancer, ulcers and other gastro-intestinal conditions,
cataracts, closed head injury, renal failure, injury to the nervous
system, and cardiovascular disease to name a few. As a result of
their high reactivity, free radicals can oxidize nucleic acids,
biological membranes, and other cell components, resulting in
severe or lethal cellular damage, mutagenesis, or carcinogenesis.
Anti-cancer radiotherapy, as well as a number of antitumor drugs,
act by generating free radicals which are toxic to tumor cells, but
are also toxic to normal cells which are exposed during cell
division causing the undesirable side-effects of cancer therapy.
Indeed, it is believed that many pathologic processes have as their
common final pathway the generation of free radicals which are the
direct, or a substantial contributing cause, of the observed
pathology. Additionally, a dramatic increase in free radical
concentrations can be observed as part of a cascade initiated by
the interruption of the flow of oxygenated blood, such as in heart
attack or stroke, often followed by the reperfusion of oxygenated
blood to the affected area. As the importance of such oxidative
stress in living systems becomes appreciated, a continuing need
exists for compounds and methods that can function as anti-oxidants
and which can be designed to interact with oxygen-derived free
radicals to alleviate their toxicity in biological systems,
particularly in humans. In other applications, the toxic free
radicals may be coincident to a beneficial treatment such as
radiation administered as part of cancer radiotherapy. For example,
since the mechanism by which ionizing radiation causes physiologic
damage to an organism involves, at least in part, a free radical
interaction with cells, compounds which possess or interact with
free radicals exhibit a localized effect on tissues exposed to
radiation therapy thereby controlling the collateral damage caused
by a therapeutic treatment. Additionally, apart from any clinically
significant function, since the unpaired electrons in free radicals
species are detectable by spectroscopy, free radical reactions may
be monitored in vivo and compounds which interact with free
radicals are observable by spectroscopic techniques.
[0006] Several therapeutic approaches have been proposed to reduce
pathologic levels of free radicals. Ideally, safe and effective
antioxidant agents would augment a patient's antioxidant capacity
and assist in blocking many pathologic free-radical based
toxicities at the stage of free radical generation. However, the
development of methods and compounds to combat oxidative stress or
the toxicity associated with oxygen-related species has enjoyed
limited success. The usefulness of many anti-oxidants is limited by
short duration of action in vivo, toxicity at effective dosage
levels, the inability of many compounds to cross cell membranes,
and an inability to counter the effects of high levels of free
radicals. For example, the administration of the enzyme superoxide
dismutase (SOD) or catalase can promote the conversion of toxic
free radical related species to a non-toxic form. However, these
enzymes do not function effectively in the intracellular space.
Procysteine as a GSH precursor, as well as vitamins and other
antioxidant chemicals, can enhance the body's natural antioxidant
capacity, but are unable to deal with the higher levels of free
radicals encountered in injury and disease and are rapidly consumed
by the body.
[0007] Free radical species are notoriously reactive and
short-lived. Such reactivity is a particularly serious hazard in
biological systems because detrimental chemical reactions between a
free radical and body tissue occurs in very close proximity to the
site where the free radial is generated. Therefore, compounds which
inherently function to reduce free radical concentrations have some
beneficial effect, although the effect may not be clinically
significant unless the therapeutic effect can be concentrated and
localized in a particular region of the body such as the brain, the
epidermis, the gut, the cardiovascular system or in discrete tissue
such as the site of radiation administration.
[0008] The difficulties encountered in creating a blood substitute
suitable for large volume intravenous administration are an acute
example of the difficulty in preventing or alleviating systemic
toxicity caused by oxygen-related species in the vascular space.
Scientists and physicians have struggled for decades to produce a
blood substitute that could be safely transfused into humans.
Persistent blood shortages and the problems of incompatible blood
types, cross-matching, and the communication of disease have led to
a broad-based effort by private industry, universities, and
governments to discover a formulation that would allow a large
volume of a blood substitute to be safely transfused without
significant physiological side effects. At present, several
companies are conducting clinical trials on experimental blood
substitutes. However, adverse physiological reactions and the
inherent complexity of the research and development process have
impeded progress through the regulatory approval stages and have
impeded the development of a clinically useful blood
substitute.
[0009] A Research Advisory Committee of the United States Navy
issued a report in August 1992 outlining the efforts by several
groups to produce a blood substitute, assessing the status of those
efforts, and generally describing the toxicity problems
encountered. The Naval Research Advisory Committee Report reflects
the current consensus in the scientific community that even though
the existing blood substitute products, often termed
"hemoglobin-based oxygen carriers" (HBOC), have demonstrated
efficacy in oxygen transport, certain toxicity issues are
unresolved. The adverse transfusion reactions that have been
observed in clinical studies of existing hemoglobin-based oxygen
carriers (HBOC) include systemic hypertension and vasoconstriction.
These adverse reactions have forced a number of pharmaceutical
companies to abandon their clinical trials or to proceed at low
dosage levels. Solving the toxicity problem in the existing
hemoglobin-based blood substitutes has been given a high priority
by the United States Government. A Naval Research Committee
recommendation has been implemented by the National Institute of
Health in the form of a Request For Proposal (PA-93-23) on the
subject of "Hemoglobin-Based Oxygen Carriers: Mechanism of
Toxicity." Therefore, the medical and scientific community suffers
from an acute and pressing need for a blood substitute that may be
infused without the side effects observed with the existing
hemoglobin-based oxygen carriers.
[0010] The red blood cells are the major component of blood and
contain the body's oxygen transport system. It has long been
recognized that the most important characteristic of a blood
substitute is the ability to carry oxygen. The red blood cells are
able to carry oxygen because the primary component of the red cells
is hemoglobin, which functions as the oxygen carrier. Most of the
products undergoing clinical testing as blood substitutes contain
hemoglobin that has been separated from the red blood cell
membranes and the remaining constituents of the red blood cells and
has been purified to remove essentially all contaminants. However,
when hemoglobin is removed from the red cells and placed in
solution in its native form, it is unstable and rapidly dissociates
into its constituent subunits. For this reason, the hemoglobin used
in a hemoglobin-based oxygen carrier (HBOC) must be stabilized to
prevent dissociation in solution. Substantial expenditures in
scientific labor and capital were necessary to develop
hemoglobin-based products that are stable in solution, and which
are stabilized in such a way that the oxygen transport function is
not impaired. The ability of the existing hemoglobin-based oxygen
carriers to transport oxygen has been well established (See U.S.
Pat. Nos. 3,925,344; 4,001,200; 4,001,401; 4,053,590; 4,061,736;
4,136,093; 4,301,144; 4,336,248; 4,376,095; 4,377,512; 4,401,652;
4,473,494; 4,473,496; 4,600,531; 4,584,130; 4,857,636; 4,826,811;
4,911,929 and 5,061,688).
[0011] In the body, hemoglobin in the red cells binds oxygen
molecules as the blood passes through the lungs and delivers the
oxygen molecules throughout the body to meet the demands of the
body's normal metabolic function. However, the atmospheric oxygen
that most living beings must breathe to survive is a scientific and
medical paradox. On the one hand, almost all living organisms
require oxygen for life. On the other hand, a variety of toxic
oxygen-related chemical species are produced during normal oxygen
metabolism.
[0012] With respect to oxidative stress resulting from the
transportation of oxygen by hemoglobin, it is known that in the
process of transporting oxygen, the hemoglobin (Hb) molecule can
itself be oxidized by the oxygen (O.sub.2) molecule it is carrying.
This auto-oxidation reaction produces two undesirable products:
met-hemoglobin (met-Hb) and the superoxide anion (.O.sub.{overscore
(2)}). The chemical reaction may be written as follows:
Hb+4O.sub.2.fwdarw.met-Hb+4.O.sub.{overscore (2)} [1]
[0013] The superoxide anion (. O.sub.{overscore (2)}) is an oxygen
molecule that carries an additional electron and a negative charge.
The superoxide anion is highly reactive and toxic.
[0014] As described in detail herein, free radical species such as
the superoxide anion are implicated as the agents of cell damage in
a wide range of pathological processes. In the case of oxygen
transport by hemoglobin, potentially damaging oxidative stress is
manifested in the vascular space and originates with the superoxide
anion being generated by the auto-oxidation of hemoglobin and
results from the subsequent conversion of the superoxide anion to
toxic hydrogen peroxide in the presence of the enzyme superoxide
dismutase (SOD) by the following reaction: 1
[0015] The reaction whereby a free radical species generates toxic
chemical species in vivo or causes cellular damage is seen
repeatedly in pathologic conditions where oxidative stress is a
factor, particularly in the instance of ischemic reperfusion injury
as in heart attack or stroke. The presence of the superoxide anion
and hydrogen peroxide in the red blood cells is believed to be the
major source of oxidative stress to the red cells.
[0016] Apart from oxygen transport by the hemoglobin contained
therein, a less recognized characteristic of the red cells is that
they contain a specific set of enzymes which are capable of
detoxifying oxygen-related chemical species produced as by-products
of oxygen metabolism. Without the protection of these specific
enzyme systems, autoxidation of hemoglobin would lead to
deterioration and destruction of the red cells. In the body,
however, the reserve capacity of the enzyme systems in the red
cells protects the body from oxygen toxicity by converting the
superoxide anion generated during normal metabolism to non-toxic
species and thereby controls the level of oxidative stress.
However, if this enzyme system breaks down, the integrity of the
red cells will be damaged. A lesion of the gene that produces one
of the enzymes in the protective system in the red blood cells will
cause an observable pathological condition. For example,
glucose-6-phosphate dehydrogenase deficiency, a genetic disorder of
red cells, is responsible for hydrogen peroxide induced hemolytic
anemia. This disorder is due to the inability of the affected cells
to maintain NAD(P)H levels sufficient for the reduction of oxidized
glutathione resulting in inadequate detoxification of hydrogen
peroxide through glutathione peroxidase (P. Hochstein, Free Radical
Biology & Medicine, 5:387 (1988)).
[0017] The protective enzyme system of the red blood cells converts
the toxic superoxide anion molecule to a non-toxic form in a
two-step chemical pathway. The first step of the pathway is the
conversion of the superoxide anion to hydrogen peroxide by the
enzyme superoxide dismutase (SOD) (See Equation [2]). Because
hydrogen peroxide is also toxic to cells, the red cells contain
another enzyme, catalase, which converts hydrogen peroxide to water
as the second step of the pathway (See Equation [3]). 2
[0018] Red cells are also capable of detoxifying hydrogen peroxide
and other toxic organoperoxides using the enzyme glutathione
peroxidase which reacts with glutathione to convert hydrogen
peroxide and organoperoxides to water. Red cells also contain an
enzyme to prevent the build up of the met-hemoglobin produced by
the auto-oxidation of hemoglobin. The enzyme met-hemoglobin
reductase converts met-hemoglobin back to the native form of
hemoglobin. Therefore, in the body, the toxic effects of the
auto-oxidation of hemoglobin are prevented by specific enzyme-based
reaction pathways that eliminate the unwanted by-products of oxygen
metabolism.
[0019] The enzymatic oxygen detoxification functions of superoxide
dismutase, catalase, and glutathione peroxidase that protect red
blood cells from oxygen toxicity during normal oxygen transport do
not exist in the hemoglobin-based oxygen carriers (HBOC) developed
to date. Without the oxygen detoxification function, the safety of
the existing HBOC solutions will suffer due to the presence of
toxic oxygen-related species.
[0020] The principle method by which the existing HBOC solutions
are manufactured is through the removal of hemoglobin from the red
cells and subsequent purification to remove all non-hemoglobin
proteins and other impurities that may cause an adverse reaction
during transfusion (See U.S. Pa. Nos. 4,780,210; 4,831,012; and
4,925,574). The substantial destruction or removal of the oxygen
detoxification enzyme systems is an unavoidable result of the
existing isolation and purification processes that yield the
purified hemoglobin used in most HBOC. Alternatively, instead of
isolating and purifying hemoglobin from red cells, pure hemoglobin
has been produced using recombinant techniques. However,
recombinant human hemoglobin is also highly purified and does not
contain the oxygen detoxification systems found in the red cells.
Thus, the development of sophisticated techniques to create a
highly purified hemoglobin solution is a mixed blessing because the
purification processes remove the detrimental impurities and the
beneficial oxygen detoxification enzymes normally present in the
red cells and ultimately contributes to oxygen-related
toxicity.
[0021] One of the observed toxic side effects resulting from
intravenous administration of the existing HBOCs is
vasoconstriction or hypertension. It is well known that the enzyme
superoxide dismutase (SOD) in vitro will rapidly scavenge the
superoxide anion and prolong the vasorelaxant effect of nitric
oxide (NO). Nitric oxide is a molecule that has recently been
discovered to be the substance previously known only as the
"endothelium-derived relaxing factor" (EDRF). The prolongation of
the vasorelaxant effect of nitric oxide by SOD has been ascribed to
the ability of SOD to prevent the reaction between the superoxide
anion and nitric oxide. (M. E. Murphy et. al., Proc. Natl. Acad.
Sci. USA 88:10860 (1991); Ignarro et.al. J. Pharmacol. Exp. Ther.
244: 81 (1988); Rubanyi Am. J. Physiol. 250: H822 (1986);
Gryglewski et.al. Nature 320: 454 (1986)).
[0022] However, in vivo, the inactivation of EDRF by the superoxide
anion has not been observed and is generally not thought to be
likely. Nevertheless, certain pathophysiological conditions that
impair SOD activity could result in toxic effects caused by the
superoxide anion (Ignarro L. J. Annu. Rev. Pharmacol. Toxicol.
30:535 (1990)). The hypertensive effect observed in preclinical
animal studies of the existing HBOC solutions suggests that the
concentration of superoxide anion in large volume transfusions of
the existing HBOCs is the cause for the destruction of EDRF and the
observed vasoconstriction and systemic hypertension.
[0023] It is, therefore, important to delineate the hypertensive
effect resulting from the reaction of the superoxide anion with
nitric oxide (NO) from that resulting from extravasation and the
binding of NO by hemoglobin. Upon transfusion of an HBOC, the
hemoglobin can also depress the vasorelaxant action of nitric oxide
by reacting with nitric oxide to yield the corresponding
nitrosylheme (NO-heme) adduct. In particular, deoxy-hemoglobin is
known to bind nitric oxide with an affinity which is several orders
of magnitude higher than that of carbon monoxide.
[0024] These hemoglobin-NO interactions have been used to assay for
nitric oxide and to study the biological activity of nitric oxide.
For example, the antagonism of the vasorelaxant effect of nitric
oxide by hemoglobin appears to be dependent on the cell membrane
permeability of hemoglobin. In intact platelets, hemoglobin did not
reverse the effect of L-arginine which is the precursor of nitric
oxide. In contrast, in the cytosol of lysed platelets, hemoglobin
is the most effective inhibitor of L-arginine induced cyclic-GMP
formation mediated by nitric oxide. These experiments demonstrated
that the hemoglobin did not penetrate the platelet membrane
effectively. (Radomski et al., Br. J. Pharmacol. 101:325 (1990)).
Therefore, one of the desired characteristics of the HBOCs is to
eliminate the interaction of nitric oxide with hemoglobin.
[0025] Hemoglobin is also known to antagonize both
endothelium-dependent vascular relaxation (Martin W. et. al. J.
Pharmacol. Exp. Ther. 232: 708 (1985)) as well as NO-elicited
vascular smooth muscle relaxation (Grueter C. A. et al., J. Cyclic.
Nucleotide Res. 5:211 (1979)). Attempts have been made to limit the
extravasation and hypertensive effect of hemoglobin by chemically
stabilizing, polymerizing, encapsulating, or conjugating the
hemoglobin in the HBOCs to prolong the circulation time. Therefore,
although the current HBOCs are relatively membrane impermeable and
able to transport oxygen, the HBOC solutions do not have the
capability of preventing the reaction between superoxide anion and
nitric oxide when transfused. The above example demonstrates the
difficulty in addressing the oxygen toxicity/stress issue, even
where the reactions mechanisms of oxygen transport are reasonably
well understood, and despite decades of research to improve the
hemoglobin production and formulation process.
[0026] An ideal solution to the toxicity problems of the existing
blood substitutes would be a hemoglobin-based formulation that
combines the oxygen-transport function of the existing HBOCs with
the oxygen detoxification function of the red cells. However, a
simple addition of the enzyme superoxide dismutase (SOD) into an
existing HBOC solution would not be desirable because, by reducing
the concentration of superoxide anion, the reaction whereby
hemoglobin is oxidized to met-hemoglobin would be encouraged,
leading to an undesirable build-up of met-hemoglobin (See Equation
[1]). Also, it is not desirable to encourage the conversion of the
superoxide anion to hydrogen peroxide in a hemoglobin solution
because the hydrogen peroxide is toxic and reactive and will
decompose to toxic hydroxyl radicals or form other toxic
organoperoxides during storage.
[0027] Because synthetic blood substitutes would ideally be
infusible in large quantities, compounds which interact with free
radicals must be able to offer sustained in vivo function and must
be stable and non-toxic. Pursuant to this invention, nitroxides and
nitroxide-labelled macromolecules, including hemoglobin, albumin
and others are used to alleviate the toxic effects of free radical
species in a living organism.
[0028] Another example of physiological damage resulting from a
free radical cascade originating in the vascular compartment is the
cerebral edema, necrosis, and apoptosis, which is associated with a
number of pathologies, including cerebrovascular occlusion and
ischemic events commonly known as a "stroke". A significant portion
of the brain damage from a stroke also arises from a reperfusion
event following the occlusion.
[0029] The brain damage resulting from stroke exacts large human
and health care costs and scientists and physicians have long
sought a treatment for preventing stroke injury to the brain. A
primary contribution to the brain damage attendant to the
ischemic/reperfusion injury in stroke is free radical formation in
the vascular space and the resulting cascade leading to cellular
injury. Oxygen free radical toxicity is linked to the edema and
neural injury resulting from stroke, as shown, for example, in
transgenic (Tg) SOD-1 mouse. Chan, P. H., C. J. Epstein, H.
Kinouchi, H. Kamii, S. Imaizumi, G. Yang, S. F. Chen, J. Gafni, and
E. Carlson (1994). SOD-1 transgenic mice are a model for studies of
neuroprotection in stroke and brain trauma. Ann. N.Y. Acad. Sci.
738: 93-103. Chan, P. H., C. J. Epstein, H. Kinouchi, S.Imaizumi,
E. Carlson, and S. F. Chen (1993). Role of superoxide dismutase in
ischemic brain injury: Reduction of edema and infarction in
transgenic mice following focal cerebral ischemia. In Molecular
Mechanisms of Ischemic Brain Damage, K. Kogure and B. K. Siesjo,
eds. Amsterdam: Elsevier. pp. 96-104. Kinouchi, H., C. J. Epstein,
T. Mizui, E. Carlson, S. F. Chen, and P. H. Chan (1991).
Attenuation of focal cerebral ischemic injury in transgenic mice
overexpressing CuZn superoxide dismutase. Proc. Natl. Acad. Sci.
USA 88: 11158-11162.
[0030] In the SOD-1 transgenic mouse, a human SOD transgene is
expressed in the mouse brain at up to three times normal level, and
provides 30% protection against stroke injury as measured by
infarction size, edema and neurological deficit following a focal
ischemic insult. Free radical damage in stroke is localized in both
the intracellular and vascular spaces, reflecting (a) damage to
cell membranes and organelles in tissue, and (b) damage
specifically to the vascular endothelium. Imaizumi, S., V.
Woolworth, and R. A. Fishman (1990). Liposome-encapsulated
superoxide dismutase reduces cerebral infarction in cerebral
ischemia in rats. Stroke 21: 1312-1317. Liu, T. H., J. S. Beckman,
B. A. Freeman, E. L. Hogan, and C. Y. Hsu (1989). Polyethylene
glycol-conjugated superoxide dismutase and catalase reduce ischemic
brain injury. Am. J. Physiol. 256: H586-H593. Chan, P. H., S.
Longar, and R. A. Fishman (1987). Protective effects of
liposome-entrapped superoxide dismutase on post-traumatic brain
edema. Ann. Neurol. 21: 540-547.
[0031] No drug therapy has yet been proven completely effective in
preventing brain damage from cerebral ischemia. A large number of
experimental neuroprotective agents, thrombolytics, and
anticoagulants have been tested, but the adverse side effects
associated with many such agents may discourage their use. Also,
the ischemia/reperfusion injury, which may also arise during
surgery, for example, following embolism by gas bubbles, from
embolism by endogenous or exogenous particles, or from hypotensive
episodes. These problems are unlikely to be addressed by
anticoagulant therapy. Given the antioxidant protective mechanism
of the nitroxide-based compounds of the invention, the invention
provides the ability to substantially alleviate reperfusion injury
by protecting the cells from damage during reperfusion.
Furthermore, given the ability to detect the compounds
spectroscopically, this invention may be used as a therapeutic and
diagnostic agent for stroke as well as a prophylactic agent for
perioperative stroke, perioperative cardiac damage and renal injury
and may be used in combination with other anti-stroke drugs such as
thrombolytics, glutamate release inhibitors, calcium influx
blockers and NMDA receptor antagonists.
[0032] The prevention of oxidative stress in the vascular system
also alleviates or reduces the development of oxidized lipids which
may lead to arterial plaques and atherosclerosis in the walls of
the cardiovascular system, particularly in arteries proximate to
the heart. The mechanism of free radical reactivity also implicates
an oxidation of plasma LDL and such oxidized lipids are also
thought to play a role in reperfusion injury in the brain and
central nervous system and in several diseases and conditions of
the cardiovascular system.
[0033] As will be appreciated by the several embodiments of the
invention described herein, the capability of nitroxides, used
together with biological macromolecules pursuant to this invention,
to control the damage caused by free radicals in vivo creates the
ability to design therapeutic and diagnostic nitroxide-containing
formulations and methods for their use which have a broad range of
applications. A large number of physiological states and processes
where oxygen-derived free radicals are present may be treated or
diagnosed by the use of the compounds described herein. The use of
membrane-permeable, low molecular weight nitroxides in combination
with biocompatible macromolecules such as hemoglobin, albumin, and
others, also allows the researcher to tailor the
nitroxide-containing formulation to fit the specific environment of
interest.
[0034] A multi-component nitroxide-based system also functions as a
radioprotective agent for use in cancer radiotherapy and in the
treatment of radiation exposure. In clinical applications, the
efficacy of radiation therapy will be enhanced by allowing higher
radiation dosages to be used safely.
[0035] There has long been a need for agents which can protect
against the ill effects of ionizing radiation encountered in the
course of medical radiotherapy or as the result of environmental
radiation exposure. Such agents would also be useful tools in
research on mechanisms of radiation cytotoxicity. Cysteamine, a
sulfur-containing compound, was one of the earliest radioprotective
agents identified. Its discovery prompted the United States
Department of Defense to sponsor the synthesis and systematic
screening of over 40,000 compounds in an attempt to find more
effective agents. This monumental undertaking resulted in the
discovery of a few radiation protectors such as the aminothiol
compound known as WR-2721. More recently superoxide dismutase,
interleukin I, and granulocyte-macrophage colony-stimulating factor
have been shown to have radioprotectant activity. In a comparison
of these agents, WR-2721 showed the most substantial and selective
protection of normal tissues. However, when used in patients
undergoing cancer radiotherapy, concern over inherent toxicity and
nonselective protection of tumor dampened enthusiasm for the use of
WR-2721. The capability to protect tissue from "ionizing" radiation
also provides the ability to protect and treat dermal tissue
suffering from exposure to UV radiation. Thus, compounds formulated
pursuant to this invention can be provided with a vehicle for
administration such as a lotion or cream which enables application
to the skin.
[0036] While certain stable nitroxides have been found to have
antioxidant and radioprotectant activities. However, these membrane
permeable nitroxides are rapidly reduced in vivo to an inactive
form and may be toxic in elevated doses. The utility of
administration of membrane permeable nitroxides can be
substantially enhanced pursuant to this invention.
SUMMARY OF THE INVENTION
[0037] This invention discloses stable nitroxides used in
connection with biologically compatible macromolecules, including
other nitroxides for therapy and diagnosis in biological systems.
In particular, this invention describes low molecular weight,
membrane-permeable nitroxides used in connection with nitroxides
bound in a high molar ratio to biocompatible macromolecules such as
albumin and hemoglobin. In certain applications, an interaction
between one form of a nitroxide and another form of nitroxide with
a differential free radical stability facilitates electron or spin
transfer between the species. The differential stability may result
from the electrochemical environment of the species or from the
inherent nature of the compound. This invention also contemplates
the use of stable nitroxide free radicals, precursors and
derivatives, hereafter referred to collectively as "nitroxide(s)",
to provide the oxygen detoxification function of the red cells to
hemoglobin-based blood substitutes and to alleviate oxidative
stress and to avoid biological damage associated with free radical
toxicity, including inflammation, radiation, head injury, shock,
post-ischemic reperfusion injury, stroke, renal failure,
endothelial damage, lipid peroxidation, sickle cell anemia,
leukocyte activation and aggregation, apoptosis, ionizing
radiation, alopecia, cataracts, sepsis, psoriasis ulcers, and the
aging process, among others.
[0038] In certain embodiments, stable nitroxides or derivatives
thereof are used to create several formulations for a blood
substitute that will possess the oxygen detoxification function of
the red cells. These formulations may be described herein as
hemoglobin-based red cell substitutes (HRCS) because the oxygen
transport capability of the hemoglobin-based oxygen carriers (HBOC)
is enhanced by providing the oxygen detoxification function of the
body's red cells. This permits the design of vasoneutral
hemoglobin-based oxygen carriers which avoid the hypertension
observed in many HBOC.
[0039] To overcome the drawbacks in the use of nitroxides alone, in
preferred embodiments of this invention, a polynitroxide-labelled
macromolecule, such as Tempo-labelled human serum albumin is
infused either alone or together with a free membrane-permeable
nitroxide to provide extended activity of the nitroxide in vivo.
One benefit of such a formulation is an improved radioprotective
agent which can be used in both diagnostic and therapeutic medical
application and to protect against exposure to radiation from any
source. In therapeutic medical applications, increased dosages of
radiation are enabled to be administered thereby improving the
possibility that radiation therapy will be successful. This
capability is particularly significant in certain tumors such as
those in the brain, and, is useful in combination with imaging and
oxygen delivery as described herein, particularly with those tumors
containing regions of hypoxia.
[0040] Also, nitroxides are detectable by electron paramagnetic
resonance spectroscopy and nuclear magnetic resonance spectroscopy.
With the development of advanced imaging instrumentation, images of
intact biological tissues and organs are available based on a
measurement and detection of the stable free radical of a
nitroxide. Pursuant to this invention, active nitroxide levels in
the body may be maintained for a prolonged period of time allowing
both improved image contrast and longer signal persistence than
seen with low molecular weight membrane permeable nitroxides alone.
Moreover, unlike certain existing image-enhancing agents, the
compositions disclosed here are capable of crossing the blood-brain
barrier.
[0041] Additionally, due to their antioxidant activity, the
compositions disclosed herein have therapeutic value which, in
combination with their diagnostic value, allows the novel
compositions and methods of this invention to be used
advantageously in a wide variety of applications.
[0042] Materials and methods are also described for the preparation
and administration of stable nitroxides in several forms. In
particular, inactive, relatively non-toxic precursors or
derivatives of membrane-permeable nitroxides are described which
are converted in vivo by other compounds described herein to
biologically active nitroxides, or antioxidant enzyme mimics. In
either case, the chemically reduced (inactive) nitroxide may be
reactivated, by other nitroxides of differential stability or by
nitroxide-labelled macromolecular species, after having been
reduced in the process of detoxifying harmful free radicals. As a
result of this regeneration effect, the nitroxides of this
invention have longer half lives in vivo compared to low molecular
weight, membrane-permeable nitroxides alone. Thus, this invention
provides compositions and methods to enhance the effectiveness of
any application where nitroxides are efficacious. Using the
multi-component system of this invention, a dynamic equilibrium is
created between low molecular weight, membrane-permeable nitroxides
and membrane permeable nitroxide-containing species of differential
stability. In particular, a nitroxide-based compound featuring a
nitroxyl group capable of accepting an electron from another
nitroxide, such as a membrane impermeable macromolecular-bound
nitroxide capable of accepting an electron from the hydroxylamine
derivative, may act as an enzyme mimic to regenerate the active
function of the membrane permeable nitroxides, or vice versa as an
electron acceptor, to convert the hydroxylamine form of a nitroxide
to the free radical form.
[0043] The capability to maintain the concentration of an active
nitroxide in vivo pursuant to this invention offers advantages in
virtually any application where administration of a nitroxide is
beneficial, but the utility is limited due to rapid reduction in
vivo, or where the optimally effective dose of a membrane preamble
nitroxide is toxic. For example, the increased active half-life of
nitroxide in vivo pursuant to this invention provides radiation
protection and improved imaging in clinical and other applications
where the effective dose of a low molecular weight membrane
permeable nitroxide is toxic or rapidly consumed.
[0044] Nitroxides, which are paramagnetic by virtue of a stable
unpaired electron, function as imaging agents in nuclear magnetic
resonance imaging (NMR/MRI) and in electron paramagnetic resonance
imaging (EPR/ERI). However, due to the rapid reduction of nitroxide
to a spectroscopically invisible species, most typically the
hydroxylamine form, the utility of such agents is limited. Because
free radical species are implicated in reperfusion injury, and are
known to accompany oxygen metabolism, ischemic tissue injury, and
hypoxia may be observed using the compositions of this invention as
imaging agents. Additionally, the antioxidant, enzyme-mimic effect
of the compositions of this invention provides the added benefit of
protection from oxidative damage.
[0045] A distinct advantage of the multi-component nitroxide based
system is the capability to deliver the antioxidant,
radioprotective, anti-ischemic, image-enhancing, enzyme-mimic, etc.
function to several regions of the body, such as the vascular
compartment, interstitial space, and intracellular regions or to a
particular region based on selective permeability of the biological
structure or utilizing known methods of administration which
provide targeted or localized effect. The researcher or clinician
can tailor the multi-component system described here to fit the
application. For example, different formulations described herein
have differing levels of vasorelaxant effect. The ability to tailor
the selection of the nitroxide-containing species of the
multi-component system of the invention provides the ability to
selectively treat or diagnose particular disease states or
conditions or to provide increases or decreases in the free radical
form of the nitroxide. For example, as will be appreciated by those
skilled in the art, the invention can be particularly applied to
the cardiovascular system by intravenous of one or more of the
components of the multi-component system described herein.
Similarly, a particular region of the skin may be selected by
topical administration of one nitroxide while administering the
other species by topical, oral, or intravenous administration
depending on the particular application of this invention.
[0046] Fundamentally, in certain embodiments, a nitroxide
(including precursors and metabolic substrates) is provided which
is selected to perform the desired function, i.e., radioprotection,
imaging, enzyme-mimic, etc., and another nitroxide-based species is
provided as a reservoir of activity. In terms of electron spin
transfer, one species may be considered an "acceptor" nitroxide and
the other a "donor nitroxide." In certain embodiments, the "donor"
and "acceptor" may remain substantially physically separated in
vivo and should have different stabilities in their free radical
moieties. In a preferred embodiment, the acceptor nitroxide is a
polynitroxide albumin which distributes predominantly in the
vascular space and acts as a storehouse of activity. The donor
species is typically a low-molecular weight, membrane permeable
species such as TPL or TPH. Alternatively, the donor species may be
membrane impermeable and the acceptor species membrane permeable
and the species selected such that the activity of a nitroxide is
inhibited.
[0047] Those of ordinary skill will appreciate that the individual
species selected as the donor or acceptor may vary as long as
substantial physical separation is maintained and differential
stability is achieved. For example, the same nitroxide species may
act as both acceptor and donor. In such an example, TPL labelled at
a number of amino groups on a macromolecular species such as
albumin provides a substantially membrane-impermeable acceptor
nitroxide. Differential stability of the macromolecular-bound TPL
is provided by labelling at the amino groups such that the
remaining carboxyl groups create an acidic microenvironment
yielding a less stable free radical state in the albumin-bound TPL.
Alternatively, different unbound nitroxide species may be provided
which, by virtue of their inherent chemical and electrical
structure, provide the requisite separation and differential
stability.
[0048] The dynamic equilibrium which is created by the compounds of
this invention is between a reduced form of a nitroxide and an
oxidized form such that one is active in vivo and the other
inactive. The fundamental mechanism is acceptance of an electron
from a first nitroxide, particularly the reduced hydroxylamine
derivative thereof, by the nitroxyl group of a second nitroxide.
The second nitroxide is capable of accepting an electron when it
contacts the first nitroxide by virtue of the differential
stability of the free radical nitroxyl group. In one example, the
free radical or "oxidized" form, e.g. TPL, becomes rapidly reduced
to TPH until regenerated to TPL by polynitroxide albumin (PNA).
3
[0049] The preferred compositions using nitroxides in connection
with biocompatible macromolecules may be varied; for example, with
a physiologically compatible solution for infusion such as a
hemoglobin-based oxygen carrier, the compositions include: 1)
nitroxide-containing compounds added to a storage container or
contained within a filter; nitroxides may be chemically attached to
an insoluble matrix used in a filter or contained therein in
several forms as an advantageous method of storage and
administration, 2) nitroxide covalently linked to hemoglobin that
is stabilized by chemical or recombinant cross-linking, 3)
nitroxide covalently linked to polymerized hemoglobin, in
particular, in 2, 4, and 8 molar equivalents of nitroxide, 4)
nitroxide coencapsulated with hemoglobin inside a liposome or
intercalated into a liposome membrane, (5) nitroxide covalently
bound to conjugated hemoglobin, (6) nitroxide covalently bound to
several forms of albumin in high molar ratios, i.e., between 6 and
95, (7) nitroxide covalently bound to immunoglobulins, and any
combination of the above in a multicomponent system.
[0050] As noted, the above compositions may be used independently
or in connection with low molecular weight, membrane permeable
nitroxides depending on the application. Moreover, the above
compositions may be specially formulated with other compounds to
alter their reactivity or stability in vivo. In particular,
cyclodextran and other recognized stabilizing agents may be used to
enhance the stability of hemoglobin-based solutions. Also, the
essential nutrient selenium is known to generate superoxide and may
be used with a polynitroxide macromolecule to promote the oxidation
thereof. These formulations may also be used with other known
compounds that provide protection from oxidative stress, which
enhance imaging, which increase or decrease sensitivity to
radiation, and other known compounds with clinical or diagnostic
utility.
[0051] Experimental results are presented below to demonstrate that
low molecular weight nitroxides may be regenerated from a reduced
inactive form to their active form by interaction with the
nitroxide-labelled macromolecules of this invention. The
experimental results and procedures below show that nitroxides may
be attached to biocompatible macromolecules, including albumin and
stabilized, polymerized, conjugated and encapsulated hemoglobin,
for diagnosis therapy, and measurement of physiological conditions
related to oxidative stress. The interaction of nitroxide-labelled
hemoglobin and nitroxide-labelled albumin, both alone and in
combination with a low molecular weight nitroxide, with free
radicals suggests that other biologically compatible macromolecules
with a substantial plasma half-life may be labelled with nitroxides
and used pursuant to this invention to advantageously provide
resistance to or protection from oxidative stress or toxicity
caused by free radical chemical species.
[0052] Experimental results are also presented to demonstrate that
the compositions and methods of this invention are
anti-hypertensive when infused with an HBOC such that the infusion
of an HBOC solution is rendered vasoneutral. Radioprotection is
demonstrated both with cell cultures and with mice exposed to
lethal doses of radiation. EPR images of the rat heart are shown
which are capable of monitoring the progress of ischemia and
reperfusion injury and which demonstrate that, in addition to
image-enhancement, the compositions disclosed herein protect the
ischemic heart from reperfusion injury. Protection from
ischemic/reperfusion injury is shown in both the cardiovascular and
cerebrovascular systems, experimental results are also presented to
show the protective effect of the invention in inhibiting lipid
oxidation and leukocyte activation and in treatment and protection
of the skin.
DESCRIPTION OF FIGURES
[0053] The file of this application contains at least one
photograph/image in color. Copies of this patent with color figures
will be provided by the Patent and Trademark Office upon request
and payment of the necessary fee.
[0054] FIGS. 1A and 1B show the electron spin resonance spectra of
4-amino-TEMPO labelled o-raffinose polymerized hemoglobin recorded
on (A) day 1 and (B) day 30 (TEMPO: 2,2,6,6
tetramethylpiperidine-1-oxyl). FIG. 1C is the spectra of the sample
in FIG. 1A diluted with equal volume of unlabelled hemoglobin
recorded on day 1. FIG. 1D is the sample in FIG. 1C recorded on day
30.
[0055] FIGS. 2A and 2B are, respectively, the electron spin
resonance spectra demonstrating covalent attachment of
4-(2-bromoacetamido) -TEMPO to .omega.-aminohexyl-agarose and
4-amino-TEMPO to 1,4-bis(2:3-Epoxypropoxy) butane-activated
agarose.
[0056] FIGS. 3A and 3B, respectively, are electron spin resonance
spectra demonstrating successful covalent attachment of
4-(2-Bromoacetamido)-TEMP- O and 3-maleimido-PROXYL to
3,5-bis-bromosilicyl-bisfumarate (DBBF) cross-linked or diaspirin
cross-linked human hemoglobin (HBOC).
[0057] FIG. 4A is an ESR spectra of 4-(2-bromoacetamido)-TEMPO.
[0058] FIG. 4B is an ESR spectra of
4-(2-bromoacetamido)-TEMPO-labelled HBOC. FIG. 4C is an ESR spectra
of .sup.15ND,.sub.7 TEMPOL in Lactated Ringer's solution recorded
at room temperature.
[0059] FIG. 5 is an ESR spectra of
4-(2-bromoacetamido)-TEMPO-labelled HBOC with different molar
ratios of nitroxides to Hb; FIG. 5A 2:1, FIG. 5B 4:1 and FIG. 5C
8:1. The instrument sensitivity were decreased proportionately from
FIG. 5A to FIG. 5B to FIG. 5C to record the spectra so that the
center peak (Mo) would be shown to have similar peak height.
[0060] FIG. 6 is an ESR spectrum of a mixture of
4-(2-bromoaceta-mido)-TEM- PO labelled HBOC and .sup.15ND.sub.7
-TEMPOL wherein the center peak (see down arrow) of the former and
the high field peak (see up-arrow) of the latter were adjusted to
similar intensity. This is a superimposition of ESR spectrum from
FIG. 4B and FIG. 4C.
[0061] FIG. 7 shows the plasma half-life of
4-(2-bromoacetamido)-TEMPO-lab- elled HBOC in a mouse. FIG. 7A is
the ESR spectrum of the nitroxide signal recorded from the mouse
tail approximately 10 minutes after intravenous infusion of 0.5 ml
of the sample shown in FIG. 6. FIG. 7B is the time dependent (scan
time 30 minutes) decrease in the center peak (Mo) signal intensity
of FIG. 7A recorded at 10 times of the instrument sensitivity. FIG.
7C is a continuation of FIG. 7B at the end of its scan.
[0062] FIG. 8 shows the plasma half-life of a mixture of
4-(2-bromoacetamido)-TEMPO-labelled HBOC (8g/dl of Hb and 8:1 TEMPO
to Hb) and .sup.15ND.sub.17 TEMPOL (0.5 ml in a 32 g. mouse)
recorded from the mouse tail with a cannula for immediate recording
of the infused nitroxides. The ESR spectrum of the sample prior to
injection is shown in FIG. 6. FIG. 8A is a series of 5 ESR spectrum
recorded at 0.5 minute intervals, the magnetic field strength was
increased by 2 Gauss in between each scan to display the decrease
in signal intensity as a function of time. FIG. 8B is the
continuation from FIG. 8A of repeated recording of a series of 6
ESR spectrum at the same time intervals except that the magnetic
field strength was decreased by 2 Gauss in between each scan.
[0063] FIGS. 9A and 9B, respectively, are electron spin resonance
spectra demonstrating 4-amino-TEMPO labelled and o-raffinose
cross-linked and polymerized human hemoglobin and
3-maleimido-PROXYL labelled DBBF-hemoglobin polymerized with
glutaldehyde.
[0064] FIGS. 10A and 10B, respectively, are electron spin resonance
spectra of liposome encapsulated human hemoglobin containing (A)
3-DOXYL-cholestane (B) 16-DOXYL-stearic acid. FIG. 10C is the
electron spin resonance spectra of both 3-DOXYL-cholestane and
16-DOXYL-stearate.
[0065] FIG. 11 is the electron spin resonance spectrum of
nitroxide-labelled hemoglobin labelled with 4-amino-TEMPO and
conjugated with dextran.
[0066] FIG. 12 is an embodiment of a filter cartridge that contains
a solid matrix to which a nitroxide is bound and through which a
hemoglobin-containing solution may be passed.
[0067] FIG. 13 shows the mean arterial pressure (MAP) response in a
rat to intravenous infusion of 7.8 g/dl 10% v/v DBBF-Hb alone in
Ringer's lactated solution (broken line) and 7.89/dl 10% v/v
DBBF-Hb+polynitroxide albumin (PNA) 5 g/dl +TPL 100 mM 10% v/v
(solid line) in conscious rats. The rats were allowed to recover
from surgery and anesthesia for approximately 7 days prior to
study.
[0068] FIG. 14 is a plot showing time dependence of rat plasma
concentrations of TPL after intravenous injections. Plasma samples
were obtained from rats described in FIG. 13. TPL concentrations
were determined from EPR spin density measurements.
[0069] FIGS. 15A, 15B, and 15C, respectively, are electron
paramagnetic resonance (EPR) spectra of: 15A, TPL (2 mM) in sodium
phosphate buffer 50 Mm, PH 7.6; 15B, TPH (2 mM) in the same buffer;
15C, polynitroxide albumin (PNA). EPR spectrometer setting
conditions as follows. Microwave power: 8mW; Receiver gain:
1.00e+03; Modulation amplitude: 0.5G; Modulation frequency: 100
KHz; Microwave frequency: 9.43 GHz; Sweep width: 200G.
[0070] FIG. 16 is a bar graph showing the surviving fraction of
Chinese Hamster V79 cells at 12 Gray radiation. The V79 cells were
pretreated 10 minutes prior to x-ray irradiation. No
radioprotection is observed with TPH or PNA alone (the bar showing
2% survival is same as the control without treatment). Increasing
radioprotection is shown by the bar corresponding to the sample
containing a combination of TPH and PNA (8% survival).
[0071] FIG. 17 shows the conversion of TPH to TPL by PNA TPH at
fixed concentration of 2SmM was mixed with increasing
concentrations of the PNA in sodium phosphate buffer 50 mM, Ph 7.6.
The ratio of TPL/TPH was plotted against 25 mM PNA concentrations.
This ratio represents conversion efficiency. TPL concentration was
determined by incubating 25 Mm of TPH and seven different
concentrations of PNA at room temperature for 30 minutes followed
by 10KD membrane centrocon separation for one hour at 5000.times.g.
The high field EPR peak intensities of TPL in the filtrate were
calibrated with TPL standard curve and plotted as shown.
[0072] FIG. 18A is a continuous recording of fifteen (15) EPR
spectra of the mouse tail recorded by manually increasing the field
strength by approximately 1 G in between scans. The scan numbers
were marked on the high field peak (M-1/2) of the .sup.1sN TPL. The
mouse was previously injected with 0.5 ml of 40 mM .sup.15N TPL,
which was reduced to TPH with a half-life of 2 minutes (results not
shown). The second injection of a mixture of PNA and .sup.15N TPL
into the mouse tail vein showed a similar rate of disappearance of
.sup.15N-TPL (see peaks 1-5 recorded at 30 sec. intervals with a
half-life of .about.2 minutes) followed by a equally rapid recovery
of the peak intensity (see peaks 5-7). The peak intensity of TPL
decays with a half-life of 13 minutes (see peaks 7-15, recorded at
1 minute intervals) The center (Mo) broad resonance peak (A) of
polynitroxide albumin (PNA) shown in between the two clusters
(M+1/2 and M-1/2) of .sup.15N-TPL peaks (TPL) also appeared to
decay at a half-life of 13 minutes. Thus, the use of .sup.15N-TPL
clearly demonstrates the spin-transfer from TPH to the .sup.14N
nitroxides on PNA in vivo. FIGS. 18B, 18C, and 18D are ESR spectra
showing activation of TOPS by three different nitroxide-labelled
human proteins. FIGS. 18B and 18D show TOPS activation by albumin
labelled with different species of nitroxide. FIG. 18C is
nitroxide-labelled hemoglobin-based oxygen carrier. FIG. 18B shows
(1) 50 Mm TOPS only; (2) 43 Mm PNA only; (3) PNA (43 mM) mixed with
TOPS (50 Mm) in sodium phosphate buffer pH 7.8 for 2 min. FIG. 18C
shows (1) 50 Mm TOPS only; (2) 15 Mm PN-HBOC; (3) PN-HBOC (15 mM)
mixed with TOPS (50 mM) in sodium phosphate buffer pH 7.8 for 2
min. FIG. 18D shows (1) 50 mM TOPS only; (2) 10 Mm B3T-labelled
albumin only; (3) B3T-labelled albumin (10 mM) mixed with TOPS (50
mM) in sodium phosphate buffer pH 7.8 for 2 min.
[0073] FIG. 19 shows pharmacokinetics of TPL and TPH in the
presence or absence of PNA in C57 mice. Plasma levels of TPL in
arbitrary units as determined by monitoring the EPR high field peak
intensity were plotted as a function of time field (min.),
(.quadrature.) TPL (2 mM alone) by intravenous administration,
(.diamond.) TPL 275 mg/kg by intraperitoneal administration and
(.largecircle.) PNA by intravenous administration followed by TPH
100 mg/kg by intraperitoneal administration.
[0074] FIG. 20 shows the 30-day survival study of C57 mice (10 mice
per group (N=10) exposed to 10 Gray irradiations after treatment
with PNA 0.5 ml/mouse by intravenous administration followed by PBS
buffer 10 minutes later (.box-solid.), 0.5 ml PBS followed by
200mg/kg of TPL 10 minutes later by intraperitoneal (ip)
administration (.circle-solid.), polynitroxide albumin 0.5 ml/mouse
by intravenous administration followed by 200mg/kg TPL 10 minutes
later (.diamond-solid.).
[0075] FIG. 21 shows the 30-day survival study of C57 mice exposed
to 10 Gray irradiations after treatment with PNA 0.5 ml/mouse by
intravenous administration followed by PBS buffer 10 minutes later
(.box-solid.), 0.5. ml PBS followed by 200 mg/kg of TPL 10 minutes
later by intraperitoneal administration (.circle-solid.),
polynitroxide albumin 0.5 ml/mouse by intravenous administration
followed by 50 mg/kg TPL 10 minutes later (.diamond.).
[0076] FIG. 22A is a 3-D spatial (25.times.25.times.25 mm.sup.3)
EPR image (full view) of the rat heart loaded with TPL and
polynitroxide albumin. The image was reconstructed using 144
projections acquired after 2.5 hours of ischemia. FIG. 22B is a
cutout view of the same image. Data acquisition parameters were as
follows: spectral window: 7.0 G; spatial window: 25 mm; maximum
gradient: 49.3 G/cm.
[0077] FIG. 23 is an EPR image (25.times.25 mm.sup.2) of the rat
heart loaded with TPL and PNA indicating time as a measure of
ischemic duration (156 min.), obtained from a 3-D spatial image.
Data acquisition parameters were as follows: acquisition time: 10
min.; spectral window: 7.0 G; spatial window: 25 mm; maximum
gradient: 49.3 G/cm.
[0078] FIG. 24 shows the intensity of .sup.15N-Tempol EPR signal in
two ischemic hearts vs. time of duration of the ischemia. The upper
line shows a heart treated with 2 mM TPL +PNA (.circle-solid.) and
the lower line shows a heart treated 2 Mm TPL alone (.box-solid.).
The solid lines are double-exponential fittings to the observed
intensity data. The half-lives are 0.4 minutes, 2.9 minutes
(.box-solid.), and 3.3 minutes, 30.1 minutes (.circle-solid.)
respectively.
[0079] FIG. 25 is a 2-D cross-sectional (25.times.25 mm.sup.2) EPR
image of transverse slices of the rat heart loaded with TPL+PNA as
a function of ischemic duration with the time indicated digitally
(min:sec.) on successive images. The images were obtained from 3-D
spatial images. Data acquisition parameters were the same as for
FIG. 23.
[0080] FIG. 26 shows a measurement of the recovery of coronary flow
in untreated control hearts (.largecircle.), treated with TPL 2 mM
(.circle-solid.), and PNA (4g/dl) +TPL 2 mM (.tangle-soliddn.) .
Hearts were subjected to 30 min. of global ischemia followed by 45
min. reflow.
[0081] FIG. 27 shows the measurement of the recovery of rate
pressure product (RPP) in untreated control hearts (.largecircle.),
treated with TPL 2 mM (.circle-solid.), and PNA 4g/dl +TPL 2 mM
(.tangle-soliddn.). Hearts were subjected to 30 min. of global
ischemia followed by 45 min. reflow.
[0082] FIG. 28 is a graph of the EPR signal intensity of TEMPOL vs.
durations of ischemia in the rat heart treated with
.sup.15N-TPL+PNA obtained from images shown in FIG. 25. The
intensity values were obtained at specific locations of the
corresponding 3-D spatial images as follows: LAD (.circle-solid.),
Aortic (.box-solid.), LV-apex (.tangle-solidup.) and tissue
(.tangle-soliddn.).
[0083] FIG. 29 shows the mean arterial pressure (MAP) response to
intravenous infusion of 7.8 g/dl 10% v/v DBBF-Hb+PNA 7.5 g/dl +TPL
100 mM 10% v/v (n=4) in conscious rats. The rats were allowed to
recover from surgery and anesthesia for approximately seven days
prior to study.
[0084] FIG. 30 shows the reduction in neurological deficit in mice
treated with PNA. Animals were subjected to 1 hour of focal
cerebral ischemia by MCA/CCA occlusion, followed by reperfusion.
Animals were treated before the onset of ischemia with PNA (Pre
PNA) or human serum albumin (HSA), or immediately after reperfusion
(Post PNA, Post HSA), and assessed for neurological status after 24
hours of reperfusion. The dose of PNA or human serum albumin was
0.5% of body weight, v/w.
[0085] FIG. 31 shows the reduction in infarct volumes in mice
treated with PNA. Animals were subjected to 1 hour of focal
cerebral ischemia by MCA/CCA occlusion followed by reperfusion.
Animals were treated before the onset of ischemia with PNA (Pre
PNA) or human serum albumin (Pre HSA), or immediately after
reperfusion (Post PNA, Post HSA), and sacrificed after 24 hrs of
reperfusion for histological analysis. The dose of PNA or albumin
was 0.5% of body weight, v/w. Infarct volumes were calculated by
integrating infarct areas in serial coronal sections taken at 0.5
mm intervals from the anterior tip of the brain. Results are
expressed as mean.+-.SEM, n=7.
[0086] FIG. 32 shows the reduction in percentage of infarction in
mice treated with PNA. Animals were subjected to 1 hour of focal
cerebral ischemia by MCA/CCA occlusion, followed by reperfusion.
Animals were treated before the onset of ischemia with PNA (Pre
PNA) or human serum albumin (Pre HSA), or immediately after
reperfusion (Post PNA, Post HSA), and sacrificed after 24 hrs of
reperfusion for histological analysis. The dose of PNA or albumin
was 0.5% of body weight, v/2. Percent infarction was calculated as
(infarct volume/ipsilateral hemisphere volume) .times.100. Results
are expressed as mean+SEM, n=7.
[0087] FIG. 32A shows quantitation of stroke injury in serial
sections of the brains of control vs. PNA-treated mice. A series of
20 .mu.m-thick coronal histologic sections was taken at 500 .mu.m
intervals from the anterior tip of the brain, and cerebral ischemia
was quantitated as infarcted area (mm.sup.2, mean.+-.S.D.) per
section. The difference in the area of the infarction in
PNA-treated mice (.circle-solid.) compared with controls
(.box-solid.)is the area between the two lines (p<0.05 by
Student's t-test).
[0088] FIG. 33 shows reduction in hemisphere enlargement in mice
treated with PNA. Animals were subjected to 1 hour of focal
cerebral ischemia by MCA/CCA occlusion, followed by reperfusion.
Animals were treated before the onset of ischemia with PNA (Pre
PNA) or human serum albumin (Pre HSA), or immediately after
reperfusion (Post PNA, Post HSA), and sacrificed after 24 hours of
reperfusion for histological analysis. The dose of PNA or albumin
was 0.5% of body weight, v/w. Percent hemisphere enlargement was
calculated as (ipsilateral hemisphere volume-contralateral
hemisphere volume contralateral hemisphere volume).times.100.
Results are expressed as mean.+-.SEM, n=7.
[0089] FIGS. 34A and 34B show representative coronal sections
showing reduction in cerebral infarction in PNA-treated vs. control
mice subjected to focal cerebral ischemia (1 hr) and reperfusion
(24 hr). 20 .mu.m-thick sections were stained with cresyl violet,
which is poorly taken up by ischemic tissue. FIG. 34A is a section
from the brain of an untreated animal, showing massive ischemic
damage (unstained area) of essentially the entire hemisphere. Edema
appears as enlargement of the infarcted hemisphere compared with
the opposite hemisphere of the same brain. FIG. 34B is a section
from the brain of a PNA-treated animal, showing normal histology
and absence of edema. This indicates that PNA provides protection
against stroke injury. FIG. 34C is representative histologic
sections showing reduction in stroke injury in PNA-treated vs.
control mice. Animals were subjected to focal cerebral ischemia (1
hour) and reperfusion (24 hours). Treated mice received intravenous
PNA before ischemia and at the onset of reperfusion. Each dose was
equivalent to 1% of body weight (v/w) . In order to demonstrate the
anatomic extent of ischemia, coronal sections 20 .mu.m thick were
made at 500 .mu.m intervals from the anterior tip of the brain, and
stained with cresyl violet. Ischemic tissue appears unstained. The
untreated animals (left-hand panel) show ischemic damage of
essentially the entire hemisphere. In striking contrast, the
PNA-treated animals (right-hand panel) show normal histology
throughout the brain.
[0090] FIG. 35 is a series of stained coronal sections of the rat
brain of an animal infused with PNA and stained after two hours of
suture occlusion of the MCA.
[0091] FIG. 36 is a series of stained coronal sections of the rat
brain of an animal infused with a saline control and stained after
two hours of suture occlusions of the MCA.
[0092] FIG. 37A and 37B are histological analyses of infarct volume
and percentage of infarction in rats following transient MCA
occlusion and administration of saline, human serum albumin, and
PNA.
[0093] FIG. 38 is the spin density of rat blood measured as a
function of time following three PNA injections at 2 hr
intervals.
[0094] FIG. 39 is a diffusion-weighted magnetic resonance image of
the rat brain during occlusion and reperfusion of the MCA.
[0095] FIGS. 40A, 40B and 40C are EPR spectra of PNA and
.sup.15N-TPL in the skin on the back of an anesthetized mouse. 100
.mu.l of PNA was injected intradermal at the back of an
anesthetized mouse and the spectrum shown in (A) was recorded.
.sup.15N-TPL (1% v/w, 20 mg/ml) was then injected intravenously and
the spectrum shown as (B) was recorded 1 min. later. Thirty minutes
later the spectrum in (C) was recorded.
[0096] FIG. 41 shows pharmacokinetics of intravenous .sup.15N-TPL
(1% v/w), measured at the patch of skin on the back of the mouse
where PNA had been injected intradermal. The intensity of the low
field .sup.15N-TPL peak is plotted against time. The .sup.15N-TPL
peak is plotted against time. The .sup.15N-TPL signal rose after
injection, then fell sharply within 5 minutes, reflecting
bioreduction. In the presence of PNA as shown here, the
.sup.15N-TPL signal stabilized after 5 minutes and remained stable
for at least 30 minutes, reflecting oxidation by PNA.
[0097] FIG. 42A and 42B show the pharmacokinetics of intravenous
.sup.15N-TPL (1% v/w) measured at a patch of skin on the abdomen of
the mouse. (A) The low field peak intensity rose and fell and fall
within 5 minutes after the injection of .sup.15N-TPL. (B) The
increase in .sup.15N-TPL signal intensity as a function of time
after intradermal injection of 100 .mu.l PNA was maintained for at
least 30 minutes.
[0098] FIG. 43 shows the rate of survival over 10 days in an animal
model of rhabdomyolysis involving injection of glycerol into a
muscle and subsequent renal failure using polynitroxide albumin
with TEMPOL and albumin as a control.
DETAILED DESCRIPTION OF THE INVENTION
[0099] Nitroxides are stable free radicals that are shown to have
antioxidant catalytic activities which mimic those of superoxide
dismutase (SOD), and which when existing in vivo, can interact with
other substances to perform catalase-mimic activity. In the past,
nitroxides have been used in electron spin resonance spectroscopy
as "spin labels" for studying conformational and motional
characteristics of biomacromolecules. Nitroxides have also been
used to detect reactive free radical intermediates because their
chemical structure provides a stable unpaired electron with well
defined hyperfine interactions. In addition, nitroxides have been
observed to act as enzyme mimics; certain low molecular weight
nitroxides have been identified to mimic the activity of superoxide
dismutase (SOD). (A. Samuni et. al. J. Biol. Chem. 263:17921
(1988)) and catalase (R. J. Mehlhorn et. al., Free Rad. Res. Comm.,
17:157 (1992)). Numerous studies also show that nitroxides that are
permeable to cell membranes are capable of short-term protection of
mammalian cells against cytotoxicity from superoxide anion
generated by hypoxanthine/xanthine oxidase and from hydrogen
peroxide exposure.
[0100] The term "nitroxide" is used herein to describe stable
nitroxide free radicals, including compounds containing nitroxyl
groups such as synthetic polymers, their precursors (such as the
N--H form), and derivatives thereof including their corresponding
hydroxylamine derivative (N--OH) where the oxygen atoms are
replaced with a hydroxyl group and exist in a hydrogen halide form.
For the purposes of this invention, the chloride salt form of the
hydroxylamine derivatives is generally preferred.
[0101] In the nitroxides described here, the unpaired electron is
stable in part because the nitrogen nucleus is attached to two
carbon atoms which are substituted with strong electron donors.
With the partial negative charge on the oxygen of the N-0 bond, the
two adjacent carbon atoms together localize the unpaired electron
on the nitrogen nucleus.
[0102] Nitroxides generally may have either a heterocyclic or
linear structure. The fundamental criterion is a stable free
radical. Structurally, nitroxides of the following formula are
preferred where R.sub.1-R.sub.4 are electron donors and A is the
remaining members of a heterocyclic ring. 4
[0103] In these heterocyclic structures, "A" represents the
remaining carbon atoms of a 5-membered (i.e., pyrrolidinyl or
PROXYL with one double bond, i.e., pyrroline) or a 6-membered
(i.e., piperidinyl or TEMPO) heterocyclic structure and in which
one carbon atoms may be substituted with an oxygen atom (oxazolinyl
or DOXYL) and certain hydrogen atoms may be substituted with up to
two bromine atoms. In such heterocyclic structures, stable isotopes
may be utilized (e g., .sub.15N, deuterium). Substitution at the a
carbons should be such that the unpaired electron is maintained
substantially in a .pi.p orbital configuration. R.sub.1 through
R.sub.4 are alkyls (straight and branched chain) or aryl groups but
are preferred to be methyl or ethyl groups. The substituent groups
on the alpha carbons in any nitroxide should be strong electron
donors to enhance stability, thus methyl (CH.sub.3) groups or ethyl
(C.sub.2H.sub.5) groups are preferred although other longer carbon
chain species could be used. See e.g., U.S. Pat. No. 5,462,946.
Alternate structures which preserve the reactivity of the free
radical are known to those of ordinary skill in the art and are
continuing to be synthesized. See e.g., Bolton et al., "An EPR and
NMR Study of some Tetramethylisoindolin-2-yloxyl Free Radicals", J.
Chem Soc. Perkin Trans. (1993); and
[0104] Gillies et al., NMR Determination of EPR Hyperfine Coupling
Constants of Some
5-(n-Alkyl)-1,1,3,3-tetrakis(trideuteriomethyl)isoindol-
in-2-yloxyls, J. Chem. Soc. Faraday Trans., 90(16), 2345-2349
(1994);
[0105] When linked with biocompatible macromolecules pursuant to
this invention, the reactivity of the nitroxide is altered due to
the microenvironment. This reactivity may be tailored by the
labelling scheme employed and by the reaction with other compounds,
such as selenium, which are known to alter the stability or
reactivity of the free radical. In practice, stearic considerations
may limit the scope of the nitroxide compounds that are practical
and economical. The preferred nitroxides used with this invention
include nitroxides having the following structure: 5
[0106] As is apparent from the above, most suitable nitroxide
compounds may be represented basically by the structural formula
6
[0107] assuming that the R group is selected from among the
configurations which preserve the stability of the free
radical.
[0108] As noted above, the structural form of the nitroxide may be
varied without departing from its essential function. The nitroxide
functional groups may also be incorporated into dimers, trimers
such as B3T described below, oligomers, or polynitroxide synthetic
polymers having a plurality of repeating subunits each containing
at least one nitroxide. A preferred synthetic polymer has
nitroxides and carboxyl groups in each subunit of the polymer. For
example, the following compound may be synthesized by known
techniques, for example as described in U.S. Pat. No. 5,407,657
from a diamine compound and a dianhydride to yield: 7
[0109] For purposes of the invention, nitroxide group of the
polymer shown in U.S. Pat. No. 5,407,657 is preferably replaced
with Bromo-TEMPO. Such synthetic polymers may be used to label
hemoglobin, particularly native hemoglobin, by synthesizing the
compound with a slight excess of the diamine to yield a free amino
group at the terminal end of the polymer. The terminal amino group
is reacted with a neutrophilic group such as bromoacetabromide
yield a nitroxide polymer derivative of Bromo-TEMPO to enable
labelling at the amino groups on the protein similar to that of PNA
and PNH.
[0110] A variety of techniques have been described to covalently
attach a nitroxide to biomacromolecules, including hemoglobin,
albumin, immunoglobulins, and liposomes. See e.g., McConnell et.
al., Quart. Rev. Biophys. 3:p.91 (1970); Hamilton et. al.,
"Structural Chemistry and Molecular Biology" A. Rich et. al., eds.
W. H. Freeman, San Francisco, p.115 (1968); Griffith et. al., Acc.
Chem. Res. 2:p.17 (1969); Smith I.C.P. "Biological Applications of
Electron Spin Resonance Spectroscopy" Swartz, H. M. et. al., eds.,
Wiley/Interscience, New York p.483 (1972). Selected nitroxides have
been covalently bound to hemoglobin molecules for the purpose of
studying cooperative oxygen binding mechanisms of hemoglobin.
[0111] With respect to the macromolecules described here, at least
two techniques for binding the nitroxides to a macromolecule, often
known as "labelling strategies" are possible. The significance of
specific labelling lies in the micro-environment in which the
nitroxide is bound to the macromolecule and the nitroxide's
resulting catalytic activity. Specific labelling at a particular
ligand binding site or sites will yield a homogeneous product with
a more consistent binding site micro-environment and thus a more
reliable compound in terms of the catalytic specificity and
activity of the nitroxide.
[0112] The term "hemoglobin" is used generally herein to describe
oxy-, carboxy-, carbonmonoxy-, and deoxy-hemoglobin except as
otherwise noted by the context of the description. The hemoglobin
used with this invention may be human, recombinant or animal in
origin and is obtained and purified by known techniques. The
hemoglobin may be covalently bound to pyridoxal groups of
pyridoxal-5'-phosphate or ring opened adenosine triphosphate
(o-ATP) by reaction with the aldehyde groups and cross-linked
derivatives of hemoglobin. The cross-linked derivatives may include
polyfunctional, heterobifunctional and homobifunctional
cross-linking regents such as dialdehyde, polyaldehyde, diepoxide,
polyepoxide, activated polycarboxyl and dicarboxyl groups, for
example, 3,5-bis-bromosilicyl-bisfumarate, and TEMPO succinate or
TOPS See (U.S. Pat. No. 4,240,797) cyclodextrans and their anionic
(e.g., sulfate) cross-linked hemoglobin as well as polymerized
hemoglobin. The stabilized hemoglobin may be formed into
microspheres having a cross-linked outer layer produced by
cross-linking cysteine residues within the protein (VivoRx
Pharmaceuticals, Inc., Santa Monica, CA). All hemoglobin solutions
described herein and each at the other nitroxide-based solutions
such as in other compounds of the invention described herein are
administered in a physiologically compatible form such as in a
physiologically accepted intravenous solution or suspended in an
acceptable carrier, solvent or base. The hemoglobin solutions are
cell-free to remove pyrogens, endotoxins, and other
contaminants.
[0113] Preferred compositions using nitroxide in connection with
albumin include:
[0114] 1) non-specific labelling of albumin with nitroxide (e.g.,
4-(2-bromoacetamido)-TEMPO at high nitroxide to albumin ratios;
[0115] 2) specific labelling of albumin at specific ligand binding
sites; and
[0116] 3) enhanced labelling of albumin by reduction and alkylation
of disulfide bonds.
[0117] As used herein, the term albumin includes human serum
albumin, animal albumin, recombinant albumin, and fragments
thereof. The albumin may be temperature or chemical treated to
increase the available labelling sites. Additionally, the albumin
may exist as a monomer, a dimer, a polymer, or may be enclosed in
microspheres. Albumin as disclosed herein may also be treated with
polyethylene glycol (PEG) by well-known techniques to increase its
immunocompatibility.
[0118] A preferred technique for alleviating oxidative stress by
augmenting the body's antioxidant capabilities is the use of
multiple component nitroxide-based system. A first component is a
membrane-permeable nitroxide, such as TEMPOL. By virtue of their
charge characteristics and small molecular size, low molecular
weight unbound nitroxides readily permeate the cell membrane and
enter the intracellular space. A second component is another
nitroxide-including species such as a biocompatible
macromolecule-labelled with a high molar ratio of nitroxide
(polynitroxide), for example, human serum albumin labeled with a
30:1 molar ratio of TEMPOL. The use of a multiple component
nitroxide system of this invention helps to alleviate toxicity
which could result from large, concentrated, or repeated doses of a
low molecular weight membrane permeable nitroxide. Because the
hydroxylamine form of the nitroxide is not active as an
antioxidant, and because nitroxide toxicity at high doses is
thought to be primarily due to the antioxidant activity causing
perturbation of the cellular redox state, the hydroxylamine state
displays much lower toxicity than the corresponding unreduced
nitroxide. Thus, one embodiment of this invention describes the use
of a non-toxic dose of a membrane-permeable nitroxide, in
conjunction with a macromolecular polynitroxide, to activate a
nitroxide in vivo which has been reduced to an inactive form. In
similar fashion, the hydroxylamine may be administered as a
non-toxic nitroxide precursor which is converted to an active
antioxidant in vivo. The result is a safe and sustained therapeutic
level of a powerful antioxidant in the body.
[0119] With regard to safety in vivo, the levels of nitroxide which
may be administered pursuant to this invention are well tolerated
in animals and are expected to be well tolerated in humans because
nitroxides are known to be relatively safe: For example, the
maximum tolerated intraperitoneal dose of TEMPO in mice is 275
mg/kg and the LD.sub.50 is 341 mg/kg. Further, a
macromolecule-bound nitroxide will be safer than a free nitroxide
in its active form. The nitroxide-labelled macromolecules of this
invention used in combination with free nitroxide will reduce the
total quantity of nitroxide that otherwise would have to be
administered to achieve an antioxidant effect. An added advantage
of nitroxide-labelled macromolecules used in antioxidant
formulations lies in the ability to achieve high active levels of
nitroxides in their active anti-oxidant state with improved
safety.
[0120] Most of the nitroxides studied to date in living organisms
have been relatively low molecular weight compounds which can
easily permeate across cell membranes into body tissues. The
macromolecular-band nitroxides of this invention may be infused
intravenously and may remain confined to the vascular compartment
due to the membrane impermeability of the macromolecular species.
In such an embodiment, a nitroxide which is covalently attached to
a macromolecule will act to alleviate free radical toxicity while
confining the nitroxide to a location, i.e., the vascular
compartment, where the utility is optimized.
[0121] When TEMPOL is injected, it diffuses rapidly into the
intracellular space, where it is reduced to the hydroxylamine form
in the process of detoxifying (oxidizing) free radicals. In the
process of scavenging the unpaired electron from a toxic free
radical, the nitroxide is reduced to an oxoammonium intermediate.
The oxoammonium intermediate can then react in either of two ways.
It can be reoxidized back to a nitroxide by spontaneously donating
the free radical-derived electron to some naturally occurring
compound; this pathway may be described as enzyme-mimetic because
the net result is that the nitroxide is unchanged. Alternatively,
the oxoammonium intermediate can be further reduced to a
hydroxylamine. The hydroxylamine is not paramagnetic (i.e., it is
silent on EPR spectroscopy) and lacks the antioxidant catalytic
activity of nitroxide. Reaction equilibria in vivo strongly favor
reduction to the hydroxylamine. By virtue of its high membrane
permeability and inert chemical backbone, the hydroxylamine also
distributes freely in the intracellular and extracellular spaces,
and persists in the body for a relatively long period of time.
However, once the nitroxide is reduced to hydroxylamine the
antioxidant activity is lost.
[0122] TEMPOL 4-hydroxyl-2,2,6,6-tetramethyl-piperidine-N-oxyl is
rapidly consumed in the process of detoxifying free radicals; it is
reduced to an oxoammonium intermediate, which can be oxidized back
to nitroxide or further reduced to a hydroxylamine. Thus, the
biotransformation of the nitroxide (in the process of free radical
detoxification) yields a hydroxylamine. The hydroxylamine is not
paramagnetic (it is silent on esr spectroscopy) and lacks the
antioxidant catalytic activity of nitroxide. The use of TEMPOL
alone is not favored therapeutically because it is rapidly
converted to the hydroxylamine and may be toxic at the dosage level
needed to achieve a meaningful antioxidant effect.
[0123] However, the hydroxylamine is chemically stable and
relatively persistent in the body (the backbone of the nitroxide
molecule is relatively inert) and, in accord with the teachings of
this invention, can be chemically converted back to the active form
of the nitroxide. This in vivo conversion enables the safe clinical
use of nitroxides to provide a sustained antioxidant activity. As
noted above, the unpaired nitroxyl electron gives nitroxides other
useful properties in addition to the antioxidant activity. In
particular, nitroxides in their free radical form are paramagnetic
probes whose EPR signal can reflect metabolic information, e.g.,
oxygen tension and information on tissue redox states, in
biological systems. Because naturally occurring unpaired electrons
are essentially absent in vivo, EPR imaging has the further
advantage that there is essentially no background noise. Nitroxides
also decrease the relaxation times of hydrogen nuclei, and are
useful as contrast agents in proton or nuclear magnetic resonance
imaging (MRI) . Nitroxides can also act as contrast agents to add
metabolic information to the morphological data already available
from MRI. For example, by substituting various functional groups on
the nitroxide, it is possible to manipulate properties including
relaxivity, solubility, biodistribution, in vivo stability and
tolerance. Contrast enhancement obtained from nitroxide can improve
the performance of MRI by differentiating isointense, but
histologically dissimilar, tissues and by facilitating localization
and characterization of lesions, such as blood brain barrier
damage, abscesses and tumors.
[0124] A macromolecular polynitroxide tends to be distributed in
the extracellular space due to its high molecular weight and low
membrane permeability and is not readily reduced by the biochemical
milieu. However, it has been discovered that the macromolecular
polynitroxide is capable of accepting an electron from the
hydroxylamine, causing an in vivo conversion back into a nitroxide
with active antioxidant capabilities. This process effectively
transfers antioxidant capacity from a high-capacity macromolecular
storehouse of antioxidant activity outside the cell, to a high
mobility membrane-permeable nitroxide which may cross the cell
membrane to provide antioxidant activity inside the cell. Once
inside the cell, the nitroxide is reduced to the hydroxylamine by
oxidizing toxic free radicals, and then cycles out to the
extracellular space, where it is reactivated by the macromolecular
polynitroxide. Moreover, the reactivity of the macromolecular
polynitroxide may be enhanced by adding other compounds such as
Selenium.
[0125] Therefore, a particularly advantageous nitroxide-containing
formulation can be prepared when a high molar ratio of a nitroxide
is bound to a macromolecule (polynitroxide) and allowed to contact
a therapeutically active amount of an unbound low molecular weight
nitroxide in vivo thereby providing a catalytically active
polynitroxide macromolecule. The interaction of the therapeutically
active nitroxide with a catalytically active nitroxide provides a
sustained antioxidant, radioprotectant, imaging agent, etc. in the
surrounding tissues. In effect, the macromolecular species is a
reservoir of antioxidant activity which can recharge the activity
of the low molecular weight species which are able to permeate
membranes. This symbiotic approach disclosed herein provides
advantageous methods of administration, such as a topical
application of a macromolecular nitroxide combined with oral
administration of a low molecular eight nitroxide to provide
localized, sustained antioxidant activity.
[0126] Based on the experimental results and formulation data
presented here, the antioxidant, radioprotectant, antihypertensive,
and spectroscopic activity of the nitroxide-containing species of
this invention has been observed in vitro and in vivo with various
formulations. Based on these results, the reaction mechanism
whereby polynitroxide-labelled macromolecules and free nitroxides
participates in the oxidation/reduction reaction of free radicals
is sufficiently demonstrated that the capability exists to
formulate novel HBOCs, and other nitroxide-containing
macromolecules to detoxify free radicals which will be
advantageously used in the diagnosis and treatment of a wide
variety of physiological conditions.
[0127] In addition, this invention describes nitroxide-containing
compounds that are associated with a container for storage or
administration of pharmaceuticals such as intravenous fluids,
topical agents and others. Nitroxide-containing compounds may be
added in solid or liquid form to the interior of a container or may
be covalently attached to the inner surface of a container. One
advantageous technique for administration is the addition of
nitroxide-containing compounds, with or without free nitroxide, to
an in-line filter used in the administration of fluids. For
example, a polynitroxide albumin may be incorporated within a
filter along with free nitroxide or are attached to an insoluble
matrix housed in a filter to be used with an intravenous fluid
administration, such as an existing HBOC to scavenge toxic
oxygen-related compounds before infusion into a patient.
[0128] The HRCS formulations and nitroxide-labelled macromolecules
described below retard the formation of toxic oxygen-related
species by causing a nitroxide to function as a "superoxide
oxidase," an enzyme-like reaction not known to occur in the red
cells. In these HRCS formulations, the nitroxide prevents the
accumulation of the undesirable superoxide anion generated from the
auto-oxidation of hemoglobin (See Equation [1]). The
nitroxide-labelled macromolecules, such as albumin and
immunoglobulins, similarly function as antioxidant enzyme mimics
whose function remains localized in the vascular and interstitial
compartments and which may react with membrane permeable nitroxides
to provide intracellular protection.
[0129] Preferred compositions using nitroxide in connection with
immunoglobulins include a nitroxide-labelled hapten or antigen
bound to an immunoglobulin specific for the hapten or antigen.
[0130] Furthermore, pursuant to this invention, the beneficial
therapeutic effects of nitroxide compounds can be controlled and
sustained. For example, the nitroxide
2,2,6,6-tetramethyl-1-oxyl-4-piperidylidene succinic acid (TOPS),
may be bound to the primary bilirubin binding site of human serum
albumin. In vivo, this binding prolongs plasma half-life and slows
the diffusion of the nitroxide into the intracellular space,
reducing the necessary dosage (and hence reducing potential
toxicity) and prolonging biological action. Thus, although
nitroxides such as TOPS alone, without a macromolecular
polynitroxide, may have utility as an antioxidant agent. Pursuant
to this invention, it is possible to select or design carriers
which can deliver nitroxides to particular sites in the body as a
means of localizing therapeutic antioxidant activity.
[0131] In view of the stable chemical nature of the nitroxides, the
compositions disclosed here can be administered by various routes.
The membrane-permeable nitroxide can be administered parenterally
or orally. In the reduced form, hydroxylamine, can act locally in
the GI system or be taken up into the blood. Thus, sustained
antioxidant activity can be provided in all body compartments. The
macromolecular polynitroxide can be administered parenterally where
it will remain localized in the extracellular space to reactivate
reduced free nitroxide, orally, or topically/transdermally where it
acts to activate circulating, reduced nitroxide thereby providing a
localized antioxidant effect.
[0132] The particular reactivity of a protein-based macromolecular
polynitroxide and a membrane-permeable nitroxide appears to be
enhanced by heating of the macromolecule and labeling at primary
amino groups in the polypeptide chain. Heating is known to alter
the conformation of the macromolecule, stretching hydrogen bonds
between amino and carboxyl groups and causing the macromolecule's
quaternary structure to be altered. Subsequent covalent labeling by
nitroxides at the amino groups may occur at relatively internal
sites on the protein which were exposed as a result of heating. In
the resulting nitroxide-labeled macromolecule, these nitroxide
moieties are more protected from reaction with the solvent. Also,
where nitroxides are attached to many amino groups on the protein,
the preponderance of remaining carboxyl groups creates an acidic
microenvironment surrounding the bound nitroxide. An acidic
environment increases the reactivity of the nitroxide by drawing
the unpaired electron in the N-O bond toward the oxygen atom.
[0133] In the embodiments of the invention directed to a red cell
substitute, the requisite property of the nitroxides is their
ability to influence the course of the superoxide anion cascade in
HRCS by mimicking the superoxide oxidase, superoxide dismutase, and
catalase activities without substantially being consumed in the
process.
[0134] In the "superoxide oxidase" reaction, the superoxide anion
is oxidized back into molecular oxygen without proceeding to the
formation of hydrogen peroxide. This is accomplished in part by
creating a storage condition wherein the concentration of nitroxide
greatly exceeds that of oxygen. Used in the manner disclosed
herein, the nitroxide prevents the cascade of undesirable oxidative
reactions that begin with the formation of the superoxide anion.
Furthermore, the physiologically compatible HRCS solutions
described here will offer advantages over the existing HBOC
solutions because the nitroxide will mimic the enzymatic activity
of superoxide dismutase and catalase after the formulations
described herein are infused into a patient.
[0135] Although a wide variety of nitroxides may be used with this
invention, the nitroxide should be physiologically acceptable at a
minimum concentration required to alleviate oxygen toxicity. In
assessing an operative species, it is noteworthy that the
relatively low toxicity of nitroxides has encouraged their
development as contrasting agent in NMR imaging (See U.S. Pat. Nos.
4,834,964; 4,863,717; 5,104,641).
[0136] A number of methods for isolating and purifying hemoglobin
solutions such that they are physiologically compatible are known
to those skilled in the art. Typically, purified hemoglobin
compositions contain at least 99% hemoglobin by weight of total
protein, a total phospholipid content of less than about 3
.mu.g/ml, less than 1 .mu.g/ml of either phosphatidylserine or
phosphatidylethanolamine and an inactive heme pigment of less than
6%. The purified hemoglobin solutions which are useful in this
invention can be prepared using a variety of conventional
techniques, including but are not limited to, those disclosed in
Cheung et. al., Anal Biochem 137:481-484 (1984), De Venuto et. al.,
J. Lab. Clin. fled. 89:509-516 (1977), and Lee, et. al., Vith
International Symposium on Blood Substitutes, San Diego, Calif.
Mar. 17-20 Abstract H51 (1993).
[0137] The purified hemoglobin solutions used in this invention
should be essentially free of oxygen. Hemoglobin in solution may be
deoxygenated by admixture with a chemical reducing agent which
causes the hemoglobin to release oxygen and to be maintained in a
substantially deoxygenated state. A preferred method for
deoxygenating a hemoglobin solution is performed by exposing a
hemoglobin solution to an inert, essentially oxygen-free gas, such
as nitrogen or carbon monoxide to cause removal of bound oxygen
from the hemoglobin and conversion of the hemoglobin in solution to
a form such as deoxy-hemoglobin or carbonmonoxy-hemoglobin that
lacks oxygen. Alternatively, hemoglobin may be exposed to a vacuum
or gas through a membrane that is permeable to oxygen yet
impermeable to hemoglobin. For example, a hemoglobin solution may
be passed through a diffusion cell having a membrane wall along
which hemoglobin flows and through which oxygen is capable of
passing, but hemoglobin is not. Inert gas is circulated along the
side of the membrane wall opposite the hemoglobin solution causing
the removal of oxygen and the conversion of the hemoglobin in
solution to the deoxygenated state. Preferably, the
deoxy-hemoglobin is maintained in an essentially oxygen-free
environment during nitroxide-labelling, cross-linking,
polymerization, or conjugation.
[0138] After removal of any bound oxygen, a nitroxide is covalently
attached to the hemoglobin. Normally at least one, and preferably
more than one, nitroxide will be covalently attached to a single
hemoglobin molecule. The nitroxide may be covalently attached to
the hemoglobin at any of several sites on the hemoglobin molecule
including:
[0139] (a) at one or more of the free sulfhydro (--SH) groups, for
example, at the .beta.-93 site;
[0140] (b) at any reactive amino (--NH.sub.2) groups, for example,
in the DPG site at Val-l of the .beta.-chain and/or lysine-82 of
the .beta.-chain and/or lysine-99 of the .alpha.-chain;
[0141] (c) at any non-specific surface amino (--NH.sub.2) or
carboxyl (--COOH) group;
[0142] A nitroxide may also be bound to any residual aldehyde,
epoxy, or activated carboxyl groups of a divalent- or a
multivalent-cross-linker involved in the cross-linking and
polymerization of hemoglobin or at any residual reactive groups on
an agent such as dextran (Dx) or hydroxylethylstarch (HES) or
polyoxyethylene (POE) used to conjugate hemoglobin.
[0143] As described in Equation [1], above, during the storage
period, the hemoglobin in an HBOC solution is slowly auto-oxidized
by oxygen to form met-hemoglobin and the superoxide anion. However,
during the storage of the HRCS that are the subject of this
invention, the superoxide anion thus formed will reduce the
nitroxide to a hydroxylamine derivative, and the superoxide anion
will be oxidized to form molecular oxygen by the following
reaction. 8
[0144] The conversion of superoxide anion to molecular oxygen
described in Equation [4] prevents the accumulation of superoxide
anion and the subsequent formation of hydrogen peroxide. This
activity, described herein as a "superoxide oxidase" activity, will
be most effective when the initial oxygen content in the
composition is kept to a minimum, the composition is stored in an
essentially oxygen free environment and the nitroxide concentration
is sufficient to prevent the formation of superoxide anion and
hydrogen peroxide. Therefore, storage of the HRCS in an essentially
oxygen-free container is preferred.
[0145] Container systems that permit a solution to be stored in an
oxygen free environment are well known because many non-hemoglobin
based intravenous solutions are sensitive to oxygen. For example, a
glass container that is purged of oxygen during the filling and
sealing process may be used. Also, flexible plastic containers are
available that may be enclosed in an overwrap to seal against
oxygen. Basically, any container that prevents oxygen from
interacting with hemoglobin in solution may be used.
[0146] To demonstrate the "superoxide oxidase" activity of a
nitroxide, samples of nitroxide-labelled hemoglobin in solution are
kept in an accelerated oxidative storage condition and the redox
state of the nitroxide is studied over time by electron spin
resonance spectroscopy. For example, an o-raffinose polymerized
hemoglobin solution that has been labelled with 4-amino-TEMPO is
stored in its oxygenated state in a sealed glass container (FIG.
1A). In such a state, the rate of superoxide anion and
met-hemoglobin formation in solution is sufficiently rapid that the
conversion of the nitroxide to its hydroxylamine derivatives may be
conveniently monitored (See Equation [4] and compare FIGS. 1A and
1B). Equation 4 represents that the conversion of nitroxide to its
diamagnetic hydroxyl derivative is coupled to the conversion of the
superoxide anion back to molecular oxygen. The experimental
evidence in support of such a conversion is shown in FIGS. 1A and
1B. The electron spin resonance spectrum of TEMPO covalently
attached to the hemoglobin (FIG. 1A) was converted to its
diamagnetic derivatives which result in the complete disappearance
of the resonance peaks after storage of the sample for 30 days at
4.degree. C. (FIG. 1B). The nitroxide is considered to have
performed a "superoxide oxidase "-like activity when it is
converted to its hydroxylamine derivative in the presence of
hemoglobin.
[0147] The "superoxide dismutase" activity of a nitroxide in an
HBOC solution is demonstrated by showing the reconversion of the
hydroxylamine derivative back to a nitroxide (See Equation [5]
together with Equation [4]). Knowing that under the experimental
conditions described in FIGS. 1A and 1B the nitroxide is fully
converted to hydroxylamine (See Equation [4]), the nitroxide may be
regenerated by simply providing more superoxide anion as shown in
Equation 5. To demonstrate this reaction mechanism, the relative
concentration of hemoglobin (and thus superoxide anion) to the
nitroxide is increased by diluting the sample in FIG. 1A with an
equal volume of unlabelled hemoglobin. A comparison of FIGS. 1A and
1C shows an approximate 50% reduction of the signal intensity of
the nitroxide due to the dilution effect. On the other hand, after
30 days of cold storage at 4.degree. C., the nitroxide was
partially regenerated (See FIG. 1D) as predicted by Equation [5].
This observation is consistent with the reconversion of the
hydroxylamine derivative to nitroxide coupled with the formation of
hydrogen peroxide from superoxide anion. 9
[0148] Summing equations [4] and [5] results in:
2.O.sub.{overscore (2)}+2H.sup.+.fwdarw.O.sub.2+H.sub.2O.sub.2
[0149] which demonstrates that the nitroxide acts as a low
molecular weight, metal-free, SOD mimic in "HBOC" solutions. The
detection of electron spin resonance spectrum of the nitroxide (in
FIG. 1D) is consistent with the reaction of superoxide anion with
the hydroxylamine (R N--OH) resulting in the formation of nitroxide
(R N--O) and hydrogen peroxide (H.sub.2O.sub.2) . Recently,
oxoammonium cation has been proposed to be involved as one
intermediate in the nitroxide catalyzed dismutation of superoxide.
(Krishna et al., Proc. Nat. Acad. Sci. USA 89 5537-5541
(1992)).
[0150] The HRCS formulations described herein will alleviate the
oxidative stress originating from the generation of the superoxide
anion in the existing HBOC solutions, and upon transfusion, will
diminish the destruction of nitric oxide, the endothelium-derived
relaxing factor (EDRF). If the destruction of EDRF is prevented,
the problem of vasoconstriction and systemic hypertension that are
observed when the existing HBOC solutions are infused into a
patient will be substantially alleviated.
[0151] The number of nitroxide molecules per hemoglobin molecule
may be in the range of approximately 1-40 or any integral value or
range of values therebetween and for specific labelling is most
preferably about 2. When used as a component of a multi-component
system, the molar ratio of nitroxide to hemoglobin may be
approximately 3 to 60 or any integral value or range of values
therebetween. For example, a nitroxide such as 3-maleimido-PROXYL
is covalently bound to hemoglobin in solution by first preparing a
100 mM solution of the nitroxide in ethanol as the carrier solvent.
Two (2) molar equivalents of the nitroxide to hemoglobin was added
directly with mixing to a DCL-Hb (8g/dl) in Lactated Ringers. The
reaction mixture was allowed to react at 22.degree. C. with
agitation until greater than 90% of the nitroxide was covalently
linked to the DCL-Hb, usually within one hour. The unreacted
nitroxide was then removed with a cross-flow membrane filtration
system having a molecular-weight cut-off of 30,000 daltons by
washing three (3) times with 10 volumes of Lactated Ringers. The
retantate hemoglobin concentration is adjusted to between 7-14
g/dl, sterile filtered, and stored at 4.degree. C. After
transfusion, when the HRCS is fully oxygenated, the nitroxide is
expected to function as a SOD-mimic and secondly as a
catalase-mimic. As an SOD-mimic it dismutates the superoxide anion
to hydrogen peroxide (See Equation [2]) and consequently protect
against the destruction of nitric oxide in the endothelium to
prevent vasoconstriction. As a catalase-mimic it prevents hydrogen
peroxide toxicity by converting the latter to harmless water (See
Equation [3]).
[0152] As noted above, nitroxides have been covalently bound to
hemoglobin to study the cooperative oxygen binding properties of
the hemoglobin molecule itself. However, nitroxides have not been
used with stabilized, i.e., cross-linked, or polymerized,
encapsulated, or conjugated hemoglobin solution that are
physiologically compatible. Nor have synthetic nitroxide-containing
polymers been used to label native hemoglobin. The known chemistry
of hemoglobin and nitroxides suggests that it is possible to
perform similar nitroxide-labelling of hemoglobin that has been
chemically cross-linked or cross-linked through recombinant
techniques by selecting an available site on the hemoglobin
molecule that is not blocked by the particular compound used to
stabilize, polymerize, or conjugate the hemoglobin molecule(s).
Because certain of the stabilized and polymerized forms of
hemoglobin described below are currently involved in clinical
trials, the attachment of nitroxides to these stabilized and
polymerized hemoglobin-based oxygen carriers is described below to
demonstrate that the oxygen detoxification function of this
invention is applicable to the existing hemoglobin solutions.
[0153] The nitroxide-labelling technology demonstrated here in the
example of nitroxide-HBOC is readily applied to the production of
other nitroxide-labelled macromolecules with useful antioxidant and
enzyme-mimetic activities, for example nitroxide-labelled serum
albumin and nitroxide-labelled immunoglobulin. Forms of serum
albumin which can readily be labelled by nitroxide by this
technology are monomeric (normal) albumin, and albumin homodimers,
oligomers, and aggregates (microspheres and microbubbles) and
polypeptide fragments of each. As described herein, the named
protein or macromolecule includes fragment, i.e., polypeptide
fragments of a protein.
[0154] Due to the differences in application, the formulations
described herein may be used together or in isolation. For example,
in the therapy and diagnoses of cardiac reperfusion injury or
ischemic injury in the cerebrovascular system, it may be desirable
to take advantage of several aspects of this invention, i.e.,
oxygen delivery, systemic protection from oxidative stress,
localized protection from reperfusion injury, and enhanced imaging.
In such a case, a combination of the formulations described herein
could be used such as an existing HBOC, nitroxide-labelled albumin
and a low molecular weight nitroxide which could be administered
simultaneously or in sequence, depending on the therapeutic or
diagnostic goal.
[0155] With respect to selecting a particular formulation and
method of administration pursuant to this invention, the
formulation and method of administration are dictated by the
particular application. The selection of a nitroxide-based compound
capable of accepting an electron from a low molecular weight
membrane permeable species for a particular application may be made
to complement, several available methods of administration and
preferred formulations may be selected based on the site specific
protection desired for the particular application. The avoidance of
oxidative stress from infusion of a hemoglobin-based oxygen carrier
as described above is a prime example of selecting formulations and
methods of administration pursuant to this invention to provide
specific protection from free radical toxicity to avoid the toxic
side effects of HBOC infusion. Where site specific protection or
activity is desired in the skin or dermal layers a preferred
compound is TOPS because it is relatively small and membrane
permeable. Where specific protection or activity is desired in the
gastrointestinal tract, a polynitroxide dextran is preferred
because such a compound is less susceptible to enzymatic digestion
while in the gastrointestinal tract. In such an application, oral
or rectal administration is preferred. Where specific protection or
activity is sought for the intravenous or intravascular regions,
such as the cerebrovascular or cardiovascular system, a
polynitroxide albumin is preferred because albumin is a major
plasma protein, is well-tolerated, easy to administer, and exhibits
an extended plasma half-life. Such applications may also include a
hemoglobin-based oxygen carrier or polynitroxide derivative
thereof. The same rationale applies for intraperitoneal or
intradermal administration.
[0156] As shown by the several examples, the administration of the
compounds of the invention may be localized in the body or may be
directed to a single group of cells or an organ. In particular, the
preservation of an organ for transplantation may be achieved by
infusion of polynitroxide albumin, either before or after the
removal of the organ from the body. For example, in a heart
transplant, PNA is used to selectively perfuse the heart, i.e., by
concentrated intravenous administration followed by removal of the
organ from the body. The organ may also be transplanted and stored
in a PNA bath to prevent deterioration by processes such as lipid
peroxidation, oxidative stress, etc. Furthermore, following
surgical transplantation, when the organ is reperfused with
oxygenated blood, the organ will be protected in the same way as is
demonstrated herein ischemic reperfusion injury of the heart and
brain. If specific protection in the lungs is desired, an aerosol
form of polynitroxide albumin is preferred to enable coating of the
pulmonary airways. As will be apparent to those skilled in the art,
these preferred formulations may be altered depending on the
particular application.
[0157] As noted, the above formulations are preferred embodiments
of the electron accepting compound. With respect to the membrane
permeable electron donating compound, the selection of a preferred
compound also depends on the application and method of
administration. The formulation and method of administration should
achieve a systemic or tissue specific distribution commensurate in
scope with the extent of the free radical species generated by, or
coincident with, the physiologic condition of interest or the
region to be imaged or treated. For example, in sepsis, a large
scale collapse of the circulatory system is observed which
accompanies a systemic increase in free radical generation.
Similarly, whole body irradiation results in free radical
generation throughout the body. In such situations, a small
molecular weight, membrane permeable, electron-donating nitroxide
such as TEMPOL may be administered orally or intravenously in a
quantity to insure systemic distribution. Accordingly, such
administration should be accompanied by administration of an
electron acceptor which is also widely distributed such as an
intravenous administration of polynitroxide albumin. Where the free
radical generation is more localized, it is preferred to provide
the localized protection or activity by selective application of
the electron accepting species because the membrane permeable
species tends to become systemically distributed upon
administration, although a topical administration of the membrane
permeable species to the skin may yield a more localized
effect.
[0158] Based on the disclosure herein, those skilled in the art can
select formulations and methods of administration to most
effectively meet the demands of the particular application where
this invention is to be used. For example, to achieve image
enhancement of the gastrointestinal system, one may select an oral
administration of polynitroxide dextran as the electron acceptor
and an oral and/or intravenous administration of membrane permeable
TEMPOL. As a further example, to treat psoriasis, a localized skin
condition where free radical generation is manifested, one may
select a topical application of TOPS as the electron acceptor and a
topic application of membrane permeable TEMPOL. By any similar
rationale, site specific protection or activity can be provided
using the formulation of this invention for other disease states,
or diagnostic or therapeutic state where free radical species are
present. Therefore, the following examples disclose a detailed
description of several formulations which can be used in any
combination depending on the application to which this invention is
applied.
Example One--Containers and Filters Containing Nitroxides and
Nitroxide-Labelled Compounds
[0159] It is possible to provide the oxygen-detoxification function
of this invention to existing intravenous solutions, such as the
HBOC solutions, without chemically modifying the existing
formulations. By including a polynitroxide macromolecule, which may
be used in connection with a free nitroxide, or by covalently
attaching nitroxides to a surface inside the vessel in which the
HBOC is stored, the adverse physiological effects caused by oxygen
toxicity that are observed with the existing formulations will be
alleviated.
[0160] The container used with the hemoglobin-containing solutions
that are the subject of this invention should be physiologically
compatible having similar characteristics as any container to be
used with intravenous fluids. Typically, glass or a biocompatible
plastic is suitable. For the embodiments of the invention where an
intravenous solution is placed in a container for any length of
time, the container should be oxygen free and capable of being
sealed to exclude oxygen. With a glass container, a traditional
stopper and closure means is sufficient. However, some of the
flexible plastic containers currently available are oxygen
permeable. In this case, a foil overwrap or other sealing mechanism
may be used to prevent oxygen from contacting the solution.
[0161] To apply a nitroxide to an inner surface of a container, a
non-leaching layer of a nitroxide polymer or a nitroxide-doped
copolymer is coated directly on the inner surface.
Nitroxide-containing polymers can be created by a number of
techniques based on generally known principles of polymerization as
long as the stability of the free radical is maintained in the
polymerization process.
[0162] Also, the interior surface of an HBOC container may be
modified to contain a coating layer of a substance that can bind a
nitroxide, such as hydrophilic hydrazide groups which will react
with the ketone or the aldehyde group of a nitroxide to form stable
hydrazone derivatives. The coating reaction is straight forward.
For example, the nitroxide (100 mM) in acetate buffer at pH 5.0 is
added to a hydrazide activated plastic container to facilitate the
formation of a hydrazone linkage.
[0163] Once the container is prepared, a physiologically compatible
solution is added. This solution may be a stabilized and purified
HBOC or the HRCS disclosed herein, and could also include any
intravenous colloid or crystalloid solution that is desirable to
co-infuse with hemoglobin. The solution is then maintained in an
essentially oxygen-free environment.
[0164] In addition to treating a surface inside a container, a
filter-type cartridge, with a luer lock inlet and outlet,
containing a gel or solid matrix upon which a nitroxide is
immobilized may be used to remove reactive oxygen-derived species
while the hemoglobin solution passes through the cartridge. For
such an administration technique, a polynitroxide macromolecule may
be added into the housing of the filter through which a solution
passes for direct infusion into a patient to react with the
solution before infusion. A low molecular weight nitroxide may also
be included. In these applications, nitroxide may also be bound to
a soft- or hard-gel matrix, thereby functioning essentially as a
sterile in-line filter, prior to infusion. A variety of methods to
attach small ligands, such as nitroxide, to a solid matrix are well
known in the art of affinity chromatography, as are the techniques
to chemically modify glass and plastic surfaces. Several types of
matrices that are compatible with sterile solutions are known
including agarose gel, polysulfone-based material, latex, and
others.
[0165] In the filter cartridge approach, the solid matrix is
covalently linked with a nitroxide and contained in a filter
housing or other such apparatus such that a solution, such as
hemoglobin can flow through the apparatus and be brought into
contact with a nitroxide while being infused into a patient. A
practical approach is to use a commonly available activated agarose
gel as the matrix and contain the gel in a sterile luer lock
cartridge. The cartridge is then simply inserted in the fluid
administration line during the transfusion of a solution containing
hemoglobin. In practice, the structure that comprises the filter
housing in which the nitroxide and through which hemoglobin is
passed can be provided by a variety of known structures. See e.g.,
U.S. Pat. No. 5,149,425. Referring now to FIG. 12, housing 1
contains a nitroxide-labelled agarose gel. For example, a
4-bromoacetamido-TEMPO labelled .omega.-aminohexyl-agarose (See
FIG. 2A) a 1,4-bis(2,3-epoxypropoxy)butane agarose coupled with
4-amino-TEMPO (See FIG. 2B). Other compounds (not shown) may be
included with the filter housing.
[0166] During the transfusion, the intravenous transfusion line
containing the solution would be connected to the luer inlet 2
allowed to enter the housing 1 wherein the hemoglobin solution
would encounter the nitroxide-containing compounds contained within
the housing or bound to the matrix 4, in this process, the
nitroxide-containing compositions will be infused and may react to
remove the toxic oxygen-related species from solution. The
hemoglobin solution would then pass out of the cartridge through
the luer outlet 3 and would be directly transfused into a patient.
The electron resonance spectrum of 4-amino-TEMPO labelled
epoxy-agarose is shown in FIG. 2A. Alternatively, an
.omega.-aminohexyl-agarose may be reacted with
4-(2-bromoacetamido)-TEMPO to form TEMPO labelled agarose. The
electron spin resonance spectrum is shown in FIG. 2B. An
alternative would be to couple the 4-carboxyl-TEMPO to the
amino-agarose with carbodiimide via a carboamide linkage.
Conversely, the 4-amino-TEMPO is readily coupled to the carboxyl
group on an agarose gel using carbodiimide, for example,
1-ethyl-3-(3-dimethylamin- o-propyl)carbodiimide.
[0167] The cartridge labelled with 4-amino-TEMPO is prepared by
circulating a 100 mM 4-amino-TEMPO (Sigma Chem. Co.) in a Lactated
Ringers solution through an aldehyde AvidChrom Cartridge (Unisyn
Tech. Inc.) at room temperature for one hour followed by the
reduction with sodium cyanoborohydride for six (6) hours. The
interior of the cartridge housing is thoroughly washed with
Lactated Ringers.
[0168] The cartridge labelled with 3-amino-PROXYL may be similarly
prepared by substituting 4-amino-TEMPO with 3-amino-PROXYL
according to the procedure described above.
Example Two--Nitroxide-Labelled Stabilized Hemoglobin
[0169] To prevent dissociation of hemoglobin into its constituent
subunits, hemoglobin is intramolecularly stabilized by chemical or
recombinant cross-linking of its subunits. "Stabilized" hemoglobin
is referred herein to describe hemoglobin monomers that are
stabilized by chemical or recombinant cross-linking and also to
describe dimers, trimers, and larger oligomers whose constituent
hemoglobin molecules are stabilized by cross-linking with
cyclodextrans and their sulfated derivatives.
[0170] A preferred technique for attaching nitroxide to stabilized
hemoglobin is by the covalent attachment of the nitroxide to the
.beta.-93 sulfhydryl groups of the two .beta.-chains of stabilized
hemoglobin. Although specific labelling at the .beta.-93 site has
been demonstrated on native human hemoglobin for conformational
studies (See review by McConnell et. al., Quart. Rev. Biophys. 3:91
(1970)), such a specific labelling of cross-linked hemoglobin has
not been reported. As noted above, several types of
hemoglobin-based oxygen carriers have been developed that are
stabilized through chemical cross-linking, for example,
DBBF-cross-linked hemoglobin and hemoglobin that is stabilized and
oligomerized with o-raffinose.
[0171] The ring-opened sugars described in my U.S. Pat. No.
4,857,636 yield polyvalent aldehydes derived from disaccharides,
oligosaccharides, or, preferably, trisaccharides such as
o-raffinose. These compounds function both to provide
intramolecular stabilization (cross-linking) and intermolecular
polymerization. By controlling the reaction disclosed in my earlier
patent, the polyvalent aldehydes may be used to produce
"stabilized" hemoglobin as defined above without polymerization. In
another case, a nitroxide may be covalently bound to the stabilized
hemoglobin or the polymerized hemoglobin. Therefore, the
hemoglobin-based solutions that are stabilized using the polyvalent
aldehydes are considered in the present embodiment as a
"stabilized" hemoglobin and in the subsequent embodiment as a
polymerized hemoglobin.
[0172] To demonstrate, that the .beta.-93 site of the chemically
modified hemoglobin has not been rendered sterically inaccessible
for nitroxide attachment, results are presented to confirm that a
nitroxide may be covalently bound to the .beta.-93 site of
DBBF-Hb.
[0173] In this embodiment, DBBF-Hb is reacted with two types of
nitroxides (TEMPO and PROXYL) which contain two types of sulfhydro
group specific functional groups and have the following structural
formula: 10
[0174] DBBF-Hb is prepared by cross-linking purified deoxygenated
hemoglobin in solution with bis(3,5 dibromosalicyl)fumarate by
known techniques, and the resulting product is purified by column
chromatography. The covalent attachment of 3-maleimido (2,2,5,5
-tetramethyl pyrrolidine-N-Oxyl) [3-maleimido-PROXYL] is
accomplished by adding 2 molar equivalents of this nitroxide using
methanol as the carrier solvent at a concentration of approximately
100 mM of 3-maleimido-PROXYL to 1 ml of DBBF-Hb at a concentration
of approximately 8 g/dl in Lactate Ringers. The DBBF-Hb is allowed
to react at 22-23.degree. C. for approximately 30 minutes with
mixing. The extent of cross-linking is estimated from the percent
disappearance of the electron spin resonance signal intensity of
the -unreacted nitroxide. To remove the unreacted nitroxide, the
reaction mixture was washed three (3) times with a 10 volume excess
of Lactated Ringers using a Filtron stire cell with a 30 kilodalton
cut-off nominal molecular weight limits (NMWL) polyethylene sulfone
(PES) membrane (Filtron Technology Co.). The electron spin
resonance measurements of the nitroxide-labelled hemoglobin was
recorded with a Bruker ESR spectrometer. FIG. 3A shows the electron
spin resonance spectra of 4-(2-bromoacetamido)-TEMPO labelled
DBBF-Hb. The electron spin resonance spectrum of DBBF-Hb that is
similarly labelled with 3-maleimido-PROXYL is shown in FIG. 3B.
[0175] In this embodiment, the nitroxide is covalently linked to
the lone sulfhydro group on the two .beta.-globin chains of
hemoglobin. Thus, the nitroxide to hemoglobin-bound oxygen ratio is
approximately 200 to 1 at 99.00% deoxy-hemoglobin because there are
two nitroxides attached to the two .beta.-globin chains of the
hemoglobin. After transfusion, however, the deoxygenated HRCS picks
up oxygen in the lung and the nitroxide to hemoglobin-bound oxygen
ratio becomes approximately 1 to 2 at 100% oxygenation because
there are four oxygen molecules bound to the four globin chains of
the hemoglobin with the two nitroxides remaining on the
.beta.-globin chains.
[0176] Using a hemoglobin-to-nitroxide ratio of 1:2, greater than
90% of the nitroxide is covalently attached to the DBBF-Hb. DBBF-Hb
may also be covalently labelled with a spacer group (e.g., an extra
methyl group) between the maleimido and PROXYL moieties (i.e.,
3-maleimidomethyl-PROXYL- ) which would exhibit a resonance
spectrum similar to that of FIG. 3B. It is noteworthy that other
nitroxides may be covalently attached to specific amino-groups in
the DPG binding site (e.g., .beta.-Val-1.beta.-Lys-82 and
.alpha.-Lys-99) or may be attached to the remaining 40-plus surface
lysine e-amino groups on hemoglobin. Isothiocyanate derivatives of
the TEMPO and PROXYL nitroxides are also reactive with the amino
group. For example, 4-isothiocyanate-TEMPO may be added to
hemoglobin at a molar ratio of approximately 10:1. Resonance
spectrum (not shown) of hemoglobin labelled with this nitroxide at
other sites is similar to that shown in FIG. 3A.
[0177] The ability to attach nitroxides at several sites of DBBF-Hb
suggests that recombinant hemoglobin that is stabilized with
alphaglobin dimers (D. Looker et.al. NATURE 356:258 (1992)) may be
similarly labelled with a nitroxide. It is also possible to prepare
a DBBF analogue of a nitroxide-labelled cross-linking agent such as
a TEMPO labelled succinate (See U.S. Pat. No. 4,240,797).
[0178] FIG. 4 is ESR spectra of (A) 2-(bromoacetamido)-TEMPO, (B)
2- (bromoacetamido) -TEMPO-labelled HBOC and (C) .sup.15ND.sub.17
TEMPOL (TEMPOL: 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) in
Lactated Ringer's solution recorded at room temperature. The
difference in FIG. 4A and 4B represents the difference in the
mobility of a small molecular weight nitroxide to that of a
nitroxide covalently attached to a macromolecule such as
hemoglobin. FIG. 4C shows that a stable isotope nitrogen .sup.15SN
with a nuclear spin of 1/2 yields two resonance peaks and that
natural-isotopic .sup.15N with a nuclear spin of 1 yields three
resonance peaks (compare (4A to 4C). In the set of experiments
described here the separation of these-resonance peaks is used to
demonstrate the enzyme-mimic and in vivo and in vitro
oxidation/reduction reactions of small and macromolecular weight
nitroxides.
[0179] Nitroxide-labelled HBOC with different molar ratios of
nitroxide to hemoglobin are prepared as follows. 2, 4, and 8 molar
equivalents of 4-(2-bromoacetamido)-TEMPO, were added as solid
powder directly into three separate 15 ml Vacutainers in a clean
hood. After replacing the rubber septum, 4-(2-bromoacetamido)-TEMPO
was subsequently dissolved in 200 ul chloroform. The Vacutainers
were then connected to high vacuum (5 mm Hg) via a 27 gauge needle
through the rubber septum and the chloroform was removed leaving a
thin film of 4-(2-bromoacetamido)-TEMPO coating the lower half of
the Vacutainer. After introducing the appropriate amount of HBOC
via sterile transfer through the rubber septum, the solutions were
allowed to react at room temperature with intermittent vortex
mixing at approximately 5 minute intervals for 1/2 hour (not all
solids were dissolved in the 4 and 8 molar ratios of nitroxide to
hemoglobin), the Vacutainers were then left at 4 degrees C in a
refrigerator over night. Vortex mixing at room temperature was
resumed the next morning for another 1/2 hour until all solids of
4-(2-bromoacetamido)-TEMPO had visually disappeared from the
surface of the Vacutainer.
[0180] The reaction mixtures and the control, were then transferred
to a sterile dialyzing tube and dialyzed against Lactated Ringers
until no unlabelled free 4-(2-bromoacetamido)-TEMPO electron spin
resonance (ESR) signals could be detected. The ESR spectra of
4-(2-bromoacetamido)-TEMPO-- labelled HBOC at 2, 4, and- 8 molar
equivalents 4-(2-bromoacetamido)-TEMPO to Hb are shown in FIGS.
5A-5C respectively. At 2 molar equivalents of
4-(2-bromoacetamido)-TEMPO to hemoglobin, the ESR spectra are
essentially the same with or without dialysis indicating the
covalent labeling is quantitative. The two SH- groups on the beta
globulin chains appear to be the site of covalent attachment in the
case of HBOC (this can be confirmed by selective blocking of
4-(2-bromoacetamido)-TEMPO labeling with N-ethyl-maleimide or
globulin chain analysis by reverse phase HPLC). It is noteworthy
that the ESR signal intensity (peak Mo) ratios for 2, 4, and 8 are
in approximately the same ratio as the spectra were recorded at
proportionately decreasing instrument sensitivity.
[0181] Furthermore, it is expected that more
4-(2-bromoacetamido)-TEMPO could be attached to Hb at even higher
molar ratios, for example as radiation-protective agents in
vivo.
[0182] A preferred molar ratio of nitroxide to hemoglobin in the
blood substitute formulations is 8:1 and a particularly preferred
molar ratio is approximately 18, prepared as described below to
yield a polynitroxide hemoglobin (PNH). A preferred formulation of
PNH is the carbonmonoxyl derivative, CO-PNH. CO binding to PNH heme
groups stabilized PNH: its has been shown that CO-PNH is stable to
heating at 600C. Therefore, CO-PNH requires no refrigeration and
can be stored at room temperature for a prolonged period of time.
Furthermore, when infused into the body, the presence of CO
enhances the vasodilatory activity of PNH, a CO is known to act as
a vasodilator in vivo. After the CO has been off-loaded from PNH,
PNH continues to function as a therapeutic agent, both by
delivering oxygen to tissue and through its nitroxide-mediated
vasodilation.
[0183] Referring to FIG. 6, an ESR spectrum of a mixture of
4-(2-bomoacetamide)-TEMPO-labelled HBOC and .sup.15ND.sub.17-TEMPOL
wherein the center peak of the 4-(2-bromoacetamido)-TEMPO
(indicated by down arrow) and the high field peak of
.sup.15ND.sub.17-TEMPOL (indicated by the up-arrow) were adjusted
to similar intensity.
[0184] The separation of the resonance peaks permits the
simultaneous monitoring of free radical or enzyme mimic activities
involving the small molecular weight nitroxide (TEMPOL) and its
macromolecular conjugate in both in vitro and in vivo (murine)
reactions. For example, the in vivo plasma half-life of the two
nitroxides was compared by referring to the unique spectral
characteristics of the different nitroxides. Specifically, the in
vivo ESR studies of hemoglobin-based solutions, on the mouse were
performed using a nitroxide to hemoglobin ratio of 8:1 (see FIG.
5C) to take advantage of its high ESR signal intensity. First, the
approximate plasma half-life of a small molecular weight nitroxide
(.sup.15ND.sub.17-TEMPOL see FIG. 4C) and a large molecular weight
4-(2-bromoacetamido)-TEMPOL-labelled HBOC (see FIG. 4B) are
determined by preparing a mixture of the two and adjusting the ESR
signal intensity to be approximately the same (see FIG. 6). 0.5 ml
of the mixture was injected under anesthesia into a distended mouse
tail vein under a heat lamp. The mouse tail was inserted into an
ESR cavity and the spectrum was recorded within 10 minutes after
injection (see FIG. 7A).
[0185] Referring to FIG. 7A, 7B, and 7C the .sup.15ND.sub.17-TEMPOL
signal could not be detected, however, the
4-(2-bromoacetamido)-TEMPO-labelled HBOC was clearly resolved (see
FIG. 7B and 7C for plasma half-life studies where 7C is a
continuation of 7B). Since the vasoconstrictive effect of HBOC is
reported to be fully developed during the first 5-15 minutes of
bolus injection of an HBOC in rats, the participation of the
nitroxide-labelled HBOC in free radical redox-reactions immediately
after transfusion in a mouse was measured. The tail vein of female
CH3 mouse was cannulated under anesthesia with 80% nitrous oxide,
20% oxygen, and 3% isofluorane. Under a heat lamp the mouse tail
vein became visibly distended, a cannula consisting of a 30 gauge
hypodermic needle attached to a one foot length of polyethylene
tubing was inserted into the tail vein and held in place by
cyanoacrylate glue. For in vivo ESR measurements, the cannulated
mouse was transferred under anesthesia to a 50 ml conical
centrifuge tube modified to allow the tail to protrude from the
conical end and to allow a continuous flow of anesthetic gas from
the opening end of the tube. The tail was inserted into a plastic
tube which was then fitted into a TE 102 cavity. The cannula was
flushed periodically with heparin (100 unit/ml) to ensure patency.
The cannula was near the root of the tail and was kept outside of
the ESR cavity so that a pure signal from the tail could be
measured immediately after bolus injection. 0.5 ml of samples (see
FIG. 8) were injected via the cannula and the spectrometer was set
for a repeat scanning mode at 1/2 min. intervals (see FIGS. 8A and
8B). In FIG. 8A the magnetic field was increased by two Gauss, and
in FIG. 8B the magnetic field was decreased by two Gauss, to
superimpose the resonance spectra. The .sup.15ND.sub.17-TEMPOL
signal disappeared within 2.5 minutes after injection. During the
same time period the 4-(2-bromoacetamido)-TEMPOL-la- belled HBOC
also decreased at a similar rate.
[0186] However, the nitroxide-HBOC signal were shown to be stable
in plasma (FIG. 8B). Therefore, FIG. 8B together with results from
FIG. 7 show that the nitroxide-labelled to macromolecules such as
HBOC has considerably longer plasma half-life as compared to small
molecular weight nitroxide (e.g., .sup.15ND.sub.17-TEMPOL).
[0187] The observed nature of the free radical reaction involves
two pathways:
[0188] 1. the rapid phase appears to involve the free radical (e.g.
superoxide) oxidation of the nitroxide to its oxoammonium cation
intermediate followed by the reduction of the oxoammonium cation to
its stable hydroxylamine derivative of the nitroxide. Such
reduction involves the participation of either one or two reducing
equivalents (e.g. NADH) present in the vascular compartment. The
reduction of nitroxide to its hydroxylamine would lead to a rapid
reduction in ESR signal intensity, in the case of 8:1 molar ratio
of 4-(2-bromoacetamido)-TEMPO-labelled HBOC represents
approximately 25% of the 4-(2-bromoacetamido)-TEMPO on the HBOC.
This phase involves both small molecule and macromolecular
nitroxide.
[0189] 2. the slow phase appears (see FIG. 8B) to represent the
antioxidant enzyme-mimic activities of the remaining 75% of
4-(2-bromoacetamido)-TEMPO on the HBOC in accordance with the
reaction mechanism wherein the nitroxide is involved in the
cyclic-free radical reactions for example the SOD-mimic reaction.
Where the nitroxide free radical is essentially unconsumed as a
SOD-mimic, the slow rate of decrease of the ESR signal intensity
can be attributable primarily to the reaction mechanism described
above and secondarily to the decrease in HBOC concentration as it
is slowly eliminated from the vascular compartment as a function of
its plasma half-life.
[0190] This result demonstrates the utility of polynitroxide
macromolecule, in this example TEMPO-labelled HBOC, in detoxifying
free radicals in vivo. This utility is defined in terms of
providing short term (in minutes) scavenging of free radicals and
persistent (in hours) protection against oxidant reactions by
nitroxides acting as enzyme mimics in vivo. In this and each of the
examples related to hemoglobin-containing solution should be
understood that unbound, low molecular weight nitroxide may be
added to the formulation to increase the concentration of active
nitroxide across the vascular membrane, into the interstitial
space, and the surrounding cellular environment. The results
presented here thereby distinguish the effect of a simple addition
of a low molecular weight nitroxide to a pharmaceutical composition
from the polynitroxide macromolecules of this invention. The
particular advantages of a multi-component system of this invention
utilizing a polynitroxide macromolecule together with molecular
weight nitroxides is highlighted below.
[0191] Based on the analysis of the spectra in FIG. 8, the
oxidation/reduction (redox) cycling reactions involve approximately
73% of 4-(2-bromoacetamido)-TEMPO-labelled HBOC remaining in its
free radical state. This indicates that TEMPOL participates in in
vivo redox-reactions within the confines of the vascular space.
[0192] To study the vasoconstrictive effects of hemoglobin-based
solutions, solutions of modified human hemoglobin are tested for
their effects in the intact rat. Humane procedures are always used
where any research animals are used.
[0193] At 7 days prior to the study, male Sprague-Dawley rats are
anesthetized with ketamine (40 mg/kg i.m.) and acetylpromazine
(0.75 mg), or with pentobarbital sodium (20 mg/kg i.p.). Medical
grade Tygon microbore (0.05 in ID, 0.03 in OD) is inserted into the
femoral artery and veins. Cannulas are exteriorized and filled with
heparinized dextrose, and sealed with stainless steel pins. After a
2-3 day recovery period, conscious animals will are in plastic
restraining cages. Seven (7) days recovery from surgical procedure
are needed to ensure healing of incisions before exchange
transfusion. Because the surgery may cause minor bleeding, it is
important to permit recovery so that minor bleeding related to
surgery is not confused with a side effect of blood replacement.
50% exchange transfusions are carried out using an infusion pump to
simultaneously infuse and withdraw the test solution and blood,
respectively, from two syringes. The volume of blood removed (12-15
ml based on total blood volume) is replaced with test solution over
approximately 20-30 minutes. The end point is the-reduction of the
hematocrit to half its initial value. The arterial blood pressure
is monitored and recorded continuously for 5 hours after the
exchange transfusion using a pressure transducer connected to a
chart recorder. Mean arterial pressure is calculated as 1/3 of the
pulse pressure. Heart rate is determined from the blood pressure
trace.
Example Three--Nitroxide-Labelled Polymers of Stabilized
Hemoglobin
[0194] While it is possible to produce dimers of stabilized
hemoglobin from cross-linked monomers, it is also possible to
produce hemoglobin polymers from stabilized or native hemoglobin.
Solutions of hemoglobin polymers contain a mixture of monomers,
dimers, trimers, tetramers, and other oligomers. Solutions
containing polymerized hemoglobins used as an HBOC generally have
longer plasma circulation times and higher oxygen carrying
capacities as compared to stabilized monomeric hemoglobin. Such
polymerized hemoglobin may be prepared by a number of pathways
using several different polymerizing agents. (See, U.S. Pat. Nos.
4,001,200, 4,857,636, and 4,826,811). The preferred method of
introducing a nitroxide to a solution of polymerized hemoglobin is
again by covalently attaching a nitroxide to the .beta.-93
sulfhydryl groups of the two .beta.-globin chains of hemoglobin.
These sulfhydryl groups are not known to be involved in the
stabilization or polymerization processes. Consequently, the
nitroxide is preferably covalently attached to hemoglobin before
the stabilization and polymerization of the hemoglobin
monomers.
[0195] For example, nitroxide is covalently attached to DBBF-Hb
according to the procedure described in the second embodiment
above, followed by polymerization with glutaldehyde according to
the procedure described in Sehgal et. al. U.S. Pat. No. 4,826,811.
FIG. 4B is an electron spin resonance spectra of the DBBF-Hb
labelled with 3-maleimido-PROXYL and polymerized with glutaldehyde.
Similarly, DEBF-Hb that is polymerized with glutaldehyde may be
labelled with 4- (2-bromoacetamido) -TEMPO by the same method.
[0196] Using a similar approach, a polymerized hemoglobin
intermediate, such as a glutaldehyde-polymerized, an
o-raffinose-polymerized, or an o-cyclodextran-polymerized
hemoglobin intermediate that contains unreacted aldehyde groups,
may be used for covalent attachment of either 4-amino-TEMPO or
3-amino-PROXYL via reductive amination to yield a
nitroxide-labelled hemoglobin polymer. With reductive amination,
the sequence and timing of the reaction are important. The
4-amino-TEMPO is added to glutaldehyde-polymerized hemoglobin after
completion of polymerization, but prior to the reduction reaction
that results in covalent attachment of the nitroxide to the
polymerized hemoglobin. Likewise, the nitroxide-labelling of a
o-raffinose polymerized hemoglobin may be accomplished by the
addition of either 4-amino-TEMPO or 3-amino-PROXYL prior to
reductive amination. For example, 4-amino-TEMPO labelled
o-raffinose polymerized hemoglobin is prepared according to the
procedure described in my U.S. Pat. No. 4,857,636 except that 6
molar equivalents of 4-amino-TEMPO is added after the completion of
the polymerization and prior to the reduction with 20 molar excess
of borane dimethylamine complex. As described therein, hemoglobin
may be cross-linked and polymerized using polyvalent aldehydes
derived from disaccharides or ring-opened sugars including,
oligosaccharides, or preferably, trisaccharides such as
o-raffinose. Likewise, monosaccharides may be used to stabilize and
polymerize hemoglobin although the higher molecular weight sugars
are preferred. The resonance spectrum of a dialyzed and washed
o-raffinose polymerized hemoglobin labelled with 4-amino-TEMPO was
shown in FIG. 9A.
[0197] To increase the yield of hemoglobin oligomers (Hb.sub.n
where n=2-4) of the polymerized hemoglobin, it is desirable to
increase the valance of the polyaldehyde of the cross-linker, with
the use of .alpha.-cyclodextran, .beta.-cyclodextran, and
.gamma.-cyclodextran, as well as their sulfate derivatives which
represents 6-, 7-, and 8-cyclized glucose molecules, the ring
opened .alpha.-cyclodextran, .beta.-cyclodextran, and
.gamma.-cyclodextran have 12, 14, and 16 reactive aldehyde groups
respectively. These ring-opened cross-linkers can be used to
cross-link and polymerize hemoglobin to produce polymerized
hemoglobin which is rich in oligomers. The unreacted aldehyde, as
described above, may be utilized to covalently attached to an
amino-nitroxide, for example, 4-amino-TEMPO or 3-amino-PROXYL.
[0198] Furthermore, the ring-opened sulfate derivatives, for
example, the sulfated a-cyclodextran will be an effective
cross-linker for two additional reasons: (1) the sulfate groups
will mimic the activity of DPG in lowering the oxygen affinity of
the cross-linked hemoglobin, thus improving oxygen transport
properties, and (2) the sulfate groups will serve as affinity
labels which will complex multiple (e.g., n=2-4) hemoglobins to
initially form a "cluster." Once the "cluster" complex is formed,
the aldehyde groups on the cyclodextran will be brought to close
proximity with the NH.sub.2 groups within the DPG binding sites,
thus promoting the covalent intrasubunit and inter-molecular
cross-linking of hemoglobin resulting in an increased yield of
hemoglobin oligomers. In addition to antioxidant enzyme-mimic
activities, the ring-opened cyclodextran polymerized and
nitroxide-labelled hemoglobin will also have improved yield and
composition as compared to o-raffinose and glutaldehyde polymerized
hemoglobin.
[0199] Example Four--Nitroxide-Labelled Liposome-Encapsulated
Hemoglobin
[0200] Liposomes are particles which are formed from the
aggregation of amphophilic molecules to form a bilayer structure in
a hollow spherical shape with the polar sides facing an internal
water compartment and external bulk water. Several acceptable
methods for forming liposomes are known in the art. Typically,
molecules form liposomes in aqueous solution like dipalmitoyl
phosphatidylcholine. Liposomes may be formulated with cholesterol
for added stability and may include other materials such as neutral
lipids, and surface modifiers such as positively or negatively
charged compounds. The preferred liposomes are small
unilamellar-bilayered spherical shells.
[0201] A method for encapsulating hemoglobin in a liposome is also
known (See Farmer et. al., U.S. Pat. No. 4,911,921). For the
purpose of this invention, a number of approaches may be used to
introduce the nitroxide-based oxygen detoxification function to a
solution of liposome-encapsulated hemoglobin. For example, it is
possible to use nitroxide-labelled native hemoglobin, or a
nitroxide-labelled stabilized hemoglobin as disclosed above, as the
starting material and then performing the process of liposome
encapsulation of the nitroxide-labelled hemoglobin by known
techniques. In the present embodiment, purified hemoglobin may also
be coencapsulated with a membrane impermeable nitroxide such as
TEMPO-choline chloride disclosed for a spin membrane immunoassay in
Hsia et. al. U.S. Pat. No. 4,235,792.
[0202] Also, any purified hemoglobin may be encapsulated with a
liposome comprised of nitroxide-labelled fatty acids (e.g.,
7-DOXYL-stearate, 12-DOXYL-stearic acid, and 16-DOXYL-stearate),
cholestane, an analogue of cholesterol (e.g., 3-DOXYL-cholestane),
or phospholipid (e.g., 12-DOXYL-stearate-labelled
phosphatidylcholine). The preparation of hemoglobin encapsulated in
a liposome comprised of 3-DOXYL-cholestane labelled may be prepared
by a method analogous to that described in Tabushi et. al., (J. Am.
Chem. Soc. 106: 219 (1984)). A 5 ml chloroform solution containing
lipid compositions, including DOXYL labelled stearic acid and/or
cholestane, as specified below were first dried in a stream of
nitrogen to remove the solvent. Next, the residues were dried in
vacuo and the resulting film was suspended in 2 ml of hemoglobin
(24 g/dl) in a Lactated Ringers solution. The lipid concentration
in the dispersion is 100 mM. The liposome encapsulated hemoglobin
is then rotated and incubated preferably at 37.degree. C. until all
lipids are dispersed to form multilamellar vesicles. The resulting
solution containing multilamellar liposome encapsulated hemoglobin
and free unencapsulated hemoglobin is then forced through a
microfluidizer to form 0.2 micron liposomes according to the
procedure of Cook et.al. (See U.S. Pat. No. 4,533,254). The molar
ratio of dipalmitoyl phosphatidylcoline: cholesterol: dipalmitidyl
phosphatidic acid: 3-DOXYL-cholestane in the liposome is
0.5:0.4:0.02:0.07. The resonance spectrum of the resulting
3-DOXYL-cholestane labelled liposome-encapsulated hemoglobin is
shown in FIG. 10A. In this configuration, the nitroxide is
intercalated into the liposome membrane and can be found at both
the inner and outer surface of the lipid bilayer water interface.
Substituting the 3-DOXYL-cholestane with 16-DOXYL-stearic acid in
the lipid composition shown in FIG. 10A results in an electron
resonance spectrum shown in FIG. 10B. The mobility of the nitroxide
as reflected from the resonance spectrum is consistent with the
interpretation that the DOXYL-moiety of the stearic acid is located
predominately in the hydrophobic interior of the lipid bilayer.
With the addition of both the 3-DOXYL-cholestane and
16-DOXYL-stearate to the lipid composition at the same molar ratio,
the resonance spectrum of the double nitroxide-labelled liposome
encapsulated hemoglobin is shown in FIG. 10C. The resonance
spectrum of FIG. 10C is a composite of FIGS. 10A and 10B because
the nitroxides in this embodiment are located at both the
membrane-water interface and its hydrophobic lipid bilayer
interior. By placing the nitroxide in both locations, this
embodiment provides the oxygen detoxification function at both the
lipid bilayer hydrophobic interior and the membrane-water interface
thus providing the added benefit of an additional reserve of
oxygen-detoxification capacity for the encapsulated hemoglobin.
Example Five--Nitroxide-Labelled Conjugated Hemoglobin
[0203] A physiologically compatible solution of conjugated
hemoglobin is produced by forming a conjugate of hemoglobin and a
biocompatible macromolecule used as a plasma expander. Plasma
expanders, such as dextran (Dx), polyoxyethylene (POE),
hydroxylethyl starch (HES), are used to increase the circulation
half life of hemoglobin in the body. In this state, the hemoglobin
molecules together with the biocompatible macromolecule are
collectively referred to as a hemoglobin conjugate. There are a
number of convenient methods to incorporate a nitroxide into a
hemoglobin conjugate. For example, one may simply substitute the
hemoglobin to be conjugated with a nitroxide-labelled hemoglobin
such as TEMPO labelled DBBF-Hb. This can be accomplished by
substituting hemoglobin or pyridoxylated hemoglobin with
3-malei-mido-PROXYL-DBBF-Hb or 4-(2-bromoacetamido)-TEMPO-DBBF-Hb
in the preparation of conjugated hemoglobin.
[0204] 4-Amino-TEMPO labelled dextran conjugated hemoglobin is
prepared in accord with the procedure described by Tam et. al.
(Proc. Natl. Acad. Sci., 73:2128 (1976)). Initially, an 8%
hemoglobin solution in 0.15 M NaCl and 5 mM phosphate buffer, pH
7.4 is conjugated to periodate-oxidized dextran to form a
Schiff-base intermediate. Twenty molar equivalents of 4-amino-TEMPO
is added to hemoglobin to form the Schiff-base between the
nitroxide and the remaining reactive aldehyde groups on the
dextran. After a 30 minute of incubation at 4.degree. C., a 50
molar equivalent of dimethylamine borane in water is added. The
solution is incubated for a further 2 hours at 4.degree. C.
Afterwards, the solution is dialyzed, reconstituted with Lactate
Ringers buffer and sterile filtered with Filtron membrane
filtration units (Filtron Technology Co.). The electron spin
resonance spectrum of the 4-amino-TEMPO labelled dextran-conjugated
hemoglobin is a sharp asymmetric triplet reflecting a high degree
of motional freedom (See FIG. 11). The increased mobility of the
TEMPO covalently attached to the Dextran is consistent with the
nitroxide linked to a flexible polysaccharide dextran chain as
compared to that of a tightly folded hemoglobin molecule (See FIGS.
3A and 3B). Thus, resonance spectrum in FIG. 11 demonstrates that a
novel nitroxide-labelled dextran conjugated hemoglobin has been
prepared.
Example Six--Nitroxide-Labelled Albumin
[0205] A preferred embodiment of this invention is the use of
nitroxide-labelled biocompatible macromolecules in connection with
low molecular weight, membrane permeable nitroxides to provide
sustained antioxidant activity in vivo. Preferably, the nitroxide
is labelled to a biocompatible protein, as used herein, the term
"protein" includes fragments and derivatives thereof. The
nitroxide-labelled protein is preferably provided by labelling at a
large portion of the amino groups of the protein. An example of
such a desirable biocompatible macromolecule is human serum albumin
(HSA). Additionally, labelling at the disulfide bonds increases the
molar ratio of nitroxide to protein. By so labelling the protein,
an acidic microenvironment is created which enhances the
interaction between the free nitroxide and the macromolecule-bound
nitroxide, facilitating election spin transfer due to the
differential stabilities of the species. Such covalently labelled
nitroxides are attached as dinitroxide, trinitroxides or
polynitroxide carboxyl copolymers with molecular weight of 5000 to
7000. Thus the molar ratio of nitroxide to albumin may be increased
to approximately 400 and may be varied by the labelling strategies
disclosed herein to reach an integral value or range of values up
to 400. In the case of albumin, it is also possible to covalently
bind a nitroxide to the primary bilirubin binding site of the
albumin, using for example an activated derivative of a nitroxide
with specific binding affinity for that site, e.g., TOPS. By
selecting the binding site on the macromolecule, the reactivity of
the nitroxide is modified and this modification can be used to
alter the reactivity of the compound.
[0206] Serum albumin is a plasma protein with multiple
ligand-binding sites and is the transport protein for many ligands
in the blood. Nitroxides can bind specifically to human serum
albumin at a number of specific ligand binding sites, or
non-specifically. This binding can be non-covalent, or covalent
through the use of activated nitroxides. Nitroxide-albumin may be
used either alone or in combination with a low molecular weight
nitroxide compound, e.g., TEMPOL, TOPS, and mono- and diesters of
TOPS for oral, parenteral, and topical therapeutic applications.
Nitroxide-labelled albumin is also available as an "improved"
version of albumin (i.e., improved by having antioxidant activity)
with utility in any application where albumin is now conventionally
used, including as red cell preservative, a parenteral colloid
solution, in biomaterials, in biocompatible surface coatings, etc.
and additionally in antioxidant applications such as cellular
preservation compound for use e.g., in preservation of organs for
transplantation and red blood cells for transfusion.
[0207] The albumin may be obtained from plasma or may be produced
by recombinant genetic means. HSA may be used in a variety of
forms, including monomers (normal plasma form), homodimers,
oligomers, and aggregates (microspheres and albumin microbubbles).
Additionally, albumin may be treated with polyethyleneglycol to
reduce its immunogenicity. Specific labelling of the albumin with a
nitroxide may be achieved at several binding sites, including
bilirubin, FFA, indole, or Cu.sup.++ binding site by using
activated derivatives of nitroxide compounds which have binding
specificity for the relevant site on the protein. A preferred
example is 2, 2, 6, 6-tetramethyl-l-oxyl-4-piperidylidene succinate
(TOPS) nitroxide covalently bound to the primary bilirubin-binding
site of HSA. Non-specific labelling of albumin may be achieved at
up to approximately 50 accessible amino groups. Temperature and
chemical treatment of the albumin permits increasing the molar
ratio of nitroxide to albumin. Using native albumin, molar ratios
above 7 and up to 60 can be achieved. Using nitroxide-labelled
albumin up to 60 moles, a molar ratio of nitroxide to albumin up to
95 may be achieved by labelling the 35 potential sulfhydrol groups
of the albumin with additional nitroxides. Such covalently labelled
nitroxides are attached as dinitroxide, trinitroxides or
polynitroxide carboxyl copolymers with molecular weight of 5000 to
7000. Thus the molar ratio of nitroxide to albumin may be increased
to approximately 400 and may be varied by the labelling strategies
disclosed herein to reach an integral value or range of values up
to 400.
[0208] To demonstrate the regeneration of the active nitroxide in
vivo, 4-hydroxyl-2,2,6,6-tetramethyl-piperdine-N-oxyl (N-TPL) in
phosphate buffered saline was injected into the tail vein of an
anesthetized mouse (body weight 40 g) as a control. The tip of the
tail was inserted into the sample tube of an electron spin
resonance (ESR) spectrometer. The mouse tail displayed no esr
signal before the injection. After the injection of 0.5 ml of 6 mM
TEMPOL, an esr signal was detected and observed to decay rapidly,
as seen in three successive scans of the spectrum at 30 second
intervals (see FIG. 18A). This result indicates that the TEMPOL was
reduced to its esr-silent, and catalytically inactive,
N-hydroxylamine (R-NOH) derivative (TEMPOL-H or TPH). To measure
the plasma half-life of TEMPOL in the mouse tail, the intensity of
the high-field peak was monitored continuously for 8 minutes (FIG.
19). After 8 minutes, a repeat injection of TEMPOL was made. The
maximum peak intensity, which corresponds to TEMPOL (TPL)
concentration, was attained in the mouse tail in approximately 10
seconds, followed by a rapid decay to base line, resulting in a
half-life in vivo of TPL of approximately 50 seconds (FIG. 19). The
half-life of TPL was confirmed by two recording methods: scanning
the entire spectrum at intervals and continuous recording of the
high-field peak intensity. The results from both methods were in
agreement.
[0209] To prepare the polynitroxide albumin (PNA), human serum
albumin (HSA, 5% solution, Baxter Healthcare Corp.) was allowed to
react with 60 molar equivalents of Br-TEMPO (Sigma) at 60.degree.
C. for 10 hours with mixing. The resulting reaction mixture,
containing 15 ml of HSA and 165 mg of Br-TEMPO in a Vacutainer
tube, was filter-sterilized with a 0.22 micron filter and
transferred into a 150 ml stirred cell (Stirred Cell Devices)
equipped with a lokda-cutoff ultrafiltration membrane (Filtron
Technology Corp.) . The filtered reaction mixture was washed with
Ringer's solution (McGaw Inc.) until the filtrate contained less
than 1 ;.mu.M of free Br-TEMPO as detected by esr spectroscopy. The
bright orange retentate was concentrated to 25% HSA and again
filter-sterilized with a 0.22 micron filter into a 10 ml sterile
vial (Abbott Laboratories) and stored -at 4.degree. C. until use.
The esr spectrum of the macromolecular polynitroxide is shown in
FIG. 15C.
[0210] To increase the molar ratio of nitroxide in a polynitroxide
albumin, the disulfide groups are reduced to sodium thioghycolate
in the presence of urea and an excess of BR-TEMPO is added to label
the broken disulfide bonds. An average increase in the molar ratio
of approximately 10 is achieved by this procedure, which is
analogous to the procedure described by Chan et al in "Potential of
Albumin Labeled with Nitroxides as a Contrast Agent for Magnetic
Resonance Imaging and Spectroscopy," Biconjugate Chemistry, 1990,
Vol. 1, pages 32-36.
[0211] The concentration of a polynitroxide albumin solution is
adjusted to 17.5 mg/ml in a flask. This solution is diluted with
urea to 8M and stirred until dissolved. The pH is adjusted to 8.2
by addition of 2% sodium carbonate. The pH-adjusted solution is
degassed completely for 15 minutes, then flashed with argon gas. In
another flask, a 3M solution of thioglycolate is prepared,
degassed, and flashed with argon gas. The thioglycolate solution is
added to the polynitroxide albumin to a final concentration of
0.3M.
[0212] The resulting solution is degassed and flashed with argon
gas to mixture and allowed to stand in the dark under argon gas for
20 hours at room temperature, followed by dialyzation against 3L of
degassed phosphate-buffer saline pH 8.4 (adjusted by 2% sodium
carbonate) for 5 hours at room temperature under argon gas. An
excess of Br-tempo is added and the mixture stirred for an
additional 24 hours at room temperature under argon gas.
[0213] The final solution is dialyzed against PBS pH 7.4 for 5
hours to remove unreacted nitroxide. EPR spectroscopy may be used
to determine spin density and protein concentration may be
determined by Biuret method.
[0214] Typical results are set forth below for the average of three
lots.:
[0215] 1. Protein concentration:
[0216] Amino group labelled polynitroxide albumin
53.0 mg/ml=0.78 mM
[0217] Amino plus disulfide
13.9 mg/ml=0.20 mM
[0218] 2. Spin density:
[0219] Amino group labelled polynitroxide albumin (PNA)
33 mM
[0220] Amino plus disulfide
10.5 mM
[0221] 3. Calculation of molar ratio of nitroxides bound to
protein.
[0222] Amino group labelled polynitroxide albumin
33 mM/0.78 mM=42 nitroxides/albumin
[0223] Amino plus disulfide
10.5 mM/0.20 mM=52 nitroxides/albumin
[0224] Reduction of the disulfide groups increased by ten the molar
ratio of nitroxide to albumin in this example.
[0225] The methods used to produce the examples of PNA described
herein and used in the examples to demonstrate the therapeutic,
diagnostic, and functional utility of the polynitroxylated
biocompatible molecules of the invention can be readily applied to
produce other compounds such as polynitroxylated HBOC (or PNH),
polynitroxylated dextran (PND) and numerous additional PNA
formulations as shown in the following chart showing the species of
nitroxide, the structure, the protein concentration, the spin
density and the binding percentage of the available groups on the
albumin protein.
1 Protein Spin Conc. Density mane structure mM mM Binding %
3-Malemido- PROXYL 11 0.67 6.9 17.2 3-(Maleimidomethyl)- PROXYL 12
0.73 7.8 17.8 3-(2-Iodoacetamido)- PROXYL 13 0.36 5.9 27.2
(B3T011996) 14 0.73 21.4 49 Ester TEMPO 15 0.46 5.5 13.3 A TEMPO 16
0.46 0 0 4-(2- Bromoacetamido)- TEMPO 17 0.67 12.3 29.8
4-Isothiocyanato- TEMPO 18 0.45 5.3 21.5 B3T(020996) 19 0.52 3.9
28.4 4-Maleimdo- TEMPO 20 0.17 1.5 14.4 4-(2-Iodoacetamido)- TEMPO
21 0.67 10.0 24.8
[0226] To demonstrate that the polynitroxide-labelled albumin
formulations described above function to enhance the in vivo
activity of the low molecular weight membrane-permeable nitroxide,
human serum albumin was covalently labelled with
4-(2-bromoacetamido) -TEMPO (BR-TEMPO) and infused after a dose of
TEMPOL had been administered and had been observed to have been
converted to its reduced state.
[0227] Two hours after the injection of TEMPOL as in the above
Example, the mouse tail showed no detectable esr signal. When 0.2
ml of the polynitroxide albumin was injected via the tail vein, the
TEMPOL signal reappeared within 4 minutes. The TEMPOL signal
intensity persisted for more than two hours, with a half-life of
approximately 40 minutes (also see FIG. 18). The TEMPOL signal was
distinguished from the polynitroxide albumin signal by their
distinctive spectral profiles. The TEMPOL signal was measured as
the intensity of its characteristic high-field peak. A nitroxide to
albumin molar ratio of 27 displays the same reactivity.
[0228] The possibility that the esr signal detected after injection
of the polynitroxide albumin was due solely to nitroxide on the
macromolecule disassociated from the macromolecule, was ruled out
by the following experiment. The experiment was performed as above,
but using [.sup.15N]-TEMPOL and albumin-[.sup.14N]-TEMPOL. The
different nitrogen species provide a method of discriminating the
esr signals of the free and the macromolecular nitroxides. The
results (FIGS. 18A) confirm that the esr signal from the
regenerated nitroxide is derived from the .sup.15N isotope and,
therefore, that the antioxidant activity of the low molecular
weight membrane permeable [.sup.15N]-TEMPOL has been regenerated
following the addition of the macromolecular polynitroxide.
[0229] As noted herein, the donor/acceptor relationship between two
nitroxide containing species provides the ability for sustained
nitroxide protection in vivo. The interaction of PNA and TPL as
described with several of the embodiments is only one example of
additional combinations provided by the invention. Referring to
FIGS. 18B(1), (2) and (3), polynitroxylated albumin (PNA) is shown
to activate TOPS. ESR spectra of 50 mM TOPS alone (FIG. 18B(1)) and
43 mM PNA alone (FIG. 18B(2)) reflect a comparatively low peak
intensity whereas the combination of TOPS with PNA, in a sodium
phosphate buffer (pH 7.8), shows a substantially greater peak
intensity (FIG. 1BB(3)). Similarly, in FIG. 18C, polynitroxylated
HBOC is shown to activate TOPS, and in FIG. 18D, B3T labelled
albumin is shown to activate TOPS. Thus, as referred to elsewhere
herein, the multiple component embodiments may be formulated with
different nitroxides or nitroxide-based compounds with different
free-electron stabilities.
[0230] Example Seven--Nitroxide-Labelled Immunoglobulin
[0231] As in the above embodiments, certain nitroxides have been
shown to have very short plasma half-life when injected
intravenously. Due to the desire to have an antioxidant enzyme
mimic with a long plasma half-life, a nitroxide compound may be
attached to an immunoglobulin to provide long-lasting antioxidant
enzyme mimic activity.
[0232] Immunoglobulins are a class of plasma proteins produced in
the B-cells of the immune system and which are characterized by two
specific ligand binding sites (the antigen-binding sites).
Nitroxides have been used in the past as probes in research on
hapten-binding specificity and affinity of immunoglobulins during
the primary and secondary immune response.
[0233] As with the above-embodiment describing nitroxide-labelled
albumin, the nitroxide-labelling technology demonstrated above in
the example of nitroxide-HBOC is readily applied to the production
of nitroxide-labelled immunoglobulins. Immunoglobulins after the
advantage of specific binding and long circulatory half-life such
that the enzyme-mimic activity of the compounds of this invention
can be targeted to specific tissues and have prolonged
activity.
[0234] Nitroxide-labelled immunoglobulins may be used in vivo to
provide protection against cellular damage by reactive oxygen
species. Nitroxide-labelled immunoglobulin may be used either alone
or in combination with a low molecular weight nitroxide compound to
provide extended antioxidant activity with an extended plasma
half-life.
[0235] Nitroxide-labelled immunoglobulin may be prepared by
specific labelling of the immunoglobulin itself or by covalently
labelling at a hapten-binding site. To avoid clearance of the
nitroxide-labelled immunoglobulin as part of the body's natural
immune response, one may use immunoglobulin fragments, for example,
(Fab).sub.2 produced by cleaving the immunoglobulin according to
known techniques with non-specific-labelling, a preferred molar
ratio of nitroxide:immunoglobulin is up to 60:1.
[0236] Nitroxide-labelled immunoglobulins are a preferred species
for use to target the enzyme-mimic effect of a particular location.
For examples by selecting antibodies specific to an antigen
implicated in inflammation or other such pathology. The image
enhancing and therapeutic benefits of this invention can be
targeted at a particular site.
[0237] Example Eight Imaging of Biological Structures and Free
Radical Reactions by EPR
[0238] Under most circumstances, free radical reactions occur so
rapidly that EPR imaging (ERI) is difficult. However, due to the
presence of stable free radicals, nitroxides are detectable by
electron paramagnetic resonance spectroscopy. With the development
of advanced imaging instrumentation images of intact biological
tissues and organs are available based on a measurement of free
radical concentration. Biocompatible nitroxides are candidates for
image-enhancing agents.
[0239] Because nitroxides are reduced in vivo to inactive
derivatives within a few minutes of administration, their utility
is limited. Pursuant to this invention, active nitroxide levels in
the body may be maintained for a prolonged period of time allowing
both improved image contrast and longer signal persistence than
seen with low molecular weight membrane permeable nitroxides
alone.
[0240] Electron paramagnetic resonance (EPR) spectroscopy is a
technique for observing the behavior of free radicals by detecting
changes in the energy state of unpaired electrons in the presence
of a magnetic field. The technique is specific for free radicals
because only unpaired electrons are detected. Using available
apparatus that measure electron paramagnetic resonance, a real-time
image of a macroscopic object, including living tissue can be
obtained. EPR imaging (ERI) provides the capability to obtain
multi-dimensional images (including spectral-spatial images) for
diagnosis or research.
[0241] Electron paramagnetic resonance imaging (ERI) with nitroxide
contrast agents is in principle a valuable method for medical
imaging, particularly the imaging of ischemic tissue. However, the
development of this technology has been limited by the fact that
nitroxides are rapidly reduced in vivo to non-paramagnetic
species.
[0242] The application for extrinsically introduced nitroxide has
demonstrated utility as a relatively low resolution in vivo EPR
imaging agent. (L.J. Berliner, "Applications of In Vivo EPR," pp.
292-304 in EPR IMAGING AND IN VIVO EPR, G. R. Eaton, S. S. Eaton,
K. Ohno, editors; CRC Press (1991). Importantly, Subramanian and
his collaborators constructed a radio frequency fourier transform
EPR spectrometer for detecting free radical species and for in vivo
imaging. J. Bourg et al., J. Mag. Res. B102, 112-115 (1993).
[0243] A polynitroxide albumin (PNA) prepared pursuant to this
invention, is administered which distributes in the vascular and
other extracellular spaces. Although the range of concentrations of
the compositions of this invention may vary. A preferred range is
5-25 g labelled PNA per deciliter and 0.1 to 200 mM TEMPOL. The
antioxidant activities and detectability by electron paramagnetic
resonance (EPR) spectroscopy and are useful for this purpose alone.
Additionally, when administered with modest doses of small
molecular weight, membrane permeable nitroxides, the nitroxide is
maintained in an active free radical state in the body for a
prolonged period of time.
[0244] A preferred formulation for ERI imaging is human serum
albumin (1.0 to 25.0 g/dl) covalently labelled with a high molar
ratio of nitroxide (7 to 95 nitroxide to albumin). As noted
elsewhere herein, the polynitroxide albumin (PNA) can accept an
unpaired electron from the hydroxylamine form of the nitroxide
(e.g., TPH) regenerating the activity of the nitroxide to its free
radical state (e.g., TPL). A fundamental advantage of this
multi-component system is that while the macromolecule or species
remains in the extracellular space, the small, membrane-permeable
nitroxide, and its reduced (hydroxylamine) derivative, distribute
freely between the intracellular and extracellular spaces. This
creates a cycle in which nitroxide free radical can be detected
within the cell prior to being reduced, followed by regeneration by
the macromolecular (extracellular) species. Thus, a desired
concentration of a spectroscopically detectable nitroxide can be
maintained in vivo for a prolonged period of time.
[0245] Extending the short half-life of nitroxides in vivo helps
overcome a major obstacle in the development of a nitroxide-based
imaging method and medical therapy. With regard to imaging, the EPR
Laboratory at the Johns Hopkins University has been developing a
continuous-wave ERI instrument utilizing nitroxide as contrast
agent to study the course of cardiac ischemia and reperfusion.
However, the limited half-life of nitroxide alone has meant that
the reperfusion phase cannot be studied. In these studies, the
three-dimensional spectral-spatial EPR imaging of nitroxide in the
rat heart suffer from the rapid decay of free radical signal due to
nitroxide reduction. Although a cross-sectional transverse 2-D
spatial EPR image of the rat heart has been reconstructed from a
3-D spectral-spatial data, this was based on an EPR signal which
was decaying continuously during the 12-minute acquisition period.
The different rates of nitroxide reduction in the epicardium,
myocardium, and endocardium, as a function of the duration of
ischemia, further reduces the definition of the cross-sectional
image of the heart. Pursuant to this invention, the ERI image may
be improved. The extended active half-life of an in vivo nitroxide
permits imaging of the reperfusion phase and provides additional
information on the progress of ischemic injury to tissues and
organs. The compositions of this invention provide a stable
nitroxide signal suitable for imaging within 10 minutes after
administration, and persists for at least approximately 2.0 hours.
(See FIG. 25). For comparison, the signal from a free nitroxide
alone effectively disappears within 20 minutes after administration
(FIG. 24). With regard to therapeutic utility, the ability to
safely maintain active nitroxide levels for prolonged periods of
time represents the ability to provide an extended antioxidant
effect aiding in the prevention of ischemia/reperfusion injury,
pathological processes where toxic oxygen-derived free radicals are
the agents of cell damage (see FIG. 26 and FIG. 27).
[0246] Using methods essentially as described in Kuppusamy et al.
Proc. Natl. Act. Sci., USA Vol. 91, pgs. 3388-3392 (1994), the rat
heart was imaged using polynitroxide albumin (PNA) at a
concentration of 4 g albumin/dl and 2 mM .sup.15N-TEMPOL.
[0247] Referring to FIG. 24, the signal intensity of
.sup.1SN-TEMPOL in the presence of polynitroxide albumin, can be
seen in the isolated rat heart. The EPR signal was stable with a
bi-phasic and gradual decline in intensity. This contrasts with the
bi-phasic rapid decline in signal intensity using a low molecular
weight membrane permeable nitroxide (TPL) alone (FIG. 24).
[0248] In an EPR imaging study, the rat heart was perfused with a
solution containing nitroxide with or without polynitroxide
albumin, followed by cessation of perfusate flow in order to create
ischemia. FIG. 24 shows the total signal intensity of [.sup.15N]
-TEMPOL in the isolated rat heart during ischemia in the presence
and absence of polynitroxide albumin. It can be seen the signal
intensity undergoes a biphasic decay and that the presence of
polynitroxide albumin greatly slows the decay. By thus stabilizing
the TEMPOL signal, polynitroxide albumin allows high-quality EPR
images to be obtained over a prolonged period of time. Referring to
FIG. 22-23, a three-dimensional EPR image of the ischemic heart can
still be obtained after 156 minutes of global ischemia. FIG. 22A
shows a cut-out view of the image. As illustrated in FIG. 23, the
three-dimensional image can also be viewed in cross-section. This
shows differential distribution of TEMPOL signal within the
ischemic heart; this is quantitated in FIG. 28. FIG. 25 shows a
series of images such as in FIG. 23, acquired at a series of
successive time points during 125 minutes of global ischemia. This
illustrates that the presence of PNA allows much better imaging
than is possible with TEMPOL alone, in terms both of resolution and
signal persistence.
[0249] A particular advantage of this invention is the ability to
localize the image enhancement by selecting compositions which are
selectively distributed in the structure of interest and/or by
selecting a means of administration which facilitates image
enhancement of a particular structure or system. For example, image
enhancement of the cardiovascular system can be provided by
intravenous infusion of a polynitroxide macromolecule having a
lengthy plasma half-life and, preferably, an intravenous infusion
of low molecular-weight membrane permeable nitroxide.
Alternatively, to enhance an image of the gastrointestinal tract,
one of these compounds may be administered orally. Similarly, an
image enhancement of a discrete region of the skin may be obtained
by a topical administration of a polynitroxide macromolecule
suspended in a suitable carrier, combined with oral or intravenous
administration of an additional nitroxide species. Alternatively,
depending on the application, multiple nitroxide-based compositions
pursuant to this invention could be dermally administered.
[0250] Example Nine--Radiation Protection
[0251] Living organisms which are exposed to ionizing radiation
suffer harmful effects which can be fatal with high doses of
radiation. Recent evidence suggest that radiation causes cellular
injury through damage to DNA. Of the total damage to DNA, as much
as 80% may result from radiation-induced water-derived free
radicals and secondary carbon-based radicals. The Department of
Defense has had screened over 40,000 aminothiol compounds looking
for an in vivo radiation protector. Although one agent (WR-2721)
showed selective radioprotective effects, WR-2721 failed to exhibit
radiation protection in human clinical trials. A nitroxide compound
(e.g., TPL) was a non-thiol radiation protective agent, but as
noted herein, (TPL) has a very short in vivo half-life. The other
compounds were macromolecules of natural origin (Superoxide
dismutase, IL-1, and GM-CSF). However, due to their molecular size,
each of these has limited capability to provide intracellular free
radical scavenging.
[0252] The National Cancer Institute attempted to use the nitroxide
compound Tempol as a radioprotective agent to allow greater dosage
levels of radiation treatment of cancer patients. The researchers
quickly found the low molecular weight nitroxide was quickly
reduced to an inactive form and safe dosages could not be
administered.
[0253] Based on the invention disclosed herein, nitroxides bound to
macromolecular compounds as enzyme mimics can be used together with
low molecular-weight nitroxides to detoxify oxygen radicals in the
vascular space by interacting with membrane-permeable nitroxide
compounds to detoxify free radicals inside cells. Extending the
duration of the radioprotective effects pursuant to this invention
allows the use of nitroxide compounds to protect against the
harmful effects of controlled radiation in medical applications
such as cancer therapy, and in accidental exposure to harmful
radioactive sources.
[0254] Based on a radiation dosage scale developed by the National
Cancer Institute, chinese hamster cell cultures are exposed to
ionizing radiation in the presence of radioprotective chemical
agents. FIG. 16 shows the survival rate of Chinese hamster V79
Cells at 12 Gray of radiation. The control, macromolecular bound
nitroxide (PNA), and reduced nitroxide (TPH) show similar survival
rates. However, TPH premixed with PNA results in the conversion to
a radioprotective TPL (see FIG. 17) which enhances the survival of
the V79 cells (see FIG. 16).
[0255] Low molecular weight, membrane-permeable nitroxides e.g.,
TPL have been demonstrated to provide radiation protection in vivo
in C3H mice. In these studies, the maximal tolerated dose of TPL
administered intraperitoneally was found to be 275 mg/kg, which
resulted in maximal TPL levels (.about.150 .mu.g/ml) in whole blood
5-10 minutes after injection. Mice were exposed to whole body
radiation in the absence or presence of TPL (275 mg/kg) 5-10
minutes after administration. The dose of radiation at which 5-10
of TPL treated mice die within 30 days was 9.97 Gray, versus 7.84
Gray for control mice.
[0256] Because the radioprotectant effect of TPL is derived from
the reactivity of the unpaired electron, when TPL is reduced to
hydroxylamine by losing its unpaired electron, it become inactive.
The effective radioprotective agent, pursuant to this invention,
maintains in vivo a therapeutic concentration of TPL in its active
(free radical) state while overcoming the fact that when TPL is
administered alone, the dosage required to maintain therapeutic
levels is high and is toxic.
[0257] FIG. 19 shows that the maximum plasma level of TPL after
intraperitoneal injection of 275 mg/kg of TPL (.diamond.) 2 mM TPL
alone by intravenous administration (.quadrature.), and TPH 100
mg/kg +PNA 1 ml (.largecircle.). The results show that the maximum
plasma level of TPL is approximately one-fifth of that observed
after the intraperitoneal injection of approximately 100 mg/kg of
TEMPOL in the presence of PNA (0.5 ml/mouse at albumin
concentration of 20 g/dl and 42 moles TPL per mole of albumin).
Therefore, the plasma level of TPL is enhanced by greater than
tenfold in the presence of PNA. This enhanced plasma level of TPL
influences the intracellular levels of TPL which are responsible
for radiation protection at the cellular and nuclear levels.
[0258] This enhance protection is demonstrated in full body
irradiation based on a 30-day survival model in mice. FIG. 20 shows
enhancement of radiation protection by the addition of PNA at a
constant TPL concentration (200 mg/kg).
[0259] The results show that TPL in the presence of PNA has a
profound radioprotective effect. Eight out of ten (80%) mice
survived the 10 Gray lethal radiation as compared to one out of ten
(10%) with TPL alone. In a control experiment, without TPL or with
PNA alone, all mice die on or about day 15. Therefore, the membrane
impermeable PNA shows no radiation protective effect and does not
protect against radiation damage at the intracellular level.
[0260] Referring to FIG. 21, the experimental data shows that PNA
enables reduction in the TPL dose to achieve similar radiation
protection. In this experiment, all ten mice died on day 15 when
PNA (0.5 ml/mouse) was used alone. At one quarter the dose of TPL
used in FIG. 20, the TPL concentration is reduced from 200 mg/kg to
50 mg/kg, the presence of PNA was able to protect two out of ten
mice from lethal radiation (10 Gray). These results demonstrate
that PNA can be used to reduce the dosage of TPL by a factor of
four to achieve the same or better radiation protection.
[0261] Due to the ability to provide a systemic or physically
localized radioprotectant effect, the use of the compositions of
this invention is particularly advantageous in protecting a patient
undergoing therapeutic radiation treatments. For example, a
systemic or localized topical administration of a membrane
permeable nitroxide can be combined with a topical administration
of a polynitroxide albumin at the site where a radiation flux
enters the body.
[0262] In one particular application, a topical ointment containing
a polynitroxide albumin is applied to the scalp of a patient
undergoing treatment for a tumor of the cranium. Concurrently
therewith, a membrane permeable nitroxide is administered by an
appropriate means, including for example suspension in the topical
ointment. When the radiation dose is applied, the skin and hair
follicles will be protected from the complete harmful effects of
the radiation thereby lessening damage to the skin and reducing
hair loss. An additional example involving brain tumors is
particularly significant because of the high mortality rate and
difficulty in successful surgical therapy. A whole body radiation
protection can be provided by systemic administration of a membrane
permeable nitroxide and topical and intravenous administration of a
polynitroxide macromolecule with decreased permeability of the
tumor. In such an example, an increased dose of radiation can be
administered due to the whole body radioprotective effect of the
invention. Due to selective permeability, the tumor region is more
susceptible to radiation than the surrounding tissue and is thereby
treated with greater efficacy. The selection of a number of
variations and modifications of the above examples is well within
the skill of those in the pertinent art as are other modifications
which do not depart from the spirit of the invention.
Example Ten--In Vivo Enzyme Mimic
[0263] As noted above, nitroxides (e.g., TEMPOL) have been shown to
have catalytic activity which mimics that of superoxide dismutase
(SOD), the metalloenzyme which dismutates superoxide to hydrogen
peroxide. Furthermore, in biological systems, nitroxides can
interact with peroxidases and pseudoperoxidases to achieve an
activity mimicking that of catalase, the enzyme which converts
hydrogen peroxide to oxygen. Demonstrated herein is the use of
nitroxides to mimic a superoxide oxidase to alleviate oxidative
stress associated with metabolism of oxygen carriers. The
biological effects of such activity derived from
nitroxide-containing compounds include contributing to protection
against cytotoxicity of reactive oxygen species by reducing
oxidative stress. Nitroxides, when administered in vivo pursuant to
this invention, display additional complex antioxidant
enzyme-mimetic activities.
[0264] As noted above, when injected intravenously, TEMPOL has been
shown to have very short plasma half-life. Due to its molecular
size and charge characteristics, it readily leaves the vascular
space. In many medical applications, it may be desirable to have an
enzyme mimic which persists in the vascular space. This is achieved
pursuant to this invention, by attaching a nitroxide compound to a
macromolecule, such as hemoglobin and albumin, which is
biologically safe and has a desirable plasma half-life.
[0265] A membrane-permeable nitroxide such as TPL, in its free
radical state has been shown to have enzyme-mimic activity both in
vitro and in vivo. However, in vivo, primarily in the intracellular
space, it is rapidly reduced to its inactive hydroxylamine
derivative (TPH) by bioreducing agents such as NADH. Previously,
the reduction of the active TPL to the inactive TPH has been
essentially irreversible on a stoichiometric basis. Thus, its
effectiveness as a therapeutic and diagnostic tool is limited.
[0266] Pursuant to this invention, a multi-component
nitroxide-containing composition has, as a first component, the
membrane-permeable nitroxide which exists in a dynamic equilibrium
between TPL (active) and TPH (inactive). 22
[0267] In vivo, the inactive TPH predominates (>90%). Both
molecular species (TPL and TPH) readily cross the cell membrane and
distribute into the intracellular and extracellular spaces.
[0268] A second component is a membrane-impermeable, macromolecular
polynitroxide which distributes in the extracellular space,
predominantly in the vascular space. The first and second
components exhibit another enzyme mimetic function previously
unknown in vivo, that of a synthetic reduced-nitroxide oxidase. For
example, the polynitroxide albumin described herein as part of a
multi-component system acts as a reduced-nitroxide oxidase by
oxidizing TPH and TPL via a spin-transfer reaction. Thus, the
macromolecular polynitroxide albumin acts as an enzyme mimic
shifting the TPL/TPH equilibrium up to .about.90% TPL in both the
intra- and extra-cellular spaces. This enzyme-mimic function is
particularly useful where a high dose of TPL necessary to produce
the requisite level of protection from radiation, ischemia, etc.
would be toxic to the cells by overwhelming their cellular redox
machinery.
[0269] For example, in the example of a dose of gamma radiation,
when the dose becomes elevated, the quantity of low molecular
weight, membrane permeable nitroxide necessary to provide
meaningful radioprotective effects can become so large that the
cells redox state is disrupted, thereby resulting in toxicity.
[0270] To overcome the toxicity hurdle, the multi-component system
of this invention regenerates the reduced TPH to TPL. This system
can be used in any application where an active nitroxide is
desirable in vivo.
[0271] The EPR spectra of TPL TPH and a polynitroxide albumin is
shown in FIGS. 15A, 15B, and 15C, respectively. Demonstration of a
reduced nitroxide oxidase activity is shown by the reoxidation
(spin-transfer) from TPH (EPR silent FIG. 15B) to the
macromolecular polynitroxide (FIG. 15C) to and its conversion to
TPL (EPR active) (FIG. 15A) is carried out as follows: (1)
equimolar ratios of TPH to a macromolecular polynitroxide are
incubated at room temperature for 30 minutes; (2) the reaction
mixture is centrifuged through a 10 kd cut-off membrane; and (3)
The EPR spectrum of the filtrate is recorded and shown in FIG. 15B.
The quantitative conversion of TPH to TPL is shown in FIG. 17.
[0272] A synthetic reduced nitroxide oxidase (polynitroxide
albumin) is prepared by allowing human serum albumin (HSA, 25%
Baxter Healthcare) to react with 40 molar equivalents of
4-(2-bromoaceamido) -TEMPO or Br-TEMPO at 60.degree. C. for ten
hours with mixing. The resulting mixture, containing 15 ml of HSA
and 165 mg or Br-TEMPO in a vacutainer tube, is sterilized with a
0.22 micron filter and transferred into a 150 ml stirred cell
equipped with a 10 kd cutoff membrane (Filtron Technology Corp.).
The filtered reaction mixture is washed with Ringers solution until
the filtrate contains less than 1 uM of Free TEMPO as detected by
ESR spectroscopy. The bright orange colored retenate is
concentrated to 25% HSA and again sterile-filtered into a 10 ml
vial and stored at 4.degree. C. until used. To demonstrate the in
vivo enzyme-like conversion of TPH to TPL, the .sup.15N stable
isotope analogue of TPL is injected into the tail vein of a
cannulated mouse and the EPR signal is directly monitored in the
tail. The direct intravenous injection of a 0.5 ml of TPL (40 mM)
solution in the anesthetized mouse demonstrates that the plasma
half-life of TPL is approximately 2 minutes. Referring to FIG. 18,
with a follow-on injection (.about.30 minutes later) of a mixture
comprising a macromolecule polynitroxide and a stable isotope
.sup.15N TPL, a biphasic change in the peak intensity of .sup.15N
TPL exists. Initially, a decrease in .sup.15N TPL signal intensity
is attributed to the diffusion of TPL out of the vascular space
followed by its intra-cellular reduction. This rate of diffusion is
initially faster than the rate of reoxidation of the TPH to TPL
based on the slower re-appearance of the .sup.1sN TPL signal
intensity in FIG. 18. Although the reoxidation of TPH to TPL is
slower than the initial diffusion/reduction rate, it is faster than
the steady state intracellular reduction rate of TPL. Thus, the
reappearance of the TPL signal shown in FIG. 18 detects the
reoxidation of TPH to TPL thereby demonstrating a synthetically
produced reduced nitroxide oxidase activity in vivo in mice. See
also FIGS. 18B-18D.
Example Eleven--Ischemia and Reperfusion Injury--Cerebrovascular
Ischemia in Stroke
[0273] As noted above, nitroxide-containing compounds can be used
in medical imaging. A particularly useful application is in
obtaining images of ischemic tissues in the heart and elsewhere,
because valuable information regarding oxygen metabolism and
reperfusion injury can be obtained. However, the rapid reduction of
free nitroxides in vivo limits the utility of free nitroxides in
this application. However, pursuant to this invention, it is
possible to enhance the imaging capability to spatially resolve
ischemic tissue in the heart, to monitor the development of
myocardial ischemia, to study the development of the myocardial
reperfusion phase, and to observe in real time the hypoxic state of
tissues or organs. Referring to FIGS. 22, 23, and 25, ERI images of
an isolated rat heart infused with .sup.15N-TEMPOL and a
polynitroxide albumin are shown.
[0274] In FIG. 24, the intensity of the EPR signal is shown as a
function of the duration of ischemia. In the lower curve, 2 mM of
TEMPOL is infused into an ischemic heart. The upper curve traces
the original intensity of 2 mM TEMPOL together with a polynitroxide
albumin (4 g/dl of albumin at 42 moles of TEMPOL per mole of
albumin). The data demonstrate that the signal intensity is
substantially greater, and is maintained, when the composition of
this invention is used. FIG. 25 shows the viability of imaging at
ischemic tissue from 3-D spatial images. The progression of images
traces the progress of ischemia over approximately 125 minutes.
[0275] Apart from demonstrating diagnostic utility, the
polynitroxide albumin and TEMPOL combination protects the heart
from ischemic reperfusion injury. FIG. 26 shows that a nitroxide
alone in combination with PNA does not affect the recovery of
coronary flow. However, FIG. 27 shows the substantially improved
recovery of RPP (rate pressure product) following 30 minutes of
global ischemia followed by 45 minutes of blood flow is only
observed in the presence of both. Furthermore, edema of the heart
as a result of ischemic/reperfusion injury was prevented.
[0276] Referring to FIG. 28, the .sup.15N-TPL concentrations in
various anatomical regions of the ischemic heart are elevated
during over 100 minutes of ischemia. Elevation of TPL tissue
concentration may contribute to the protection of cardiac function
(FIG. 27) by PNA.
[0277] To demonstrate that PNA provides protection from
ischemia/reperfusion injury in the brain and central nervous system
as well as in the heart and cardiovascular system (as described
above), CD-1 mice at 3 months of age, body weight 35-40 g were
anesthetized with 1.5% isoflurane in a continuous flow of 30%
oxygen/70% nitrous oxide. The animals were subjected to middle
cerebral artery (MCA) occlusion and reperfusion by the method of
Yang et al. (Stroke 25: 165-170, 1994) with occlusion of the
ipsilateral common carotid artery (CCA) PNA or a control solution
of human serum albumin was injected intravenously just before one
hour of MCA occlusion (pre-ischemia group) or just after
reperfusion following one hour of ischemia (post-reperfusion
group). The dose was 0.5of body weight, v/w). Body temperature was
maintained within the range 36.5 - 37.5.degree. C. during the
surgery. Mean arterial pressure and blood gasses were measured in
four animals to insure a valid control group.
[0278] Twenty-four hours after reperfusion, animals were assessed
for neurological deficit as described by Bederson et al. (Stroke
17: 472-476, 1986). Neurological deficits were scored on the
following scale: 0+ no observable deficit, 1+ failure to extend
right forepaw, 2+ circling to the right, 3+ falling to the right, 4
+cannot walk spontaneously. Following the neurological assessment,
the animals were decapitated and the brains rapidly removed and
frozen for histological analysis. Coronal sections 20 .mu.m thick
were made at intervals of 0.5 mm from the anterior tip of the brain
and strained with cresyl violet in order to evaluate infarction
(n+7 animals). Infarct volume was calculated by integrating infarct
size (measured in mm.sup.2 in the serial sections.
[0279] Referring to FIG. 30, PNA treatment either before the onset
of ischemia or immediately after reperfusion significantly reduces
neurological deficit as measured 24 hrs. post-reperfusion (n=7).
Referring now to FIG. 31, PNA treatment either before cerebral
ischemia or immediately after reperfusion significantly reduced
infarct volumes as measured 24 hrs. post-reperfusion. In animals
treated before the onset of ischemia, infarct volumes were 6.956
mm.sup.3.+-.4.910 in PNA-treated animals, vs. 70.378
mm.sup.3+12.812 in control animals. In animals treated immediately
after reperfusion, infarct volumes were 13.710 mm.sup.3.+-.6.644 in
PNA-treated animals, vs. 62.636 mm.sup.3.+-.12.372 in control
animals.
[0280] FIGS. 32 and 32A show % infarction values and infarct area
respectively, which were calculated from infarct volume and
ipsilateral hemisphere volume. In animals treated with PNA
pre-ischemia, the % infarction value was 5.148% .+-.3.237, compared
with 42.462% 5.001 in pre-ischemia controls. In animals treated
immediately post-reperfusion, these values were 9.210.+-.4.080 in
PNA-treated animals and 37.782%.+-.8.030 in controls (mean.+-.SEM,
N+7). FIG. 33 shows that PNA treatment either before the onset of
ischemia or immediately post-reperfusion significantly reduced
hemisphere enlargement as measured 24 hrs. post-reperfusion.
Animals treated with pre-ischemia showed essentially no enlargement
(0.094% +0.094), while pre-ischemic controls showed
13.916%.+-.3.491 enlargement. Animals treated with PNA at the onset
of reperfusion showed 3.056%.+-.2.053 enlargement, compared with
the control value of 22.994%.+-.8.562.
[0281] The data in FIGS. 30-33 shows that PNA treatment either
before the onset of focal cerebral ischemia or immediately after
reperfusion gave statistically significant protection against
stroke injury as measured by neurological deficit, infarct volume,
% infarction, and hemisphere enlargement. As can be seen, the
neurological deficit scores correlated well with histological
measures of cerebral edema and infarction.
[0282] To determine the comparative level of protection provided by
PNA in comparison with another compound which functions by
alleviating a free radical cascade in the vascular space, a study
was performed to compare the protection provided by PNA in the
normal mouse with the comparatively high degree of protection
provided by overexpressed SOD in the SOD-1 Tg mouse. Chan, P. H.,
C. J. Epstein, H. Kinouchi, H. Kamii, G. Yang, S. F. Chen, J.
Gafni, and E. Carlson (1994). SOD-1 transgenic mice as a model for
studies of neuroprotection in stroke and brain trauma. Ann. N.Y.
Acad. Sci. 738:93-103. Chan, P. H., C. J. Epstein, H. Kinouchi, S.
Imaizumi, E. Carlson, and S. F. Chen (1993). Role of superoxide
dismutase in ischemic brain injury: Reduction of edema and
infarction in transgenic mice following focal cerebral ischemia. In
Molecular Mechanisms of Ischemic Brain Damage, K. Kogure and B. K.
Siesjo, eds. Amsterdam: Elsevier. pp. 97-104. Kinouchi, H., C. J.
Epstein, T. Mizui, E. Carlson, S. F. Chen and P. H. Chan (1991).
Attenuation of focal cerebral ischemic injury in transgenic mice
overexpressing CuZn superoxide dismutase. Proc. Natl. Acad. Sci.
USA 88:11158-11162. Imaizumi, S., V. Woolworth, and R. A. Fishman
(1990). Liposome-encapsulated superoxide dismutase reduces cerebral
infarction in cerebral ischemia in rats. Stroke 21: 1312-1317. Liu,
T. H., J. S. Beckman, B. A. Freeman, E. L. Hogan, and C. Y. Hsu
(1989). Polyethylene glycol-conjugated superoxide dismutase and
catalase reduce ischemic brain injury. Am. J. Physiol. 256:
H586-H593. Chan, P.H., S. Longar, and R.A. Fishman (1987).
Protective effects of liposome-entrapped superoxide dismutase on
post-traumatic brain edema. Ann. Neurol. 21: 540-547. PNA at 1of
body weight v/w (.sup.200 .mu.l in a 20 g mouse) was injected
intravenously into an anesthetized animal. Ten minutes later, the
middle coronary artery (MCA) was occluded by luminal blockade using
a nylon suture. After one hour of ischemia, reperfusion, and after
24 hours of reperfusion, animals were assessed for neurological
function and sacrificed for histological measurement of infarction
and edema. Referring to FIGS. 34A and 34B, a representative coronal
section from the brain of a control mouse treated mouse were
stained with cresyl violet, which stains normal tissue such that
areas of ischemic damage do not take up the stain. The control
brain shows massive ischemic injury of the involved hemisphere and
comparison of that hemisphere with the contralateral one in the
same brain showing substantial edema. In contrast, in the brain of
the treated mouse, the involved hemisphere is normal and symmetric
with the contralateral one in terms of size, shape, and staining
density. Serial sections as shown in FIG. 34C indicate the anatomic
distribution of ischemic injury throughout the brain of a
representative control animal, and the normal appearance of the
brain of a representative treated animal. The areas of infarction
in the control animal vs. the PNA treated animal are quantitated in
FIG. 32A, showing that PNA treatment gives essentially complete
protection against infarction in this model. The following table
summarizes data on the reduction in neurological deficit, percent
infarction (calculated from infarction areas in serial sections),
and hemisphere enlargement in the treated animals. Thus, the
infusion of PNA provides a greater degree of protection than does
threefold-overexpressed transgenic SOD.
2 Protection against stroke injury in the mouse Control PNA
Neurological Deficit Score.sup.1 5.5 2.0 % Infarction.sup.2 58.0
.+-. 9.4 1.9 .+-. 1.9 % Hemisphere Enlargement.sup.2 24.1 .+-. 6.2
1.7 .+-. 0.9 PNA (1% of body weight v/w, 200 .mu.l per 20 g mouse)
was administered both 10 minutes before the onset of 1 hour of
ischemia and at the onset of reperfusion. Animals were assessed for
neurological function and sacrificed for histology after 24 hours
of reperfusion. .sup.1Neurological deficit is expressed as mean
rank of deficits divided into four groups; the PNA group showed
significantly less deficit (p < 0.05 by Mann-Whitney U test).
.sup.2Percent infarction and hemisphere enlargement in animals that
survived to 24 hours are expressed as mean .+-. S.E.M.; the PNA
group showed reductions which were significant at p < 0.05 by
Student's t-test.
[0283] There is also increasing evidence of significant incidence
of ischemic injury in surgical patients undergoing cardiopulmonary
bypass and other surgical procedures resulting from embolism and/or
hypotension, with subsequent reperfusion injury. The principal
target organs are the brain, the heart, and the kidney.
[0284] As a model of prophylactic therapy in the
surgical/perioperative stroke setting, PNA was administered to mice
both before MCA occlusion and after reperfusion. Because albumin
alone may provide modest protection against cerebral ischemia, PNA
was tested with both HSA and saline as controls. Referring to FIGS.
35 and 36, the saline-treated rat of FIG. 35 shows massive
infarction, while the PNA rat of FIG. 36 shows no grossly visible
infarction. Histologic analysis (FIGS. 37A and 37B) shows that
saline-treated rats had a mean infarct volume of 192.+-.17
mm.sup.3. Treatment with albumin gave a significantly smaller
(p<0.01) mean infarct volume of 81.+-.26 mm.sup.3, and treatment
with PNA gave approximately twice as much reduction as albumin
alone, with a mean infarct volume of 18.+-.22.sup.3 (p<0.05 vs.
albumin, p<O.01 vs. saline)
[0285] In parallel with the histological analysis in the rat MCAO
model, a magnetic resonance imaging study shows that when present
at the onset of MCA occlusion, PNA slows the development of the
diffusion-weighted imaging (DWI) hyperintensity during cerebral
ischemia (FIG. 39). In addition, the protection offered by PNA
against reperfusion injury is indicated by the rapid disappearance
of DWI hyperintensity following reperfusion.
[0286] In addition to the results herein demonstrating protection
for cerebral ischemia in mice, the compounds of the invention have
shown efficacy in a rat model of stroke. Referring to FIGS. 35a and
35b, representative coronal sections of the brain of a rat treated
with normal saline (FIG. 35) and a rat treated with PNA (FIG. 36)
reveal significant tissue necrosis in the saline group and
significantly less necrosis in the PNA treated group. Rats were
subjected to 2 hours of suture occlusion of the MCA, followed by 22
hours of reperfusion. Drug or saline was administered IV in a total
dose of 1% of body weight (v/w), divided as follows: 1/2 the total
dose was given 5 minutes before MCA occlusion; 1/4 of the total
dose was given five minutes before the suture was removed to allow
reperfusion of the MCA; and the final 1/4 of the total dose was
given 2 hours after reperfusion. Brain sections were stained with
TTC.
[0287] Referring to FIGS. 37A and 37B, histological analysis of
infarct volume and percent infarction in rats subjected to
transient MCA occlusion and treated with saline, human serum
albumin (administered as a 25 g/dl solution, total dose l of body
weight), or PNA shows protection of the brain tissue against
ischemic/reperfusion injury. Animals treated with PNA showed
approximately 2.+-.3% infarction; animals treated with albumin
showed approximately 11.+-.4% infarction; and animals treated with
saline showed approximately 22.+-.4% infarction. The fact albumin
alone, has approximately 50% protective effort as compared to that
of PNA, suggest the potential role of plasma expansion in stroke
therapy. Therefore, PNA in hypertonic saline may further improve
its efficiency in stroke therapy. The fact that albumin gave
substantial protection in this experiment suggests a potentially
beneficial role for plasma expansion in stroke therapy. The fact
that PNA gave approximately twice as much protection as albumin
suggests that polynitroxylated compounds may be useful in
increasing efficacy of hypertonic or hyperoncotic therapies in
stroke.
[0288] Referring to FIG. 38, due to the absence of significant
quantities of naturally occurring paramagnetic species in vivo, the
concentration of active PNA-bound nitroxide can be measured by in
vivo EPR spectroscopy. In this experiment, a femoral arteriovenous
shunt was created in the rat using PESO polyethylene tubing. Part
of the tubing was placed in the cavity of an EPR spectrometer,
allowing measurement of the spin density of the blood flowing
through the shunt. PNA was injected via a catheter in the
contralateral femoral vein, and the spin density of the blood was
measured as a function of time following PNA injection. Comparison
with a standard curve (not shown) indicated that, with three
injections at two-hour intervals, the EPR signal of PNA was
maintained in the blood at levels in the range of 1-6 mM nitroxide
for five hours.
[0289] Referring to FIG. 39, Diffusion-weighted magnetic resonance
imaging (DWI) has been shown to indicate changes in the apparent
diffusion coefficient (ADC) of water. In stroke, hyperintensity on
DWI is thought to be a real-time indicator of the course of
cerebral damage during ischemia. Perfusion-weighted imaging (PWI)
was used in this experiment to confirm the occlusion and
reperfusion of the MCA (images not shown). The figure shows DWI
images made before, during, and after a 1.5-hour period of MCA
occlusion in the rat, each row shows images made in three planes of
the same brain. Row A shows that before MCA occlusion there is no
DWI hyperintensity. Row B shows that 30 minutes after MCA
occlusion, an area of DWI hyperintensity begins to appear, and is
clearly defined at 1 hour of MCA occlusion (Row C). For comparison,
in control images (not shown) a distinct DWI image typically
appears within 5-10 minutes of MCA occlusion. Thus, when present
before the onset of ischemia, PNA is capable of substantially
slowing the progression of ischemic injury in the brain. Row D
shows images made 8 minutes after reperfusion; the DWI image has
essentially disappeared, indicating that the ADC change which
occurred during ischemia resolved quickly upon reperfusion in the
presence of PNA. For comparison, control images typically show
progression of the DWI image after reperfusion, reflecting
reperfusion injury. Rows E and F show images made, respectively, 1
hour and 2 hours post-reperfusion, confirming the absence of a DWI
image. This animal was sacrificed 24 hours post--MCA occlusion and
found histologically to have a small infarct in the basal ganglia,
and no cortical infarct.
[0290] Example Twelve--Vasodilatory and Vasoneutral formulation of
Hemoglobin-based Red Cell Substitute (HRCS)
[0291] FIG. 13 shows the effect of the compositions of this
invention on the vasoconstrictive effect of hemoglobin-based oxygen
carriers (HBOC), specifically DBBF-Hb. This vasoconstriction is
demonstrated in conscious rat models by measuring the increase in
mean arterial pressure (MAP) when a 10% v/v top load of this
solution is infused. Referring to FIG. 13, the dotted line
indicates the mean arterial pressure as a function of time
following infusion of an HBOC. Pursuant to this invention, the same
PNA and TPL solution used for radiation protection, ERI, and
ischemia/reperfusion injury protection is shown to possess a broad
range of enzyme mimic and radical detoxification functions. PNA or
TPL, when injected alone with HBOC were found to have no
antihypertensive effect. Further, PNA (5 g/dl) or TPL (100 mM)
alone, 10% v/v top load, in conscious rats produces no significant
vasodilatory effect. However, PNA (5%/dl)/TPL (100 mM) as top
loaded at 10% v/v produces a significant and sustained vasodilatory
effect was observed (FIG. 29). This vasodilatory effect coincides
with the sustained plasma TPL levels in these rats (FIG. 14). In
the absence of PNA, the plasma half-life of TPL in these rats is
less than 60 seconds. Therefore, by mixing equal volumes of PNA (5
g/dl)/100 mM TPL with DBBF-Hb 7.8 g/dl and top load a 20% v/v in
these rats a vasoneutral HRCS formulation is produced (FIG. 13).
The hypotensive affect observed in FIG. 29 coincides with the
sustained elevation of TPL (FIG. 14) in the vascular smooth
muscles, which prevent the destruction of nitric oxide (i.e.,
endothelium derived relaxing factor (EDRF) by superoxide), thus
enhancing the vasodilation and lowering the MAP in the rat (FIG.
29). In the case of a vasoneutral HRCS formulation (FIG. 13), the
vasoconstrictive and vasodilatory activities of the HBOC and
PNA/TPL cancelled each other's effect on the nitric oxide levels in
vivo. Therefore, this vasoneutral formulation of HRCS is a
significant improvement of the HBOC currently in clinical
development, based on the global protection of free radical and
oxidative stress.
Example Thirteen--Inhibition of Lipid Oxidation
[0292] Oxidation of lipids is implicated in reperfusion injury and
atherosclerosis as well as tissue injury to the brain and central
nervous system. The nitroxide-labelled macromolecules of the
invention are demonstrated to alleviate lipid oxidation.
[0293] Approximately 60 mL of heparinized blood was obtained from
healthy, non-smoking men. Plasma LDL was prepared from EDTA (1
mg/mL) -anticoagulated venous blood after centrifugation of blood
at 2000 g at 4.degree. C. for 20 minutes. Blood, plasma, and LDL
samples were processed in subdued light on ice to inhibit
photoxidation of LDL antioxidants. LDL was isolated from plasma by
rate zonal density gradient ultra-centrifugation. The density of
plasma was raised to 1.3 g/mL by adding 5 g KBr to 10 mL of plasma.
The KBr was dissolved in plasma by gentle mixing on a tipper for 10
to 15 minutes. Each KBr-plasma sample was layered underneath 23 mL
of ice-cold 0.9% NaCl in an EasySeal polyallomer ultracentrifuge
tube. The sealed tubes were centrifuged at 50,000 rpm (302,000 g)
for 3 hours at 4.degree. C. After centrifugation, the density
gradients were fractionated by piercing the bottom of the tube with
a needle and pumping Fluorinert into the tube at a rate of 5
mL/minute. The contents were collected from the top of the tube and
diverted to a fraction collector. The first 12 mL of the gradient,
containing very low density lipoproteins was discarded.
Subsequently, six fractions of 1.2 mL each were collected. The dark
yellow LDL peak was generally found in the fourth fraction. LDL was
pooled from the peak fraction plus one fraction after the peak and
two fractions before the peak. This LDL gave a single
.beta.-migrating band after agarose electrophoresis, indicating
that the LDL was free from contamination by other lipoproteins. LDL
samples were dialyzed against 6L of HBSS buffer, three times before
use. The isolated LDL samples were stored on ice in the dark. LDL
protein content was determined by the Peterson-Lowry method with
bovine serum albumin as a standard.
[0294] Lipid oxidation was measured by oxidation of LDL with hemin
and H.sub.2O.sub.2. LDL 200 .mu.g/mL was added to cuvette that
contained 5 .mu.mol hemin and 50 .mu.mol H.sub.2O.sub.2. The final
assay volume was 2 to 3 mL in HBSS buffer at pH 7.4 The assays were
started by the addition of H.sub.2O.sub.2. In some experiments, PNA
plus TPL was diluted in HBSS at 1:100, 1:1000 and 1:10,000. At
various time points samples were removed and measurement of
thiobarbituric acid reducing substances (TBARS) were measured. This
was done by adding the sample to 500 .mu.L of thiobarbituric acid
reagent which included thiobarbituric acid, hydrochloric acid,
trichlorocetic acid and distilled water. Butylated hydroxytoluene
in DSMO was added at a final concentration of 0.1 mg/mL to inhibit
spontaneous formation of TBARS during the subsequent heating step.
After heating at 100.degree. for 15 minutes, the samples were
cooled to room temperature and centrifuged at 10,000 g for 10
minutes. The clear supernatants were analyzed
spectrophotometrically at 532 nm with an extinction coefficient of
1.56.times.10.sup.5 mol/L.sup.-1 cm.sup.-1, and the results are
presented as nanomoles TBARS per milligram LDL protein. FIGS.
35a-35d illustrate a time course of LDL oxidation in response to
hemin H.sub.2O.sub.2. In these four separate experiments, the time
to oxidation of LDL in the presence of PNA plus TPL at 1:100,
1:1,000, 1:10,000 is potently inhibited. The increase in optical
density which reflects TBARS formation is blocked in experiments
one and two by 1:100 and 1:10,000 PNA plus TPL.
Example Fourteen--Inhibition of Leukocyte Activation
[0295] The interactions of leukocytes with endothelial cells and
platelets are an indicator of the microcirculatory response to
several adverse physiological conditions, ranging from localized
and systemic ischemia/reperfusion injury, rejection of organ
grafts, and the response to the organism to various oxidizing
novae. See also Lehr et al., PNAS, 791:7688 (August 1984). The
mediator role of reactive oxygen species has been documented in
many in vitro and in vivo models and, as a consequence,
antioxidants and radical scavengers have been applied to counteract
leukocyte activation, aggregation, and adhesion to endothelium and
the subsequent microcirculatory dysfunction. Using a dorsal
skinfold chamber model in Syrian Golden hamsters to study the
inhibitory effect of two different polynitroxylated carriers on
cigarette smoke induced leukocyte rolling and adhesion to
endothelial cells of arterioles and venules, as well as on the
formation of leukocyte/platelet aggregates in the blood stream
(Lehr et al., PNAS 91:7688, 1994), it is demonstrated that a HBOC
(DBBF-crosslinked hemoglobin) had no effect on leukocyte activation
when compared with saline-treated controls. However,
polynitroxylated HBOC inhibited leukocyte activation essentially
completely. It is known that albumin provides a degree of
protection, as is observed in the following table, where at the
dose used it inhibited activation. However, the Table shows that
PNA also inhibited activation, and the HBOC and polynitroxyl-HBOC
results shown in the Table predict that PNA will give better
protection than albumin at lower protein concentrations.
Polynitroxylated albumin and polynitroxylated, stabilized
hemoglobin based oxygen carrier, as described above, effectively
prevent detrimental leukocyte interactions with endothelial cells
and platelets as shown in the following table.
3 Time points BASELINE 15 MINUTES 60 MINUTES A. Human serum
albumin, 25 g/dl arteriolar rolling (/mm) 0.0 0.1 .+-. 0.2 0.1 .+-.
0.2 arteriolar sticking (/mm.sup.2) 0.0 0.0 .+-. 0.0 0.0 .+-. 0.0
venular rolling (%) 4.9 .+-. 0.2 5.5 .+-. 0.9 6.9 .+-. 2.1 venular
sticking (/mm.sup.2) 0.0 .+-. 0.0 0.0 .+-. 0.0 0.0 .+-. 0.0
aggregate formation 0.6 .+-. 0.7 0.7 .+-. 0.7 1.3 .+-. 1.1 (/30
sec) B. Polynitroxylated Albumin arteriolar rolling (/mm) 0.0 0.0
.+-. 0.0 0.0 .+-. 0.0 arteriolar sticking (/mm.sup.2) 0.0 0.0 .+-.
0.0 0.0 .+-. 0.0 venular rolling (%) 5.2 .+-. 0.8 5.8 .+-. 0.8 6.3
.+-. 1.8 venular sticking (/mm.sup.2) 0.0 .+-. 0.0 0.0 .+-. 0.0 0.0
.+-. 0.0 aggregate formation 0.4 .+-. 0.5 0.9 .+-. 1.7 1.2 .+-. 0.8
(/30 sec) C. Hemoglobin-based oxygen carrier (HBOC), DBBF-Hb
arteriolar rolling (/mm) 0.3 1.9 .+-. 2.0 3.8 .+-. 2.9 arteriolar
sticking (/mm.sup.2) 0.0 31.9 .+-. 26.4 42.0 .+-. 32.1 venular
rolling (%) 5.8 .+-. 1.2 7.9 .+-. 2.8 9.2 .+-. 3.8 venular sticking
(/mm.sup.2) 0.0 .+-. 0.0 66.0 .+-. 43.2 101.5 .+-. 76.9 aggregate
formation 0.3 .+-. 0.5 3.0 .+-. 1.7 4.5 .+-. 2.6 (/30 sec) D.
Polynitroxylated hemoglobin- based oxygen carrier arteriolar
rolling (/mm) 0.0 0.2 .+-. 0.3 0.1 .+-. 0.3 arteriolar sticking
(/mm.sup.2) 0.0 0.0 .+-. 0.0 1.3 .+-. 2.5 venular rolling (%) 5.0
.+-. 0.2 6.7 .+-. 1.0 6.8 .+-. 1.2 venular sticking (/mm.sup.2) 0.0
.+-. 0.0 0.0 .+-. 0.0 0.0 .+-. 0.0 aggregate formation 0.2 .+-. 0.2
1.9 .+-. 0.2 1.4 .+-. 0.3 (/30 sec) E. Isovolemic normal saline
(control) arteriolar rolling (/mm) 0.3 .+-. 0.2 2.5 .+-. 2.9 3.3
.+-. 2.0 arteriolar sticking (/mm.sup.2) 0.0 .+-. 0.0 36.0 .+-.
27.6 40.0 .+-. 32.7 venular rolling (%) 4.5 .+-. 1.3 9.3 .+-. 1.7
10.3 .+-. 2.5 venular sticking (/mm.sup.2) 0.0 .+-. 0.0 68.0 .+-.
43.8 90 .+-. 56.7 aggregate formation 0.3 .+-. 0.3 3.5 .+-. 0.6 5.1
.+-. 1.1 (/30 sec)
Example Fifteen--Topical Application to Skin Diseases Particularly
Psoriasis
[0296] Much research has been directed at the underlying causes of
several diseases at the skin where free radical levels are elevated
or where a free radical cascade is manifested in the dermis. In
psoriasis, the causes are multi-faceted, however, the end effectors
of the inflammatory process appear to be superoxide and other toxic
oxygen radicals. Both skin cells and leukocytes have been reported
to contribute to pathologic free radical levels in psoriasis.
Dermal fibroblasts from both lesional and uninvolved skin of
psoriatic patients have been reported to be higher (by 150% and
100% respectively) than normal controls. Er-Raki A, Charveron M,
and Bonafe JL 1993. Increased superoxide anion production in dermal
fibroblasts of psoriatic patients. Skin Pharmacol. 6:253-258.
Psoriatic sera have been reported to increase superoxide generation
by polymorphonuclear leukocytes (PMNs). Miyachi Y and Niwa Y 1983.
Effects of psoriatic sera on the generation of oxygenintermediates
by polymorphonuclear leukocytes. Arch. Dermatol. Res. 275:23-26.
SOD levels in PMNs from psoriatic patients were reported to be
significantly lower than normal. Dogan P, Soyuer U, and Tanrikulu G
1989. Superoxide dismutase and myeloperoxidase activity in
polymorphonuclear leukocytes, and serum ceruloplasmin and copper
levels, in psoriasis. Br. J. Dermatol. 120:239-244. Superoxide
dismutase mRNA and immunoreactive enzyme have been reported to be
increased in psoriatic skin, perhaps reflecting an attempt to
compensate for elevated free radical levels. Kobayashi T, Matsumoto
M, Iizuka H, Suzuki K, and Taniguchi N 1991. Superoxide dismutase
in psoriasis, squamous cell carcinoma and basal cel epithelioma: an
immunohistochemical study. Br. J. Dermatol. 124:555-559. Since
free-radical scavengers have been reported to offer benefit in
experimental studies on psoriasis, it follows that an agent which
detoxifies free radicals may block the inflammatory process by
preventing the cytotoxic effects of free radicals at the site of
the psoriatic lesion. Haseloff RF, Blasio IE, Meffert H, and Ebert
B. 1990. Hydroxyl radical scavenging and antipsoriatic activity of
benzoic acid and derivatives. Free Radical Biol. Med.
9:111-115.
[0297] Using in vivo EPR spectroscopy in the mouse, the
effectiveness of the compounds of the invention can be determined
from an analysis of the pharmacokinetics of intradermal PNA and
intravenous .sup.15N-TPL.
[0298] A custom-built bridged loop-gap surface resonator,
absorption microwave bridge was used for in vivo EPR spectroscopy
at 1.25 GHz (L-band) in the mouse. This instrumentation was used to
measure local TPL and PNA concentrations at specific sites on the
body of the mouse. The anesthetized mouse was placed in a
`sandwich` of two templates which allowed it to be placed on the
loop-gap surface resonator, absorption microwave bridge in either
the supine position, for spectroscopy of the back of the head, or
in the prone position for spectroscopy of the abdomen. A tail vein
cannula was used for intravenous injection of .sup.15N-TEMPOL; this
injection site was remote from the resonator.
[0299] FIG. 40A shows the EPR spectrum of intradermally injected
PNA (100 .mu.l of a 10 g/dl PNA solution), measured at the site of
injection on the animal's back.
[0300] FIG. 40B shows the composite spectrum of intradermal PNA and
intravenous .sup.15N-TPL, measured at the site of intradermal PNA
injection on the animal's back. The broad, asymmetric central peak
reflects only PNA concentration, while the sharp low-field peak to
the left reflects only .sup.15N-TPL concentration. The spectrum was
made 1 minute after .sup.15N-TPL was injected via the tail vein.
The .sup.15N-TPL dose was 1% of body weight (w/w) of a stock
solution of .sup.15N-TPL (20 mg/ml or .about.115 mM, in lactated
Ringer's solution), giving a blood concentration of approximately
1.15 mM. FIG. 40B was recorded at approximately 1/5 of the
instrument gain used in FIG. 40A in order to accommodate the
1sN-TPL signal. FIG. 40C shows the EPR spectrum made 30 minutes
after tail vein injection of .sup.15N-TPL. The specific
.sup.15N-TPL and PNA peaks remain clearly distinguishable.
[0301] The pharmacokinetics of .sup.15N-TPL at the site of
intradermal PNA injection is shown in FIG. 41, a plot of the
intensity of the .sup.15N-TPL-specific peak vs. time. The
.sup.15N-TPL signal increased immediately after the tail vein
injection, reaching a maximum at less than 2 minutes after
injection, then decayed to a steady-state level. The residual
steady-state level corresponds with 17% of the initial .sup.15N-TPL
concentration, assuming complete distribution in the total body
water. The initial decay reflects the well-known reduction of TPL
to the diamagnetic (EPR-silent) TPH. The persistence of the
residual .sup.15N-TPL signal is attributable to reoxidation of
.sup.15N-TPH to .sup.15N-TPL by the intradermal PNA.
[0302] Referring to FIGS. 42A and 42B, to verify the intradermal
reoxidation by PNA, the local pharmacokinetics of .sup.15N-TPL were
tested and showed a half-life of 30 seconds. In the absence of
intradermal PNA, the decay of .sup.15N-TPL to .sup.15N-TPH is
complete.
[0303] The presence of .sup.15N-TPH in the skin is demonstrated by
the intradermal injection of PNA at the belly. The intradermal PNA
(100 .mu.l, 10 g/dl) regenerates and sustains the .sup.15N-TPL
locally at the site of injection. The pharmacokinetics of the in
vivo reoxidation of .sup.15N-TPH is shown by comparing FIGS. 42A
and 42B. A +5 minutes after the intradermal injection of PNA, the
reoxidation of .sup.15N-TPH is essentially complete.
Example Sixteen--Free Radical Toxicity--Renal Injury
[0304] In an animal model of rhabdomyolysis, a bolus injection of
glycerine into a major muscle causes heme-induced renal injury.
Polynitroxide albumin is shown to reduce glycerol-induced renal
injury in a survival study with albumin as a control. Male
Sprague-Dawley rats weighing approximately 275 g were dehydrated
overnight (16 h) after having baseline serum creatinine determined.
On the following morning, 1.25 ml of either polynitroxide albumin
and TEMPOL (10 g/dl) or albumin (10 g/dl) was given via tail vein
injection. Immediately following the injection, an IM injection of
glycerol (50% in water v/v, 10 ml/Kg) was given, 1/2 of the dose
into each anterior thigh muscle. Two hours after the first
injection another identical dose was given via tail vein injection.
Referring to FIG. 43, for the next 10 days, survival and serum
creatinine were monitored. At the end of this time, all rats tested
had either suffered mortality or had showed signs of recovery
(falling serum creatinine).
[0305] The particular examples set forth herein are instructional
and should not be interpreted as limitations on the applications to
which those of ordinary skill are able to apply this invention.
Modifications and other uses are available to those skilled in the
art which are encompassed within the spirit of the invention as
defined by the scope of the following claims. All references and
publications referred to above are specifically incorporated by
reference.
* * * * *