U.S. patent application number 15/662663 was filed with the patent office on 2018-03-15 for method of treating or preventing pathologic effects of acute increases in hyperglycemia and/or acute increases of free fatty acid flux.
The applicant listed for this patent is Albert Einstein College of Medicine, Geoffrey C. Gurtner. Invention is credited to Michael A. Brownlee, Geoffrey C. Gurtner.
Application Number | 20180071265 15/662663 |
Document ID | / |
Family ID | 38123438 |
Filed Date | 2018-03-15 |
United States Patent
Application |
20180071265 |
Kind Code |
A1 |
Gurtner; Geoffrey C. ; et
al. |
March 15, 2018 |
Method of Treating or Preventing Pathologic Effects of Acute
Increases in Hyperglycemia and/or Acute Increases of Free Fatty
Acid Flux
Abstract
One aspect of the present invention relates to a method of
treating or preventing pathologic effects of hyperglycemia and/or
increased fatty acid flux in a subject in need of such treatment or
preventive therapy. This method involves administering a
composition containing a therapeutically effective amount of a ROS
inhibitor to a subject in need thereof.
Inventors: |
Gurtner; Geoffrey C.;
(Stanford, CA) ; Brownlee; Michael A.; (New York,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gurtner; Geoffrey C.
Albert Einstein College of Medicine |
Stanford
Bronx |
CA
NY |
US
US |
|
|
Family ID: |
38123438 |
Appl. No.: |
15/662663 |
Filed: |
July 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11297808 |
Dec 7, 2005 |
9737511 |
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15662663 |
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11136254 |
May 24, 2005 |
8829051 |
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11297808 |
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60573947 |
May 24, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/496 20130101;
A61K 31/4196 20130101; A61P 3/10 20180101; A61K 31/5377
20130101 |
International
Class: |
A61K 31/4196 20060101
A61K031/4196; A61K 31/496 20060101 A61K031/496; A61K 31/5377
20060101 A61K031/5377 |
Claims
1-19. (canceled)
20. A composition of matter comprising the compound R-L-C-M or a
pharmaceutically acceptable salt thereof, wherein R is H or a
biocompatible moiety capable of facilitating or hindering the
penetration of said composition of matter in target cells, L is a
linker connecting R to C, wherein L is capable of facilitating a
rapid cellular intake and delaying a cellular exit of said
composition of matter, C is an iron chelating moiety, and M is a
functional-masking group susceptible to cleavage by (OH.).
21. (canceled)
22. A therapeutic composition comprising: (a) a compound of the
formula R-L-C-M or a pharmaceutically acceptable salt thereof;
wherein R is H or a biocompatible moiety capable of facilitating or
hindering the penetration of said therapeutic composition in target
cells, L is a linker connecting R to C, wherein L is capable of
facilitating a rapid cellular intake and delaying a cellular exit
of said therapeutic composition, C is an iron chelating moiety, and
M is a functional-masking group susceptible to cleavage by (OH.),
and (b) a pharmaceutical carrier.
23. (canceled)
Description
[0001] This application is a continuation in part of U.S. Ser. No.
11/136,254 filed May 24, 2005, which claims benefit to and priority
from U.S. Provisional Patent Application Ser. No. 60/573,947, filed
May 24, 2004.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of treating or
preventing pathologic effects of acute increases in hyperglycemia
and/or acute increases of fatty acid flux in a subject.
BACKGROUND OF THE INVENTION
[0003] Cardiovascular Complications Associated with Diabetes are a
Major Public Health Problem.
[0004] Diabetes mellitus is an epidemic in the United States
(Brownlee, "Biochemistry and Molecular Cell Biology of Diabetic
Complications," Nature 414:813-20 (2001); Nishikawa et al.,
"Normalizing Mitochondrial Superoxide Production Blocks Three
Pathways of Hyperglycaemic Damage," Nature 404:787-90 (2000);
Zimmet et al., "Global and Societal Implications of the Diabetes
Epidemic," Nature 414:782-7 (2001)). Currently 15-17 million adults
(5% of the adult population) in the U.S. are affected by Type I and
Type II diabetes (Harris et al., "Prevalence of Diabetes, Impaired
Fasting Glucose, and Impaired Glucose Tolerance in U.S. Adults. The
Third National Health and Nutrition Examination Survey, 1918-1994,"
Diabetes Care 21:518-24 (1998); AD Association, "Economic Costs of
Diabetes in the U.S. in 2002," Diabetes Care 26:917-932 (2003)). By
the year 2020, the diabetic population is expected to increase by
another 44% (AD Association, "Economic Costs of Diabetes in the
U.S. in 2002," Diabetes Care 26.917-932 (2003)). In addition to
those with diabetes mellitus, an additional number of people
display the metabolic syndrome, with impaired glucose and insulin
tolerance and altered vascular reactivity.
[0005] The greatest impact of diabetes is on the vascular system
(Caro et al., "Lifetime Costs of Complications Resulting From Type
2 Diabetes in the U.S. Diabetes Care 25:476-81 (2002)). Diabetic
patients have an increased risk for vascular disease affecting the
heart, brain, and peripheral vessels (Howard et al., "Prevention
Conference VI: Diabetes and Cardiovascular Disease: Writing Group
I: Epidemiology," Circulation 105:e132-7 (2002)). The relative risk
of cardiovascular disease in diabetics is 2-8 times higher than
age-matched controls (Howard at al., "Prevention Conference VI:
Diabetes and Cardiovascular Disease: Writing Group I:
Epidemiology," Circulation 105:e132-7 (2002)). Diabetes accounts
for 180 billion dollars in annual health costs in the U.S., with
85% of this amount attributable to vascular complications (Caro at
al., "Lifetime Costs of Complications Resulting From Type 2
Diabetes in the U.S. Diabetes Care 25:476-81 (2002)). Indeed, if
macrovascular complications (stroke, MI, TIA, angina) and
microvascular complications (nephropathy, neuropathy, retinopathy,
wound healing) are considered together, the vast majority of
diabetes related healthcare expenditures result from
vasculopathies.
One or the Reasons why Diabetic Patients have Poor Outcomes is
Because of Impaired Compensatory Vascular Growth.
[0006] The recognition that diabetes impairs survival after
ischemic events dates back to the last century and has been
independently confirmed by two landmark epidemiologic studies (The
Framingham Study and The Diabetes Control and Complications Trial)
(Garcia et al, "Morbidity and Mortality in Diabetics in the
Framingham Population. Sixteen Year Follow-Up Study," Diabetes
23:105-11 (1974); TDCaCTR Group, "The Effect of Intensive Treatment
of Diabetes on the Development and Progression of Long-Term
Complications in Insulin-Dependent Diabetes Mellitus," N Engl J Med
329:977-86 (1993)). These prospective studies substantiated a
relationship between poor glycemic control and decreased survival
after myocardial infarction. Of note, these trials demonstrated
that in addition to an increased incidence of ischemic episodes
(Kannel at al., "Diabetes and Cardiovascular Risk Factors: the
Framingham Study," Circulation; 59:8-13 (1979)), diabetic patients
have higher rates of post-infarct complications, such as cardiac
failure and secondary ischemic events (Haffner et al., "Mortality
From Coronary Heart Disease in Subjects With Type 2 Diabetes and in
Nondiabetic Subjects With and Without Prior Myocardial Infarction,"
N Engl J Med 339:229-34 (1998); Zuanetti et al., "Influence of
Diabetes on Mortality in Acute Myocardial Infarction: Data From the
GISSI-2 Study," J Am Coll Cardiol 22:1788-94 (1993)). These
disparities were not due to increased infarct size in the diabetic
population (Wilson, "Diabetes Mellitus and Coronary Heart Disease,"
Am J Kidney Dis 32:S89-100 (1998)), suggesting that an impairment
existed in the compensatory response of the diabetic myocardium.
Similar impairments have been described in other diabetic tissues,
including the extremities and brain (Uusitupa at al., "5-Year
Incidence of Atherosclerotic Vascular Disease in Relation to
General Risk Factors, Insulin Level, and Abnormalities in
Lipoprotein Composition in Non-Insulin-Dependent Diabetic and
Nondiabetic Subjects," Circulation 82:27-36 (1990); Jude et al.,
"Peripheral Arterial Disease in Diabetic and Nondiabetic Patients:
a Comparison of Severity and Outcome," Diabetes Care 24:1433-7
(2001); Tuomilehto et al., "Diabetes Mellitus as a Risk Factor for
Death From Stroke. Prospective Study of the Middle-Aged Finnish
Population," Stroke 27:210-5 (1996)).
[0007] The concept that these impairments result from a poorly
adapting diabetic vasculature has both clinical and experimental
support. Since angiogenesis and collateral development are the
processes that restore blood flow to watershed areas of the heart,
the rapid restoration of a normal vascular density in the
microvasculature ultimately determines patient outcome following
ischemia (Helfant et al., "Functional Importance of the Human
Coronary Collateral Circulation," N Engl J Med 284:1277-81 (1971);
Chilian at al., "Microvascular Occlusions Promote Coronary
Collateral Growth," Am J Physiol 258:H1103-1 (1990)). Indeed, the
theoretical basis for therapeutic angiogenesis is the belief that
augmenting the microvascular network in ischemic and watershed
areas of the heart would be beneficial. Clinical as well as
experimental studies provide conclusive evidence that diabetes
impairs ischemia-driven neovascularization (Abaci at al., "Effect
of Diabetes Mellitus on Formation of Coronary Collateral Vessels,"
Circulation 99.2239-42 (1999); Tooke, "Microvasculature in
Diabetes," Cardiovasc Res 32:764-71 (1996); Waltenberger, "Impaired
Collateral Vessel Development in Diabetes: Potential Cellular
Mechanisms and Therapeutic Implications," Cardiovasc Res 49554-60
(2001); Yarom et al., "Human Coronary Microvessels in Diabetes and
Ischaemia. Morphometric Study of Autopsy Material," J Pathol
166:265-70 (1992)). In animal studies, diabetic animals demonstrate
a decreased vascular density following hindlimb ischemia (Rivard et
al., "Rescue of Diabetes-Related Impairment of Angiogenesis By
Intramuscular Gene Therapy With Adeno-VEGF," Am J Pathol 154:355-63
(1999); Taniyama et al., "Therapeutic Angiogenesis Induced By Human
Hepatocyte Growth Factor Gene in Rat Diabetic Hind Limb Ischemia
Model: Molecular Mechanisms of Delayed Angiogenesis in-Diabetes,"
Circulation 104:2344-50 (2001); Schatteman et al., "Blood-Derived
Angioblasts Accelerate Blood-Flow Restoration in Diabetic Mice" J
Clin Invest 106:571-8 (2000)). Human angiographic studies have
demonstrated that diabetic patients have fewer collateral vessels
than non-diabetic controls (Abaci et al., "Effect of Diabetes
Mellitus on Formation of Coronary Collateral Vessels," Circulation
99:2239-42 (1999)). Moreover, revascularization via coronary
angioplasty, coronary artery bypass surgery, or lower extremity
revascularization has a significantly lower success rate in
diabetic patients even in the presence of a patient bypass conduit,
suggesting the existence of a defect at the microcirculatory level
(Kip et al., "Coronary Angioplasty in Diabetic Patients. The
National Heart, Lung, and Blood Institute Percutaneous Transluminal
Coronary Angioplasty Registry," Circulation 94:1.818-25 (1996);
Palumbo et al., "Diabetes Mellitus: Incidence, Prevalence,
Survivorship, and Causes of Death in Rochester, Minn., 1945-1970,"
Diabetes 25:366-73 (1976); Schwartz et al., "Coronary Bypass Graft
Patency in Patients With Diabetes in the Bypass Angioplasty
Revascularization Investigation (BART)," Circulation 10:2652-8
(2002); Kip et al., "Differential Influence of Diabetes Mellitus on
Increased Jeopardized Myocardium After Initial Angioplasty or
Bypass Surgery: Bypass Angioplasty Revascularization
Investigation," Circulation 105:1914-20 (2002)).
TABLE-US-00001 TABLE 1 Published Studies Supporting Impaired
Ischemic Responsiveness in Diabetes Type of Study Study Major
Findings Abaci et al.sup.(a) Clinical Angiographic demonstration of
decreased collaterals in the hearts of diabetic patients Abaci et
al.sup.(b) Clinical Cardiac failure is more Common following an M1
in diabetic patients Altavilla et Experimental Diabetic mice have
less VEGF, less angiogenesis and impaired wound al.sup.(c) healing
compared to normal mice Arora et al.sup.(d) Clinical Diabetics
undergoing lower-extremity bypass maintain an impaired vascular
reactivity even after successful surgical grafting, highlighting
the limits of surgical interventions Bradley et Clinical Diabetic
patients have worse survival after an M1 al.sup.(e) Chou et
al.sup.(f) Experimental First demonstration that myocardial tissue
and cells from diabetic animals express less VEGF and its receptors
Frank et al.sup.(g) Experimental Diabetic mice express much less
VEGF RNA and protein in their wounds Goova et al.sup.(h)
Experimental Blockade of the RAGE receptor accelerated wound
healing, augmented VEGF expression, and increased angiogenesis in
diabetic mice. Guzik et al.sup.(i) Clinical Blood vessels from
diabetic patients produce augmented levels of superoxide, a
marker/cause of oxidative stress Haffner et Clinical Diabetic
patients have a greatly increased incidence of experiencing an M1
al.sup.(j) and dying from an M1 Hiller et al.sup.(k) Clinical
Epidemiologic study suggesting that diabetic microangiopathy is
greatly increased in diabetics Jude et al.sup.(l) Clinical Diabetic
patients have an increased incidence, severity, and poorer outcomes
in peripheral arterial disease of the lower extremities Kip et
al.sup.(m) Clinical Angiographic and epidemiologic study
demonstrating that diabetic patients have more diffuse
atherosclerotic disease, and worm outcomes after seemingly
successful interventional revascularization Lerman et Experimental
First demonstration that cells isolated from diabetic animals and
patients al.sup.(n) produce attenuated levels of VEGF in hypoxia
Marsh et al.sup.(o) Experimental Monocytes from diabetic patients
without retinopathy express less VEGF in hypoxia compared to
monocytes from patients with diabetic retinopathy Partamian et
Clinical Diabetic patients have increased peri-infarct
complications and decreased al.sup.(p) long-term survival Rivard et
al.sup.(q) Experimental Diabetes decreases reactive angiogenesis
and tissue survival following hindlimb ischemia Schatteman
Experimental Angioblasts from diabetic humans show decreased
proliferation and et al.sup.(r) differentiation to mature
endothelial cells in culture. Also, diabetic mice have less
tolerance hindlimb ischemia than nondiabetic mice Tepper et
al.sup.(s) Experimental First demonstration that endothelial
progenitor cells from diabetic patients show decreased function
with assays that measure functions important for angiogenesis Yarom
et al.sup.(t) Clinical Autopsy pathologic study demonstrating that
diabetic patients have decreased ischemia-induced reactive
angiogenesis .sup.(a)Abaci et al., "Effect of Diabetes Mellitus on
Formation of coronary Collateral Vessels," Circulation 99: 2239-42
(1999). .sup.(b)Abbort et al., The Impact of Diabetes on Survival
Following Myocardial Infarction in Men vs Women. Framiogham Study,"
Jama 260: 3456-60 (1988). .sup.(c)Altavilla et at., "Inhibition of
Lipid Peroxidation Restores Impaired Vascular Endothelial Growth
Factor Expression and Stimulates Wound Healing and Angiogenesis in
the Genetically Disbetic Mouse," Diabetes 50: 667-74 (2001).
.sup.(d)Arora et al., "Cutaneous Microcirculation in the
Neuropathic Diabetic Foot Improves: Significantly But Not
Completely After Successful Lower Extremity Revascularization," J
Yasc Surg 35: 501-5 (2002) .sup.(e) Bradley et al., "Survival of
Diabetic Patients After Myocardial Infarction," Am J Med 20:
207-216 (1956). .sup.(f)Chou et al., "Decreased Cardiac Expression
of Vascular Endothelial Growth Factor and its Receptors in
Insulin-Resistant and Diabetic States. A Possible Explanation for
Impaired Collateral Formation in Cardiac Tissue." Circulation 105:
373-9 (2002). .sup.(g)Frank et al., "Regulation of Vascular
Endothelial Growth Factor Expression in Cultured Keratinocytes
Implications for Normal and Impaired Wound Healing," J Biol Chem
270: 12607-13 (1995). .sup.(h)Goova et at., "Blockade of Receptor
for Advanced Glycation End-Products Restores Effective Wound
Healing in Diabetic Mice," Am J Pathol 159: 513-25 (2001).
.sup.(i)Guzik et al., "Mechanisms of Increased Vascular Superoxide
Production in Human Diabetes Mellitus. Role of NAD(P)H Oxidase and
Endothelial Nitric Oxide Synthase," Circulation 105: 1656-62 (2002)
.sup.(j)Haffner et al., "Mortality From Coronary Heart Disease in
Subject With Type 2 Diabetes and in Nondiabetic Subjects With and
Without Prior Myocardial Infarction," N Engl J Med 339: 229-34
(1998). .sup.(k)Hiller et al., "Diabetic Retinopathy and
Cardiovascular Disease in Type II Diabetics. The Framingham Heart
Study and the Framingham Eye Study," Am J Epidemiol 128: 402-9
(1988). .sup.(l)Jude et al., "Peripheral Arterial Disease in
Diabetic and Nondiabetic Patients; a Comparison of Severity and
Outcome," Diabetes Care 24: 1433-7 (2001). .sup.(m)Kip et al.,
"Coronary Angioplasty in Diabetic Patients. The National Heart,
Lung, and Blood Institute Percutaneous Transluminal Coronary
Angioplasty Registry," Circulation 94: 1818-25 (1996)
.sup.(n)Lerman et al., "Cellular Dysfunction in the Diabetic
Fibroblast; Impairment in Migration, Vascular Endtothelial Growth
Factor Production, and Response to Hypoxin," Am JPathol 162: 303-12
(2003). .sup.(o)Marsh et al., "Hypoxic Induction or Vascular
Endothelial Growth Factor is Markedly Decreased in Diabetic
Individuals Who Do Not Develop Retinopathy," Diabetes Care 23:
1375-80 (2000). .sup.(p)Partamian et al., "Acute Myocardiol
Infarction in 258 Cases of Diabetes, Immediate Mortality and
Five-Year Survival." N Engl J Med 273: 455-61 (1965).
.sup.(q)Rivard et al., "Rescue of Diabetes-Related Impairment of
Angiogenesis By Intramuscular Gene Therapy With Adeno-VEGF," Am J
Pathol 154: 355-63 (1999) .sup.(r)Schatteman et al., "Blood-Derived
Angioblasts Accelerate Blood-Flow Restoration in Diabetic Mice," J
Clin Invest 106: 571-8 (2000). .sup.(s)Tepper et al., "Human
Endothelial Progenitor Cells From Type II Diabetics Exhibit
Impaired Proliferation, Adhesion, and Incorporation Into Vascular
Structures," Circulation 106: 2781-6 (2002). .sup.(t)
Despite the preponderance of these observations, the mechanisms
underlying impaired neovascularization in diabetes remain unclear.
Impaired VEGF expression has been implicated as a significant
contributing factor (Rivard et al., "Rescue of Diabetes-Related
Impairment of Angiogenesis By Intramuscular Gene Therapy With
Adeno-VEGF," Am J Pathol 154.355-63 (1999); Schratzberger, et al.,
"Reversal of Experimental Diabetic Neuropathy by VEGF Gene
Transfer," J Clin Invest 107:108392 (2001); Aiello et al., "Role of
Vascular Endothelial Growth Factor in Diabetic Vascular
Complications," Kidney Int Suppl 77:S113-9 (2000)). A detailed
understanding of the mechanism of reduced VEGF expression would
provide a useful framework for new approaches to improve diabetic
outcomes following ischemic events.
Ischemia-Induced Neovascularization Occurs by Two Mechanisms:
Angiogenesis and Vasculogenesis.
[0008] After the appropriate hypoxic signaling cascade is
initiated, compensatory vascular growth in response to ischemic
insult occurs by two different mechanisms (FIG. 1). In
angiogenesis, mature resident endothelial cells proliferate and
sprout new vessels from an existing vessel in response to an
angiogenic stimulus. In a more recently described mechanism, termed
vasculogenesis, circulating cells with characteristics of vascular
stem cells (endothelial progenitor cells, or EPCs) are mobilized
from the bone marrow in response to an ischemic event, and then
home specifically to ischemic vascular beds and contribute to
neovascularization (Asahara et al., "Isolation of Putative
Progenitor Endothelial Cells for Angiogenesis," Science 275:964-7
(1997); Shi et al., "Evidence for Circulating Bone Marrow-Derived
Endothelial Cells" Blood 92:362.7 (1998); Asahara at al., "Bone
Marrow Origin of Endothelial Progenitor Cells Responsible for
Postnatal Vasculogenesis in Physiological and Pathological
Neovascularization," Circ Res 85:2214 (1999); Isner et al.,
"Angiogenesis and Vasculogenesis as Therapeutic Strategies for
Postnatal Neovascularization," J Clin Invest 103:1231-6 (1999);
Crosby et al., "Endothelial Cells of Hematopoietic Origin Make a
Significant Contribution to Adult Blood Vessel Formation," Circ Res
87:728-30 (2000); Pelosi et al., "Identification of the
Hemangioblast in Postnatal Life," Blood 100:3203-8 (2002)).
Hypoxia-Inducible Factor-4 (HIF-1) is the Central Mediator of the
Hypoxia Response Including Subsequent Blood Vessel Growth.
[0009] The observation that ischemia regulates blood vessel growth
has been known for many years, yet the responsible factor eluded
identification until 1992, when Semenza and colleagues described a
hypoxia-responsive transcription factor (HIF-1) which mediates
erythropoietin gene upregulation (Semenza at al., "A Nuclear Factor
Induced by Hypoxia via de Novo Protein Synthesis Binds to the Human
Erythropoietin Gene Enhancer at a Site Required for Transcriptional
Activation," Mol Cell Biol 12:5447-54 (1992); Semenza at al.,
"Hypoxia-Inducible Nuclear Factors Bind to an Enhancer Element
Located 3' to the Human Erythropoietin Gene," Proc Natl Acad Sci
USA 88:5680-4 (1991)). HIF-1 proved to be a novel transcription
factor conserved in all metazoan phyla and is ubiquitously present
in all cells examined thus far (Carmeliet et al., "Abnormal Blood
Vessel Development and Lethality in Embryos Lacking a Single VEGF
Allele," Nature 380:435-9 (1996)). Evidence for its involvement in
angiogenesis stemmed from the initial observation that VEGF was
strongly upregulated by hypoxic conditions (Shweiki et al.,
"Vascular Endothelial Growth Factor Induced by Hypoxia May Mediate
Hypoxia-Initiated Angiogenesis," Nature 359:843-5 (1992)). Soon
thereafter, HIF-1 was shown to be the transcription factor
responsible for VEGF upregulation by hypoxia and hypoglycemia
(Forsythe et al., "Activation of Vascular Endothelial Growth
Factor, Gene Transcription by Hypoxia-Inducible Factor 1," Mol Cell
Biol 16:4604-13 (1996)). It is now clear that HIF-regulated VEGF
expression is essential for vascular development during both
embryogenesis and postnatal neovascularization in physiologic and
pathologic states (Carmeliet et al., "Abnormal Blood Vessel
Development and Lethality in Embryos Lacking a Single VEGF Allele,"
Nature 380:435.9 (1996); Carmeliet at al., "Abnormal Blood Vessel
Development and Lethality in Embryos Lacking a Single VEGF Allele,"
Nature 380:435-9 (1996); Iyer et al., "Cellular and Developmental
Control of O2 Homeostasis by Hypoxia-Inducible Factor I Alpha,"
Genes Dev 12:149-62 (1998)). HIF-1 consists of the oxygen-regulated
HIF-1.alpha. subunit and the HIF-1.beta. subunit, which is not
regulated by oxygen. HIF-1 is now believed to be the master
transcription factor directing the physiologic response to hypoxia
by upregulating pathways essential for adaptation to ischemia,
including angiogenesis, vasculogenesis, erythropoiesis and glucose
metabolism (FIG. 2).
Regulation of HIF-1.alpha. Transcriptional Activation.
[0010] The HIF-1 transcriptional complex is comprised of
HIF-1.alpha./.beta. and more than seven other factors that modulate
gene transcription. The two predominant functional components of
this complex are HIF-1.alpha. and CBP/p300, which directly interact
to transactivate gene expression. HIF-1.alpha. function is
predominantly regulated by oxygen via protein stabilization and
post-translational modification. Recent reports demonstrate that
HIF-1.alpha. is activated by phosphorylation in vitro, enhancing
HIF-mediated gene expression (Richard et al., "p42/p44
Mitogen-Activated Protein Kinases Phosphorylate Hypoxia-Inducible
Factor I alpha (HIF-1 alpha) and Enhance the Transcriptional
activity of HIF-1," J Biol Chem 274:32631-7 (1999)). Whether this
modification results in a direct stimulation of the transactivation
function of HIF-1.alpha. itself or facilitates recruitment of
co-activators is not clear (Richard t al, "p42/p44
Mitogen-Activated Protein Kinases Phosphorylate Hypoxia-Inducible
Factor I alpha (HIF-1 alpha) and Enhance the Transcriptional
activity of HIF-1," J Biol Chem 274:32631-7 (1999); Sang et al.,
"Signaling Up-Regulates the Activity of Hypoxia-Inducible Factors
by Its Effects on p300," J Biol Chem 278:14013-9 (2003)).
[0011] It has also been recently demonstrated that CBP/p300 also
undergoes phosphorylation in vitro, enhancing its ability to
function as a transcriptional activator in association with
HIF-1.alpha. (Sang et al., "Signaling Up-Regulates the Activity of
Hypoxia-Inducible Factors by Its Effects on p300," J Biol Chem
278:14013-9 (2003)). Thus, cellular states that promote
phosphorylation of these two factors likely increase
hypoxia-induced gene expression, while those that favor
dephosphorylation have the opposite effect. Although HIF-1 mediated
gene expression is essential for both angiogenesis and
vasculogenesis, the role of its regulation in diabetic states has
not been previously examined.
Both Angiogenesis and Vasculogenesis are Modulated by VEGF.
[0012] It is well known that angiogenesis is mediated by VEGF and
this mechanism has been extensively investigated (Ferrara et al.,
"The Biology of VEGF and its Receptors," Nat Med 9:669-76 (2003)).
Recently, VEGF has also been implicated in regulation of
vasculogenesis (FIG. 2). Ischemia is a potent mobilizer of
endothelial progenitor cells from the bone marrow. This appears to
be mediated through VEGF signaling, as EPCs express both VEGF
receptor I and 2 on their cell surface (Asahara et al., "VEGF
Contributes to Postnatal Neovascularization by Mobilizing Bone
Marrow-Derived Endothelial Progenitor Cells," Embo J 18:3964-72
(1999); Takahashi et al., "Ischemia- and Cytokine-Induced
Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells
for Neovascularization," Nat Med 5:434-8 (1999); Kalka et al.,
"Vascular Endothelial Growth Factor(165) Gene Transfer Augments
Circulating Endothelial Progenitor Cells in Human Subjects," Circ
Res 86:1198-202 (2000); Gill at al., "Vascular Trauma Induces Rapid
but Transient Mobilization of VEGFR2(+)AC133(+) Endothelial
Precursor Cells," Circ Res 88:167-74 (2001); Hattori et al.,
"Vascular Endothelial Growth Factor and Angiopoietin-1 Stimulate
Postnatal Hematopoiesis by Recruitment of Vasculogenic and
Hematopoietic Stem Cells," J Exp Med 193:1005-14 (2001)). Given
that VEGF production is impaired in diabetes mellitus, it seems
likely that various aspects of vasculogenesis, including EPC
mobilization, may also be impaired. Indeed, recent evidence has
demonstrated that the incorporation of these vascular progenitors
into blood vessels is decreased in diabetic states.
VEGF Expression May be Regulated in a Tissue-Specific Manner.
[0013] It also clear that various tissues and organs in diabetic
patients exhibit different pathologies. The retina is often
characterized by excessive angiogenesis, while skin, muscle, and
nerves in diabetic patients suffer from a paucity of new vessel
formation. Similarly, diabetic retinopathy has been characterized
by increased levels of ocular VEGF levels, (Aiello et al.,
"Vascular Endothelial Growth Factor in Ocular Fluid of Patients
with Diabetic Retinopathy and Other Retinal Disorders," N Engl J
Med 331:1480-7 (1994); Adamis t al., "Increased Vascular
Endothelial Growth Factor Levels in the Vitreous of Eyes with
Proliferative Diabetic Retinopathy," Am J Ophthalmol 118:445-50
(1994)), while impaired wound healing has been characterized by
severely decreased levels of VEGF (Frank et al., "Regulation of
Vascular Endothelial Growth Factor Expression in Cultured
Keratinocytes. Implications for Normal and Impaired Wound Healing,"
J Biol Chem 270:12607-13 (1995); Peters et al., "Vascular
Endothelial Growth Factor Receptor Expression During Embryogenesis
and Tissue Repair Suggests a Role in Endothelial Differentiation
and Blood Vessel Growth," Proc Natl Acad Sci USA 90:8915-9 (1993);
Silhi, N., "Diabetes and Wound Healing," J Wound Care 7:47-51
(1998); Brown, L. F., "Expression of Vascular Permeability Factor
(Vascular Endothelial Growth Factor) by Epidermal Keratinocytes
During Wound Healing," J Exp Med 176:1375-9 (1992); Nissen et al.,
"Vascular Endothelial Growth Factor Mediates Angiogenic Activity
During the Proliferative Phase of Wound Healing." Am J Pathol
152:1445-52 (1998)). This so-called "diabetic paradox," by which
the diabetic phenotype exhibits both excessive and impaired new
blood vessel formation in different tissues, leads to different
types of complications. It is believed this phenomenon represents a
cell- and tissue-specific difference in the transcriptional
regulation of VEGF.
Hyperglycemia Results in Specific Impairments of Cellular Function
Through Overproduction of Reactive Oxygen Species: a Potential Link
to VEGF.
[0014] The cellular mechanism that accounts for impaired
hypoxia-induced VEGF and SDF-1 expression has not yet been
determined. Recently, the biochemical basis for
hyperglycemia-induced cellular damage was described, demonstrating
that many of the effects of high glucose are mediated through four
specific cellular pathways (FIG. 3) (Brownlee, "Biochemistry and
Molecular Cell Biology of Diabetic Complications," Nature
414:813-20 (2001); Nishikawa et al. "Normalizing Mitochondrial
Superoxide Production Blocks Three Pathways of Hyperglycaemic
Damage," Nature 404:787-90 (2000)). Intracellular elevations in
glucose increase flux of metabolites through glycolysis and the
Kreb's cycle, resulting in overproduction of ROS by the
mitochondria. Overproduction of ROS inhibits GAPDH activity,
resulting in accumulation of early glucose metabolites in the
initial phases of glycolysis. The abundance of these metabolites
and their inability to progress through glycolysis causes shunting
of these intermediates into alternative pathways of glucose
utilization (polyol pathway, hexosamine pathway, protein kinase C
pathway, and AGE pathway, FIG. 3). Accumulation of end products in
each of these pathways leads to specific changes in cellular
function, including gene expression (Nissen et al., "Vascular
Endothelial Growth Factor Mediates Angiogenic Activity During the
Proliferative Phase of Wound Healing" Am J Pathol 152:1445-52
(1998)), and are implicated in the pathophysiology of diabetic
complications (Brownlee, "Biochemistry and Molecular Cell Biology
of Diabetic Complications," Nature 414:813-20 (2001)). Indeed,
specific blockade of one, several, or all of these pathways has
been shown to prevent diabetic complications in an animal model,
including those complications that result from ischemic injury
(Hammes at al., "Benfotiamine Blocks Three Major Pathways of
Hyperglycemic Damage and Prevents Experimental Diabetic
Retinopathy," Nat Med 9:294-9 (2003); Obrosova et al., "Aldose
Reductase Inhibitor Fidarestat Prevents Retinal Oxidative Stress
and Vascular Endothelial Growth Factor Overexpression in
Streptozotocin-Diabetic Rats," Diabetes 52:864-71 (2003)).
[0015] Hyperglycemia-induced reactive oxygen species also impair
the ability of HIF-1.alpha. to mediate appropriate upregulation of
VEGF and the chemokine SDF-1 that are required for
neovascularization in ischemic settings. This impairment also
affects hypoxia-specific functions of vascular effector cells. This
results in impaired angiogenesis, vasculogenesis, and diminished
tissue survival in diabetic states. Increased free fatty acid flux
has been shown to increase ROS by identical mechanisms (Du t al.,
"Insulin Resistance Causes Proatherogenic Changes in Arterial
Endothelium by Increasing Fatty Acid Oxidation-Induced Superoxide
Production" J. Clin. Invest. in press).
[0016] The present invention is directed to treating or preventing
the pathologic sequelae of acute hyperglycemia and/or increased
fatty acid flux in a subject, thus, preventing metabolite-induced
reactive oxygen-species mediated injury.
SUMMARY OF THE INVENTION
[0017] One aspect of the present invention relates to a method of
treating or preventing pathologic sequelae of acute hyperglycemia
and/or increased fatty acid flux in non-diabetic subjects,
metabolic syndrome/insulin resistance subjects, impaired fasting
glucose subjects, impaired glucose tolerance subjects, and diabetic
subjects. This method involves administering an ROS inhibitor to
the subject under conditions effective to treat or prevent
pathologic sequelae of acute hyperglycemia and/or increased fatty
acid flux in the subject.
[0018] Another aspect of the present invention relates to a method
of promoting neovascularization in a subject prone to hyperglycemia
or increased fatty acid flux. This method involves administering an
ROS inhibitor to the subject under conditions effective to promote
neovascularization in the subject.
[0019] A further aspect of the present invention pertains to a
method of inhibiting oxidation or excessive release of free fatty
acids in a subject. This method involves administering to the
subject certain compounds under conditions effective to inhibit
excessive release of free fatty acids in the subject. These
compounds include thiazolidinedione nicotinic acid, etomoxir, and
ranolazine.
[0020] A further aspect of the present invention is directed to a
method of identifying compounds suitable for treatment or
prevention of ROS-mediated injury. This method involves providing a
diabetic animal model and inducing diabetes in the animal model. A
compound to be tested is then administered to the animal model.
Compounds which achieve recovery of local oxygen tension, blood
flow, increase in vessel density, and tissue survival in the animal
model as therapeutic candidates for treating or preventing
ROS-mediated injury are then recovered.
[0021] The present invention provides a means of restoring
deficient angiogenesis in response to ischemia in patients with
disorders of glucose and fatty acid metabolism. This would
drastically reduce the rate of lower limb amputation, and reduce
the extent of cardiac and brain damage due to heart attacks and
strokes. In addition, it would result in healing of intractable
diabetic foot ulcers, a major clinical problem for which there is
currently no available effective medical treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows a schematic of angiogenesis and
vasculogenesis.
[0023] FIG. 2 shows the central role of HIF and VEGF in the
ischemic response.
[0024] FIG. 3 shows pathways of hyperglycemic damage.
[0025] FIG. 4 shows an overall experimental plan.
[0026] FIG. 5 shows a murine model of graded cutaneous ischemia.
Regions A, B, and C reflect increasingly ischemic tissue regions,
as measured by direct tissue oxygen tension at reference points p1
(27 mm Hg)-p5 (6 mm Hg).
[0027] FIG. 6 shows tissue survival in diabetic mice.
[0028] FIG. 7 shows oxygen tension measurements in ischemic tissue
from least ischemic (p1) to most ischemic (p5) compared to normal
skin oxygen tension (NL Skin).
[0029] FIG. 8 shows the number of blood vessels identified by CD31
staining in areas A, B, and C of ischemic flaps.
[0030] FIG. 9 shows oxygen tension measurements post-operatively in
wild type and MnSOD transgenic mice with streptozotocin-induced
diabetes.
[0031] FIG. 10 shows JC-1 staining of C2Cl2 myoblasts cultivated in
normal glucose (5 mM), as well as acute and chronic high glucose
(25 mM).
[0032] FIG. 11 shows VEGF mRNA in high and low glucose in response
to hypoxia.
[0033] FIG. 12 shows VEGF mRNA half life in cells cultivated in
normal glucose (.circle-solid.) or high glucose (.smallcircle.)
conditions.
[0034] FIG. 13 shows VEGF promoter activity in C2Cl2 myoblasts in
normal (5 mM, grey) and high glucose (25 mM, black) after hypoxic
stimulus.
[0035] FIG. 14 shows HIF-Id is preferentially glycosylated in high
glucose conditions (HG, 25 mM) compared to normal glucose (NG, 5
mM).
[0036] FIGS. 15 A-B show high glucose (30 mM) impairs the
association between p300 and PPAR.gamma.. This effect was abolished
by inhibiting GFAT.
[0037] FIGS. 16A-C show pathways of cellular damage resulting from
reactive oxygen species can be selectively targeted and
prevented.
[0038] FIGS. 17A-B show fibroblasts from diabetic mice do not
demonstrate a normal hypoxia-induced increase in migration seen in
non-diabetic cells (p<0.05).
[0039] FIGS. 18A-B show fibroblasts from diabetic mice produce more
proMMP-9, but not active MMP-9 (p<0.001).
[0040] FIGS. 19A-D show EPCs from Type II diabetics proliferate
less during expansion (A) which inversely correlated with HbA1c
levels (B). Fewer EPC clusters formed in culture (C), which was
also inversely correlated to the total number of years with
diabetes.
[0041] FIGS. 20A-C show EPCs from Type II diabetic patients are
impaired in their ability to adhere, migrate, and proliferate in
response to hypoxic stimuli (&=p<0.001,
&&=p<0.05).
[0042] FIG. 21 shows the effect of deferoxamine on
hyperglycemia-induced ROS.
[0043] FIG. 22 shows the free intracellular iron measurement in
bovine aortic endothelial cells after infection with UCP-1, Mn-SOD
or empty adenoviral vectors and subsequent treatment with 5 mM or
30 mM glucose. The x-axis shows the different treatments. The
y-axis shows fluorescence units indicating the amount of free
iron.
[0044] FIGS. 23A-C show the free intracellular iron measurement in
bovine aortic endothelial cells after incubation with 5 mM or 30 mM
glucose (FIGS. 23A and 238, respectively) or 30 mM glucose plus 100
.mu.M deferoxamine (FIG. 23C) for 24 hours. Detection of free iron
was accomplished by visualizing the fluorescent marker fura-2
AM.
[0045] FIGS. 24A-D show, respectively, DNA strand breakage in
aortic endothelial cells after incubation with 5 mM or 30 mM
glucose or 30 mM glucose plus 100 .mu.M deferoxamine for 7
days.
[0046] FIG. 25 shows PARP activity in aortic endothelial cells
after incubation with 5 mM or 30 mM glucose, 30 mM glucose plus 100
.mu.M deferoxamine, or 30 mM glucose plus 100 .mu.M DMSO for 6
days. .sup.3H NAD incorporation was used to assess PARP activity.
The x-axis shows the different treatments. The y-axis shows the
PARP activity as measured in pmol/mg protein.
[0047] FIG. 26 shows prostacyclin synthase activity in aortic
endothelial cells after 24 hour incubation with 5 mM or 30 mM
glucose, or 30 mM glucose plus 100 .mu.M deferoxamine. The x-axis
shows the different treatments. The y-axis shows the prostacyclin
synthase activity expressed as concentration of the prostacyclin
synthase product PGF-1.alpha..
[0048] FIG. 27 shows prostacyclin synthase activity in aortas of
diabetic and control mice after daily deferoxamine injections for 7
days. The x-axis shows the different treatments. The y-axis shows
the prostacyclin synthase activity as measured by the concentration
of the prostacyclin synthase product PGF-1.alpha..
[0049] FIG. 28 shows eNOS activity in aortic endothelial cells
after incubation with 5 mM or 30 mM glucose or 30 mM glucose plus
100 .mu.M deferoxamine for 24 hours. The x-axis shows the different
treatments. The y-axis shows the eNOS activity as a function of
.sup.3H-citrulline generated per minute per 10.sup.5 cells.
[0050] FIG. 29 shows eNOS activity in aortas of diabetic and
control mice after daily deferoxamine injections for 7 days. The
x-axis shows the different treatments. The y-axis shows the eNOS
activity as a function of .sup.3H-citrulline generated per minute
per mg of protein.
[0051] FIGS. 30A-F show a diabetes-induced defect in mouse
angiogenic response to ischemia. FIGS. 30A-B show oxygenation
levels in non-diabetic and diabetic mice, respectively. P1-P4 on
the y-axis designate adjacent quadrants of the ischemic skin flap
starting closest to the site of attachment to the animal, i.e. P1,
and proceeding distally to P4. FIGS. 30C-D show mobilization of
bone marrow-derived endothelial cells in response to ischemia.
Flk-1 on the y-axis is a marker for ischemic bone-marrow-derived
endothelial precursor cells. CDI lb on the x-axis is a general
marker for bone marrow-derived endothelial precursor cells. FIGS.
30E-F show the amount of capillary formation in non-diabetic and
diabetic mice, respectively. NI on the y-axis represents capillary
density of a non-ischemic control. Area C on the y-axis represents
the capillary density in an ischemic skin flap after 7 days.
[0052] FIGS. 31A-C show that deferoxamine treatment corrects the
diabetes-induced defect in mouse angiogenic response to ischemia.
The treatment groups were wild-type mice (WT),
streptozotocin-induced diabetic mice (shown as STZ in FIG. 31 C,
and DM in FIGS. 31 A-B), and deferoxamine-treated
streptozotocin-induced diabetic mice (shown as STZ+deferox in FIG.
31 C, and DM+DEF in FIGS. 31A-B).
[0053] FIG. 32 shows CD 31 positive blood vessel counts in
wild-type mice (WT C), wild-type mice treated with deferoxamine (WT
Def C), streptozotocin-induced diabetic mice (STZ C), and
streptozotocin-induced diabetic mice treated with deferoxamine (STZ
Def C). The y-axis shows the CD 31 positive blood vessel counts per
hpf (high powered field).
[0054] FIGS. 33A-B show that deferoxamine treatment corrects the
diabetes-induced defect in mouse angiogenic response to ischemia.
Diabetes was induced in the mice by streptozotocin (abbreviated STZ
in FIG. 33 A-B). The mouse shown in FIG. 33A was treated with
vehicle alone, while the diabetic mouse shown in FIG. 33B was
treated with deferoxamine (labeled as STZ Deferoxamine in FIG. 33
B).
[0055] FIGS. 34 A,C, and E show that diabetes-induces defects in
mouse angiogenic response to ischemia. FIGS. 34B,D, and F that
these defects are corrected by treatment with deferoxamine. FIGS.
34 A-B show oxygenation levels in streptozotocin-induced diabetic
(FIG. 33 A) and deferoxamine-treated streptozotocin-induced
diabetic mice (FIG. 33 B) respectively. P1-P4 on the y-axis
designate adjacent quadrants of the ischemic flap starting closest
to the site of attachment to the animal, i.e., P1, and proceeding
distally to P4. FIGS. 34 C-D show mobilization of bone marrow
derived endothelial cells in response to ischemia. Flk-1 on the
y-axis is a marker for bone-marrow derived endothelial precursor
cells. CD11b on the x axis is a general marker for bone-marrow
derived cells of the myeloid, macrophage, and granulocytic lines.
FIGS. 34 E-F show the amount of capillary formation in
vehicle-treated streptozotocin-induced diabetic and
deferoxamine-treated streptozotocin-induced diabetic mice
respectively. NI on the y-axis represents capillary density of a
non-ischemic control. Area C on the y-axis represents the capillary
density at the most distal third of an ischemic skin flap alter 7
days.
[0056] FIG. 35 is a bar chart showing EPC mobilization for control,
STZ, and STZ-deferoxamine mice. EPC mobilization was determined at
day 7 postischemic insult. Diabetes resulted in a 3-fold decrease
in EPC mobilization. Deferoxamine restores ischemia specific EPC
mobilization as compared to untreated STZ mice (p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0057] One aspect of the present invention relates to a method of
treating or preventing pathologic effects of acute increases in
hyperglycemia and/or acute increases of fatty acid flux in
non-diabetic subjects, metabolic syndrome/insulin resistance
subjects, impaired fasting glucose subjects, impaired glucose
tolerance subjects, and diabetic subjects. This method involves
administering an ROS inhibitor to the subject under conditions
effective to treat or prevent pathologic effects of acute increases
in hyperglycemia and/or acute increases in fatty acid flux in the
subject.
[0058] As noted above, in this aspect of the present invention, the
claimed method can be applied to non-diabetic subjects, metabolic
syndrome/Insulin resistance subjects, impaired fasting glucose
subjects, impaired glucose tolerance subjects, and diabetic
subjects. In each case, the subject has a base line level of
hyperglycemia and/or fatty acid flux. The present invention is
directed to the prevention or treatment of pathologic conditions in
subjects whose base line levels of hyperglycemia and/or fatty acid
flux undergo a rapid and relatively short-term (i.e. acute)
increase.
[0059] Subjects where acute increases in hyperglycemia and/or acute
increases of fatty acid flux take place may be suffering from any
of the following conditions: diabetes-specific microvascular
pathology in the retina (i.e. diabetic retinopathy), renal
glomerulus (i.e. diabetic nephropathy), peripheral nerve (i.e.
diabetic neuropathy), accelerated atherosclerotic macrovascular
disease affecting arteries that supply the heart, brain, and lower
extremities (i.e. diabetic macrovasular disease), or nonalcoholic
fatty liver disease ("NAFLD") which includes a wide spectrum of
liver injury ranging from simple steatosis to steatohepatitis
("NASH"), fibrosis, and cirrhosis. The pathologic effect of acute
increases in hyperglycemia and/or acute increases of fatty acid
flux may also be prevented or treated where the subject has a
critical care illness, an acute myocardial infarction, an acute
stroke, or who has undergone arterial bypass or general
surgery.
[0060] Acute increases in hyperglycemia and/or acute increases of
fatty acid flux impairs mobilization of vascular endothelial cell
precursors from the bone marrow. This may take the form of
impairing mobilization of vascular endothelial cell precursors from
the bone marrow, impairing HIF-1.alpha.- and SDF-1-mediated
upregulation of vascular endothelial growth fact, and/or
ROS-mediated injury which inhibits neovascularization. The subject
can also have an ischemic condition which includes coronary artery
disease, peripheral vascular disease, cerebral vascular disease,
non-healing foot ulcers, or a wound (acute or chronic).
[0061] The ROS generation by hyperglycemia or increased fatty acid
flux takes place in the mitochondria. The most common ROS are
hydrogen peroxide (H.sub.2O.sub.2), hydroxyl radicals (OH.), lipid
peroxy radicals (LOO.sup.-), and peroxynitrites (ONOO.sup.-).
[0062] H.sub.2O.sub.2 is relatively stable and can diffuse through
membranes. In most cells, H.sub.2O.sub.2 is detoxified by enzymatic
reduction to H.sub.2O and O.sub.2. In mitochondria, the enzyme
glutathione peroxidase is primarily responsible for this reaction.
In the cytosol and peroxisomes, both glutathione peroxidase and the
enzyme catalyse mediate this reaction. However, in the presence of
free d-block transition metals, such as iron, the oxidized form of
the metal is thought to react with superoxide, producing the
oxidized from of the metal and molecular oxygen (O.sub.2). The
reduced metal then reacts with H.sub.2O.sub.2 to regenerate the
initial oxidized metal, hydroxyl ions (OH.sup.+) and hydroxyl
radicals (OH.). It is important to note, however, that this
chemistry is still far from being understood.
[0063] It is well known that iron and other d-block transition
metals can function as free-radical catalysts, potentially
generating toxic species such as hydroxyl radicals. Transition
metals are the large block of elements in the Periodic Chart that
have group numerical designations ending with B, such as IB, IIB,
IIIB, and so on. They are the four rows of ten elements located in
the heart of the chart. They are also the elements whose final
electron enter the d orbital (called d-block metals). All first row
d-block metals (except zinc) have unpaired electrons (Sc, Ti, V,
Cr, Mn, fe, Co, Ni, and Cu) which removes spin restrictions and
allows then to function in free radical catalysis, both as elements
bound at the active site of enzymes, and free in solution.
[0064] Iron has been the focus of many chemical studies because
this chemistry was first demonstrated by H. J. H. Fenton in 1876
using unchelated Fe.sup.2+/H.sub.2O.sub.2 mixture in aqueous
solution. To distinguish between the iron (II) and iron (III)
combinations, the convention is to use Fenton-like reagent for the
Fe.sup.3+/H.sub.2O.sub.2 mixture and restrict the use of Fenton's
reagent to denote the Fe.sup.2+/H.sub.2O.sub.2 The Fenton-like
reagent is also capable of oxidizing organic substrates, but it is
somewhat less reactive than Fenton's reagent. As iron(III) can be
produced in applications of Fenton's reagent, Fenton chemistry and
Fenton-like chemistry often occur simultaneously.
[0065] Fenton reagent chemistry is still far from being fully
understood, and Fenton-like reagent chemistry even less well
understood. Numerous reaction mechanisms have been proposed for
Fenton reagent chemistry based on different active intermediates
such as OH. and OOH. radicals and high-valent iron species. Haber
and Weiss's OH. radical mechanisms (citation) is probably the most
popular candidate for the Fenton reaction:
Fe.sup.2++H.sub.2O.sub.2.fwdarw.Fe.sup.3++OH.sup.-+OH.sup.-
[0066] A popular alternative mechanistic candidate is that first
suggested by Bray and Gorin (citation), in which the ferryl ion,
[Fe.sup.IV O].sup.2+, is supposed to be the active
intermediate:
Fe.sup.2+H.sub.2O.sub.2.fwdarw.[Fe.sup.IVO].sup.2++H.sub.2O
[0067] In addition to reducing hydrogen peroxide, ferrous iron can
also react with alkyl hydroperoxides, to produce alkoxyl radicals.
These alkoxyl radicals can then initiate the oxidation of
polyunsaturated lipids by a flee radical chain reaction
(citation).
[0068] Despite its appearance in numerous biochemistry textbooks,
the biological significance of Fenton chemistry has been questioned
by many free radical chemists in part because the rate constants
for the reaction of reduced metals and their complexes with
H.sub.2O.sub.2 are not rapid, and their in vivo metal ligands are
unknown (citation). However, it has been shown recently that
HCO.sub.3.sup.- and CO.sub.2 greatly accelerate the rate of
H.sub.2O.sub.2 reduction by Mn.sup.2+ and other transition metals
(citation). It is probable that many of the proposed mechanisms
compete with each other in complex and unpredictable ways,
depending on the reaction conditions, such as the metal ligands,
their valence, the solvent, the pH and the organic substrate to be
oxidized (citation)
[0069] The ROS inhibitor can be alpha lipoic acid, a superoxide
dismutase mimetic, or a catalase mimetic. The superoxide dismutase
mimetic or the catalase mimetic can be MnTBAP
(Mn(III)tetrakis(4-benzoic acid)porphyrin chloride)(produced by
Calbiochem), ZnTBAP (Zn(III)tetrakis(4-benzoic acid)porphyrin
chloride), SC-55858 (manganese (11) dichloro
(2R,3R,8R,9R-bis-cyclohexano-1,4,7,10,13-pentaazacyclopentadecane)]
Euk-134 (3,3'-methoxysalenMn(III)) (produced by Eukarion).
[0070] Alternatively, the ROS inhibitor can be an iron chelator or
a composition comprising a mixture of iron chelators. Chelators are
small molecules that bind very tightly to metal ions. The key
property shared by all chelators is that the reactivity of the
metal ion bound to the chelator is greatly reduced, although in
some cases and under certain conditions, chelator metal-complexes
themselves can generate reactive oxygen free radicals. Clinically
useful chelators must be highly specific for one d-block transition
metal such as iron. Chelators which are non-specific are highly
toxic.
[0071] The basic property of a chelator consists in having the
ability of forming a heterocyclic ring structure with a metal ion
as the closing member. The chelator must possess two or more
functional groups (ligands) with atoms which can donate a pair of
electrons for the formation of a bond with the metal ion. Donor
atoms are usually N, O and S, which can function either as members
of an acidic group such as: --COOH, OH (phenolic, enolic), --SH,
--NH.dbd.O, --NOH in which case the proton is displaced by the
metal ion, or as lone pair of electron donors (Lewis base) such as
--C.dbd.O, --NH.sub.2, --O--R, --OH (alcoholic), --S-thioether, as
described in Current Medicinal Chemistry, 2004, 11, 2161-2183,
incorporated herein by reference in its entirety.
[0072] Ideally, tight binding of iron to a chelator should
completely inhibit its ability to function as a free-radical
catalyst. Iron chelators are classified according to the
stoichiometry of binding with iron. Iron ions have six
electrochemical coordination sites. Thus, a chelator molecule that
binds to all six sites in a 1:1 ratio is called `hexidentate." A
chelator molecule that binds to only two of the six sites is called
"bidentate,` and chelators that bind to three of the six sites are
called "tridentate.` In theory, three molecules of a bidentate
chelator should reduce free iron reactivity as completely as one
molecule of a hexidentate chelator. However, with bidentate iron
chelators, formation of fee-radical catalyzing partial reduction
products often occurs. Practically, this means that a large
chemical excess of such chelators is needed in order to avoid the
formation of these reactive chelator iron complexes.
[0073] Iron chelators can be classified using a number of criteria
such as their origin (synthetic versus biologically produced
molecules), their interaction with solvents such as water
(hydrophobic versus hydrophilic) or their stoichiometric
interaction (bidentate versus hexadentate).
[0074] A general structure of an effective iron chelator comprises
the generic structure R-L-C-M and all the combinations thereof.
[0075] For example, C can represent the iron-chelating moiety bi,
tri or hexidentate characterized by selective iron-binding affinity
and avidity as described in Current Medicinal Chemistry, 2004, 11,
2161-2183. The combination C-M can represent a bi-functional drug
structure containing an iron-chelating moiety C bound to a masking
group M which could be an electron-donor atom. Intracellular
hydroxyl radicals OH. can be reduced by the electrons of M, cleave
M from C which, once unmasked, can bind free iron ions.
[0076] The combination R--C can represent the iron chelating moiety
C bound to a back bone side-chain R, wherein R can be H, a linear
aliphatic chain structure, or an alifatic chain including aromatic,
alifatic and/or heteroaromatic rings. Because the relative potency
of chelators appears to be related to the hydrophilicity of the
molecule, the chemical structure of R can facilitate or hinder the
penetration of the chelators in target cells and/or target cell
compartments. Finally, the combination R-L-C can represent the iron
chelating moiety C bound to a side-chain R through a linker L. The
linker L can facilitate a rapid cellular intake and delay the
cellular exit of C as described in J. Am. Chem. Soc. 2002, 124,
12666-12667, incorporated herein by reference in its entirety. For
example, R-L-C can represent a prohydrophilic drug (a pro-drug). L
can be an ester bond, R an ester moiety and C an iron chelating
moiety. Upon entrance in the cell, R-L-C can turn highly
hydrophilic upon esterase-mediated hydrolysis of the lipophilic
moiety R. Thus, L is hydrolyzed, R is chemically detached from the
molecule and the more hydrophilic C is retained inside the cell
where it can perform its chelating function.
[0077] Other general structures of effective iron chelators
comprise the family of 3,5-diphenyl-1,2,4-triazoles of the formula
I described in U.S. Pat. No. 6,465,504 incorporated herein by
reference in its entirety.
[0078] Of the iron chelators, deferoxamine or DFO may be the most
important, because it is FDA-approved for treatment of iron excess
in thallasemia.
[0079] When deferoxamine is employed, a patient (e.g., a patient
with an acute myocardial infarction) can be treated with
intramuscular injections of 1,000 to 10,000 mg of deferoxamine or
with intravenous injections of 100 to 10,000 mg of deferoxamine.
Such patients can be treated within 24 hours of symptoms by
intravenous injection of deferoxamine in liquid form at a
concentration between 100 to 10,000 mg/liter of deferoxamine.
Deferoxamine can also be administered together with DFP, ICL-670, a
poly (ADP-ribose) polymerase inhibitor, and a glucagon-like
peptide-1 fragment that prevents hyperglycemia-induced ROS
production, for example, GLP-1 (9-36 amide), and GLP-1 9-37).
Alternatively, deferoxamine can be administered together with a
poly (ADP-ribose) polymerase inhibitor including, but not limited
to, nicotinamide, 3-aminobenzamide, P134
(N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N N-dimethylacetamide),
and mixtures thereof.
[0080] While deferoxamine can provide life-saving treatment for
patients in iron overload situations, numerous deferoxamine
derivatives can also be employed. Aliphatic, aromatic, succinic,
and methylsulphonic analogs of DFO have been synthesized to enhance
the lipophilicity of DFO (Ihnat et al., "Solution Equilibria of
Deferoxamine Amides," J. Pharm Sci. 91:1733-1741 (2002), which is
hereby incorporated by reference in its entirety). Specifically,
these derivatives include formamide-deferoxamine,
acetamide-deferoxamine, propylamide deferoxamine,
butylamide-deferoxamine, benzoylamide-deferoxamine
succinamide-deferoxamine, and methylsulfonamide-deferoxamine.
Hydroxylethyl starch (HES)-deferoxamine has been synthesized which
was shown to have a greater plasma half-life than deferoxamine
(Pedchenko et al., "Desferrioxamine Suppresses Experimental
Allergic Encephalomyelitis Induced by MBP in SJL mice," J.
Neuroimmunol. 84:188-197 (1998), which is hereby incorporated by
reference in its entirety). An aminooxyacetyl-ferrioxamine has also
been prepared allowing for site specific conjugation to antibodies
(Pochon et al., "A novel Derivative of the Chelon Desferrioxamine
for Site-specific Conjugation to Antibodies," Int. J. Cancer.
43:1188-1194 (1989), which is hereby incorporated by reference in
its entirety. Fluorescent deferoxamine derivatives have also been
synthesized for free iron measurements in a range of biological
experimental conditions (Al-Mehdi et al.,
"Depolarization-associated iron release with abrupt reduction in
pulmonary endothelial shear stress in situ," Antioxid. Redox
Signal. 2:335-345 (2000), which is hereby incorporated by reference
in its entirety.
[0081] Other suitable iron chelators include those set forth in
Table 2:
TABLE-US-00002 PHARMACOLOGY Name Formula Chem. structure MW Dent
Route DFO 4-[3,5- bis- [hy- droxy- phenyl]- 1,2,4- triazol- 1-yl]-
benzoic acid ##STR00001## 560 6 par- ent- al HBED N,N'- bis(o- hy-
droxy- benzyl) ethyl- ene- damine- N,N'- diacetic acid ##STR00002##
388 6 oral/ par- ent- al PIH pyri- doxal isanico- tinoyl hydra-
zone ##STR00003## 262 3 oral DFT 4'-hy- droxy- (S)- desaza des-
methyl- desferri- thiocin; (S)-4,5- di- hydro-2- (2,4- dihydro-
phenyl)- 4- thiazo- fecar- boxylic acid ##STR00004## 238 3 oral DFP
(L1) 1,2- di- methyl- 3-hy- droxy- pyridin- 4-one ##STR00005## 139
2 oral S- hydroxy- -- 250.000 6 i.v. DFO ethyl- starch- bound-
deferox- amine ICL- 670 4-[3,5- bis- (hy- droxy- phenyl)- 1,2,4-
triazol- 1-yl]- benzoic acid ##STR00006## 373 3 oral GT56- 252
4,5-di- hydro-2- (2,4- dihy- droxl- phenyl)- 4- methyl- thiazole-
4(S)- car- boxylic acid ##STR00007## 252 3 oral
[0082] HEBED is a synthetic chelator that appears to have higher
efficacy than DFO, and fewer adverse effects. However, in primate
studies, it still had to be administered by subcutaneous infusion
(Chaston, et. al., "Iron Chelators for the Treatment of Iron
Overload Disease: Relationship Between Structure, Redox Activity,
and Toxicity" Am J Hematol. 73:200-210 (2003), which is hereby
incorporated by reference in its entirety.
[0083] PIH is an orally active, triedentate chelator which crosses
membranes much better than does DFO. PCHI (i.e. analogues of
2-pyridylcarboxaldehyde isonicotinoyl hydrazone) compounds (which
are not shown in Table 2) are substantially similar to PIH. This
class of chelators can also access mitochondrial iron pools, making
it a potential drug for the rare genetic disease Friedrich's Ataxia
(caused by a mutation in the mitochondrial iron-sulfur complex
chaperone frataxin).
[0084] Like HBED, DFT and GT56-252 are both second generation
hydroxypyridones that are in preclinical or phase I trials.
[0085] DFP or Deferipone, is approved for clinical use in Europe
under the trade name Ferriprox. It is a bidentate chelator that is
administered orally. However, the efficacy and toxicity of the drug
are still controversial. Combined use of DFO and DFP has been
proposed.
[0086] S-DFO is a starch-bound DFO derivative that has a longer
half-life after intravenous administration.
[0087] ICL-670 is a tridentate chelator of the triazole family
currently in phase II trials. It is orally available and is
administered once a day (Hershko, C., et al., Blood 97:1115-1122
(2001), which is hereby incorporated by reference in its
entirety).
[0088] Another class of iron chelator is the biomimetic class
(Meijier, M M, et al. "Synthesis and Evaluation of Iron Chelators
with Masked Hydrophilic Moieties" J. Amer. Chem. Soc. 124:1266-1267
(2002), which is hereby incorporated by reference in its entirety).
These molecules are modified analogues of such naturally produced
chelators as DFO and ferrichrome. The analogues allow attachment of
lipophilic moieties (e.g., acetoxymethyl ester) which greatly
enhance passage through membranes. The lipophilic moieties are then
cleaved intracellularly by endogenous esterases, converting the
chelators back into hydrophilic molecules which cannot leak out of
the cell. These compounds appear to be highly effective, and reduce
free-iron mediated oxidative damage much more efficiently than does
DFO.
[0089] Lastly, a number of compounds developed as inhibitors of
advanced glycation endproduct (AGE) formation and/or degradation
and tested in animal models of diabetic complications appear to act
via chelation (Price, D L, et al., JBC 276:48967-72 (2001), which
is hereby incorporated by reference in its entirety). These include
(in order from weakest to strongest copper chelation):
aminoguanidine and pyridoxamine; carnosine, phenazinediamine,
OPB-9195, and tenilsetam. The so-called AGE-breakers,
phenacylthiazoloum and phenacyldimethythiazolium bromide, and their
hydrolysis products, were among the most potent inhibitors of
copper-catalyzed autoxidation of ascorbate. Aminoguanidine has been
through Phase II/III trials, pyridoxamine has been through Phase II
trials, and the AGE breakers are currently in Phase II trials.
[0090] The inhibitors can be administered orally, parenterally,
transdermally, subcutaneously, intravenously, intramuscularly,
intraperitoneally, by intraversal instillation, intracularly,
intranasally, intraarterially, intralesionally, or by application
to mucous membranes, such as that of the nose, throat, and
bronchial tubes. The inhibitors can be administered alone or with a
pharmaceutically acceptable salt, carrier, excipient, or
stabilizer, and can be in solid or liquid form, including, for
example, tablets, capsules, powders, solutions, suspensions, or
emulsions.
[0091] The solid unit dosage forms can be of the conventional type.
The solid form can be a capsule, such as an ordinary gelatin type
containing the inhibitors of the present invention and a carrier,
for example, lubricants and inert fillers such as lactose, sucrose,
or cornstarch. In another embodiment, the inhibitors are tableted
with conventional tablet bases such as lactose, sucrose, or
cornstarch in combination with binders like acacia, cornstarch, or
gelatin, disintegrating agents such as, cornstarch, potato starch,
or alginic acid, and a lubricant like stearic acid or magnesium
stearate.
[0092] In another aspect, the inhibitors of the present invention
may be orally administered, for example, with an inert diluent, or
with an assimilable edible carrier, or they may be enclosed in hard
or soft shell capsules, or they may be compressed into tablets, or
they may be incorporated directly with the food of the diet. For
oral therapeutic administration, the inhibitors of the present
invention may be incorporated with excipients and used in the form
of tablets, capsules, elixirs, suspensions, syrups, and the like.
In one aspect, such formulations should contain at least 0.1% of
the inhibitors of the present invention. The percentage of the
inhibitors in the formulations of the present invention may, of
course, be varied and may conveniently be between about 2% to about
60% of the weight of the unit. The amount of inhibitors in the
formulations of the present invention is such that a suitable
dosage will be obtained. As one example, formulations according to
the present invention are prepared so that an oral dosage unit
contains between about 1 and 250 mg of the inhibitors.
[0093] The tablets, capsules, and the like may also contain a
binder such as gum tragacanth, acacia, corn starch, or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch, alginic acid; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose,
lactose, or saccharin. When the dosage unit form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a fatty oil.
[0094] Various other materials may be present as coatings or to
modify the physical form of the dosage unit. For instance, tablets
may be coated with shellac, sugar, or both. A syrup may contain, in
addition to active ingredient, sucrose as a sweetening agent,
methyl and propylparabens as preservatives, a dye, and flavoring
such as cherry or orange flavor.
[0095] As described above, in one aspect of the present invention,
the formulations containing the inhibitors may be administered
parenterally. Solutions or suspensions of the inhibitors can be
prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof in
oils. Illustrative oils are those of petroleum, animal, vegetable,
or synthetic origin, for example, peanut oil, soybean oil, or
mineral oil. In general, water, saline, aqueous dextrose and
related sugar solution, and glycols such as, propylene glycol or
polyethylene glycol, are preferred liquid carriers, particularly
for injectable solutions. Under ordinary conditions of storage and
use, these preparations contain a preservative to prevent the
growth of microorganisms.
[0096] When the inhibitor is deferoxamine, deferoxamine
compositions for parental use can be in the form of a solution or a
suspension. Such solutions or suspenions may also include sterile
diluents such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents. Parenteral formulations may also include
antibacterial agents such as benzyl alcohol or methyl parabens, or
antioxidants such a sodium bisulfite. Buffers such as acetates,
citrates, or phosphates and agents for the adjustment of tonicity
such as sodium chloride or dextrose may also be added. The
parenteral preparation can be enclosed in ampules, disposable
syringes, or multiple dose vials made of glass or plastic.
[0097] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0098] Slow-release deferoxamine compositions for intramuscular
administration may be formulated by standard methods, such as a
microcrystalline composition. Deferoxamine preparations with longer
half-lives may be formulated by conjugation of deferoxamine with,
for example, dextrans or polyethylene glycols. In addition,
deferoxamine derivatives with great ability to permeate cell
membranes can be made by linking deferoxamine to a lipophilic ester
moiety such as acetyoxymethyl ester, which is then removed by
intracellular esterases once the compound is inside the cell
(Meijler et al., "Synthesis and Evaluation of Iron Chelators with
Masked Hydrophilic Moieties", J. Am. Chem. Soc. 124:12666-12667
(2002)).
[0099] The formulations containing the inhibitors of the present
invention may also be administered directly to the airways in the
form of an aerosol. For use as aerosols, the inhibitors of the
present invention in solution or suspension may be packaged in a
pressurized aerosol container together with suitable propellants,
for example, hydrocarbon propellants like propane, butane, or
isobutane with conventional adjuvants. The inhibitors of the
present invention also may be administered in a non-pressurized
form such as in a nebulizer or atomizer.
[0100] In carrying out this method, an ROS-mediated injury can be
treated or prevented. Hyperglycemic conditions which can be so
treated or prevented include chronic hyperglycemia. This includes
hyperglycemic diabetes or acute hyperglycemia (such as stress
hyperglycemia). Resistance to insulin is another form of a
metabolite-induced excessive ROS production in accordance with this
aspect of the present invention. This can be where there is
resistance to insulin resulting in increased free fatty acid flux
and increased free fatty acid oxidation by vascular cells.
[0101] Another aspect of the present invention relates to a method
of promoting neovascularization in a subject prone to hyperglycemia
or increased fatty acid flux. This method involves administering an
ROS inhibitor to the subject under conditions effective to promote
neovascularization in the subject.
[0102] Here, neovascularization can be in response to hypoxic
signaling, and involve both angiogenesis (e.g. cardiac or lower
limb) or vasculogenesis. The subject can have an ischemic
condition, such as coronary artery disease, peripheral vascular
disease, cerebral vascular disease, or a wound which is either
chronic or acute.
[0103] The ROS inhibitor, its formulation, and its modes of
administration for this embodiment of the present invention are the
same as those described above.
[0104] Here the subject is preferably a human prone to
hyperglycemia or fatty acid flux.
[0105] A further aspect of the present invention pertains to a
method inhibiting oxidation or excessive release of free fatty
acids in a subject. This method involves administering to the
subject certain compounds under conditions effective to inhibit
oxidation excessive release of free fatty acids in the subject.
These compounds include thiazolidinedione, nicotinic acid,
etomoxir, and ranolazine.
[0106] In this embodiment of the present invention, the
above-identified compounds are formulated and administered in
substantially the same way as noted above.
[0107] In this aspect of the present invention, the subject is a
mammal, preferably a human.
[0108] A further aspect of the present invention is directed to a
method of identifying compounds suitable for treatment or
prevention of ROS-mediated injury. This method involves providing a
diabetic animal model and inducing diabetes in the animal model. A
compound to be tested is then administered to the animal model.
Compounds which achieve recovery of local oxygen tension, blood
flow increase in vessel density, and tissue survival in the animal
model as therapeutic candidates for treating or preventing
ROS-mediated injury are then recovered.
EXAMPLES
Example 1--Three Different Murine Models of Diabetes Exhibit
Increased Tissue Necrosis in Response to Ischemia
[0109] It is well recognized that diabetic tissues have a reduced
tolerance to ischemia (Haffner et al., "Mortality From Coronary
Heart Disease in Subjects With Type 2 Diabetes and in Nondiabetic
Subjects With and Without Prior Myocardial Infarction," N Engl J
Med 339:229-34 (1998); Jude et al., "Peripheral Arterial Disease in
Diabetic and Nondiabetic Patients: a Comparison of Severity and
Outcome," Diabetes Care 24:1433-7 (2001); Tuomilehto et al.,
"Diabetes Mellitus as a Risk Factor for Death From Stroke.
Prospective Study of the Middle-Aged Finnish Population," Stroke
27:210-5 (1996); Waltenberger, "Impaired Collateral Vessel
Development in Diabetes: Potential Cellular Mechanisms and
Therapeutic Implications," Cardiovasc Res 49:554-60 (2001); Rivard
at al., "Rescue of Diabetes-Related Impairment of Angiogenesis By
Intramuscular Gene Therapy With Adeno-VEGF," Am J Pathol 154:355-63
(1999); Kip at al., "Differential Influence of Diabetes Mellitus on
Increased Jeopardized Myocardium After initial Angioplasty or
Bypass Surgery: Bypass Angioplasty Revascularization
Investigation," Circulation 105:1914-20 (2002); Partamian at al.,
"Acute Myocardial Infarction in 258 Cases of Diabetes. Immediate
Mortality and Five-Year Survival," N Engl J Med 273:455-61 (1965);
Simovic et al., "Improvement in Chronic Ischemic Neuropathy After
Intramuscular phVEGF165 Gene Transfer in Patients With Critical
Limb Ischemia," Arch Neurol 58:761-8 (2001): Margolis at al., "Risk
Factors for Delayed Healing of Neuropathic Diabetic Foot Ulcers: A
Pooled Analysis," Arch Dermatol 136:1531-5 (2000), which are hereby
incorporated by reference in their entirety). Clinically, this
results in increased rates of heart failure, increased mortality
and prolonged wound healing. While this relationship has been
studied in animal models of cardiac and hindlimb ischemia (Rivard
at al., "Rescue of Diabetes-Related Impairment of Angiogenesis By
Intramuscular Gene Therapy With Adeno-VEGF," Am J Pathol 154:355-63
(1999); Schratzberger, et al., "Reversal of Experimental Diabetic
Neuropathy by VEGF Gene Transfer," J Clin Invest 107:1083-92
(2001), which are hereby incorporated by reference in their
entirety), there are limitations to these models. Due to the
variations in large vessel anatomy, the resultant pattern of
necrosis is unpredictable, leading to discrepancies in the
experimental results. In addition, it is not possible to determine
tissue survival except at sacrifice. Furthermore, indirect measures
of perfusion such a laser doppler must often be utilized to
estimate ischemia, but these techniques do not provide direct
information regarding tissue oxygenation.
[0110] To address these problems, a novel model of graded ischemia
in the dorsal soft tissue of mice has been created (FIG. 5) (Tepper
et al., "Human Endothelial Progenitor Cells From Type II Diabetics
Exhibit Impaired Proliferation, Adhesion, and incorporation Into
Vascular Structures," Circulation 106:2781-6 (2002), which is
hereby incorporated by reference in its entirety). Since the
vascular anatomy of the mouse dorsum is precisely known, and the
major axial vessels can be easily visualized, one can create a
reliable zone of ischemia with a reproducible oxygen gradient in
the tissue. This has been confirmed with direct tissue oxygen
tension measurements utilizing five reference points (p1-p5) spaced
0.5 cm apart proceeding from the least to most ischemic regions.
This also allows for the study of discrete microenvironments of
ischemia (Areas A, B, C), with Area A being the least ischemic and
Area C being the most ischemic portion of the soft tissue. The
design of this model facilitates direct dynamic measurement of
oxygen tension, quantitation of tissue survival, with a degree of
reproducibility that allows correlation of specific oxygen tensions
with changes in gene expression.
[0111] Using this model, it has been observed that the response too
ischemia is dramatically impaired in three different murine models
of diabetes, all characterized by significant hyperglycemia. In the
db/db mouse, a leptin receptor deficient model of Type U diabetes,
it has been demonstrated that ischemia produces significant
necrosis of nearly all of the tissue, whereas all the tissue
survived in non-diabetic animals. Similar results were noted in the
streptozotocin-induced diabetic mouse model (Stz), as well as an
Akita mouse model of Type I diabetes with tissue survival
approximately 30% of that observed in non-diabetic mice (FIG. 6).
Importantly, oxygen tensions and vascular density (as determined by
CD31 staining and FITClectin perfusion) were identical in all four
groups prior to surgery, suggesting that the differences in tissue
survival were due to an impaired response to ischemia rather than
baseline differences in vascular density.
Example 2--Diabetic Mice have a Diminished Neovascular Response to
Ischemia
[0112] The decrease in tissue survival observed in this model was
also associated with diminished neo-vascularization in the
surviving tissue. Seven days following surgery, the oxygen tension
in ischemic soft tissue of non-diabetic mice approaches that of
normal skin (FIG. 7, grey plot), while the diabetic mice
demonstrate a significant reduction in oxygen tension at the same
reference points (black plot). These findings correlated with a
reduction in the number of blood vessels observed in the surviving
tissue in diabetic mice (FIG. 8, black plot) as determined by CD31
staining. This suggests that ischemia-induced neovascularization is
impaired in diabetic mice.
Example 3--Prevention of Hyperglycemia-Induced Reactive Oxygen
Species Restores Tissue Survival in a Diabetic Animal Model
[0113] It has been examined whether increased oxidative damage was
an upstream modulator of the impaired tissue response to ischemia
in diabetic animals. To address this question, a transgenic mouse
that overexpress mitochondrial manganese superoxide dismutase
(MnSOD) was used. MnSOD catalyzes the formation of molecular oxygen
from superoxide, preventing the generation of ROS, and effectively
blocks all four pathways of hyperglycemic damage. Diabetes was
induced in wild type and MnSOD transgenic mice via streptozotocin
injection, and hyperglycemia (>400 mg/dl) was maintained for one
month. Following ischemic surgery, tissue was monitored by direct
oxygen tension measurements on days 1, 3, and 7. Compared to wild
type diabetic mice, MnSOD diabetic mice demonstrated a rapid
recovery of local tissue oxygen tensions, neovascularization, and
increased tissue survival that was similar to that observed in
non-diabetic mice (FIG. 9). Non-diabetic MnSOD control mice were
similar to wild type mice. This suggests that the prevention of
hyperglycemia-Induced ROS improves tissue survival in diabetic
animals following ischemic events.
Example 4--Chronic High Glucose Levels Also Correlate with
Increased Mitochondrial Membrane Potential
[0114] The effects of high glucose culture on mitochondrial
membrane potential were also examined in C2CI 2 cells exposed to
acute or chronic high glucose using the potential-dependent
cationic dye JC-1. This has been used as an indicator of oxidative
stress. In concordance with recent reports (Du et al.,
"Hyperglycemia Inhibits Endothelial Nitric Oxide Synthase Activity
by Posttranslational Modification at the Akt Site," J Clin Invest
108:1341-8 (2001), which is hereby incorporated by reference in its
entirety), chronic high glucose profoundly increases the
mitochondrial proton electrochemical gradient (evidenced by a shift
to orange-red fluorescence) compared to normal glucose culture or
acute exposure to high glucose (FIG. 10) (Du et al., "Hyperglycemia
Inhibits Endothelial Nitric Oxide Synthase Activity by
Posttranslational Modification at the Akt Site," J Clin Invest
108:1341-8 (2001), which is hereby incorporated by reference in its
entirety). Thus, a correlation exists between hyperglycemia,
oxidative stress, and VEGF impairment in vitro.
Example 5--Impaired VEGF Production Lies at the Level of RNA
Transcription
[0115] With evidence implicating decreased VEGF production as a
contributor to impaired angiogenesis in hyperglycemic states, the
mechanism by which high glucose alters VEGF expression was
examined. Analysis of VEGF mRNA transcripts present in normal and
high glucose culture under hypoxic conditions revealed a
substantial reduction in VEGF mRNA production in cells cultivated
in high glucose (FIG. 11). Possible explanations for this finding
included abnormal mRNA stabilization or decreased promoter activity
in high glucose. To address the issue of mRNA stabilization, the
RNA 1/2-life in C2Cl2 myoblasts was examined by inhibiting
transcription with actinomycin D. Results of these experiments
showed no differences in VEGF mRNA stability between normal an
hyperglycemic cells despite significant differences in VEGF protein
levels (FIG. 12). VEGF promoter activity was then examined using a
reporter construct containing the full length VEGF promoter fused
to a luciferase gene. This construct was transiently co-transfected
into C2Cl2 myoblasts cultivated in normal and high glucose with a
constitutively expressed Renilla plasmid to control for
transfection efficiency. Hypoxia-induced luciferase production was
significantly impaired in high glucose conditions compared to
normal glucose controls (FIG. 13). This demonstrates that the
impaired VEGF protein production in hypoxia resulted from decreased
VEGF transcription in vivo.
Example 6--p300 and HIF-1a are Substrates for 0-linked
Glycosylation, Potentially Linking the Hexosamine Pathway of
Hyperglycemic Oxidative Damage to Impairments in Hypoxia-Induced
VEGF Expression
[0116] Based on findings implicating impaired HIF-1.alpha.
transactivation in high glucose as the mechanism for impaired
hypoxia-induced VEGF expression, potential post-translational
modifications of HIF-1.alpha. were examined under these conditions.
It was initially examined whether HIF-1.alpha. is a substrate for
O-linked glycosylation. HIF-1.alpha. was immunoprecipitated from
cells grown in normal or high glucose conditions, and Western blots
were probed with an antibody that specifically recognizes residues
containing the O-linked glycosylation modification. While no
glycosylated HIF-1.alpha. was present under normal glucose
conditions, there was significant glycosylation in high glucose
(FIG. 14). This is the first demonstration that HIF-1.alpha. is a
substrate for O-linked glycosylation, and is preferentially
glycosylated under conditions of high glucose.
[0117] Since the HIF-1 transcriptional complex is comprised of
several coactivators, it was also examined whether p300, the major
co-activator of the HIF-1, was also glycosylated. While many
transcription factors have been found to associate with p300
constitutively, some cases have been identified where this
interaction is modulated by post-translational modification (Zanger
at al., "CREB Binding Protein Recruitment to the Transcription
Complex Requires Growth Factor-Dependent Phosphorylation of its OF
Box," Mol Cell 7:551-8 (2001); Soutoglou at al., "Acetylation
Regulates Transcription Factor Activity at Multiple Levels," Mol
Cell 5:745-51 (2000), which are hereby incorporated by reference in
their entirety). Repeating the HIF-1 experiments, it was also found
that p300 also serves as a substrate for post-translational
O-linked glycosylation in conditions of high glucose. This was
physiologically significant since the association of p300 with the
transcription factor peroxisome proliferator-activated receptor
gamma (PPAR.gamma.) was reduced in conditions of high glucose
compared to normal glucose by co-immunoprecipitation assays (FIG.
15A-B). Interestingly, blockade of the rate-limiting enzyme of
hexosamine biosynthesis, glutamine:fructose-6-phosphate
amidotransferase (GFAT) with antisense oligonucleotides reduced the
amount of Olinked glycosylation of p300 in high glucose nearly
three-fold, and restored the p300/PPAR.gamma. interaction to levels
comparable to cells grown in normal glucose (FIG. 15A-B). This
suggests that the recruitment of p300 to transcriptional complexes
is impaired in conditions of high glucose, which can be reversed by
preventing glucose-induced O-linked glycosylation. The physiologic
relevance of O-linked glycosylation of HIF-1.alpha. is unclear.
However, the demonstration that glycosylation modifies p300
function suggests a possible mechanism by which the HIF-1
transcriptional complex fails to upregulate VEGF expression, due to
its inability to recruit and/or associate with co-activators
required for transcriptional activation (i.e. p300).
Example 7--Hyperglycemia-Induced Reactive Oxygen Species Activate
Pathways of Cellular Damage, Impairing Endothelial Cell
Function
[0118] The data presented thus far have examined the mechanisms
responsible for initial observations that high glucose levels, both
in vivo and in vitro, produce profound deficits in the ability to
upregulate VEGF under hypoxic conditions.
[0119] Although there is significant literature examining
hyperglycemia-induced vascular damage in non-ischemic settings,
very few studies have examined the effect of hyperglycemia-induced
cellular damage on vascular functions in ischemic settings. This is
of clinical importance, as most situations requiring new vascular
growth occur in scenarios characterized by significant tissue
hypoxia. It has been demonstrated that endothelial cells grown in
high glucose in vitro show increased mitochondrial production of
ROS. This results in increased hexosamine pathway activity with
increased glycosylation of certain transcription factors (SPI) and
signaling molecules (eNOS), increased PKC activity resulting in
part in increased NFkB activity, greater accumulation of AGEs, and
increased flux through the sorbitol pathway (FIG. 16A-C). The
downstream consequences of these intracellular events likely result
in impaired neovascularization observed in vivo, but the
intermediate steps remain unclear.
Example 8--Diabetic Cells are Impaired in Functions Critical for
Angiogenesis
[0120] While it is clear that VEGF expression is altered in
diabetic states, it has also been demonstrated that diabetic cells
are impaired in other ways. Fibroblasts isolated from diabetic mice
(db/db) show dramatic decreases in migration (four-fold less) than
normal fibroblasts on collagen and fibronectin using a gold salt
phagokinetic migration assay. When the haplotactic response of
these cells was examined using a modified Boyden chamber migration
assay, a similar decrease of 77% in migration in response to serum
and PDGF was observed (Lerman et al., "Cellular Dysfunction in the
Diabetic Fibroblast: Impairment in Migration, Vascular Endothelial
Growth Factor Production, and Response to Hypoxia," Am J Pathol
162-303-12 (2003), which is hereby incorporated by reference in its
entirety).
[0121] Once again, this difference was accentuated by hypoxia (FIG.
17A-B). Whereas migration in normal cells was upregulated by
hypoxia (two-fold), diabetic cells showed no difference in the rate
of migration in hypoxia. These assays again emphasize the profound
impact that diabetes has on cellular function, and that this impact
is magnified under hypoxic conditions.
[0122] These migration differences may be due to differential
expression of members of the matrix metalloproteinase (MMP) family
in diabetic fibroblasts. It has been demonstrated that diabetic
fibroblasts have greater levels of pro-MW-9 than normal
fibroblasts, but no differences in active MMP-9 or
active/pro-MMP-2. (FIG. 18A-B). This confirms similar findings in
endothelial cells cultured in high glucose (Uemura at al.,
"Diabetes Mellitus Enhances Vascular Matrix Metalloproteinase
Activity: Role of Oxidative Stress," Circ Res 88:1291-8 (2001),
which is hereby incorporated by reference in its entirety).
Furthermore, these findings suggest that diabetic cellular
dysfunction is not characterized by a simple downregulation of all
cellular proteins or functions, but involves selective modulation
of specific genes and proteins.
Example 9--Endothelial Progenitor Cells from Type II Diabetic
Patients are Impaired in their Ability to Proliferate, Adhere, and
Incorporate into Vascular Structures
[0123] Hyperglycemic alterations in the effector cells of
vasculogenesis, the endothelial progenitor cell or precursor cell
remain poorly defined. Recently, it was demonstrated that
endothelial progenitor cells (EPCs) harvested from Type 1 diabetic
patients exhibit reduced proliferation, adhesion, and incorporation
into vascular structures as compared to age matched controls under
normoxic conditions (Tepper et al., "Human Endothelial Progenitor
Cells From Type II Diabetics Exhibit Impaired Proliferation.
Adhesion, and incorporation Into Vascular Structures," Circulation
106:2781-6 (2002), which is hereby incorporated by reference in its
entirety). Diabetic cultures contained significantly fewer EPCs
after 7 days of expansion (FIG. 19A-D), and this was inversely
correlated with HbA.sub.1c. Additionally, significantly fewer
EPC-bearing clusters were noted in the cultures of diabetic
patients. This was inversely correlated with the number of years of
clinical diabetes (R=-0.471, P<0.01). Functionally, these cells
were found to adhere less to TNF-.alpha. activated endothelial
monolayers but exhibited normal adhesion to quiescent endothelial
monolayers, which suggests that their ability to respond to
environmental cues is deficient. This was confirmed with in vitro
angiogenesis assays, which demonstrated that fewer diabetic EPCs
were incorporated into tubules on Matrigel when compared to
age-matched controls.
Example 10--Endothelial Progenitor Cells from Diabetic Patients
have an Impaired Ability to Respond to Hypoxia
[0124] Given preliminary data suggesting that diabetic cells have
an impaired response to hypoxia, studies in EPCs have been to
specifically examine the response of these cells to an ischemic
environment. It was demonstrated that EPCs from Type II diabetic
patients were impaired in their ability to adhere to hypoxic
endothelial monolayers, migrate towards conditioned media from
hypoxic endothelial cells, and proliferate a hypoxic environment
(FIG. 10A, B, C, respectively). This may be reflective of an
impaired ability of these cells to sense and respond appropriately
to hypoxic environmental cues, resulting in poor
neovascularization.
Example 11--Deferoxamine Prevents Hyperglycemia-Induced Reactive
Oxygen Production in Vascular Endothelial Cells
[0125] Cultured vascular endothelial cells were treated with
deferoxamine to determine the effect of deferoxamine on
hyperglycemia-induced reactive oxygen production by those
cells.
[0126] Cell culture conditions: For ROS measurement, bovine aortic
endothelial cells (BAECs, passage 4-10) were plated in 96 well
plates at 100,000 cells/well in Eagle's MEM containing 10% FBS,
essential and nonessential amino acids, and antibiotics. Cells were
incubated with either 5 mM glucose, 30 mM glucose, 30 mM glucose
plus 100 micromolar deferoxamine, 30 mM glucose plus 250 micromolar
deferoxamine. The deferoxamine was freshly prepared and added to
the cells on three consecutive days. The ROS measurements were
performed 72 hrs after the initial treatment.
[0127] Intracellular reactive oxygen species measurements: The
intracellular formation of reactive oxygen species was detected
using the fluorescent probe CMH2DCFDA (Molecular Probes). Cells
(1.times.105 ml-l) were loaded with 10 .mu.M CM-H2DCFDDA, incubated
for 45 rain at 37.degree. C., and analyzed in an HTS 7000 Bio Assay
Fluorescent Plate Reader (Perkin Elmer) using the HTSoft program.
ROS production was determined from an H202 standard curve (10-200
nmol ml-l).
[0128] As shown in FIG. 21 deferoxamine inhibited production of ROS
in vascular endothelial cells in culture. Diabetic levels of
hyperglycemia cause increased ROS (superoxide) production in these
cells (FIG. 21, bar 2). Adding 250 .mu.M deferoxamine completely
prevents this damaging effect (FIG. 21, bar 4).
[0129] Thus, the iron chelator deferoxamine has a profound effect
on vascular endothelial cells--i.e. it prevents completely
hyperglycemia-induced overproduction of hydroxyl radicals (FIG.
21).
Example 12--Normalizing Excess Mitochondrial Superoxide Production
Inhibits Hyperglycemia-Induced Increases in Intracelluar Free Iron
in Aortic Endothelial Cells
[0130] For free intracellular iron measurement, bovine aortic
endothelial cells ("BAECs", passage 4-10) were plated in 24 well
plates at 500,000 cells/well in Eagle's MEM containing 10% FBS,
essential and nonessential amino acids, and antibiotics. Cells were
infected with UCP-1, Mn-SOD or empty adenoviral vectors,
respectively, for 48 hours. 30 mM glucose was added to each well
that was infected with the adenovirus Uninfected cells were
incubated with 5 mM and 30 mM glucose as controls. The free
intracellular iron was detected after 24 hours.
[0131] In order to detect intracellular free iron, cells were
loaded with fura-2 AM in the dark at 37.degree. C. for 15 min in 1
ml of TBSS containing 5 .mu.M furs-2 AM. After loading, cells were
incubated with TBSS with 1 ml of 20 .mu.M EDTA for 5 min. (Kress et
al., "The Relationship between Intracellular Free Iron and Cell
Injury in Cultured Neurons, Astrocytes, and Oligodendrocytes", J.
Neuro., 22(14):5848-5855 (2002), which is hereby incorporated by
reference in its entirety). Fluorescence was detected using an
Olympus IX70 with 10.times. planapo objectives, run by I.P. Lab
Spectrum on a Power PC computer. Analysis was performed with I.P.
Lab Spectrum.
[0132] As shown in FIG. 22, bar 2, hyperglycemia, increased the
amount of free iron by nearly 3-fold. Since the probe fura-2 AM
specifically detects Fe.sup.3+ iron, this shows that it is free
Fe.sup.3+ iron which is increased. Inhibition of this effect by
overexpression of uncoupling protein-1, a mitochondrial protein
that prevents superoxide formation by the electron transport chain
(bar 3) demonstrates that the mitochondria are the origin of the
hyperglycemia-induced-superoxide. Inhibition of this effect by
overexpression of MnSOD, the mitochondrial isoform of the enzyme
superoxide dismutase (bar 4), demonstrates that mitochondrial
superoxide is the reactive oxygen species that induces increased
intracellular free iron.
Example 13--Deferoxamine Inhibits Hyperglycemia-Induced Increases
in Intracellular Free Iron in Aortic Endothelial Cells
[0133] Bovine aortic endothelial cells ("BAECs", passage 4-10) were
plated in 24 well plates at 500,000 cells/well in Eagle's MEM
containing 10% FBS, essential and nonessential amino acids, and
antibiotics. Cells were incubated with either 5 mM glucose, 30 mM
glucose, or 30 mM glucose plus 100 .mu.M deferoxamine. Free
intracellular iron measurement was performed 24 hours later. To
detect intracellular free iron, cells were loaded with fura-2 AM as
described above in Example 12.
[0134] As shown in FIG. 23B, hyperglycemia (accomplished by 30 mM
glucose incubation) dramatically increases intracellular free iron
in the Fe.sup.3+ form compared to normal glycemia, as shown in FIG.
23A (accomplished by 5 mM glucose treatment), as it did in Example
12. As shown in FIG. 23C, the Fe.sup.3+-specific iron chelator
deferoxamine (100 .mu.M) completely prevents this effect of
hyperglycemia.
Example 14--Deferoxamine and the Hydroxyl Radical Scavenger DMSO
Both Inhibit Hyperglycemia-Induced Increases in DNA Strand Breakage
in Aortic Endothelial Cells
[0135] Bovine aortic endothelial cells ("BAECs", passage 4-10) were
plated in 10 mm cell culture plates until confluent. Cells were
incubated with either 5 mM glucose, 30 mM glucose, 30 mM glucose
plus 100 .mu.M deferoxamine (DFO), or 30 mM glucose plus 100 .mu.M
DMSO, a hydroxyl radical scavenger, for 7 days. Medium with
reagents was changed daily. DNA strand breakage was detected using
the Comet assay method.
[0136] DNA breakage detection was performed using the Comet Assay
kit (Trevigen Gaithersburg Md.). Briefly, single cell
electrophoresis was performed on the cometslide for 10 min at 1
volt/cm (measured from one electrode to another). After air-drying,
the cometslide was stained with SYBR green. Fluorescence was
detected using the Olympus IX70 fluorescent microscope and analysis
of the fluorescent density of DNA breakage (length of tail) was
performed using Image J software.
[0137] It has previously been shown that hyperglycemia-Induced
superoxide production by the mitochondrial electron transport chain
causes DNA strand breakage in aortic endothelial cells, as
demonstrated in FIG. 24B. The data shown in FIG. 24C prove that
this effect requires the superoxide-induced increase in free Fe
3.sup.+. Similarly, the data shown in FIG. 24D show that this
effect requires superoxideinduced hydroxyl radical production.
Together, these data show that deferoxamine treatment prevents
hydroxyl radical generation and subsequent DNA strand breakage,
despite the continued overproduction of superoxide by the
mitochondrial electron transport chain.
Example 15--Deferoxamine and the Hydroxyl Radical Scavenger DMSO
Both Inhibit Hyperglycemia-Induced Increases in PARP Activity in
Aortic Endothelial Cells
[0138] Bovine aortic endothelial cells ("BAECs", passage 4-10) were
plated in 10 mm cell culture plates until confluent. Cells were
incubated with either 5 mM glucose, 30 mM glucose, 30 mM glucose
plus 100 .mu.M deferoxamine, or 30 mM glucose plus 100 .mu.M DMSO
for 6 days and medium changed daily.
[0139] The 3H-NAD incorporation method was used to assess PARP
activity. BAECs were incubated with buffer which was composed of 56
mM Hepes (pH 7.5), 28 mM KCl, 28 mM NaCl, 2 mM MgCl.sub.2, 0.01%
digitonin, 25 mM NAD.sup.+, and 1 .mu.Ci/ml .sup.3HNAD.sup.+ for 10
min at 37.degree. C. TCA was added to precipitate ribosylated
protein and cells were lysed in 2% NaOH. Detection of incorporated
.sup.3H-NAD was performed using a scintillation counter, and PARP
activity determined according to the number of .sup.3H-NAD dpm.
[0140] It has previously been shown that hyperglycemia-induced
superoxide production by the mitochondrial electron transport chain
causes DNA strand breakage which then activates the enzyme poly
(ADP-ribose)polymerase (PARP) in aortic endothelial cells, as shown
in FIG. 25. The data shown in bar 3 prove that this effect requires
the superoxide-induced increase in free Fe.sup.3+. Similarly, the
data shown in bar 4 show that this effect requires
superoxide-induced hydroxyl radical production. Together, these
data show that deferoxamine treatment prevents hydroxyl radical
generation, subsequent DNA strand breakage, and resultant PARP
activation, despite the continued overproduction of superoxide by
the mitochondrial electron transport chain.
Example 6--Deferoxamine Prevents Hyperglycemia-Induced Inhibition
of Prostacyclin Synthase (PGF-1a) in Aortic Endothelial Cells
[0141] Bovine aortic endothelial cells ("BAECs", passage 4-10) were
plated in a 24-well plate (50,000 cell/well). Cells were incubated
with either 5 mM glucose, 30 mM glucose, or 30 mM glucose plus 100
.mu.M deferoxamine. The prostacyclin synthase product,
PGF-1.alpha., was measured 24 hours later.
[0142] Prostacyclin synthase activity measured as the concentration
of the stable product of prostacyclin synthase, PGF-1a. A
competitive immunoassay method (Correlate-EIA) was used for the
quantitative determination of 6-keto-PGFI.alpha.. Samples (100
.mu.l) collected from BAECs culture medium were added to the assay
plate, which was precoated with antibody (6-keto-PGF1.alpha., EIA
conjugate solution). PGFI.alpha. concentration was calculated
according to a standard curve, and data analysis performed using
AssayZap software.
[0143] It has previously been shown that hyperglycemia-induced
superoxide production by the mitochondrial electron transport chain
completely inactivates the endothelial enzyme prostacyclin
synthase, which is a major natural defense against the development
of atherosclerosis. In bar 2 of FIG. 26, hyperglycemia is shown to
decrease the activity of this enzyme by over 90%. In contrast, bar
3 shows that hyperglycemia does not inhibit the activity of this
important antiatherogenic enzyme at all when the superoxide-induced
increase in free Fe.sup.3+ is prevented by deferoxamine.
Example 17--Deferoxamine Prevents Diabetes-Induced Inhibition of
Prostacyclin Synthase (PGF-1a) In Aortas of Diabetic Mice
[0144] Male C57B16 mice (6-8 weeks old) were made diabetic by daily
injections of S0 mg/kg streptozotocin in 0.05 M NaCitrate pH 4.5
after an eight hour fast, for five consecutive days. Two weeks
after the initial injection the blood glucose was determined and
the diabetic mice were randomized into two groups with equal mean
blood glucose levels. Deferoxamine (10 mg/kg) was injected
subcutaneously once per day for 7 days in one group of diabetic
animas. The aortas were collected, for prostacyclin synthase
activity measurement.
[0145] Prostacyclin synthase activity measurement A competitive
immunoassay method (Correlate-EIA) was used for the quantitative
determination of 6-keto-PGF.sub.1.alpha.. Mouse aortas were washed
with PBS and incubated at 37.degree. C. for 3 hours in 400 .mu.l
incubation buffer containing 20 mM TRIS buffer (pH 7.5), and 15
.mu.M arachidonic acid. 100 .mu.l of sample was used to measure the
PGF1.alpha..
[0146] It has previously been shown that diabetes-induced
superoxide production by the mitochondrial electron transport chain
completely inactivates the endothelial enzyme prostacyclin synthase
in aortas of diabetic mice. In bar 2 of FIG. 27, hyperglycemia is
shown to decrease the activity of this enzyme in vivo by over 90%.
In contrast, bar 3 of FIG. 27 shows that hyperglycemia does not
inhibit the activity of this important antiatherogenic enzyme at
all when the superoxide-induced increase in free Fe.sup.3+ is
prevented by deferoxamine.
Example 18--Deferoxamine Prevents Hyperglycemia-Induced Inhibition
or Endothelial Nitric Oxide Synthase (eNOS) in Aortic Endothelial
Cells
[0147] Bovine aortic endothelial cells ("BAECs", passage 4-10) were
plated in 24-well plate (50,000 cell/well). Cells were incubated
with either 5 mM glucose, 30 mM glucose alone, or 30 mM glucose
plus 100 .mu.M deferoxamine, for 24 hour. Six hours before eNOS
activity determination, media without arginine was added to the
cells to deplete endogenous arginine.
[0148] Measurement of eNOS activity was accomplished as follows.
BAECs were incubated with 400 .mu.l of PBS-.sup.3H-arginine (1.5
.mu.ci/ml) buffer for 30 min at 37.degree. C. The reaction was
stopped by adding IN TCA (500 .mu.l/well, ice cold), the cells were
freeze fractured in liquid nitrogen for 2 min and thawed at
37.degree. C. for 5 min to obtain the cell lysate. After extraction
with ether, the cell lysate was adjusted to pH 5.5 using Hepes
buffer containing 2 mM EDTA and 2 mM of EGTA, then loaded onto
Trisformed DOWEX 50WX8 ion-exchange columns and .sup.3H-citrulline
collected. Detection of .sup.3H-citrulline was performed using a
liquid scintillation counter, and eNOS activity was calculated from
the amount of .sup.3H-citrulline generated.
[0149] It has previously been shown that hyperglycemia-induced
superoxide formation significantly inactivates another critical
endothelial enzyme, endothelial nitric oxide synthase (eNOS). This
enzyme plays a critical role in acute dilation of blood vessels in
response to hypoxia, and a chronic role as another major defense
against development and progression of atherosclerosis. In bar 2 of
FIG. 28, hyperglycemia is shown to decrease eNOS activity by 65%.
In contrast, bar 3 shows that hyperglycemia does not inhibit the
activity of this important antiatherogenic enzyme at all when the
superoxide-induced increase in free Fe 3.sup.+ is prevented by
deferoxamine.
Example 19--Deferoxamine Prevents Diabetes-Induced Inhibition of
Endothelial Nitric Oxide Synthase (eNOS) in Aortas of Diabetic
Mice
[0150] Male C57B16 mice (6-8 weeks old) were made diabetic by daily
injections of 50 mg/kg streptozotocin in 0.05 M NaCitrate pH 4.5
after an eight hour fast, for five consecutive days. Two weeks
after the initial injection, the blood glucose was determined and
the diabetic mice were randomized into two groups with equal mean
blood glucose levels. Deferoxamine (10 mg/kg) was injected
subcutaneously once per day for 7 days in one group of diabetic
animals. The aortas were collected for endothelial nitric oxide
synthase (eNOS) activity measurement.
[0151] Measurement eNOS activity was accomplished as follows.
Aortas were collected in liquid-nitrogen and tissue proteins
isolated. Immunoprecipitation methods were used to purify the eNOS
from tissue lysates. The purified eNOS immuno-complex was incubated
with 100 .mu.l of reaction buffer (3 .mu.M Tetrahydrobiopterin, 1
mM NAPDH, 2.5 mM CaCl.sub.2, 200 U Calmodulin, .sup.3H-L-arginine
0.2 .mu.Ci) for 45 min at 37.degree. C. with rolling. After the
incubation, samples were loaded onto Tris-formed DOWEX 50WX8
ion-exchange column and .sup.3H-citrulline was collected.
.sup.3H-citrulline was quantitated using a liquid scintillation
counter and eNOS activity was calculated from the amount of
.sup.3H-citrulline generated.
[0152] It has previously been shown that diabetes-induced-induced
superoxide production by the mitochondrial electron transport chain
inactivates the endothelial enzyme eNOS in aortas of diabetic mice.
In bar 2 of FIG. 29, diabetic hyperglycemia is shown to decrease
the activity of this enzyme in vivo by 65%. In contrast, bar 3
shows that hyperglycemia does not inhibit the activity of this
important antiatherogenic enzyme at all when the superoxide-induced
increase in free Fe.sup.3+ is prevented by deferoxamine.
Example 20--Diabetes-Induced Defect in Angiogenic Response to
Ischemia
[0153] FIGS. 30A-B show that diabetic animals do not increase
oxygenation by forming new vessels the way non-diabetic animals do.
FIGS. 30C-D show that diabetic animals only mobilize 0.22 vs. 1.83%
of bone marrow-derived endothelial precursor cells in response to
ischemia. FIGS. 30E-F show (black bar) that diabetics do not
increase capillary formation in ischemic tissue.
[0154] Researchers have created a novel model of graded ischemia in
the dorsal soft tissue of mice. Since the vascular anatomy of the
mouse dorsum is precisely known, and the major axial vessels can be
easily visualized, this model creates a reliable zone of ischemia
with a reproducible oxygen gradient in the tissue. This has been
confirmed with direct tissue oxygen tension measurements utilizing
four reference points (p1-p4) spaced 0.5 cm apart, proceeding from
the least to most ischemic regions.
[0155] The mechanisms underlying this diabetes-induced defect are
complex and incompletely understood, but appear to involve
mitochondrial superoxide overproduction, since the defect is
significantly prevented in diabetic transgenic mice which
overexpress the mitochondrial isoform of SOD.
Example 21--Deferoxamine Treatment Corrects the Diabetes-Induced
Defect in Angiogenic Response to Ischemia
[0156] The effect of deferoxamine, an iron chelator, on ischemic
neovascularization in streptozotocin-induced diabetic (STZ) and
wild type C57 (WT) mice was examined. Male C57B16 mice (6-8 weeks
old) were made diabetic by daily injections of 50 mg/kg
streptozotocin in 0.05 M NaCitrate pH 4.5 after an eight hour fast,
for five consecutive days. Two weeks after the initial injection
the blood glucose was determined, the diabetic mice were randomized
into two groups with equal mean blood glucose levels.
[0157] The treatment group was pretreated 7 days prior to having an
ischemic flap created on their dorsum and throughout the experiment
with daily injections of deferoxamine (10 mg/kg) subcutaneously
once per day for 7 days in one group of diabetic animals.
[0158] On day 7, it was found that blood flow was restored to
normal in the STZ-deferoxamine group (DM+DEF) when compared with
the non-diabetic untreated group (WT), end the severely impaired
STZ-untreated group (DM), a assessed by Doppler and as shown in
FIG. 31A. FIG. 31B shows tissue survival was restored to normal in
the STZ-deferoxamine group (DM+DEF) when compared with the
non-diabetic untreated group (WT), and the severely impaired
STZ-untreated group (DM). CD31 positive blood vessel counts
demonstrate that post-ischemic neovascularization was restored in
the STZ-deferoxamine group (STZ DefC), as shown in FIG. 32.
Interestingly, deferoxamine in the wild type mice (WT Def C) also
improved neovascularization. EPC mobilization was also improved in
the STZ-deferoxamine group when compared with the untreated STZ
mice. Migration of diabetic bone marrow derived, lineage depleted
cell population migration toward SDF was restored to normal when
STZ mice were treated with deferoxamine. See FIG. 35.
[0159] These results show that treatment of diabetic animals with
deferoxamine completely prevents the diabetes-induced defect in the
normal angiogenic response to ischemia.
Example 22--Deferoxamine Normalizes Diabetic Wound Healing and
Diabetes-Induced Defect in Angiogenic Response to Ischemia
[0160] The effect of reducing the wound healing in diabetic mice
(db/db) by treating them with deferoxamine was also studied in the
novel model of graded ischemia in the dorsal soft tissue of mice of
Example 20. The animals were made diabetics as described in Example
21. Deferoxamine-treated diabetic mice demonstrated complete would
closure at day 16, whereas untreated db mice did not close their
wounds until day 26 (FIGS. 33A-B). FIGS. 34 A-B show that diabetic
animals do not increase oxygenation by forming new vessels the way
deferoxamine-treated diabetic mice do. FIGS. 34C-D show that
deferoxamine-treated diabetic animals mobilize 1.12% of bone
marrow-derived endothelial precursor cells in response to ischemia
compared to 0.22% of diabetic deferoxamine-untreated mice. FIG.
348-F show (black bar) that deferoxamine-treated diabetics mice
substantially increase capillary formation in ischemic tissue
compared to diabetic deferoxamine-untreated mice
[0161] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow.
* * * * *