Method of Treating or Preventing Pathologic Effects of Acute Increases in Hyperglycemia and/or Acute Increases of Free Fatty Acid Flux
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.
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 INVENTIONThe 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 INVENTIONCardiovascular Complications Associated with Diabetes are a Major Public Health Problem.
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.
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.
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)).
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)).
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.
After the appropriate hypoxic signaling cascade is initiated, compensatory vascular growth in response to ischemic insult occurs by two different mechanisms (
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-1α subunit and the HIF-1β 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 (
The HIF-1 transcriptional complex is comprised of HIF-1α/β and more than seven other factors that modulate gene transcription. The two predominant functional components of this complex are HIF-1α and CBP/p300, which directly interact to transactivate gene expression. HIF-1α function is predominantly regulated by oxygen via protein stabilization and post-translational modification. Recent reports demonstrate that HIF-1α 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-1α 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)).
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-1α (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.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 (
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.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 (
Hyperglycemia-induced reactive oxygen species also impair the ability of HIF-1α 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).
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 INVENTIONOne 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.
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.
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.
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.
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.
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.
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.
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.
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-1α- 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).
The ROS generation by hyperglycemia or increased fatty acid flux takes place in the mitochondria. The most common ROS are hydrogen peroxide (H2O2), hydroxyl radicals (OH.), lipid peroxy radicals (LOO−), and peroxynitrites (ONOO−).
H2O2 is relatively stable and can diffuse through membranes. In most cells, H2O2 is detoxified by enzymatic reduction to H2O and O2. 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 (O2). The reduced metal then reacts with H2O2 to regenerate the initial oxidized metal, hydroxyl ions (OH+) and hydroxyl radicals (OH.). It is important to note, however, that this chemistry is still far from being understood.
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.
Iron has been the focus of many chemical studies because this chemistry was first demonstrated by H. J. H. Fenton in 1876 using unchelated Fe2+/H2O2 mixture in aqueous solution. To distinguish between the iron (II) and iron (III) combinations, the convention is to use Fenton-like reagent for the Fe3+/H2O2 mixture and restrict the use of Fenton's reagent to denote the Fe2+/H2O2 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.
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:
Fe2++H2O2→Fe3++OH−+OH−
A popular alternative mechanistic candidate is that first suggested by Bray and Gorin (citation), in which the ferryl ion, [FeIV O]2+, is supposed to be the active intermediate:
Fe2+H2O2→[FeIVO]2++H2O
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).
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 H2O2 are not rapid, and their in vivo metal ligands are unknown (citation). However, it has been shown recently that HCO3− and CO2 greatly accelerate the rate of H2O2 reduction by Mn2+ 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)
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).
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.
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═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═O, —NH2, —O—R, —OH (alcoholic), —S-thioether, as described in Current Medicinal Chemistry, 2004, 11, 2161-2183, incorporated herein by reference in its entirety.
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.
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).
A general structure of an effective iron chelator comprises the generic structure R-L-C-M and all the combinations thereof.
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.
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.
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.
Of the iron chelators, deferoxamine or DFO may be the most important, because it is FDA-approved for treatment of iron excess in thallasemia.
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.
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.
Other suitable iron chelators include those set forth in Table 2:
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.
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).
Like HBED, DFT and GT56-252 are both second generation hydroxypyridones that are in preclinical or phase I trials.
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.
S-DFO is a starch-bound DFO derivative that has a longer half-life after intravenous administration.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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)).
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.
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.
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.
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.
The ROS inhibitor, its formulation, and its modes of administration for this embodiment of the present invention are the same as those described above.
Here the subject is preferably a human prone to hyperglycemia or fatty acid flux.
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.
In this embodiment of the present invention, the above-identified compounds are formulated and administered in substantially the same way as noted above.
In this aspect of the present invention, the subject is a mammal, preferably a human.
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 IschemiaIt 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.
To address these problems, a novel model of graded ischemia in the dorsal soft tissue of mice has been created (
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 (
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 (
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 (
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 (
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 (
Based on findings implicating impaired HIF-1α transactivation in high glucose as the mechanism for impaired hypoxia-induced VEGF expression, potential post-translational modifications of HIF-1α were examined under these conditions. It was initially examined whether HIF-1α is a substrate for O-linked glycosylation. HIF-1α 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-1α was present under normal glucose conditions, there was significant glycosylation in high glucose (
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 (PPARγ) was reduced in conditions of high glucose compared to normal glucose by co-immunoprecipitation assays (
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.
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 (
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).
Once again, this difference was accentuated by hypoxia (
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. (
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 (
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 CellsCultured vascular endothelial cells were treated with deferoxamine to determine the effect of deferoxamine on hyperglycemia-induced reactive oxygen production by those cells.
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.
Intracellular reactive oxygen species measurements: The intracellular formation of reactive oxygen species was detected using the fluorescent probe CMH2DCFDA (Molecular Probes). Cells (1×105 ml-l) were loaded with 10 μM CM-H2DCFDDA, incubated for 45 rain at 37° 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).
As shown in
Thus, the iron chelator deferoxamine has a profound effect on vascular endothelial cells—i.e. it prevents completely hyperglycemia-induced overproduction of hydroxyl radicals (
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.
In order to detect intracellular free iron, cells were loaded with fura-2 AM in the dark at 37° C. for 15 min in 1 ml of TBSS containing 5 μM furs-2 AM. After loading, cells were incubated with TBSS with 1 ml of 20 μM 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× planapo objectives, run by I.P. Lab Spectrum on a Power PC computer. Analysis was performed with I.P. Lab Spectrum.
As shown in
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 μM 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.
As shown in
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 μM deferoxamine (DFO), or 30 mM glucose plus 100 μM DMSO, a hydroxyl radical scavenger, for 7 days. Medium with reagents was changed daily. DNA strand breakage was detected using the Comet assay method.
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.
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
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 μM deferoxamine, or 30 mM glucose plus 100 μM DMSO for 6 days and medium changed daily.
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 MgCl2, 0.01% digitonin, 25 mM NAD+, and 1 μCi/ml 3HNAD+ for 10 min at 37° C. TCA was added to precipitate ribosylated protein and cells were lysed in 2% NaOH. Detection of incorporated 3H-NAD was performed using a scintillation counter, and PARP activity determined according to the number of 3H-NAD dpm.
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
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 μM deferoxamine. The prostacyclin synthase product, PGF-1α, was measured 24 hours later.
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-PGFIα. Samples (100 μl) collected from BAECs culture medium were added to the assay plate, which was precoated with antibody (6-keto-PGF1α, EIA conjugate solution). PGFIα concentration was calculated according to a standard curve, and data analysis performed using AssayZap software.
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
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.
Prostacyclin synthase activity measurement A competitive immunoassay method (Correlate-EIA) was used for the quantitative determination of 6-keto-PGF1α. Mouse aortas were washed with PBS and incubated at 37° C. for 3 hours in 400 μl incubation buffer containing 20 mM TRIS buffer (pH 7.5), and 15 μM arachidonic acid. 100 μl of sample was used to measure the PGF1α.
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
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 μM deferoxamine, for 24 hour. Six hours before eNOS activity determination, media without arginine was added to the cells to deplete endogenous arginine.
Measurement of eNOS activity was accomplished as follows. BAECs were incubated with 400 μl of PBS-3H-arginine (1.5 μci/ml) buffer for 30 min at 37° C. The reaction was stopped by adding IN TCA (500 μl/well, ice cold), the cells were freeze fractured in liquid nitrogen for 2 min and thawed at 37° 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 3H-citrulline collected. Detection of 3H-citrulline was performed using a liquid scintillation counter, and eNOS activity was calculated from the amount of 3H-citrulline generated.
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
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.
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 μl of reaction buffer (3 μM Tetrahydrobiopterin, 1 mM NAPDH, 2.5 mM CaCl2, 200 U Calmodulin, 3H-L-arginine 0.2 μCi) for 45 min at 37° C. with rolling. After the incubation, samples were loaded onto Tris-formed DOWEX 50WX8 ion-exchange column and 3H-citrulline was collected. 3H-citrulline was quantitated using a liquid scintillation counter and eNOS activity was calculated from the amount of 3H-citrulline generated.
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
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.
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 IschemiaThe 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.
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.
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
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 IschemiaThe 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 (
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.
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)
Type: Application
Filed: Jul 28, 2017
Publication Date: Mar 15, 2018
Inventors: Geoffrey C. Gurtner (Stanford, CA), Michael A. Brownlee (New York, NY)
Application Number: 15/662,663
