WO2000023568A2 - Procedes et compositions permettant de reduire la surproduction de mitochondries des especes oxygene reactives dans les cellules - Google Patents

Procedes et compositions permettant de reduire la surproduction de mitochondries des especes oxygene reactives dans les cellules Download PDF

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WO2000023568A2
WO2000023568A2 PCT/US1999/015734 US9915734W WO0023568A2 WO 2000023568 A2 WO2000023568 A2 WO 2000023568A2 US 9915734 W US9915734 W US 9915734W WO 0023568 A2 WO0023568 A2 WO 0023568A2
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cells
glucose
insulin
oxygen species
reactive oxygen
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Michael Brownlee
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Albert Einstein College Of Medicine Of Yeshiva University
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Definitions

  • the field of the invention is related to methods and compositions for decreasing accumulation of reactive oxygen species (ROS) that damage cells, such as those involved in vascular and/or neurological disease and/or dysfunction, with intracellular hyperglycemia as a means of preventing and/or treating diabetic and hyperglycemic tissue damage or organ damage as well as age-related diseases in which tissues are damaged due to intracellular ROS production by mitochondria.
  • ROS reactive oxygen species
  • the invention is exemplified by the use of carbonyl cyanide m- chlorophenylhydrazone, theonyltrifluoroacetone and Mn (111) tetrakis (benzoic acid) porphyrin to decrease ROS generation and consequences, such as PKC activation, in insulin- independent cells in the presence of high glucose.
  • Diabetic complications are divided into those affecting the small blood vessels in the retina, kidney, and nerve, as well as nerve cells (microvascular complications), and those affecting the large blood vessels supplying the heart, brain, and lower limbs (macrovascular complications).
  • Diabetic microvascular complications are the leading cause of new blindness in people 20-74 years old, and account for 35% of all new cases of end-stage renal disease.
  • neuropathy includes distal symmetrical sensory poly neuropathy, mononeuropathies, and a variety of autonomic neuropathies causing erectile dysfunction, urinary incontinence, gastroparesis and nocturnal diarrhea.
  • Diabetes accounts for 50% of all non-traumatic amputations in the USA, primarily as a result of diabetic macrovascular disease, and diabetics have a death rate from coronary artery disease that is 2.5 times that of non-diabetics.
  • Hyperglycemia is believed to initiate and accelerate progression of diabetic microvascular complications based upon the fact that target tissues of complications tend to be those that do not require insulin for glucose transport into cells and/or which do not down- regulate glucose transport with hyperglycemia, thus putting mechanistic focus on the consequences of intracellular hyperglycemia.
  • Various intracellular mechanisms have been proposed to explain the damage that occurs and used as a basis for treatment of hyperglycemia induced complications.
  • Vascular abnormalities associated with this are normalized by pyruvate, which produces NAD by the action of lactate dehydrogenase.
  • pyruvate is a substrate for mitochondria and this treatment therefore has the disadvantage that it would generate free radicals in the mitochondria.
  • the focus is on diacylglycerol (DAG)/protein kinase C (PKC) activation: increased glucose results in the production of DAG and the subsequent activation of various PKC isoforms by DAG.
  • DAG diacylglycerol
  • PKC protein kinase C
  • Various vascular abnormalities are corrected by a specific PKC isoform inhibitor.
  • the disadvantages associated with the use of PKC inhibitors, even those that are theoretically specific, is that it is unlikely that the inhibitor is completely specific and/or it could inhibit essential PKC functions.
  • nonenzymatic glycation/advanced glycation endproducts is the focus: glucose and glucose metabolites covalently attach to amino groups of proteins and form a variety of complex AGE adducts, including cross-links.
  • Aminoguanidine prevents the formation of AGEs, in part by trapping reactive sugar-derived intermediates, and prevents most diabetic microvascular complications in animal models.
  • Aminoguanidine has a number of effects, including inhibiting an indue ible enzyme that makes nitric oxide in macrophages to fight infection. This treatment therefore has the disadvantage that it would impair immune function.
  • aminoguanidine exhibits tissue toxicity.
  • oxidative stress/lipid peroxidation hyperglycemia causes increased intracellular reactive oxygen species and lipid peroxidation.
  • a variety of deleterious consequences of these processes has been documented, including activation of NF K B.
  • a disadvantage of using non-catalytic antioxidants such as Vitamin E, ascorbate and glutathione is their finite ability to titrate continuously produced ROS.
  • ROS scavengers such as superoxide dismutases and catalase, as proteins, have a number of delivery and other shortcomings.
  • ROS reactive oxygen species
  • Antioxidants prevent aldose reductase activation (Ou et al, Free Radical Res 25:337-346, 1996), normalize altered redox state (SOD/catalase, desferoxamine) (Thies and autor, Arch Biochem Biophys 286:353-363, 1991), inhibit PKC activation (taurine, N-acetylcysteine) (Wolf et al, J Clin Invest 87:31-38, 1991; Sruder et al, Metabolism: Clinical and Experimental 46:918-25, 1997), prevent AGE formation (deferoxamine, alpha tocopherol, DMSO, penicillamine, catalase) (Giardino et al, J Clin Invest 97:1422-28, 1996; Fu et al, Diabetes -.43:616-83, 1994; Elgawish et al, J Biol Chem 271 :
  • Aldose reductase inhibitors also prevent loss of the cellular antioxidant taurine and chelate ROS-forming divalent metal ions (Ou et al, Free Radical Res 25:337-346, 1996; Malone et al, Pediatric Research 27:293-6, 1990).
  • the redox-correcting compound pyruvate is also a peroxide/hydroxyl scavenger (O'Donnell-Tormey et al, J Exp Med 165:500-14, 1987; Lee et al, J Biol Chem 273:5294-9, 1998).
  • the AGE-inhibitor aminoguanidine prevents hyperglycemia-induced ROS formation in vitro and in vivo (Giardino et al, Diabetes 47:1114-1120, 1998).
  • This invention is directed to methods and compositions for preventing and/or treating hyperglycemic or diabetic complications in a patient in need thereof by directly or indirectly decreasing the mitochondrial reactive oxygen species (ROS) generated in insulin-independent cells of the patient, particularly vascular cells and cells comprising peripheral neurons, or by counteracting the effects of ROS-induced mitochondrial DNA mutations.
  • ROS mitochondrial reactive oxygen species
  • Embodiments of the methods for preventing and/or treating tissue or organ abnormalities associated with diabetes and hyperglycemia include (1) providing the patient with a composition comprising an agent that decreases the level of hyperglycemia induced reactive oxygen species produced in insulin- independent cells, particularly by normal mitochondria; (2) reducing the number of mitochondria with DNA mutations that result from production of reactive oxygen species in insulin-independent cells by transfecting a cell with peptide-nucleic acids having sequences complementary to mitochondrial DNA that contain a deletion breakpoint or single base mutation as compared to wild-type mitochondrial DNA; and (3) decreasing the effects of an abnormal mitochondrial DNA gene product in a patient by introducing into the patient an expression cassette comprising as operatively linked components a transcriptional initiation region, a nucleotide sequence encoding an amino-terminal mitochondrial targeting sequence and a normal counterpart of the abnormal mitochondrial gene under conditions whereby the normal gene is expressed.
  • the invention additionally encompasses methods of screening for pharmaceutical agents that can be used for treating diabetic and hyperglycemic patients.
  • the method includes screening for agents that (1) decrease the level of or inhibit the formation of hyperglycemia- induced reactive oxygen species under high glucose conditions, including agents effective in partially uncoupling oxidative phosphorylation from electron transport in mitochondria;
  • the pharmaceutical agents identified find use in treating symptoms associated with diabetes and hyperglycemia in a patient in need thereof.
  • a patient's risk for development or progression of diabetic or hyperglycemic complications is determined by evaluating whether there is an increase in either (1) the levels of reactive oxygen species generated in fusion cells obtained by fusing non-nucleated or enucleated insulin-independent cells from a patient with nucleus donor cells lacking mitochondrial DNA, as compared to levels of reactive oxygen species in control cells or (2) the presence of risk-associated homoplastic polymorphisms in mitochondrial DNA in cells obtained from the patient as compared to a risk index for complications obtained by correlating the mitochondrial DNA sequences of homoplastic polymorphisms in insulin-independent cells obtained from individual members of a group of diabetics with development or progression of diabetic complications.
  • the invention finds use in the treatment and/or prevention of hyperglycemic or diabetic complications as well as symptoms of other diseases in which tissue is damaged due to elevated intracellular ROS production by mitochondria.
  • diseases include Alzheimer's Disease, Parkinson's Disease, and other age-related, tissue-degenerative diseases.
  • Figure 1 shows the effect of specific metabolic inhibitors on hyperglycemia-induced reactive oxygen species (ROS) formation in bovine aortic endothelial cells.
  • ROS reactive oxygen species
  • Figure 2 shows the effect of different mitochondrial blockades on hyperglycemia- induced cellular lipid peroxidation in BAE cells determined as thiobarbiruric acid reactive substances (TBARS).
  • BAE cells were incubated for 168 hours in media containing the following: (i) 5mM glucose (low glucose); (ii) 30mM glucose (high glucose); (iii) 30mM glucose plus 2mM azide; (iv) 30mM glucose plus 0.5 ⁇ M CCCP; (v) 30mM glucose plus 10 ⁇ M TTFA. The results are expressed as mean + SE of three experiments.
  • Figure 3 shows the effect of different mitochondrial blockades on hyperglycemia- induced activation of NF K B in BAE cells as measured by an electrophoretic mobility shift assay.
  • Figure 3 A are shown the results of incubating BAE cells for 2 hours in media containing the following: (i) 5mM glucose (low glucose); (ii) 30mM glucose (high glucose); (iii) 30mM glucose plus 2mM azide; (iv) 30mM glucose plus 0.5 ⁇ M CCCP; (v) 30mM glucose plus 10 ⁇ M TTFA; (vi) 30mM glucose plus 0.5 ⁇ M oligomycin.
  • the results are expressed as mean + SE of three experiments.
  • Figure 3B shows the effect of electron transport Complex II inhibition or oxidative phosphorylation uncoupling on hyperglycemia- induced activation of NF K B.
  • Cells were incubated 7 days in 5mM glucose alone, and in 30mM glucose, either alone, or with either thenoyltrifluoracetone (TTFA) or carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an electrophoretic mobility shift assay was performed.
  • TTFA thenoyltrifluoracetone
  • CCCP carbonyl cyanide m-chlorophenylhydrazone
  • Figure 4 shows the effect of different mitochondrial blockades on hyperglycemia- induced formation of methyglyoxal-derived advanced glycation endproducts (AGEs) in BAE cells determined by scanning densitometry of IH7G5 antibody immunoblots.
  • BAE cells were incubated for 168 hours in media containing the following: (i) 5mM glucose (low glucose); (ii) 30mM glucose (high glucose); (iii) 30mM glucose plus 2mM azide; (iv) 30mM glucose plus 0.5 ⁇ M CCCP; (v) 30mM glucose plus 10 ⁇ M TTFA. The results are expressed as mean + SE of three experiments.
  • Figure 5 shows the effect of different mitochondrial blockades on hyperglycemia- induced formation of glyoxal and/or fructosyllysine-derived advanced glycation endproducts (AGEs) in BAE cells determined by scanning densitometry of 6D12 antibody immunoblots.
  • AGEs glyoxal and/or fructosyllysine-derived advanced glycation endproducts
  • BAE cells were incubated for 168 hours in media containing the following: (i) 5mM glucose (low glucose); (ii) 30mM glucose (high glucose); (iii) 30mM glucose plus 2mM azide; (iv) 30mM glucose plus 0.5 ⁇ M CCCP; (v) 30mM glucose plus 10 ⁇ M TTFA.
  • the results are expressed as mean + SE of three experiments.
  • Figure 6 shows the effect of different mitochondrial blockades on PKC activity in BAE cells.
  • BAE cells were incubated for 5 days in media containing the following: (i) 5mM glucose (low glucose); (ii) 30mM glucose (high glucose); (iii) 30mM glucose plus 10 ⁇ M TTFA; (iv) 30mM glucose plus 0.5 ⁇ M CCCP.
  • the results are expressed as mean + SE of four experiments. *p ⁇ 0.01 as compared to 5mM glucose.
  • Figure 7 shows the effect of electron transport Complex II inhibition on hyperglycemia-induced sorbitol accumulation resulting from aldose reductase activity.
  • Figure 8 shows the effect of electron transport Complex II inhibition on hyperglycemia-induced overexpression of the tyrosine kinase vascular endothelial growth factor receptor flk-1 in BAE cells determined by scanning densitometry of immunoblots.
  • BAE cells ere incubated for 168 hours in media containing the following: (1) 5mM glucose (low glucose); (2) 30mM glucose (high glucose); (3) 30mM glucose plus 10 ⁇ M TTFA. The results are expressed as mean + SE of three experiments.
  • Figure 9 shows the effect of a superoxide dismutase mimetic on hyperglycemia- induced reactive oxygen species production (micromoles/ml).
  • BAE cells were incubated for 24 hours in 5mM glucose alone and in 30mM glucose alone, and with one of the indicated concentrations of Mn (111) tetrakis (benzoic acid) porphyrin (MnTBAP). The results are expressed as mean + SE of 8 experiments.
  • Figure 10 shows the effect of different mitochondrial blockades on hyperglycemia- induced reactive oxygen species accumulation (micromoles/ml) in cultured rat hepatocytes.
  • Methods and compositions are provided for preventing and/or treating hyperglycemic or diabetic complications in a diabetic patient in need thereof by directly or indirectly decreasing the mitochondrial reactive oxygen species (ROS) generated in insulin-independent hyperglycemic cells of the patient by mitochondria, particularly normal mitochondria, or by counteracting the effects of ROS-induced mitochondrial DNA mutations.
  • ROS mitochondrial reactive oxygen species
  • hyperglycemic or diabetic complications is intended any of a series of diabetes-induced abnormalities that cause morbidity and premature mortality. These include circulatory abnormalities, neurologic abnormalities and hepatic abnormalities. Circulatory abnormalities in large vessels affect peripheral arteries (causing intermittent claudication, gangrene, and in men, organic impotence), coronary arteries (causing myocardial infarction), and arteries supplying the brain
  • Circulatory abnormalities in small vessels affect the retina (causing blindness), the kidney (causing renal failure), and the heart (causing heart failure).
  • Neurologic abnormalities affect every part of the nervous system with the possible exception of the brain, causing pain, numbness, autonomic dysfunction, and foot ulcers leading to amputation.
  • Hepatic abnormalities include non-physiologic overproduction of glucose and production of a pro-atherogenic secreted protein profile.
  • a diabetic patient is intended a patient with a degree of glucose intolerance sufficient to produce diabetic complications as defined by the American Diabetes Association in Diabetes Care (1998) 21:(Supplement 1).
  • insulin-independent cells By insulin-independent cells is intended cells that either do not require insulin for glucose transport and/ or do not down-regulate glucose transport in response to increasing glucose concentrations, i.e., those cells which develop intracellular hyperglycemia in response to high blood glucose, above the normal range, to levels occurring in diabetics.
  • a diabetic patient can be treated in one of several ways.
  • a diabetic patient is provided with a composition which comprises an agent that decreases the level of hyperglycemia induced reactive oxygen species in insulin-independent cells.
  • compositions used include compounds that partially uncouple oxidative phosphorylation from electron transport in mitochondria, inhibit mitochondrial electron transport Complexes I and/or II, dismutate superoxide and/or hydrogen peroxide, or inhibit binding and activation of hexokinase isoforms to or by the mitochondrial membrane.
  • a second embodiment of treating and/ or preventing diabetic and hyperglycemia-related complications is to reduce the number of mitochondria with DNA mutations associated with diabetes that result from production of ROS in insulin-independent cells by transfecting cells of a diabetic patient with peptide nucleic acids having sequences complementary to mitochondrial DNA that contains a deletion breakpoint or single base mutation associated with diabetes as compared to wild-type mitochondrial DNA complexed to mitochondrial targeting peptides.
  • the effects of an abnormal mitochondrial DNA gene product in a patient are ameliorated by introducing into the patient an expression cassette comprising as operatively linked components a transcriptional initiation region, a nucleotide sequence encoding an amino-terminal mitochondrial targeting sequence and a normal counterpart of the abnormal mitochondrial gene.
  • This invention also pertains to methods for identification of a patient's risk for development or progression of diabetic or hyperglycemic complications by comparing the levels of reactive oxygen species in fusion cells obtained by fusing non-nucleated or enucleated insulin-independent cells from a patient with nucleus donor cells lacking mitochondrial DNA, with levels of reactive oxygen species in control fusion cells including cells from patients having hemoplactic polymorphisms in the mitochondrial DNA.
  • the control fusion cells generally are prepared using cells from non-diabetics.
  • the subject invention has several advantages over existing methods for treating diabetics to prevent or retard diabetic complications. Because the invention centers upon the identification of mitochondrial genetics that predispose the individual patient to diabetic or hyperglycemic complications, it is superior to any prior art method for identification of those at risk for such complications. For the same reason it is a superior method for development of mitochondrial gene therapy and elimination of mitochondria which predispose cells to diabetes instigated tissue or organ damage.
  • the invention identifies the mitochondrial genetics of predisposition to hyperglycemic injury by measuring the chemical species causing the cellular pathology, namely mitochondrial ROS and/or species either caused or correlated to elevated ROS, therefore the invention also is superior to existing methods for identifying chemical or pharmacologic agents which reduce ROS and therefore have the potential for prophylactics and/or treatment of diabetic complications.
  • mitochondrial production of ROS is the central event that leads to tissue or organ damage of cells with intracellular hyperglycemia. If this theory is correct, then mechanisms proposed by others and the various dysfunctions related to intracellular damage are not causes but are instead consequences of mitochondrial ROS production.
  • the instant treatments based upon reducing mitochondrial production of ROS therefore are treating the primary abnormality and can be used to prevent not only the known consequences of increased ROS, but also consequences not yet known to be involved with the pathogenesis of organ dysfunction in diabetic patients.
  • the subject invention also offers advantages over the use of antioxidants such as Vitamin E; such treatments generally are not effective because they cannot penetrate into the mitochondrial compartment or because they are used up before they have had the desired effect.
  • Vitamin E interferes with absorption of Vitamin K. It therefore is more efficient to inhibit mitochondrial production of ROS.
  • hyperglycemia-induced reactive oxygen species in hepatocytes induces increased expression of the enzyme glucose-6-phosphatase and/or products in the liver that increase very low density lipoprotein (VLDL) and low density lipoprotein (LDL), increased oxidized lipoproteins, or decrease high density lipoprotein (HDL).
  • VLDL very low density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • Overproduction of glucose by the liver is the major cause of fasting hyperglycemia in both insulin-dependent and non-insulin-dependent diabetes mellirus.
  • Atherogenic molecule appears to be an oxidatively- modified form of low density lipoprotein (Hajjar and Haberland, J. Biol. Chem. (1997) 272:22975-8).
  • LDL modified by advanced glycation endproducts may also be more atherogenic (Wang et al. , Proc. Natl. Acad. Sci. (1998) 95:7643-7; Zhang et al. , Thrombosis and Vascular Biology (1998) 18: 1140-8).
  • Such lipoprotein modification may occur extracellularly, e.g., by extracellular exposure to ROS or AGE-precursors produced by endothelial cells.
  • An unexplored possibility is that lipoprotein modification occurs inside the hepatocytes before the lipoproteins are secreted.
  • hyperglycemia- generated ROS and its consequences could explain the increased amount of atherogenic oxidized and age-modified lipoproteins observed in diabetic patients, as well as atherogenic changes in the production levels of various lipoproteins. Decreasing hyperglycemia-induced ROS in hepatocytes therefore can play a major role in preventing diabetic complications.
  • Agents useful in reducing ROS in insulin-independent cells are identified using a screening assay which uses cultures of any of a variety of mammalian insulin-independent cells, preferably primary cells or cells which have been passaged for only a few passages and retain the intrinsic phenotype of cells, such as endothelial cells, from a particular tissue.
  • a screening assay which uses cultures of any of a variety of mammalian insulin-independent cells, preferably primary cells or cells which have been passaged for only a few passages and retain the intrinsic phenotype of cells, such as endothelial cells, from a particular tissue.
  • the cells are isolated from tissues which contain insulin-independent cells, including the eye (retinal endothelial cells and pericytes); kidney (glomerulus endothelial cells, epithelial cells, and mesangial cells); artery (arterial endothelial cells and smooth-muscle cells); heart (endothelial cells, and cardiac myocytes); nerve (neurons and Schwann cells); blood (monocytes, macrophages, lympocytes, and platelets); and hepatoctytes.
  • the cell cultures generally are cultured in medium containing either physiologic levels of glucose, 5mM glucose, which is equivalent to a 90 mg/dl blood level, or an amount of glucose which is equivalent to typical blood levels of glucose seen in diabetic patients, generally >5-50mM glucose, preferably 20-40mM glucose, most preferably 30mM glucose, with or without the agent to be tested.
  • Agents which are candidates for testing include those which inhibit the activity or expression of a molecule, generally an enzyme, in one or more of the pathways for glucose- derived ROS produced by the mitochondria or act as mitochondrial superoxide dismutase mimetics.
  • ROS are produced as a byproduct of electron transport, primarily at the interface between the flavin dehydrogenases and ubiquinone (CoQ), and at Complex III (Wallace, Annu.Rev. Biochem. 61 : 1175-1212, 1992; Forman and Boveris, Free Radicals in Biology, Pryor, WA (ed) vol. V, pp 65-90. Academic Press, 1982; Kwong and Sohal, Arch. Biochem. Biophys. (1998) 350:118-126). The principal site of ROS generation differs markedly among various cell types (Kwong and Sohal, Arch. Biochem. Biophys. (1998) 350:118-126).
  • Electron flow through the mitochondrial electron transport assembly is carried out by 4 inner membrane enzyme Complexes (I-IV), plus cytochrome c and the mobile carrier CoQ. Electrons enter the transport chain from NADH (Complex I) or FADH2 (Complex II). Electrons from both complexes are transferred to CoQ, and then to Complex III,
  • agents that can be used to inhibit electron flow generated ROS include, e.g., amytal and rotenone (Complex I); TTFA (Complex II), and partial uncouplers of oxidative phosphorylation, such as CCCP.
  • Inhibitors of Complex I such as amytal and rotenone, prevent ROS generation in some systems, for example, the J774J6 macrophage-like cell line (Kiyotaki et al. , Oxygen metabolism in cloned macrophage cell lines: glucose dependence of superoxide production, metabolic and spectral analysis. Journal of Immunology (1984) 132:857-66), and L929 cells (Schulze-Osthoff et al. , Annu. Rev. Biochem. (1992) 61 : 1175-1212).
  • TTFA Thenoyltrifluoroacetone
  • partial uncouplers that can be used include protein uncouplers from other cell types.
  • Nucleic acid, particularly cDNA, coding for uncoupling proteins (UCP 1, 2, 3), expression of which is preferably driven by a glucose responsive promoter element to specifically act during periods of hyperglycemia can be used. Odagiri et al, J. Biol. Chem. 271 : 1909-15, 1996; Fleury et al, Nature Genetics 15:269-72, 1997.
  • Rate limiting electron acceptors such as CoQ and analogues of CoQ also can be used (Langsjven et al. , PNAS (1985) 82:4240-4044; Nishikawa et al.
  • Electrons channeled via Complex II produce about 4 times more superoxide than those channeled via Complex I (Forman and Boveris, supra) and agents that are Complex II inhibitors are therefore preferred as compared to agents that inhibit Complex I.
  • agents that can inhibit Complex I include amytal and rotenone.
  • agents that can be used to scavenge superoxide include superoxide/catalase mimetics such as the salen-manganese complexes exemplified by EUK-8 described in Doctrow et al, Advances in Pharmacology (1997) 38:247-269 and manganese porphyrins described in Day et al, Archives Biochem Biophys (1997) 347:256-262 and Patel, J. Neurochem (1998) 71 : 1068-1074.
  • NADH (Complex I) substrate and FADH2 (Complex II) substrate are generated by mitochondrial TCA-cycle oxidation of cytosolic glycolysis-derived pyruvate (3 moles of NADH to 1 mole of FADH2), with additional glucose oxidation-derived NADH equivalent(s) transferred from the cytosol into the mitochondria via the malate (mitochondrial NADH) and/or glycerol phosphate (mitochondrial FADH2 ) shuttles. These shuttles utilize both cytosolic and mitochondrial flavin dehydrogenases (Wallace, Annu.Rev. Biochem.
  • agents that can be used to inhibit the malate shuttle include aminooxy acetate; and to inhibit the glycerol phosphate shuttle include analogues of glycerol-3- phosphate that are not metabolized.
  • Increased cytosolic glucose accelerates mitochondrial electron transport by (a) increasing the glucose oxidation rate by inducing mitochondrial binding and activation of hexokinase; (b) increasing cytosolic NADH through glycolysis and other glucose oxidative pathways, and (c) increasing pyruvate formation through glycolysis (Wallace, supra; and
  • agents that can be used to inhibit mitochondrial binding and activation of hexokinase include glycerol and analogues of glycerol.
  • Agents of interest are tested by contacting a predetermined quantity of a test compound with the cultured primary or low passage (generally ⁇ 14 passages) insulin-independent cells and comparing the results of incubating the cells in high glucose with low and high glucose alone.
  • a phenotypic trait in the cells is measured that is related to production of ROS either directly or indirectly and whether and to what extent the agent of interest can cause the trait to become more similar to that of the cells evaluated in physiologic glucose is determined.
  • the screening assays include the following. Effects on the level of inhibition of hyperglycemia- induced ROS formation in the cell culture system is measured using the fluorescent ROS- detector DCF and FACS as a readout (Giardino et al, J Clin Invest 97: 1422-28, 1996). Partial inhibition of Complex II, Complex I, or partial uncoupling action on oxidative phosphorylation, which converts state 4 to a pseudo state 3, is measured by the resultant decrease in ROS production (Skulachev, Q. Rev. Biophys. 29: 169-2021, 1996; Korshunov et al. , FEBS Letters 416:15-18, 1997).
  • Complex Il-activated antioxidant function (Suno and
  • hexokinase isoforms The effect of inhibitors of activation of hexokinase isoforms can be evaluated by separating cytosol and mitochondria by standard techniques after exposure to high glucose and assessing enzyme activity (Adams et al. , Nature Genetics 15:212-215, 1997).
  • cultured fusion cells For identifying a patient at risk for development or progression of diabetic complications, cultured fusion cells, cybrids, are used.
  • the cybrids are obtained by fusing non-nucleated insulin-independent cells from the patient with nucleus donor cells which lack mitochondrial DNA (mtDNA).
  • the non-nucleated insulin independent cells can be either circulating or non-circulating cells. Insulin independent cells which naturally do not include a nucleus include platelets. Other non-nucleated insulin independent cells can be obtained by enucleation of the cells using techniques known to those skilled in the art. See for example
  • Circulating cells for enucleation are conveniently obtained from a blood sample and include, for example, monocytes, lymphocytes and circulating endothelial cells.
  • Non-circulating cells that are insulin independent can be obtained by standard biopsy techniques from an appropriate tissue.
  • the nucleus donor cells can be any mammalian cell line, preferably a primate cell line, and more preferably a human cell line which lacks mitochondrial DNA.
  • mtDNA of the nucleus donor cells can be removed, for example, by treatment with ethidium bromide.
  • the risk analysis is carried out by comparing the levels of ROS formed in cybrids produced using the patient cells either under high glucose conditions alone or under both high and normal glucose conditions.
  • the levels of ROS formed are then compared to the level of ROS in control cybrids obtained by fusing non-nucleated insulin independent cells from non-diabetics.
  • An increased level of ROS in the patient cybrids as compared to the control cybrids identifies a patient at risk for the development or progression of diabetic or hyperglycemic complications.
  • the ROS is measured by fluorescence activated cell sorting. Giardino et al, supra.
  • a second method for predicting the risk of diabetic or hyperglycemic complications in a patient involves preparing a risk index for development of complications.
  • the risk index is obtained by correlating the mtDNA sequences of homoplastic polymorphisms in insulin independent cells obtained from a plurality of diabetics with developmental progression of diabetic complications in each individual of the plurality of diabetics.
  • the mtDNA sequences of homoplastic polymorphisms in an individual patient can then be compared with the risk index as a means of predicting the risk of diabetic or hyperglycemic complications.
  • the mtDNA sequences of homoplastic polymorphisms can be determined according to the method of Merriwether et al. , Journal of Molecular Evolution 33:543-555, 1991.
  • abnormal mitochondrial function in diabetic patients can be corrected by transfer of normal mitochondrial genes into the nucleus of a patient's cells, particularly cells which are subject to diabetic complications and/or already compromised by diabetic complications.
  • An expression cassette is prepared which includes an amino-terminal mitochondrial targeting sequence and an appropriate promoter together with the first exon and upstream sequences of a nuclear-encoded oxidative phosphorylation gene (for example, ATP synthase) and the mtDNA gene.
  • a nuclear-encoded oxidative phosphorylation gene for example, ATP synthase
  • Appropriate promoters capable of directing the transcription of a cloned gene or cDNA include both viral promoters and cellular promoters.
  • Viral promoters include the immediate early cytomegalovirus (CMV) promoter (Boshart et al, (1985) Cell 41:521- 530), the SV40 promoter (Subramani et al, (1981) Mol. Cell. Biol. 1:854-864) and the major late promoter from Adenovirus 2 (Kaufman and Sharp, (1982) Mol. Cell. Biol. 2:1304-13199).
  • CMV immediate early cytomegalovirus
  • SV40 promoter Subramani et al, (1981) Mol. Cell. Biol. 1:854-864
  • Adenovirus 2 Kaufman and Sharp, (1982) Mol. Cell. Biol. 2:1304-13199.
  • Promoters for tissue specific expression in vivo include the following.
  • the alpha-myosin heavy chain promoter can be
  • Neuron specific expression can be obtained using the neuron-specific enolase (NSE) promoter (Klein et al, (1998) Exp. Neurol. 150: 183-194 and Rondi-Reig et al, (1997) Neuroreport 8:2429-2432).
  • NSE neuron-specific enolase
  • the expression cassette can be inserted into a viral vector with the appropriate tissue trophism.
  • viral vectors include adenovirus, Herpes simplex virus, adeno- associated virus, retrovirus, lentivirus, and the like. See Blomer et al. , Human Molecular Genetics 5 Spec No: 1397-404, 1996; and Robbins et al , Trends in Biotechnology 16:35-40, 1998.
  • Adenovirus-mediated gene therapy has been used successfully to transiently correct the chloride transport defect in nasal epithelia of patients with cystic fibrosis. See Zabner et al, Cell 75:207-216, 1993.
  • the invention provides for a method of selecting non-mutated mitochondria by transfecting a cell with peptide-nucleic acids having sequences complimentary to mitochondrial DNA that contain a deletion breakpoint or single base mutation as compared to wild-type mitochondrial DNA so that replication of the mitochondrial DNA is blocked and mitochondria with DNA mutations are eliminated.
  • transfection in vivo is obtained by introducing a therapeutic transcription or expression vector into the mammalian host, either as naked DNA, complexed to lipid carriers, particularly cationic lipid carriers, or inserted into a viral vector, for example a recombinant adenovirus.
  • a therapeutic transcription or expression vector into the mammalian host, either as naked DNA, complexed to lipid carriers, particularly cationic lipid carriers, or inserted into a viral vector, for example a recombinant adenovirus.
  • the introduction into the mammalian host can be by any of several routes, including intravenous or intraperitoneal injection, intratracheally, intrathecally, parenterally, intraarticularly, intranasally, intramuscularly, topically, transdermally, application to any mucous membrane surface, corneal installation, etc.
  • a therapeutic expression vector into a circulating bodily fluid or into a body orifice or cavity, such as the heart.
  • intravenous administration and intrathecal administration are of particular interest since the vector may be widely disseminated following such routes of administration, and aerosol administration finds use with introduction into a body orifice or cavity.
  • Particular cells and tissues can be targeted, depending upon the route of administration and the site of administration. For example, a tissue which is closest to the site of injection in the direction of blood flow can be transfected in the absence of any specific targeting.
  • An arterial catheter can be used to introduce the expression vector into an organ such as the heart or kidney. The eye can be accessed directly either by the use of ocular drops or by injecting into the eye.
  • lipid carriers For accessing nerves, this can be by injection into the nerve or injection into the region of the cell body.
  • lipid carriers are used, they can be modified to direct the complexes to particular types of cells using site-directing molecules.
  • antibodies or ligands for particular receptors or other cell surface proteins may be employed, with a target cell associated with a particular surface protein.
  • An amino terminal mitochondrial targeting sequence joined to a nucleic acid can be used to target the nucleic acid to the mitochondria. See Taylor et al, Nature Genetics 15:212-
  • any physiologically acceptable medium may be employed for administering the DNA, recombinant viral vectors or lipid carriers, such as deionized water, saline, phosphate-buffered saline, 5 % dextrose in water, and the like as described above for the pharmaceutical composition, depending upon the route of administration.
  • Other components may be included in the formulation such as buffers, stabilizers, biocides, etc. These components have found extensive exemplification in the literature and need not be described in particular here. Any diluent or components of diluents that would cause aggregation of the complexes should be avoided, including high salt, chelating agents, and the like.
  • the amount of therapeutic vector used will be an amount sufficient to provide for a therapeutic level of expression in a target tissue susceptible to diabetic complications or for adequate dissemination to a variety of tissues after entry into the bloodstream and to provide for a therapeutic level of expression in susceptible target tissues.
  • a therapeutic level of expression is a sufficient amount of expression to prevent, treat, or palliate one or more diabetic complication or the symptoms of diabetic complications.
  • the dose of the nucleic acid vector used must be sufficient to produce a desired level of transgene expression in the affected tissue or tissues in vivo.
  • Other DNA sequences, such as adenovirus VA genes can be included in the administration medium and be co-transfected with the gene of interest. The presence of genes coding for the adenovirus VA gene product may significantly enhance the translation of mRNA transcribed from the expression cassette if this is desired.
  • a number of factors can affect the amount of expression in transfected tissue and thus can be used to modify the level of expression to fit a particular purpose. Where a high level of expression is desired, all factors can be optimized, where less expression is desired, one or more parameters can be altered so that the desired level of expression is attained. For example, if high expression would exceed the therapeutic window, then less than optimum conditions can be used.
  • the level and tissues of expression of the recombinant gene may be determined at the mRNA level as described above, and/or at the level of polypeptide or protein. Gene product may be quantitated by measuring its biological activity in tissues.
  • protein activity can be measured by immunoassay as described above, by biological assay such as inhibition of ROS, or by identifying the gene product in transfected cells by immunostaining techniques such as probing with an antibody which specifically recognizes the gene product or a reporter gene product present in the expression cassette.
  • the therapeutic cassette is not integrated into the patient's genome. If necessary, the treatment can be repeated on an ad hoc basis depending upon the results achieved. If the treatment is repeated, the patient can be monitored to ensure that there is no adverse immune or other response to the treatment.
  • compositions comprising agents identified as directly or indirectly inhibiting ROS in insulin independent cells, the mtDNA and polynucleotide compositions of the present invention are useful in treatment and prevention of complications related to hyperglycemia and diabetes. They also can be used to treat other diseases in which mtDNA is damaged due to elevated intracellular ROS. These diseases include Alzheimer's Disease, Parkinson's Disease, and other age-related, tissue-degenerative diseases.
  • Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. Pharmaceutically acceptable carriers and formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985), which is incorporated herein by reference. For a brief review of methods for drug delivery, see Langer, (1990) Science 249: 1527-1533, which is incorporated herein by reference.
  • compositions of the present invention it may be desirable to modify the compositions of the present invention to alter their pharmacokinetics and biodistribution.
  • pharmacokinetics see Remington's Pharmaceutical Sciences, supra, Chapters 37-39.
  • methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art (See, e.g.,
  • agents of the present invention can be incorporated into liposomes in order to enhance their pharmacokinetics and biodistribution characteristics.
  • methods are available for preparing liposomes, as described in, e.g., Szoka et al,
  • the agents of the present invention can be used in pharmaceutical compositions that are useful for administration to humans.
  • the pharmaceutical compositions of the invention are intended for parenteral, topical, oral or local administration.
  • the pharmaceutical compositions can be administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • the invention provides compositions that comprise a solution of the agents described above dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • a variety of pharmaceutically acceptable aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered.
  • the resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the compositions can contain as pharmaceutically acceptable carriers, substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanol amine oleate, and the like.
  • nontoxic pharmaceutically acceptable carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25% -75% .
  • the pharmaceutical compositions are preferably supplied in finely divided form along with a surfactant and propellant as pharmaceutically acceptable carriers.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides, can be employed.
  • a carrier also can be included, as desired, as with, for example, lecithin for intranasal delivery.
  • compositions of the invention can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include powder, tablets, pills, and capsules.
  • the effective amount of a particular agent in a pharmaceutical composition depends on, for example, the chemical nature of the agent, the manner of administration, the weight and general state of health of the patient, the severity of the disease being treated and the judgment of the prescribing physician. Dosages, formulations and administration schedules can vary in these patients as compared to normal individuals. In general, dosages range from about 100 ⁇ g to about 500 mg or more, with dosages of from about 250 ⁇ g to about 50 mg being more commonly used. It must be kept in mind that the materials of the present invention may be employed in serious disease or injury states, and in such cases it is possible and may be felt desirable by the treating physician to administer substantial excesses of these compositions.
  • the effect of treatment of diabetic complications can be evaluated as follows. Where it is desired to treat a particular diabetic complication, both the biological efficacy of the treatment modality as well as the clinical efficacy are evaluated, if possible.
  • the biological efficacy of the treatment can be evaluated, for example, by observation of return of the impaired left-ventricular relaxation to normal or cessation of progression.
  • the clinical efficacy, whether treatment of the underlying defect is effective in changing the course of disease, can be more difficult to measure.
  • diabetic retinopathy therapy can be evaluated by before and after treatment observations of changes in the disease manifestations.
  • Diabetic retinopathy is divided into three levels— (1) no diabetic retinopathy, (2) nonproliferative diabetic retinopathy, and (3) proliferative diabetic retinopathy.
  • macular edema can be present at any level of retinopathy and is also graded.
  • No retinopathy refers to a clinically normal fundus.
  • Nonproliferative retinopathy is subdivided into four groups: mild, moderate, severe and very severe.
  • Each level is precisely defined by the presence and degree of specific clinical features— hemorrhages with or without micoaneurysms, venous bleeding, and intraretinal microvascular abnormalities (see Early Treatment Diabetic Retinopathy Study Report Number 10: Grading diabetic retinopathy from stereoscopic color fundus photographs— an extension of the modified Airlie House classification. Opthalmology 98:786-806, 1991).
  • Proliferative retinopathy indicates the presence of any retinal neovascularization. The extent and progression or regression of diabetic retinopathy are evaluated and monitored by fundus photographs, direct or indirect ophthalmoscopy, and fluorescein angiography of the retina. The presence of retinal thickening within the macula constitutes macular edema.
  • kits for use in one or more operations can be provided for use in one or more operations.
  • the kits will include a composition comprising an effective agent either as concentrates (including lyophilized compositions), which may be further diluted prior to use or they may be provided at the concentration of use, where the vials may include one or more dosages.
  • Kits for genetic therapy usually will include the therapeutic mtDNA construct either as naked mtDNA with or without mitochondrial targeting sequence peptides, as a recombinant viral vector or complexed to lipid carriers.
  • lipid carriers and mitochondrial targeting peptides can be provided in separate containers for complexing with the provided mtDNA.
  • the therapeutic mtDNA can be present as concentrates which may be further diluted prior to use or they may be provided at the concentration of use, where the vials may include one or more dosages.
  • single dosages can be provided in sterile vials so that the physician may employ the vials directly, where the vials will have the desired amount and concentration of agents.
  • the subject compositions can be contained in packaging material, which comprises a label indicating that the subject compositions can be used to treat diabetic complications in humans. The following examples are offered by way of illustration and not by way of limitation. EXAMPLES
  • Bovine aorta endothelial (BAE) cells were obtained from the N.I.A. cell repository in Camden, N.J.. The cells were cultured in Eagles Minimal Essential Medium (MEM) containing 10% fetal bovine serum (FBS). Replicate cultures of 6-8 x 10 6 cells in 300 mm dishes were prepared. The experimental medium is MEM plus 0.4% FBS to put the cells in a growth-arrested state. Cytosolic NADH is transferred into mitochondria predominantly through the malate-aspartate shuttle, while mitochondrial NADH and FADH 2 are generated by the TCA cycle from cytosolic pyruvate.
  • MEM Eagles Minimal Essential Medium
  • FBS fetal bovine serum
  • bovine aortic endothelial cells were incubated in the presence of aminooxyacetate, a well-characterized inhibitor of the malate-aspartate shuttle.
  • the intracellular formation of reactive oxygen species was detected by using the fluorescent probe CM-H 2 DCFDA (Molecular Probes Inc., Eugene, OR).
  • Cells (5 x lOVml) were loaded with 10 ⁇ M CM-H 2 DCFDDA, incubated for 45min at 37°C, and analyzed in a HTS 7000 Bio
  • BAE were incubated with either amytal (500 ⁇ M), an inhibitor of Complex I, TTFA (10 ⁇ M), an inhibitor of Complex II, carbonyl cyanide m- chlorophenylhydrazone (CCCP) (0.5 ⁇ M), an uncoupler of oxidative phosphorylation that abolishes the mitochondrial membrane proton gradient, 4-OHCA (250 ⁇ M), or AOAC
  • Example 2 Effect of different mitochondrial blockades on cellular lipid peroxidation.
  • BAE cells were obtained as described in Example 1. Lipid peroxides were determined fluorimetrically (Giardino et al, J. Clin. Invest. (1996) 97: 1422-28).
  • Figure 2 shows levels of lipid peroxidation as a percentage of the value of cells incubated in low glucose determined as TBARS in BAE cells incubated for 168 hours in medium containing either 5 mM glucose, 30 mM glucose or 30 mM glucose plus 2mM azide, 10 ⁇ M TTFA or 0.5 ⁇ M CCCP.
  • Lipid peroxide (LP) levels in cells grown in media containing 30mM glucose and TTFA are approximately equivalent to LP levels in cells grown in media containing only 5mM glucose.
  • LP levels in cells grown in media containing 30mM glucose and CCCP are decreased as compared to cells grown in 30mM glucose alone to approximately 120% of LP levels in cells grown in media containing only 5mM glucose; cells grown in 30mM glucose have approximately 150% the LP levels seen in cells grown in 5mM glucose.
  • the levels of LP are decreased as compared to cells grown in 30mM glucose alone to approximately the level in cells grown in 5mM glucose.
  • Cells grown in 30mM glucose and azide have LP levels higher than for cells grown in 30mM glucose alone (approximately 180% of LP levels measured in cells grown in 5mM glucose). Lipid peroxides were determined fluorimetrically as described in Giardino et al, J. Clin. Invest. (1996) 97:422-28.
  • Example 3 Hyperglycemia activates NF K B, in part by activation of PKC (Pieper and Haq,
  • FIG. 3A shows levels of NF K B activation (percent of low glucose) as measured using an electrophoretic mobility shift assay kit (Gel Shift Assay Kit) from Promega (Madison, WI); the kit was used according to the manufacturer's instructions. Gels were transferred to
  • NF K B activation levels in cells grown in media containing 30mM glucose and TTFA or CCCP are reduced to a level approximately equivalent to NF K B activation levels in cells grown in media containing only 5mM glucose, whereas cells grown in 30mM glucose alone have nearly 200% the NF K B activation levels seen in cells grown in 5mM glucose.
  • Cells grown in 30mM glucose and azide or oligomycin have NF K B activation levels that are increased above the levels for cells grown in 30mM glucose alone to between approximately 250-280% of NF K B activation levels measured in cells grown in 5mM glucose. This activation by glucose was specific for the NF K B p50 subunit (lower band of NF K B binding activity, Figure 3B).
  • NF K B activation by 30mM glucose was completely inhibited by both TTFA and CCCP. This observation indicates that glucose activates endothelial cell NF K B by inducing mitochondrial ROS overproduction, rather than by inducing activation of ROS-producing cytosolic NADPH oxidases.
  • Example 4 Effect of different mitochondrial blockades on levels of methylglyoxal-derived AGEs.
  • FIG. 4 shows levels of methylglyoxal-derived AGEs (MAGEs) as determined by scanning densitometry (Maytin et al, Anal Biochem 194: 284 294, 1991) of immunoblots made with a 1 :4000 dilution of 1H7G5 antibody (Giardino et al, Diabetes 47 (Supp 1) A: 123, 1998) in BAE cells incubated for 168 hours in medium containing either 5 mM glucose, 30 mM glucose or 30 mM glucose plus 2mM azide, 10 ⁇ M TTFA or 0.5 ⁇ M CCCP. Cells grown in 30mM glucose have approximately 2 times the MAGE levels seen in cells grown in 5mM glucose.
  • MAGEs methylglyoxal-derived AGEs
  • MAGE levels in cells grown in 30mM glucose and azide have MAGE levels approximately 2.5 times those of cells grown in 5mM glucose.
  • MAGE levels in cells grown in media containing 30mM glucose and TTFA or CCCP are equivalent to MAGE levels in cells grown in media containing only 5mM glucose. Since methylglyoxal is formed by fragmentation of glyceraldehyde-3-phosphate, this dependency on increased mitochondrial ROS production may reflect the well-described reversible inhibition of glyceraldehyde-3-phosphate dehydrogenase by ROS, which increases glyceraldehyde-3-phosphate levels (Knight et al, Cardiovasc. Res. (1996) 32:1016-1023).
  • Figure 5 shows levels of glyoxal and/or fructosyllysine-derived AGEs (GFAGEs) as determined by scanning densitometry (Maytin et al , Analytical Biochem (1991) 194:284-294) of immunoblots made with 6D12 antibody (Ikeda et al, Biochem 35:8075-83, 1996) in BAE cells incubated for 168 hours in medium containing either 5 mM glucose, 30 mM glucose or 30 mM glucose plus
  • GFAGE levels in cells grown in media containing 30mM glucose and TTFA or CCCP are reduced to approximately equivalent to GFAGE levels in cells grown in media containing only 5mM glucose as compared to cells grown in 30mM glucose alone which have approximately twice the GFAGE levels seen in cells grown in 5mM glucose.
  • Cells grown in 30mM glucose and azide have GFAGE levels approximately 2.5 times of those measured in cells grown in 5mM glucose.
  • Example 6 Effect of different mitochondrial blockades on PKC activity.
  • BAE cells were obtained as in Example 1.
  • Figure 6 shows PKC activity (pmol/min/ mg protein) in BAE cells incubated for 5 days in medium containing either 5 mM glucose, 30 mM glucose or 30 mM glucose plus either 10 ⁇ M TTFA or 0.5 ⁇ M CCCP.
  • Subcellular fractions were prepared and PKC activities in soluble and detergent-solubilized preparations were determined as the phosphorylation of acetylated myelin basic protein amino acids 4-14 ((Ac-MBP) 4-14).
  • soluble fraction cytosol
  • TTFA acetylated myelin basic protein amino acids 4-14
  • CCCP acetylated myelin basic protein amino acids 4-14
  • Example 7 Effect of electron transport Complex II inhibition on hyperglycemia-induced sorbitol accumulation. Increased flux through the polyol pathway is a third mechanism that appears to play a major role in the pathogenesis of diabetic complications (Lee et al, Proc. Nat 'I Sci USA
  • BAE cells obtained as described in Example 1 were incubated for 240 h. 30 ⁇ l of a neutralized perchloric acid cell extract was added to 100 ⁇ l of working reagent containing 23 Units Diaphorase, 40 mg NAD and 50 ⁇ l resazurin/50 ml triethanolamine buffer pH 8.5. 0J5U of sorbitol dehydrogenase in 10 ⁇ l was then added. The assay was done in a 96 well flourescence plate and incubated at room temperature with an opaque cover and mixed on an orbital shaker for 60 minutes.
  • Example 8 Effect of electron transport Complex II inhibition on tyrosine kinase vascular endothelial growth factor receptor.
  • expression of the tyrosine kinase vascular endothelial growth factor receptor flk-1 was examined.
  • Flk-1 mRNA and protein are both increased in retinae of diabetic rats (Hammes et al, Diabetes (1998) 47:401-6).
  • 30mM glucose increased flk-1 protein levels by 2-fold compared to levels at 5mM glucose (637 + 52 vs. 329+ 523 AU).
  • Equal amounts of cell extract protein were used for quantitative immunoblotting (Giardino et al. , J. Clin. Invest. (1996) 97:1422-
  • BAE cells were obtained as described in Example 1.
  • Figure 9 shows the results of incubating the cells for 24 hours in medium containing either 5mM glucose, 30mM glucose, or 30mM glucose plus lOnM Mn (111) tetrakis (benzoic acid) porphyrin (MnTBAP), 25nM TBAP, 50nM TBAP, lOOnM TBAP, 150nM TBAP, or 200nM TBAP.
  • Intracellular reactive oxygen species were measured as described in Example 1. At a concentration of 50nM or higher TABP, inhibition of hyperglycemia-induced ROS is complete.
  • Example 10 Effect of mitochondrial inhibitors on hyperglycemia-induced ROS levels in rat hepatocytes.
  • the purpose of this experiment was to determine whether agents which inhibited accumulation of hyperglycemia-induced ROS accumulation in aortic endothelial cells (see Example 1) would also inhibit accumulation of hyperglycemia-induced ROS in hepatocytes.
  • AOAC AOAC
  • CM- H2DCFDA fluorescent probe CM- H2DCFDA
  • endothelial cells high glucose induced a 4-fold increase in ROS.
  • Inhibition of NADH transport into mitochondria by AOAC had no effect, while inhibition of pyruvate transport with 4-OHCA completed inhibition hyperglycemia-induced ROS .
  • Example 11 The effects of TTFA or MnTBAP on intracellular lipoprotein modification in human hepatocytes.
  • the purpose of this experiment is to evaluate whether inhibitors of mitochondrial electron transport complexes or superoxide dismutase/catalase mimetics can be used to modify intracellular lipoproteins.
  • Cultured human hepatocytes are prepared and maintained as described by Davis (Davis, Methods in Enzymology (1986) 129:227-283). Cultured hepatocytes are incubated in 5mM glucose, 30mM glucose, and 30mM glucose plus an inhibitor of a mitochondrial electron transport complex, such as TTFA (10 ⁇ M), or a superoxide dismutase/catalase mimetic, such as MnTBAP (50-100 ⁇ M).
  • TTFA TTFA
  • MnTBAP 50-100 ⁇ M
  • RNA-6-phosphatase apolipoproteins
  • MnTBAP abnormal hepatocyte gene expression of glucose-6-phosphatase, apolipoproteins, and other complication-promoting genes.
  • Total RNA is isolated and quantitative competitive RT-PCR is performed using the Access RT-PCR system (Promega).
  • the competitor RNA is constructed using the RT-PCR Competitor Construction Kit (Ambion), which generates an RNAse- resistant competitor.
  • Glucose-6-phosphate protein levels are assessed using Western blots with an anti-glucose-6-phosphate antibody according to the method of Lange et al. (J. Biol. Chem. 261 : 101-107, 1986). Similar methods are used to assess the effects of TTFA or
  • MnTBAP on abnormal hepatocyte gene expression of apolipoproteins and other proteins influencing the atherogenic process negatively or positively.
  • Probes are designed for doing quantitative RT-PCR from appropriate genebank sequences, and antibodies for Western blotting are available from commercial sources.
  • Cells are incubated in 5mM glucose, 30mM glucose, or 30mM glucose plus either an inhibitor of a mitochondrial electron complex, such as TTFA (10 ⁇ M) or a superoxide dismutase/catalase mimetic, such as MnTBAP (50-100 ⁇ M), as described in Example 10.
  • TTFA a mitochondrial electron complex
  • MnTBAP 50-100 ⁇ M
  • the increased ROS is substantially normalized by the oxidative phosphorylation uncoupler m-chlorophenylhydrazone (CCCP), the Complex II inhibitor theonyltrifluoroacetone (TTFA), and the superoxide dismutase mimetic, MnTBAP.
  • CCCP oxidative phosphorylation uncoupler m-chlorophenylhydrazone
  • TTFA Complex II inhibitor theonyltrifluoroacetone
  • MnTBAP superoxide dismutase mimetic
  • azide an inhibitor of mitochondrial electron transport Complex IV
  • a Complex V inhibitor oligomycin is shown to aggravate the high glucose effect in the example where ROS are directly measured, and the example where NF K B activation is measured.

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Abstract

L'invention concerne un moyen d'identification d'agents permettant de réduire les taux d'espèces oxygène réactive. Ces agents peuvent également réduire les indicateurs corrélés au taux d'espèces oxygène réactives, tels que les taux de LP, AGE, NFλB, PKC ou d'activation de l'aldose réductase dans les cellules insulino-dépendantes développées dans des conditions de forte concentration de glucose.
PCT/US1999/015734 1998-10-06 1999-07-12 Procedes et compositions permettant de reduire la surproduction de mitochondries des especes oxygene reactives dans les cellules WO2000023568A2 (fr)

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EP1442747A1 (fr) * 1993-10-15 2004-08-04 Duke University Superoxyde-dismutase et ses mimétiques
US8217026B2 (en) 1999-01-25 2012-07-10 Aeolus Sciences, Inc. Substituted porphyrins
US8461093B2 (en) 2008-02-29 2013-06-11 Buck-Chemie Gmbh Adhesive agent for application on a sanitary object
US8470808B2 (en) 1999-01-25 2013-06-25 Jon D. Piganelli Oxidant scavengers for treatment of type I diabetes or type II diabetes
US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions
US9750705B2 (en) 2012-08-31 2017-09-05 The Regents Of The University Of California Agents useful for treating obesity, diabetes and related disorders
US11382895B2 (en) 2008-05-23 2022-07-12 National Jewish Health Methods for treating injury associated with exposure to an alkylating species

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WO2003052067A2 (fr) * 2001-12-13 2003-06-26 University Of Virginia Patent Foundation Transfection d'organelle induite par un vecteur
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US20090123468A1 (en) 2003-10-24 2009-05-14 Gencia Corporation Transducible polypeptides for modifying metabolism
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CA2550217A1 (fr) * 2003-12-12 2005-07-07 Michael A. Brownlee Glp-1 (9-36): methodes et compositions
US9737511B2 (en) * 2004-05-24 2017-08-22 Geoffrey C. GURTNER Method of treating or preventing pathologic effects of acute increases in hyperglycemia and/or acute increases of free fatty acid flux
US8829051B2 (en) 2004-05-24 2014-09-09 Geoffrey C. GURTNER Method of treating or preventing pathologic effects of acute increases in hyperglycemia and/or acute increases of free fatty acid flux
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US10098857B2 (en) 2008-10-10 2018-10-16 The Board Of Trustees Of The Leland Stanford Junior University Topical and transdermal delivery of HIF-1 modulators to prevent and treat chronic wounds
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1442747A1 (fr) * 1993-10-15 2004-08-04 Duke University Superoxyde-dismutase et ses mimétiques
US8217026B2 (en) 1999-01-25 2012-07-10 Aeolus Sciences, Inc. Substituted porphyrins
US8470808B2 (en) 1999-01-25 2013-06-25 Jon D. Piganelli Oxidant scavengers for treatment of type I diabetes or type II diabetes
US8546562B2 (en) 1999-01-25 2013-10-01 James D. Crapo Substituted porphyrins
US8946202B2 (en) 1999-01-25 2015-02-03 Aeolus Sciences, Inc. Substituted porphyrins
US9289434B2 (en) 1999-01-25 2016-03-22 Aeolus Sciences, Inc. Substituted porphyrins
US8461093B2 (en) 2008-02-29 2013-06-11 Buck-Chemie Gmbh Adhesive agent for application on a sanitary object
US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8809312B2 (en) 2008-04-02 2014-08-19 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions
US11382895B2 (en) 2008-05-23 2022-07-12 National Jewish Health Methods for treating injury associated with exposure to an alkylating species
US9750705B2 (en) 2012-08-31 2017-09-05 The Regents Of The University Of California Agents useful for treating obesity, diabetes and related disorders

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