WO2000056403A1 - Regulation positive de l'oxyde nitrique synthase des cellules endotheliales de type iii par des inhibiteurs de la hmg-coa reductase - Google Patents

Regulation positive de l'oxyde nitrique synthase des cellules endotheliales de type iii par des inhibiteurs de la hmg-coa reductase Download PDF

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WO2000056403A1
WO2000056403A1 PCT/US2000/007221 US0007221W WO0056403A1 WO 2000056403 A1 WO2000056403 A1 WO 2000056403A1 US 0007221 W US0007221 W US 0007221W WO 0056403 A1 WO0056403 A1 WO 0056403A1
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nitric oxide
endothelial cell
oxide synthase
cell nitric
subject
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PCT/US2000/007221
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James K. Liao
Ulrich Laufs
Matthias Endres
Michael A. Moskowitz
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The Brigham And Women's Hospital, Inc.
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Priority to JP2000606302A priority Critical patent/JP2003511347A/ja
Priority to AU37603/00A priority patent/AU3760300A/en
Priority to CA002368187A priority patent/CA2368187A1/fr
Priority to EP00916511A priority patent/EP1175246A4/fr
Publication of WO2000056403A1 publication Critical patent/WO2000056403A1/fr

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Definitions

  • This invention describes the new use of HMG-CoA reductase inhibitors as upregulators of Type III endothelial cell Nitric Oxide Synthase. Further, this invention describes methods that employ HMG-CoA reductase inhibitors to treat conditions that result from the abnormally low expression and/or activity of endothelial cell Nitric Oxide Synthase in a subject.
  • Nitric oxide has been recognized as an unusual messenger molecule with many physiologic roles, in the cardiovascular, neurologic and immune systems (Griffith. TM et al., J Am Coll Cardiol, 1988, 12:797-806). It mediates blood vessel relaxation, neurotransmission and pathogen suppression. NO is produced from the guanidino nitrogen of L-arginine by NO Synthase (Moncada, S and Higgs, EA, Eur J Clin Invest, 1991 , 21(4):361-374) . In mammals, at least three isoenzymes of NO Synthase have been identified.
  • endothelial-derived NO inhibits several components of the atherogenic process including monocyte adhesion to the endothelial surface (Tsao, PS et al., Circulation, 1994, 89:2176-2182), platelet aggregation (Radomski, MW, et al., Proc Natl Acad Sci USA, 1990, 87:5193-5197), vascular smooth muscle cell proliferation (Garg, UC and Hassid, A, J Clin Invest, 1989, 83:1774-1777), and vasoconstriction (Tanner, FC et al., Circulation, 1991 , 83:2012-2020).
  • LDL low-density lipoprotein
  • hypoxia downregulates ecNOS expression and/or activity via decreases in both ecNOS gene transcription and mRNA stability (Liao, JK et al., J Clin Invest, 1995, 96:2661-2666, Shaul, PW et al., Am J Physiol, 1997, 272: L1005-L1012).
  • ischemia-induced hypoxia may produce deleterious effects, in part, through decreases in ecNOS activity.
  • HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is the microsomal enzyme that catalyzes the rate limiting reaction in cholesterol biosynthesis (HMG- CoA6Mevalonate).
  • An HMG-CoA reductase inhibitor inhibits HMG-CoA reductase, and as a result inhibits the synthesis of cholesterol.
  • a number of HMG-CoA reductase inhibitors has been used to treat individuals with hypercholesterolemia. Clinical trials with such compounds have shown great reductions of cholesterol levels in hypercholesterolemic patients.
  • HMG-CoA reductase inhibitors restore endothelial function is primarily attributed to the inhibition of hepatic HMG-CoA reductase and the subsequent lowering of serum cholesterol levels, little is known on whether inhibition of endothelial HMG-CoA reductase has additional beneficial effects on endothelial function.
  • Pulmonary hypertension is a major cause of morbidity and mortality in individuals exposed to hypoxic conditions (Scherrer, U et al., N Engl J Med, 1996, 334:624-629). Recent studies demonstrate that pulmonary arterial vessels from patients with pulmonary hypertension have impaired release of NO (Giaid, A and Saleh, D, N Engl J Med, 1995, 333:214-221, Shaul, PW, Am J Physiol, 1997, 272: L1005-L1012).
  • mice lacking ecNOS gene or newborn lambs treated with the ecNOS inhibitor develop progressive elevation of pulmonary arterial pressures and resistance (Steudel, W et al., Circ Res, 1997, 81 :34-41, Fineman, JR et al, J Clin Invest, 1994, 93:2675-2683).
  • LNMA N-omega- monomethyl-L-arginine
  • N-omega-nitro-L-arginine develop progressive elevation of pulmonary arterial pressures and resistance (Steudel, W et al., Circ Res, 1997, 81 :34-41, Fineman, JR et al, J Clin Invest, 1994, 93:2675-2683).
  • hypoxia causes pulmonary vasoconstriction via inhibition of endothelial cell nitric oxide synthase (ecNOS) expression and activity (Adnot, S et al., J Clin Invest, 1991, 87:155-162, Liao, JK et al., J Clin Invest, 1995, 96, 2661-2666).
  • ecNOS endothelial cell nitric oxide synthase
  • stroke has been defined as the abrupt impairment of brain function caused by a variety of pathologic changes involving one or several intracranial or extracranial blood vessels. Approximately 80% of all strokes are ischemic strokes, resulting from restricted blood flow. Mutant mice lacking the gene for ecNOS are hypertensive (Huang, PL et al., Nature, 1995, 377:239-242, Steudel, W et al., Circ Res, 1997, 81 :34-41) and develop greater intimal smooth muscle proliferation in response to cuff injury.
  • HMG-CoA reductase inhibitors can upregulate endothelial cell Nitric Oxide Synthase (Type III) activity other than through preventing the formation of oxidative LDL. It previously was believed that such reductase inhibitors functioned by lowering serum cholesterol levels by blocking hepatic conversion of HMG-CoA to L-mevalonate in the cholesterol biosynthetic pathway. It has been discovered, surprisingly, that HMG-CoA reductase inhibitors can increase Nitric Oxide Synthase activity by effects directly on endothelial rather than hepatic HMG-CoA reductase.
  • This upregulation of activity does not depend upon a decrease in cholesterol synthesis and in particular does not depend upon a decrease in the formation of ox-LDL.
  • the invention therefore, is useful whenever it is desirable to restore endothelial cell Nitric Oxide Synthase activity or increase such activity in an affected cell or tissue.
  • the tissue is defined as to include both the cells in the vasculature supplying nutrients to the tissue, as well as cells of the tissue that express endothelial cell Nitric Oxide Synthase.
  • Nitric Oxide Synthase activity is involved in many conditions, including impotence, heart failure, gastric and esophageal motility disorders, kidney disorders such as kidney hypertension and progressive renal disease, insulin deficiency, etc.
  • a method for increasing endothelial cell Nitric Oxide Synthase activity in a nonhypercholesterolemic subject who would benefit from increased endothelial cell Nitric Oxide Synthase activity in a tissue.
  • the method involves administering to a nonhypercholesterolemic subject in need of such treatment a HMG-CoA reductase inhibitor that increases endothelial cell Nitric Oxide Synthase activity in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject.
  • HMG-CoA reductase inhibitor when the subject in need of such treatment has an abnormally elevated risk of an ischemic stroke use of HMG CoA reductase inhibitors is excluded.
  • HMG-CoA reductase inhibitors do not affect cholesterol levels in a subject.
  • reduction in serum cholesterol is correlated with improved endothelium-dependent relaxation in atherosclerotic vessels (Treasure, et al., N. Engl. J. Med., 1995, 332:481-487).
  • HMG-CoA reductase inhibitors have been demonstrated to reduce serum cholesterol in a matter of weeks, and maximum level of cholesterol reduction can be achieved after a few months of chronic administration.
  • the effect of HMG-CoA reductase inhibitors on up-regulation of ecNOS occurs within a few days.
  • treatment according to the present invention provides significant advantages, e.g., when administered to address short term increases in risk of stroke or other embolic events, such as that due to surgical intervention, even for hypercholesterolemic patients.
  • the subject is not hypercholesterolemic or not hypertriglyceridemic or both (i.e., nonhyperlipidemic).
  • the amount is sufficient to increase endothelial cell Nitric Oxide Synthase activity above normal baseline levels established by age-controlled groups, described in greater detail below.
  • the HMG-CoA reductase inhibitor is administered in an amount which alters the blood LDL cholesterol levels in the subject by less than 10%. The alteration may even be less than 5%.
  • the amount is sufficient to increase endothelial cell Nitric Oxide Synthase activity above normal baseline levels established by age-controlled groups, described in greater detail below.
  • the subject can have a condition characterized by an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is hypoxia-induced. In other embodiments the subject can have a condition comprising an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is chemically induced. In still other embodiments the subject can have a condition comprising an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is cytokine induced. In certain important embodiments, the subject has pulmonary hypertension or an abnormally elevated risk of pulmonary hypertension. In other important embodiments, the subject has experienced an ischemic stroke or has an abnormally elevated risk of an ischemic stroke. In still other important embodiments, the subject has heart failure or progressive renal disease. In yet other important embodiments, the subject is chronically exposed to hypoxic conditions.
  • the preferred HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin.
  • the subject has experienced a thrombotic event or has an abnormally elevated risk of thrombosis.
  • the subject has an abnormally elevated risk of arteriosclerosis or has arteriosclerosis.
  • the subject has an abnormally elevated risk of developing a myocardial infarction or has experienced a myocardial infarction.
  • the subject has an abnormally elevated risk of reperfusion injury.
  • the subject with an elevated risk of reperfusion injury is an organ transplant recipient (e.g., heart, kidney, liver, etc.).
  • the subject has homocystinuria.
  • the subject has Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) syndrome.
  • CADASIL Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy
  • the subject has a degenerative disorder of the nervous system.
  • the subject with a degenerative disorder of the nervous system has Alzheimer's disease.
  • HMG-CoA reductase inhibitors are excluded as treatments for such subjects.
  • the method can further comprise co-administering an endothelial cell Nitric Oxide Synthase substrate (L-arginine preferred) and/or co-administering a nonHMG-CoA reductase inhibitor agent that increases endothelial cell Nitric Oxide Synthase activity.
  • a preferred such agent is selected from the group consisting of estrogens and angiotensin-converting enzyme (ACE) inhibitors.
  • ACE angiotensin-converting enzyme
  • the agents may be administered to a subject who has a condition or prophylactically to a subject who has a risk, and more preferably, an abnormally elevated risk, of developing a condition.
  • the inhibitors also may be administered acutely.
  • a method for increasing endothelial cell Nitric Oxide Synthase activity in a subject to treat a nonhyperlipidemic condition favorably affected by an increase in endothelial cell Nitric Oxide Synthase activity in a tissue.
  • Such conditions are exemplified above.
  • the method involves administering to a subject in need of such treatment a HMG-CoA reductase inhibitor in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject.
  • Important conditions are as described above.
  • the method can involve co-administration of substrates of endothelial cell Nitric Oxide Synthase and/or a nonHMG-CoA reductase inhibitor agent that increases endothelial cell Nitric Oxide Synthase activity.
  • a nonHMG-CoA reductase inhibitor agent that increases endothelial cell Nitric Oxide Synthase activity.
  • the reductase inhibitor can be administered, inter alia, acutely or prophylactically.
  • HMG CoA reductase inhibitors are excluded from treating such subjects.
  • a method for reducing brain injury resulting from stroke.
  • the method involves administering to a subject having an abnormally high risk of an ischemic stroke a HMG-CoA reductase inhibitor in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the brain of the subject.
  • a HMG-CoA reductase inhibitor in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the brain of the subject.
  • important embodiments include the inhibitor being selected from the group consisting of simvastatin and lovastatin.
  • important embodiments include co-administering a substrate of endothelial cell Nitric Oxide Synthase (L-arginine preferred) and/or a nonHMG-CoA reductase inhibitor agent that increases endothelial cell Nitric Oxide Synthase activity.
  • important embodiments include prophylactic and acute administration of the inhibitor.
  • HMG CoA reductase inhibitors are excluded from treating such subjects.
  • a method is provided for treating pulmonary hypertension. The method involves administering to a subject in need of such treatment a HMG-CoA reductase inhibitor in an amount effective to increase pulmonary endothelial cell Nitric Oxide Synthase activity in the subject. Particularly important embodiments are as described above in connection with the methods for treating brain injury. Another important embodiment is administering the inhibitor prophylactically to a subject who has an abnormally elevated risk of developing pulmonary hypertension, including subjects that are chronically exposed to hypoxic conditions.
  • a method for treating heart failure involves administering to a subject in need of such treatment a HMG-CoA reductase inhibitor in an amount effective to increase vascular endothelial cell Nitric Oxide Synthase activity in the subject.
  • important embodiments include prophylactic and acute administration of the inhibitor.
  • Another important embodiment is treating a subject that is nonhyperlipidemic. Preferred compounds and co-administration schemes are as described above.
  • a method is provided for treating progressive renal disease.
  • the method involves administering to a subject in need of such treatment a HMG-CoA reductase inhibitor in an amount effective to increase renal endothelial cell Nitric Oxide Synthase activity in the kidney of the subject.
  • a HMG-CoA reductase inhibitor in an amount effective to increase renal endothelial cell Nitric Oxide Synthase activity in the kidney of the subject.
  • Important embodiments and preferred compounds and schemes of co-administration are as described above in connection with heart failure.
  • a method for increasing blood flow in a tissue of a subject involves administering to a subject in need of such treatment a HMG-CoA reductase inhibitor in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject.
  • the tissue in which blood flow is increased includes tissue in the brain.
  • cerebral blood flow is enhanced.
  • important embodiments include prophylactic and acute administration of the inhibitor. Preferred compounds and co- administration schemes are also as described above.
  • Other important embodiments include co- administering a second agent to the subject with a condition treatable by the second agent in an amount effective to treat the condition, whereby the delivery of the second agent to a tissue of the subject is enhanced as a result of the increased blood flow.
  • the tissue is brain and the second agent comprises an agent having a site of action in the brain.
  • a method of screening for identifying an inhibitor of HMG-CoA reductase for treatment of subjects who would benefit from increased endothelial cell Nitric Oxide Synthase activity in a tissue involves identifying an inhibitor of HMG-CoA reductase suspected of increasing endothelial cell Nitric Oxide Synthase activity, and determining whether or not the inhibitor of HMG-CoA reductase produces an increase in endothelial cell Nitric Oxide Synthase activity in vivo or in vitro.
  • the subject who would benefit from increased endothelial cell Nitric Oxide Synthase activity has an abnormally elevated risk of stroke.
  • the invention also involves the use of HMG-CoA reductase inhibitors in the manufacture of medicaments for treating the above-noted conditions. Important conditions, compounds, etc. are as described above.
  • the invention further involves pharmaceutical preparations including the HMG-CoA reductase inhibitors for treating the above-noted conditions.
  • the preparations can include other agents such as second agents, ecNOS substrates, ecNOS cofactors, as described above, or can be cocktails of the HMG-CoA reductase inhibitors together with a nonHMG-CoA reductase inhibitor agent that increases ecNOS activity in a cell, directly or indirectly (synergistically, cooperatively, additively, etc.).
  • compositions and pharmaceutical preparations that are cocktails of a HMG-CoA reductase inhibitor and L-arginine are provided.
  • the HMG-CoA reductase inhibitor and the L-arginine are in amounts effective to increase blood flow.
  • the HMG-CoA reductase inhibitor and the L-arginine are in amounts effective to increase blood flow in brain tissue.
  • administration of the HMG-CoA reductase inhibitor and the L-arginine results in increased blood flow.
  • administration of the HMG-CoA reductase inhibitor and the L-arginine results in increased blood flow to the brain.
  • Any of the above cocktail compositions may also include other cofactors that enhance ecNOS substrate connversion by ecNOS to nitric oxide, the preferred cofactors being NADPH and tetrahydrobiopterin.
  • the invention also involves methods for increasing ecNOS activity in a cell by contacting the cell with an effective amount of a HMG-CoA reductase inhibitor, alone, or together with any of the agents co-administered as described above, or as a cocktail as described above.
  • a HMG-CoA reductase inhibitor alone, or together with any of the agents co-administered as described above, or as a cocktail as described above.
  • Any of the above cocktails may also include a substrate for endothelial cell Nitric Oxide Synthase, the preferred substrate being L-arginine, and/or other cofactors that enhance ecNOS substrate connversion by ecNOS to nitric oxide, the preferred cofactors being NADPH and tetrahydrobiopterin.
  • Figure 1 Western blots showing the effects of oxidized (ox)-LDL on ecNOS protein levels in the presence and absence of simvastatin.
  • Figure 1 A depicts the effects of increasing concentrations of simvastatin on ecNOS protein levels.
  • Figure IB depicts the effects of increasing concentrations of simvastatin on ecNOS protein levels in a time-dependent manner.
  • Figure 2. Northern blots showing the effects of ox-LDL on ecNOS mRNA levels in the presence and absence of HMG-CoA reductase inhibitors.
  • Figure 2A depicts the effects of simvastatin on ecNOS mRNA levels.
  • Figure 2B depicts the effects of lovastatin on ecNOS mRNA levels.
  • FIG. 3 Western blots showing the concentration-dependent effects of simvastatin (Figure 3A) and lovastatin (Figure 3B) on ecNOS protein levels after 48 hours.
  • Figure 4 Volume of cerebral infarction after 2 h filamentous middle cerebral artery occlusion and 22 h reperfusion as % of control, with and without simvastatin treatment.
  • Figure 4 A depicts the cerebral infarction in wild-type SV-129 mice.
  • Figure 4B depicts the cerebral infarction in ecNOS-deficient mice.
  • Figure 5 Bar graph showing regional CBF changes in wild type and eNOS null mice for 40 min after L-arginine or saline infusion. Figure 6. Bar graph showing regional CBF changes in simvastatin-treated mice for
  • Nitric Oxide Synthase is the enzyme that catalyzes the reaction that produces nitric oxide from the substrate L-arginine.
  • endothelial cell nitric oxide Synthase refers to the Type III isoform of the enzyme found in the endothelium.
  • ecNOS activity it is meant the ability of a cell to generate nitric oxide from the substrate L-arginine.
  • Increased ecNOS activity can be accomplished in a number of different ways. For example, an increase in tlie amount of ecNOS protein or an increase in the activity of the protein (while maintaining a constant level of the protein) can result in increased “activity”.
  • An increase in the amount of protein available can result from increased transcription of the ecNOS gene, increased stability of the ecNOS mRNA or a decrease in ecNOS protein degradation.
  • the ecNOS activity in a cell or in a tissue can be measured in a variety of different ways.
  • a direct measure would be to measure the amount of ecNOS present.
  • Another direct measure would be to measure the amount of conversion of arginine to citrulline by ecNOS or the amount of generation of nitric oxide by ecNOS under particular conditions, such as the physiologic conditions of the tissue.
  • the ecNOS activity also can be measured more indirectly, for example by measuring mRNA half-life (an upstream indicator) or by a phenotypic response to the presence of nitric oxide (a downstream indicator).
  • One phenotypic measurement employed in the art is detecting endothelial dependent relaxation in response to a acetylcholine, which response is affected by ecNOS activity.
  • the level of nitric oxide present in a sample can be measured using a nitric oxide meter. All of the foregoing techniques are well known to those of ordinary skill in the art, and some are described in the examples below.
  • the present invention by causing an increase in ecNOS activity, permits not only the re-establishment of normal base-line levels of ecNOS activity, but also allows increasing such activity above normal base-line levels.
  • Normal base-line levels are the amounts of activity in a normal control group, controlled for age and having no symptoms which would indicate alteration of endothelial cell Nitric Oxide Synthase activity (such as hypoxic conditions, hyperlipidemia and the like). The actual level then will depend upon the particular age group selected and the particular measure employed to assay activity. Specific examples of various measures are provided below. In abnormal circumstances, e.g. hypoxic conditions, pulmonary hypertension, etc., endothelial cell Nitric Oxide Synthase activity is depressed below normal levels.
  • increasing activity means any increase in endothelial cell Nitric Oxide Synthase activity in the subject resulting from the treatment with reductase inhibitors according to the invention, including, but not limited to, such activity as would be sufficient to restore normal base-line levels and such activity as would be sufficient to elevate the activity above normal base-line levels.
  • Nitric Oxide Synthase activity is involved in many conditions, including stroke, pulmonary hypertension, thrombosis, arteriosclerosis, myocardial infarction, reperfusion injury (e.g., in an organ transplant recipient), impotence, heart failure, gastric and esophageal motility disorders, kidney disorders such as kidney hypertension and progressive renal disease, insulin deficiency, hypoxia-induced conditions, homocystinuria, neurodegenerative disorders, CADASIL syndrome, etc.
  • the decrease in endothelial cell Nitric Oxide Synthase activity is cytokine induced. Cytokines are soluble polypeptides produced by a wide variety of cells that control gene activation and cell surface molecule expression.
  • cytokine induced endothelial cell Nitric Oxide Synthase activity
  • Ischemic stroke ischemic cerebral infarction
  • Ischemic stroke is an acute neurologic injury that results from a decrease in the blood flow involving the blood vessels of the brain. Ischemic stroke is divided into two broad categories, thrombotic and embolic.
  • a surprising finding was made in connection with the treatment of ischemic stroke.
  • treatment according to the invention can reduce the brain injury that follows an ischemic stroke.
  • Brain injury reduction as demonstrated in the examples below, can be measured by determining a reduction in infarct size in the treated versus the control groups.
  • functional tests measuring neurological deficits provided further evidence of reduction in brain injury in the treated animals versus the controls. Cerebral blood flow also was better in the treated animals versus the controls.
  • An important embodiment of the invention is treatment of a subject with an abnormally elevated risk of an ischemic stroke.
  • subjects having an abnormally elevated risk of an ischemic stroke are a category determined according to conventional medical practice; such subjects may also be identified in conventional medical practice as having known risk factors for stroke or having increased risk of cerebrovascular events.
  • the risk factors associated with cardiac disease are the same as are associated with stroke.
  • the primary risk factors include hypertension, hypercholesterolemia, and smoking.
  • atrial fibrillation or recent myocardial infarction are important risk factors.
  • subjects having an abnormally elevated risk of an ischemic stroke also include individuals undergoing surgical or diagnostic procedures which risk release of emboli, lowering of blood pressure or decrease in blood flow to the brain, such as carotid endarterectomy, brain angiography, neurosurgical procedures in which blood vessels are compressed or occluded, cardiac catheterization, angioplasty, including balloon angioplasty, coronary by-pass surgery, or similar procedures.
  • Subjects having an abnormally elevated risk of an ischemic stroke also include individuals having any cardiac condition that may lead to decreased blood flow to the brain, such as atrial fibrillation, ventrical tachycardia, dilated cardiomyopathy and other cardiac conditions requiring anticoagulation.
  • Subjects having an abnormally elevated risk of an ischemic stroke also include individuals having conditions including arteriopathy or brain vasculitis, such as that caused by lupus, congenital diseases of blood vessels, such as cadasil syndrome, or migraine, especially prolonged episodes.
  • the subject is not hypercholesterolemic or not hypertriglyceridemic or both (i.e., nonhyperlipidemic) .
  • the treatment of stroke can be for patients who have experienced a stroke or can be a prophylactic treatment. Short term prophylactic treatment is indicated for subjects having surgical or diagnostic procedures which risk release of emboli, lowering of blood pressure or decrease in blood flow to the brain, to reduce the injury due to any ischemic event that occurs as a consequence of the procedure.
  • prophylactic Longer term or chronic prophylactic treatment is indicated for subjects having cardiac conditions that may lead to decreased blood flow to the brain, or conditions directly affecting brain vasculature. If prophylactic, then the treatment is for subjects having an abnormally elevated risk of an ischemic stroke, as described above. If the subjects have an abnormally elevated risk of an ischemic stroke because of having experienced a previous ischemic event, then the prophylactic treatment for these subjects excludes the use ofHMG Co A reductase inhibitors. If the subject has experienced a stroke, then the treatment can include acute treatment. Acute treatment for stroke subjects means administration of the HMG-CoA reductase inhibitors at the onset of symptoms of the condition or at the onset of a substantial change in the symptoms of an existing condition.
  • Pulmonary hypertension is a disease characterized by increased pulmonary arterial pressure and pulmonary vascular resistance. Hypoxemia, hypocapnia, and an abnormal diffusing capacity for carbon monoxide are almost invariable findings of the disease. Additionally, according to the present invention, patients with pulmonary hypertension also have reduced levels of ecNOS expression in their pulmonary vessels. Traditionally, the criteria for subjects with, or at risk for pulmonary hypertension are defined on the basis of clinical and histological characteristics according to Heath and Edwards (Circulation, 1958, 18:533-547).
  • Subjects may be treated prophylactically to reduce the risk of pulmonary hypertension or subjects with pulmonary hypertension may be treated long term and/or acutely. If the treatment is prophylactic, then the subjects treated are those with an abnormally elevated risk of pulmonary hypertension.
  • a subject with an abnormally elevated risk of pulmonary hypertension is a subject with chronic exposure to hypoxic conditions, a subject with sustained vasoconstriction, a subject with multiple pulmonary emboli, a subject with cardiomegaly and/or a subject with a family history of pulmonary hypertension.
  • Hypoxia as used herein is defined as the decrease below normal levels of oxygen in a tissue. Hypoxia can result from a variety of circumstances, but most frequently results from impaired lung function. Impaired lung function can be caused by emphysema, cigarette smoking, chronic bronchitis, asthma, infectious agents, pneumonitis (infectious or chemical), lupus, rheumatoid arthritis, inherited disorders such as cystic fibrosis, obesity, ⁇ ,-antitrypsin deficiency and the like. It also can result from non-lung impairments such as from living at very high altitudes. Hypoxia can result in pulmonary vasoconstriction via inhibition of ecNOS activity.
  • Heart failure is a clinical syndrome of diverse etiologies linked by the common denominator of impaired heart pumping and is characterized by the failure of the heart to pump blood commensurate with the requirements of the metabolizing tissues, or to do so only from an elevating filling pressure.
  • the invention involves treatment of the foregoing conditions using HMG-CoA reductase inhibitors.
  • HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme A)
  • HMG-CoA reductase is the microsomal enzyme that catalyzes the rate limiting reaction in cholesterol biosynthesis (HMG-CoA6Mevalonate).
  • HMG-CoA reductase inhibitor inhibits HMG-CoA reductase, and therefore inhibits the synthesis of cholesterol.
  • compounds described in the art that have been obtained naturally or synthetically, which have been seen to inhibit HMG-CoA reductase, and which form the category of agents useful for practicing the present invention.
  • these agents have been used to treat individuals with hypercholesterolemia. Examples include some which are commercially available, such as simvastatin (U.S. Patent No. 4, 444,784), lovastatin (U.S. Patent No. 4,231,938), pravastatin sodium (U.S. Patent No. 4,346,227), fluvastatin (U.S. Patent No.
  • Patent No. 5,302,604 U.S. Patent No. 5,166,171 , U.S. Patent No. 5,202,327, U.S. Patent No. 5,276,021, U.S. Patent No. 5,196,440, U.S. Patent No. 5,091,386, U.S. Patent No. 5,091,378, U.S. Patent No. 4,904,646, U.S. PatentNo. 5,385,932, U.S. Patent No. 5,250,435, U.S. Patent No. 5,132,312, U.S. Patent No. 5,130,306, U.S. Patent No. 5,116,870, U.S. Patent No. 5,112,857, U.S. PatentNo. 5,102,91 1, U.S. Patent No.
  • Important embodiments of the invention involve populations never before treated with an HMG-CoA reductase inhibitor.
  • the invention involves in certain aspects treatments of individuals who are otherwise free of symptoms calling for treatment with an HMG-CoA reductase inhibitor. It is believed that the only clinically accepted such condition is hypercholesterolemia, wherein the reductase inhibitor is administered for the purpose of preventing the biosynthesis of cholesterol.
  • the treated population is nonhypercholesterolemic.
  • the subject is nonhypertriglyceridemic.
  • the subject is nonhypercholesterolemic and/or nonhypertriglyceridemic, i.e., nonhyperlipidemic.
  • a nonhypercholesterolemic subject is one that does not fit the current criteria established for a hypercholesterolemic subject.
  • a nonhypertriglyceridemic subject is one that does not fit the current criteria established for a hypertriglyceridemic subject (See, e.g., Harrison's Principles of Experimental Medicine, 13th Edition, McGraw-Hill, Inc., N.Y., hereinafter "Harrison's").
  • Hypercholesterolemic subjects and hypertriglyceridemic subjects are associated with increased incidence of premature coronary heart disease.
  • a hypercholesterolemic subject has an LDL level of > 160 mg/dL or > 130 mg/dL and at least two risk factors selected from the group consisting of male gender, family history of premature coronary heart disease, cigarette smoking (more than 10 per day), hypertension, low HDL ( ⁇ 35 mg/dL), diabetes mellitus, hyperinsulinemia, abdominal obesity, high lipoprotein (a), and personal history of cerebrovascular disease or occlusive peripheral vascular disease.
  • a hypertriglyceridemic subject has a triglyceride (TG) level of >250 mg/dL.
  • TG triglyceride
  • a hyperlipidemic subject is defined as one whose cholesterol and triglyceride levels equal or exceed the limits set as described above for both the hypercholesterolemic and hypertriglyceridemic subjects.
  • thromboembolism is the collective term used for diseases characterized by the formation, development, or presence of a thrombus and the blocking of a vessel by a thrombus brought to a thrombotic vascular site by the blood current.
  • Thromboembolism can reduce blood flow to almost all organs including the brain and myocardium.
  • Thromboembolism involving the brain is otherwise known as an ischemic stroke and is described elsewhere in this application.
  • Thromboembolism involving the heart is otherwise known as a myocardial infarction and is also described elsewhere in this application.
  • certain patient groups have been identified who are particularly prone to thrombosis and embolism. These include patients: (1) immobilized after surgery; (2) with chronic congestive heart failure; (3) with atherosclerotic vascular disease; (4) with malignancy; or (5) who are pregnant.
  • An important embodiment of the invention is treatment of subjects with an abnormally elevated risk of thrombosis (or thromboembolism).
  • subjects having an abnormally elevated risk of thrombosis are a category determined according to conventional medical practice.
  • prethrombotic patients can be identified by a careful history.
  • Subjects may be treated prophylactically to reduce the risk of a thrombotic episode or subjects with thrombosis may be treated long-term and/or acutely. If the treatment is prophylactic, then the subjects treated are those with an abnormally elevated risk of thrombosis.
  • Myocardial infarction is the diseased state which occurs with the abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by artheosclerosis. Such injury is produced or facilitated by factors such as cigarette smoking, hypertension and lipid accumulation.
  • An important embodiment of the invention is treatment of a subject with an abnormally elevated risk of myocardial infarction.
  • subjects having an abnormally elevated risk of myocardial infarction are the category of patients that include those with unstable angina, multiple coronary risk factors (similar to those described for stroke elsewhere herein), and Prinzmetal's variant angina. Less common etiologic factors include hypercoagulability, coronary emboli, collagen vascular disease, and cocaine abuse.
  • Subjects may be treated prophylactically to reduce the risk of myocardial infarction, or subjects with myocardial infarction, may be treated long-term and/or acutely. If the treatment is prophylactic, then the subjects treated are those with an abnormally elevated risk of myocardial infarction.
  • a subject with an abnormally elevated risk of myocardial infarction is a subject that falls in the above-described categories.
  • Another important embodiment of the invention is the treatment of subjects with an abnormally elevated risk of reperfusion injury damage.
  • Preferred subjects are about to receive or have received a transplant.
  • increase in ecNOS expression and/or activity in the vessels of the transplanted organ is believed to reduce reperfusion injury damage.
  • Reperfusion injury is the functional, metabolic, or structural change that includes necrosis in ischemic tissues, thought to result from reperfusion to ischemic areas of the tissue.
  • the most common example involves myocardial reperfusion injury. In myocardial reperfusion injury, changes in ischemic heart muscle are thought to result from reperfusion to the ischemic areas of the heart.
  • Changes can be fatal to muscle cells and may include oedema with explosive cell swelling and disintegration, sarcolemma disruption, fragmentation of mitochondria, contraction and necrosis, enzyme washout and calcium overload.
  • Another important embodiment of the invention is the treatment of subjects with a homocystinuria.
  • the homocystinurias are seven biochemically and clinically distinct disorders, each characterized by increased concentration of the sulfur-containing amino acid homocysteine in blood and urine. This is because the enzyme cystathione synthetase that converts homocysteine and serine into cystathione, a precursor of cysteine, is missing.
  • Subjects with a homocystinuria are also likely to suffer from thrombosis, and can benefit from increased ecNOS expression and/or activity.
  • Another important embodiment of the invention is the treatment of subjects with Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) syndrome.
  • the disorder is characterized by relapsing strokes with neuropsychiatric symptoms and affects relatively young adults of both sexes.
  • CT scans have demonstrated occlusive cerebrovascular infarcts in the white matter, which was usually reduced.
  • Subjects with CADASIL syndrome can also benefit from increased ecNOS expression and/or activity.
  • Another important embodiment of the invention is the treatment of subjects with a neurodegenerative disease.
  • neurodegenerative disease is meant to include any pathological state involving neuronal degeneration, including Parkinson's Disease, Huntington's Disease, Alzheimer's Disease, and amyotrophic lateral sclerosis (ALS).
  • the neurodegenerative disease is Alzheimer's Disease.
  • Alzheimer's Disease is a progressive, neurodegenerative disease characterised by loss of function and death of nerve cells in several areas of the brain leading to loss of cognitive function such as memory and language.
  • the cause of nerve cell death is unknown but the cells are recognised by the appearance of unusual helical protein filaments in the nerve cells (neurofibrillary tangles) and by degeneration in cortical regions of brain, especially frontal and temporal lobes.
  • Increase of cerebral blood flow mediated by an increase in ecNOS expression and/or activity can also be of benefit to subjects suffering from a neurodegenerative disease.
  • a method of screening for identifying an inhibitor of HMG-CoA reductase inhibitor for treatment of subjects who would benefit from increased endothelial cell Nitric Oxide Synthase activity in a tissue is provided.
  • the method involves identifying an inhibitor of HMG-CoA reductase suspected of increasing endothelial cell Nitric Oxide Synthase activity, and determining whether or not the inhibitor of HMG-CoA reductase produces an increase in endothelial cell Nitric Oxide Synthase activity in vivo or in vitro.
  • HMG-CoA reductase inhibitors according to this invention can be identified by confirming that the inhibitor produces increased ecNOS activity in a model system compared to a control, using any of the model systems described herein, and also inhibits at least one other HMG-CoA reductase dependent function as determined in any of the model systems described herein and/or other model systems known in the art.
  • the invention also involves the co-administration of agents that are not HMG-CoA reductase inhibitors but that can act cooperatively, additively or synergistically with such HMG-CoA reductase inhibitors to increase ecNOS activity.
  • agents that are not HMG-CoA reductase inhibitors but that can act cooperatively, additively or synergistically with such HMG-CoA reductase inhibitors to increase ecNOS activity.
  • ecNOS substrates which are converted by ecNOS to nitric oxide and cofactors enhancing such conversion, can be co-administered with the HMG-CoA reductase inhibitors according to the invention.
  • Such ecNOS substrates e.g. L-arginine
  • cofactors e.g., NADPH, tetrahydrobiopterin, etc.
  • HMG-CoA reductase inhibitors that are not substrates of ecNOS and that can increase ecNOS activity.
  • Agents belonging to these categories are therefore nonHMG-CoA reductase inl ibitors and can be used in co-administrations with rho GTPase function inhibitors in cocktails.
  • categories of such agents are estrogens and ACE inhibitors.
  • Estrogens are a well defined category of molecules known by those of ordinary skill in the art, and will not be elaborated upon further herein. All share a high degree of structural similarity.
  • ACE inhibitors also have been well characterized, although they do not always share structural homology.
  • Angiotensin converting enzyme is an enzyme which catalyzes the conversion of angiotensin I to angiotensin II.
  • ACE inhibitors include amino acids and derivatives thereof, peptides, including di and tri peptides and antibodies to ACE which intervene in the renin-angiotensin system by inhibiting the activity of ACE thereby reducing or eliminating the formation of pressor substance angiotensin II.
  • ACE inhibitors have been used medically to treat hypertension, congestive heart failure, myocardial infarction and renal disease.
  • Classes of compounds known to be useful as ACE inhibitors include acylmercapto and mercaptoalkanoyl prolines such as captopril (US Patent Number 4,105,776) and zofenopril (US Patent Number 4,316,906), carboxyalkyl dipeptides such as enalapril (US Patent Number 4,374,829), lisinopril (US Patent Number 4,374,829), quinapril (US Patent Number 4,344,949), ramipril (US Patent Number 4,587,258), and perindopril (US Patent Number 4,508,729), carboxyalkyl dipeptide mimics such as cilazapril (US Patent Number 4,512,924) and benazapril (US Patent Number 4,410,520), phosphinylalkanoyl prolines such as fosinopril (US Patent Number 4,337,201) and trandolopril.
  • captopril US Patent Number 4,105,776) and
  • This invention also contemplates co-administration of agents that increase the production of NO by ecNOS without affecting ecNOS expression, as do ACE inhibitors or administration of ecNOS substrate and or ecNOS cofactors.
  • Estrogens upregulate Nitric Oxide Synthase expression whereas ACE inhibitors do not affect expression, but instead influence the efficiency of the action of Nitric Oxide Synthase on L-arginine.
  • activity can be increased in a variety of ways. In general, activity is increased by the reductase inhibitors of the invention by increasing the amount of the active enzyme present in a cell versus the amount present in a cell absent treatment with the reductase inhibitors according to the invention.
  • an effective amount is any amount that can cause an increase in Nitric Oxide Synthase activity in a desired tissue, and preferably in an amount sufficient to cause a favorable phenotypic change in the condition such as a lessening, alleviation or elimination of a symptom or of a condition.
  • an effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently or delaying the onset of or preventing the disease or condition from occurring. This can be monitored by routine methods.
  • doses of active compounds would be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that doses ranging from 50-500 mg/kg will be suitable, preferably orally and in one or several administrations per day.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • Lower doses will result from certain forms of administration, such as intravenous administration.
  • higher doses or effectively higher doses by a different, more localized delivery route
  • Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • the reductase inhibitors useful according to the invention may be combined, optionally, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular drug selected, the severity of the condition being treated and the dosage required for therapeutic efficacy.
  • the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of admimstration include oral, rectal, topical, nasal, interdermal, or parenteral routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of reductase inhibitors, which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di- glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the active compound, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides
  • hydrogel release systems such as those described in U.S. Patent Nos. 4,452,775, 4,675, 189 and 5,736,152
  • peptide based systems such as those described in U.S. Patent Nos. 4,452,775, 4,675, 189 and 5,736,152
  • diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S.
  • a long-term sustained release implant may be desirable.
  • Long-term release are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • a method for increasing blood flow in a tissue of a subject involves administering to a subject in need of such treatment a HMG-CoA reductase inhibitor in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject.
  • a second agent is co-administered to a subject with a condition treatable by the second agent in an amount effective to treat the condition, whereby the delivery of the second agent to a tissue of the subject is enhanced as a result of the increased blood flow from administering the first agent of the invention (a HMG CoA reductase inhibitor).
  • the "second agent” may be any pharmacological compound or diagnostic agent, as desired.
  • Preferred second agents are agents having a site of action in the brain.
  • Such agents include analeptic, analgetic, anesthetic, adrenergic agent, anti-adrenergic agent, amino acids, antagonists, antidote, anti-anxiety agent, anticholinergic, anticolvunsant, antidepressant, anti-emetic, anti-epileptic, antihypertensive, antifibrinolytic, antihyperlipidemia, antimigraine, antinauseant, antineoplastic (brain cancer), antiobessional agent, antiparkinsonian, antipsychotic, appetite suppressant, blood glucose regulator, cognition adjuvant, cognition enhancer, dopaminenergic agent, emetic, free oxygen radical scavenger, glucocorticoid, hypocholesterolemic, holylipidemic, histamine H2 receptor antagonists, immunosuppressant, inhibitor, memory adjuvant, mental performance enhancer, mood regulator, mydriatic, neuromuscular blocking agent, neuroprotective, NMD A antagonist
  • examples of categories of other pharmaceutical agents that can be used as second agents include: adrenergic agent; adrenocortical steroid; adrenocortical suppressant; alcohol deterrent; aldosterone antagonist; amino acid; ammonia detoxicant; anabolic; analeptic; analgesic; androgen; anesthesia, adjunct to; anesthetic; anorectic; antagonist; anterior pituitary suppressant; anthelmintic; anti-acne agent; anti-adrenergic; anti-allergic; anti-amebic; anti-androgen; anti-anemic; anti-anginal; anti-anxiety; anti-arthritic; anti-asthmatic; anti-atherosclerotic; antibacterial; anticholelithic; anticholelithogenic; anticholinergic; anticoagulant; anticoccidal; anticonvulsant; antidepressant; antidiabetic; antidiar
  • the reductase inhibitor is "co-administered," which means administered substantially simultaneously with another agent.
  • substantially simultaneously it is meant that the reductase inhibitor is administered to the subject close enough in time with the administration of the other agent (e.g., a nonHMG-CoA reductase inhibitor agent, a "second agent”, etc.), whereby the two compounds may exert an additive or even synergistic effect, i.e. on increasing ecNOS activity or on delivering a second agent to a tissue via increased blood flow.
  • the antibody detection kit (Enhanced Chemiluminescence) and the nylon nucleic acid (Hybond) and protein (PVDF) transfer membranes were purchased from Amersham Corp. (Arlington Heights, IL). Simvastatin and lovastatin were obtained from Merck, Sharp, and Dohme, Inc. (West Point, PA). Since endothelial cells lack lactonases to process simvastatin and lovastatin to their active forms, these HMG-CoA reductase inhibitors were chemically activated prior to their use as previously described (Laufs, U et al., J Biol Chem, 1997, 272:31725-31729).
  • Human endothelial cells were harvested from saphenous veins and cultured as described (15). For transfection studies, bovine aortic endothelial cells of less than 3 passages were cultured in a growth medium containing DMEM (Dulbecco's Modified Eagle's Medium), 5 mmol/L L-glutamine (Gibco), and 10% fetal calf serum (Hyclone Lot#l 114577). For all experiments, the endothelial cells were placed in 10% lipoprotein-deficient serum (Sigma, Lot#26H94031) for 48 h prior to treatment conditions.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Libco 10% fetal calf serum
  • endothelial cells were preheated with actinomycin D (5 mg/ml) for 1 h prior to treatment with ox-LDL and/or simvastatin. Cellular viability as determined by cell count, morphology, and Trypan blue exclusion was maintained for all treatment conditions.
  • the LDL was prepared by discontinuous ultracentrifugation according to the method of Chung et al. with some modification (Methods Enzymol, 1984, 128:181-209). Fresh plasma from a single donor was anticoagulated with heparin and filtered through a Sephadex G-25 column equilibrated with PBS. The density was adjusted to 1.21 g/ml by addition of KBr (0.3265 g/ml plasma).
  • a discontinuous NaCl/KBr gradient was established in Beckman Quick-Seal centrifuge tubes (5.0 ml capacity) by layering 1.5 ml of density-adjusted plasma under 3.5 ml of 0.154 M NaCl in Chelex-100-treated water (BioRad, Hercules, CA). After ultracentrifugation at 443,000 x g and 7°C for 45 min in a Beckman Near Vertical Tube 90 rotor (Beckman L8-80M ultracentrifuge), the yellow band in the upper middle of the tube corresponding to LDL was removed by puncturing with a needle and withdrawing into a syringe. The KBr was removed from the LDL by dialyzing with three changes of sterile PBS, pH 7.4, containing 100 :g/ml polymyxin B.
  • LDL samples The purity of the LDL samples was confirmed by SDS/polyacrylamide and cellulose acetate gel electrophoresis. Cholesterol and triglyceride content were determined as previously described (Liao, JK et al., J Biol Chem, 1995, 270:319-324.). The LDL protein concentration was determined by the method of Lowry et al., (J Biol Chem, 1951, 193:265-275.). For comparison, commercially-available LDL (Biomedical Technologies Inc., Stoughton, MA; Calbiochem, San Diego, CA) were characterized and used in selected experiments.
  • Oxidation of LDL Oxidized LDL was prepared by exposing freshly-isolated LDL to CuSO 4 (5-10 mM) at
  • the reaction was stopped by dialyzing with three changes of sterile buffer (150 :mol/L NaCI, 0.01% EDTA and 100 :g/ml polymyxin B, pH 7.4) at 4°C.
  • the degree of LDL oxidation was estimated by measuring the amounts of thiobarbituric acid reactive substances (TBARS) produced using a fluorescent assay for malondialdehyde as previously described (Yagi, KA, Biochem Med, 1916, 15:212-21.).
  • TBARS thiobarbituric acid reactive substances
  • the extent of LDL modification was expressed as nanomoles of malondialdehyde per mg of LDL protein. Only mild to moderate ox-LDL with TBARS values between 12 and 16 nmol/mg LDL protein (i.e. 3 to 4 nmol/mg LDL cholesterol) were used in this study. All oxidatively-modified LDL samples were used within 24 h of preparation.
  • RNA Equal amounts of total RNA (10-20 mg) were separated by 1.2% formaldehyde-agarose gel electrophoresis and transferred overnight onto Hybond nylon membranes. Radiolabeling of human full-length ecNOS cDNA (Verbeuren, TJ et al, Circ Res, 1986, 58:552-564, Liao, JK et al., J Clin Invest, 1995, 96:2661-2666) was performed using random hexamer priming, [a- 32 P]CTP, and Klenow (Pharmacia).
  • the membranes were hybridized with the probes overnight at 45°C in a solution containing 50% formamide, 5 X SSC, 2.5 X Denhardt's Solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA. All Northern blots were subjected to stringent washing conditions (0.2 X SSC/0.1% SDS at 65°C) prior to autoradiography. RNA loading was determined by rehybridization with human GAPDH probe.
  • endothelial cells were treated for 24 h with ox-LDL in the presence and absence of simvastatin (0.1 to 1 mM). After treatment, the medium was removed, and the cells were washed and incubated for 24 h in phenol red-free medium. After 24 h, 300 :1 of conditioned medium was mixed with 30 :1 of freshly prepared 2,3-diaminonaphthalene (1.5 mmol/L DAN in 1 mol/L HCl).
  • Confluent endothelial cells ( ⁇ 5 x 10 7 cells) grown in LPDS were treated with simvastatin
  • Hybridization of radiolabeled mRNA transcripts to the nitrocellulose membranes was carried out at 45°C for 48 h in a buffer containing 50% formamide, 5 X SSC, 2.5 X Denhardt's solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA. The membranes were then washed with 1 x SSC/0.1% SDS for 1 h at 65°C prior to autoradiography for 72 h at -80°C.
  • bovine rather than human endothelial cells were used because of their higher transfection efficiency by the calcium-phosphate precipitation method (12% vs ⁇ 4%) (Graham, FL and Van der Erb, AJ, Virology, 1973, 52:456-457).
  • Fl.LUC human ecNOS promoter construct
  • Bovine endothelial cells (60%-70% confluent) were transfected with 30 mg of the indicated constructs: p.LUC (no promoter), pSV2.LUC (SV40 early promoter), or Fl.LUC.
  • p.LUC no promoter
  • pSV2.LUC SV40 early promoter
  • Fl.LUC Fl.LUC
  • pCMV.bGal plasmid 10 mg was co-transfected in all experiments. Preliminary results using b-galactosidase staining indicate that cellular transfection efficiency was approximately 10% to 14%.
  • Endothelial cells were placed in lipoprotein-deficient serum for 48 h after transfection and treated with ox-LDL (50 mg/ml, TBARS 12.4 nmol/mg) in the presence and absence of simvastatin (1 mM) for an additional 24 h.
  • ox-LDL 50 mg/ml, TBARS 12.4 nmol/mg
  • simvastatin 1 mM
  • the luciferase and b-galactosidase activities were determined by a chemiluminescence assay (Dual-Light, Tropix, Bedford, MA) using a Berthold L9501 luminometer.
  • the relative promoter activity was calculated as the ratio of luciferase-to ⁇ -galactosidase activity. Each experiment was performed three times in triplicate.
  • TBARS values of >30 nmol/mg) caused vacuolization and some detachment of endothelial cells after 24 h.
  • simvastatin (0.01 to 0.1 mmol/L) nor lovastatin (10 mmol/L) produced any noticeable adverse effects on human endothelial cell for up to 96 h.
  • higher concentrations of simvastatin (>15 mmol/L) or lovastatin (>50 mmol/L) caused cytotoxicity after 36 h, and therefore, were not used.
  • Example 2 Characterization of LDL SDS/polyacrylamide gel electrophoresis of native or unmodified LDL revealed a single band (-510 kD) corresponding to ApoB-100 (data not shown). Similarly, cellulose acetate electrophoresis revealed only one band corresponding to the presence of a single class of low-density lipids (density of 1.02 to 1.06 g/ml).
  • the LDL had a protein, cholesterol, and triglyceride concentration of 6.3 ⁇ 0.2, 2.5 ⁇ 0.1, and 0.5 ⁇ 0.1 mg/ml, respectively.
  • lipoprotein-deficient serum was devoid of both apoB-100 protein and low-density lipid bands, and had non-detectable levels of cholesterol. There was no detectable level of endotoxin ( ⁇ 0.10 EU/ml) in the lipoprotein-deficient serum or ox-LDL samples by the chromogenic Limulus amebocyte assay.
  • Example 3 Effect of ox-LDL and HMG-Co ⁇ Reductase Inhibitors on ecNOS Protein
  • ox-LDL 50 mg/ml downregulates ecNOS expression
  • ox-LDL 50 mg/ml, TBARS 12.2 nmol/mg
  • Example 4 Effect of ox-LDL and HMG-CoA Reductase Inhibitors on ecNOS mRNA
  • Treatment with lovastatin (10 mmol/L) not only reversed the inhibitory effects of ox-LDL on ecNOS mRNA, but also caused a 40 ⁇ 9% increase in ecNOS mRNA level compared to that of untreated cells. Compared to ox-LDL alone, co-treatment with lovastatin caused a 3.6-fold increase in ecNOS mRNA levels after 24 h. Treatment with lovastatin alone, however, produced 36% increase in ecNOS mRNA levels compared to untreated cells (p ⁇ 0.05, n 3). Each experiment was performed three times with comparable results. The corresponding ethidium bromide-stained 28S band intensities were used to standardize loading conditions.
  • Example 5 Effect of ox-LDL and Simvastatin on ecNOS Activity
  • ecNOS The activity of ecNOS was assessed by measuring the LNMA-inhibitable nitrite production from human endothelial cells (Liao, JK et al., J Clin Invest, 1995, 96:2661-2666). Basal ecNOS activity was 8.8 ⁇ 1.4 nmol/500,000 cells/24 h. Treatment with ox-LDL (50 mg/ml, TBARS 16 nmol/mg) for 48 h decreased ecNOS-dependent nitrite production by 94 ⁇ 3% (0.6 ⁇ 0.5 nmol/500,000 cells/24 h, p ⁇ 0.001).
  • ecNOS mRNA The post-transcriptional regulation of ecNOS mRNA was determined in the presence of the transcriptional inhibitor, actinomycin D (5 mg/ml).
  • Oxidized LDL 50 mg/ml, TBARS 13.1 nmol/mg
  • the blots shown are representative of four separate experiments.
  • ecNOS 5'-promoter construct linked to a luciferase reporter gene (FI .LUC) (Zhang, R et al, JBiol Chem, 1995, 270:15320-15326).
  • This promoter construct contains putative cis-acting elements for activator protein (AP)-l and -2, sterol regulatory element- 1 , retinoblastoma control element, shear stress response element (SSRE), nuclear factor-1 (NF-1), and cAMP response element (CRE).
  • endothelial cells were treated with ox-LDL (50 mg/ml, TBARS 15.1 nmol/mg), simvastatin (1 mmol/L), alone or in combination, in the presence of L-mevalonate (100 mmol/L).
  • ox-LDL 50 mg/ml, TBARS 15.1 nmol/mg
  • simvastatin (1 mmol/L) alone or in combination
  • L-mevalonate 100 mmol/L
  • HMG-CoA Reductase Inhibitors Reduce Cerebral Infarct Size by Upregulating endothelial cell Nitric Oxide Synthase
  • Human endothelial cells were harvested from saphenous veins using Type II collagenase (Worthington Biochemical Corp., Freehold, NJ) as previously described. Cells of less than three passages were grown to confluence in a culture medium containing Medium 199, 20 mM HEPES, 50 mg/ml ECGS (Collaborative Research Inc., Bedford, MA), 100 mg/ml heparin sulfate, 5 mM L-glutamine (Gibco), 5% fetal calf serum (Hyclone, Logan, UT), and antibiotic mixture of penicillin (100 U/ml)/ streptomycin (100 mg/ml)/Fungizone (1.25 mg/ml). For all experiments, the endothelial cells were grown to confluence before any treatment conditions. In some experiments, cells were pretreated with actinomycin D (5 mg/ml) for 1 h prior to treatment with HMG-CoA reductase inhibitors.
  • actinomycin D 5
  • Confluent endothelial cells grown in 100 mm culture dishes were treated with HMG-CoA reductase inhibitors and then placed without culture dish covers in humidified airtight incubation chambers (Billups-Rothenberg, Del Mar, CA).
  • the chambers were gassed with 20%) or 3% O 2 , 5% CO 2 , and balanced nitrogen for 10 min prior to sealing the chambers.
  • the chambers were maintained in a 37°C incubator for various durations (0-48 h) and found to have less than 2% variation in O 2 concentration as previously described (Liao, JK et al., J Clin Invest, 1995, 96:2661-2666).
  • Cellular confluence and viability were determined by cell count, morphology, and trypan blue exclusion.
  • Confluent endothelial cells (5 x 10 7 cells were treated with simvastatin (1 mM) in the presence of 20%> or 3% O 2 for 24 h. Nuclei were isolated and in vitro transcription was performed as previously described (Liao, JK et al., J Clin Invest, 1995, 96:2661-2666). Equal amounts (1 mg) of purified, denatured full-length human ecNOS, human ⁇ -tubulin (ATCC #37855), and linearized pGEM-3z cDNA were vacuum-transferred onto nitrocellulose membranes using a slot blot apparatus (Schleicher & Schuell).
  • Hybridization of radiolabeled mRNA transcripts to the nitrocellulose membranes was carried out at 45°C for 48 h in a buffer containing 50% formamide, 5 X SSC, 2.5 X Denhardt's solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml salmon sperm DNA.
  • the membranes were then washed with 1 x SSC/0.1% SDS for 1 h at 65°C prior to autoradiography for 72 h at -80°C. Band intensities were subjected to analyses by laser densitometry.
  • Nitrite Accumulation The amount of NO produced by ecNOS was determined by nitrite accumulation in the conditioned medium. Nitrite accumulation was determined by measuring the conversion of 2,3-diaminonaphthalene (1.5 mM of DAN in 1 M of HCl) and nitrite to l-(H)-naphthotriazole as previously described (13,24). Nonspecific fluorescence was determined in the presence of LNMA (5 mM). Previous studies with nitrate reductase indicate that the nitrite to nitrate concentration in the medium was approximately 5:1 and that this ratio did not vary with exposure to 20% > or 3%
  • mice (Taconic farm, Germantown, NY) and ecNOS mutant mice (Huang, PL et al., Nature, 1995, 377:239-242.) were subcutaneously- injected with 0.2, 2, or 20 mg of activated simvastatin per kg body weight or saline (control) once daily for 14 days.
  • Ischemia was produced by occluding the left middle cerebral artery (MCA) with a coated 8.0 nylon monofilament under anesthesia as described (Huang, Z et al, J Cereb Blood Flow Metab, 1996, 16:981-987, Huang, Z et al., Science, 1994, 265:1883-1885, Hara, H et al., J Cereb Blood Flow Metab, 1997, 1 :515-526).
  • mice were either sacrificed or tested for neurological deficits using a well-established, standardized, observer-blinded protocol as described (Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987, Huang, Z et al., Science, 1994, 265:1883-1885, Hara, H et al, J Cereb Blood Flow Metab, 1997 ', 1:515-526).
  • the motor deficit score range from 0 (no deficit) to 2 (complete deficit).
  • mice aortae and brains were measured by the conversion of [ 3 H]arginine to [ 3 H]citrulline in the presence and absence of LNMA (5 mM) as described earlier.
  • RNA from mouse aortae and brains was isolated by the guanidinium isothiocyanate method and reverse transcribed using oligo-dT (mRNA Preamplification reagents; Gibco BRL) and Taq ploymerase (Perkin-Elmer).
  • oligo-dT mRNA Preamplification reagents; Gibco BRL
  • Taq ploymerase Perkin-Elmer
  • One tenth of the sDNA was used as template for the PCR reaction.
  • Approximately 0.2 nmol of the following primers amplifying a 254-bp fragment of murine ecNOS cDNA were used: 5 'Primer: 5'-GGGCTCCCTCCTTCCGGCTGCCACC-3' (SEQ ID NO. 1) and 3'Primer: 5'-GGATCCCTGGAAAAGGCGGTGAGG-3' (SEQ ID NO.
  • Example 1 1 Effects of HMG-CoA Reductase Inhibitors on ecNOS Activity
  • the activity of ecNOS was assessed by measuring the LNMA-inhibitable nitrite accumulation from human endothelial cells (Liao, JK et al, J Clin Invest, 1995, 96:2661-2666).
  • the ratio of nitrite to nitrate production under our culture condition was approximately 5:1 and was similar for hypoxia and normoxia.
  • Basal ecNOS activity at 20% O 2 was 6.0 ⁇ 3.3 nmol/500,000 cells/24 h.
  • Example 12 Effects of HMG-CoA Reductase Inhibitors on ecNOS Protein and mRNA Levels
  • Treatment with simvastatin (0.1 mM) increased ecNOS protein levels in a time-dependent manner by 4 ( 6%, 21 ( 9%, 80 ( 8%, 90 ( 12%, and 95 ( 16% after 12 h, 24 h, 48 h, 72 h, and 84 h, respectively (p ⁇ 0.05 for all time points after 12 h, n 4).
  • HMG-CoA reductase inhibitor lovastatin
  • ecNOS protein levels in a time-, and concentration-dependent manner.
  • lovastatin has a higher IC50 value for HMG-CoA reductase compared to that of simvastatin, it was 10-fold less potent in upregulating ecNOS protein levels than simvastatin at equimolar concentrations.
  • Co-treatment with L-mevalonic acid 400 mM completely blocked simvastatin-induced increase in ecNOS protein levels after 48 h (35 ⁇ 2.4 %).
  • simvastatin which was not chemically-activated had no effect on ecNOS expression.
  • Example 13 Effects of HMG-CoA Reductase Inhibitors on ecNOS mRNA Half-life
  • each ecNOS band was standardized to the density of its corresponding ( ⁇ -tubulin band, relative intensity).
  • GAPDH another gene, GAPDH. Similar relative indices were obtained when ecNOS gene transcription was standardized to GAPDH gene transcription. The specificity of each band was determined by the lack of hybridization to the nonspecific pGEM cDNA vector.
  • Example 15 Effect of HMG-CoA Reductase Inhibitors on Mouse Physiology
  • SV-129 wild-type and ecNOS knockout mice were treated with 2 mg/kg simvastatin or saline subcutaneously for 14 days.
  • the mean arterial blood pressures of wild-type and ecNOS mutant mice were as reported previously (Huang, PL et al., Nature, 1995,
  • the ecNOS mutants were relatively hypertensive. There was no significant change in mean arterial blood pressures of wild-type mice after 14 days of simvastatin treatment (81 ⁇
  • control 147 ⁇ 10 vs. simvastatin 161 ⁇ 5.2 mg/dl
  • creatinine and transaminases after treatment with simvastatin compared to control values.
  • Example 16 Effect of HMG-CoA Reductase Inhibitors on ecNOS Expression and Function in Mouse Aorta
  • the activity of ecNOS in the aortae of simvastatin-treated (2 mg/kg, s.c, 14 days) and saline-injected mice was determined by measuring the LNMA-inhibitable conversion of arginine to citrulline (C 14 ).
  • the ecNOS mRNA expression in the aortae of simvastatin-treated and -untreated mice was examined by quantitative RT-PCR.
  • Example 17 Effect of HMG-CoA Reductase Inhibitors on Cerebral Ischemia in Mice
  • Endothelium-derived NO protects against ischemic cerebral injury (Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16:981-987). Therefore we examined, wether the observed upregulation of ecNOS by simvastatin in vivo has beneficial effects on cerebral infarct size. Following treatment for 14 days with 2 mg/kg of simvastatin, cerebral ischemia was produced by occluding the left middle cerebral artery for 2 hours. After 22 hours of reperfusion, mice were tested for neurological deficits using a well-established, standardized, observer-blinded protocol.
  • Example 18 Effect of HMG-CoA Reductase Inhibitors on ecNOS Expression in Mouse Brain
  • the ecNOS mRNA expression in the ischemic and contralateral (non-ischemic) hemispheres of mouse brain was examined by quantitative RT-PCR with respect to GAPDH mRNA levels.
  • I ipsilateral
  • C contralateral
  • rCBF regional cerebral blood flow
  • 450 mg/kg or saline was infused at a constant rate of 100 microliter/kg/min over 15 minutes into wild type mice, mutant mice deficient in endothelial nitric oxide synthase (eNOS null), and mice which had received chronic daily administration of simvastatin (2 mg/kg).
  • Regional cerebral blood flow (rCBF) was monitored by laser-Doppler flowimetry in groups of urethane-anesthetized, ventilated mice.
  • mice were also monitored in the mice, including mean arterial blood pressure (MABP), heart rate, blood pH, PaO 2 , and PaCO 2 .
  • MABP mean arterial blood pressure
  • results Physiological variables during laser-Doppler flowimetry in urethane-anesthetized ventilated wild type, simvastatin-treated and eNOS null mice infused with L-arginine or saline are shown in Table 1. Number of mice in each group is shown in parenthesis. Values are reported as mean +/- SEM. * denotes statistically significant difference (PO.05) compared with eNOS null mice; # denotes statistically significant difference (P ⁇ 0.05) compared with baseline by one-way ANOVA followed by Scheffe test.
  • MABP indicates mean arterial blood pressure
  • sim indicates mice chronically administered simvastatin.
  • Figure 5 is a bar graph showing regional CBF changes in wild type and eNOS null mice for 40 min after L-arginine (450 mg/kg) or saline infusion at a constant rate of 100 micro liter/kg/min over 15 min. The number of mice in each group is indicated in parenthesis. Error bars denote standard error of the mean (SEM), and an asterisk (*) denotes statistically significant difference (PO.05) compared with baseline control by one-way ANOVA followed by Fisher's protected least-squares difference test.
  • L-arginine infusion (450 mg/kg, i.v.) increased rCBF in parietal cortex in wild type mice, as shown in Figure 1 (Fig. 1).
  • the increase in rCBF began at 5-10 minutes and achieved statistical significance at 10-15 minutes after infusion. Maximum values achieved at 20-25 min reached 26% above, after which values decreased to control levels.
  • L-arginine did not increase rCBF in eNOS null mice. Values in these mutants ranged from -4 to +5% during the 40 minute recording period. Saline infusion in wild type mice did not increase rCBF significantly.
  • Figure 6 is a bar graph showing regional CBF changes in simvastatin-treated mice for 40 min after L-arginine or saline infusion at the same dose. The number of mice in each group is indicated in parenthesis; sim indicates simvastatin. Error bars denote SEM and an asterisk (*) denotes statistically significant difference (P ⁇ 0.05) compared with baseline control by one-way ANOVA followed by Fisher's protected least-squares difference test.

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Abstract

L'invention concerne une nouvelle utilisation d'inhibiteurs de la HMG-CoA réductase. Dans la présente invention, on a découvert que les inhibiteurs de la HMG-CoA réductase font une régulation positive de l'activité de l'oxyde nitrique synthase des cellules endothéliales par un mécanisme autre que celui consistant à empêcher la formation de LDL oxydantes. Par conséquent, les inhibiteurs de la HMG-CoA réductase sont utilisés dans le traitement ou la prévention des états pathologiques résultant d'une expression et/ou d'une activité anormalement faibles de l'oxyde nitrique synthase des cellules endothéliales. Ces états pathologiques comprennent l'hypertension artérielle pulmonaire, l'infarctus cérébral, l'impuissance, l'insuffisance cardiaque, les états pathologiques induits par l'hypoxie, la carence insulinique, la néphropathie évolutive, le syndrome de la motricité gastrique ou oesophagienne, etc. Les sujets susceptible de bénéficier le plus de ces traitements sont ceux atteints de non-hyperlipidémie et de non-hypercholestérolémie, sans exclure expressément les sujets atteints d'hyperlipidémie ou d'hypercholestérolémie.
PCT/US2000/007221 1999-03-19 2000-03-17 Regulation positive de l'oxyde nitrique synthase des cellules endotheliales de type iii par des inhibiteurs de la hmg-coa reductase WO2000056403A1 (fr)

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AU37603/00A AU3760300A (en) 1999-03-19 2000-03-17 Upregulation of type iii endothelial cell nitric oxide synthase by hmg-coa reductase inhibitors
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EP1370210A2 (fr) * 2001-02-07 2003-12-17 The McLean Hospital Corporation Hypocholesterolemiants utilises pour traiter les troubles psychologiques et cognitifs
WO2004096278A1 (fr) * 2003-04-28 2004-11-11 Sankyo Company, Limited Activateur de la production d'adiponectine
US6818669B2 (en) 1999-03-19 2004-11-16 Enos Pharmaceuticals, Inc. Increasing cerebral bioavailability of drugs
JP2006510640A (ja) * 2002-12-09 2006-03-30 フレセニウス・カビ・ドイチュランド・ゲーエムベーハー 胃腸に投与することができる配合物、およびその使用
EP1729761A1 (fr) * 2004-03-05 2006-12-13 Eisai Co., Ltd. Traitement de cadasil avec des inhibiteurs de la cholinesterase
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US7381731B2 (en) 1994-10-05 2008-06-03 Angiogenix, Inc. Pharmaceutical composition comprising citrulline
WO2009000592A1 (fr) * 2007-06-25 2008-12-31 Nicox S.A. Utilisation de statines a liberation d'oxyde nitrique dans le traitement de l'hypertension arterielle pulmonaire
US7772272B2 (en) 2003-04-28 2010-08-10 Daiichi Sankyo Company, Limited Method for enhancing glucose uptake into warm-blooded animal adipocytes
US7893050B2 (en) 2005-10-26 2011-02-22 Asahi Kasei Pharma Corporation Fasudil in combination therapies for the treatment of pulmonary arterial hypertension
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WO2019067844A1 (fr) * 2017-09-28 2019-04-04 University Of Massachusetts Facilitation endothéliale dans des maladies neurodégénératives par amélioration du flux sanguin cérébral
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JP4949661B2 (ja) * 2004-09-21 2012-06-13 第一三共株式会社 ＨＭＧ−ＣｏＡリダクターゼ阻害剤とグルタチオンを含有する医薬組成物
WO2006033287A1 (fr) * 2004-09-21 2006-03-30 Sankyo Company, Limited PRÉPARATION PHARMACEUTIQUE CONTENANT UN INHIBITEUR DE LA HMG-CoA RÉDUCTASE ET DE LA GLUTATHIONE

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US7381731B2 (en) 1994-10-05 2008-06-03 Angiogenix, Inc. Pharmaceutical composition comprising citrulline
US6465516B1 (en) 1997-04-10 2002-10-15 Nitrosystems, Inc. Method of stimulating nitric oxide synthase
US6239172B1 (en) 1997-04-10 2001-05-29 Nitrosystems, Inc. Formulations for treating disease and methods of using same
US6818669B2 (en) 1999-03-19 2004-11-16 Enos Pharmaceuticals, Inc. Increasing cerebral bioavailability of drugs
JP2017019876A (ja) * 2000-11-17 2017-01-26 ノバルティス アーゲー 循環器系疾患のためのレニン阻害剤を含む相乗的組合せ剤
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EP1175246A4 (fr) 2004-12-15
CA2368187A1 (fr) 2000-09-28
AU3760300A (en) 2000-10-09
JP2003511347A (ja) 2003-03-25

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