JP2003511347A - Upregulation of type III endothelial cell nitric oxide synthase by HMG-CoA reductase inhibitor - Google Patents

Abstract

  (57) [Summary] Novel uses of HMG-CoA reductase inhibitors are provided. In the present invention, HMG-CoA inhibitors are found to up-regulate endothelial cell nitric oxide synthase activity through a mechanism other than preventing the formation of oxidized LDL. As a result, HMG-CoA reductase inhibitors are useful for treating or preventing abnormalities resulting from abnormally low expression and / or activity of endothelial cell nitric oxide synthase. Such abnormalities include pulmonary hypertension, ischemic stroke, impotence, heart failure, hypoxia-induced abnormalities, insulin deficiency, progressive renal disease, gastric or esophageal motility syndrome, and the like. Subjects that would benefit most from such treatment include, but do not necessarily exclude, hyperlipidemia and hypercholesterolemia.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to HMG-CoA as an upregulator of type III endothelial cell nitric oxide synthase.
New uses of reductase inhibitors are described. Furthermore, the present invention describes methods of using HMG-CoA reductase inhibitors to treat conditions resulting from abnormally low expression and / or activity of endothelial cell nitric oxide synthase in a subject.

BACKGROUND OF THE INVENTION Nitric oxide (NO), cardiovascular, in the nervous and immune system has many physiological roles, it has been recognized as unique messenger molecules (Griffith, TM et al.
, J. Am Coll Cardiol, 1988, 12: 797-806). Nitric oxide mediates vasorelaxation, 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). At least three isozymes of NO synthase have been identified in mammals. Neurons (nNOS) and endothelial cells (type III-ecNO
The two expressed in S) are calcium-dependent, while the third is calcium-independent and expressed by macrophages and other cells after induction with cytokines (type II-iNOS ) (Bredt, DS and Snyder, SH, Proc Natl Acad
Sci USA, 1990, 87: 682-685, Janssens, SP et al., J Biol Chem, 1992, 267: 229.
64, Lyons, CR et al., J Biol Chem, 1992, 267: 6370-6374). The diverse physiological and pathological effects of NO are due to its reactivity and different pathways of formation and metabolism.
It is possible to explain.

Recent studies suggest that loss of endothelium-derived NO activity may contribute to the atherogenic process (O'Driscoll, G et al., Circulation, 1997, 95: 1126-1).
131). For example, NO derived from endothelium is used for monocyte adhesion to the surface of endothelium (Tsao, PS et al., Circulati
on, 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., Circ
ulation, 1991, 83: 2012-2020), which inhibits several components of the atherogenic process. Moreover, NO, especially in its oxidised form, is able to interfere with the oxidative modification of low density lipoprotein (LDL), a major contributor to atherosclerosis (Cox, DA and Cohen, ML, Pharm Rev, 1996, 48: 3-19).

In the prior art, hypoxia has been shown to down-regulate ecNOS expression and / or activity through a reduction 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 Ph.
ysiol, 1997, 272: L1005-L1012). Therefore, ischemia-induced hypoxia can have deleterious effects, in part through a decrease in ecNOS activity.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is a microsomal enzyme that catalyzes a rate-limiting reaction in cholesterol biosynthesis (HMG-Co
A6 mevalonate). HMG-CoA reductase inhibitors inhibit HMG-CoA reductase and, consequently, cholesterol synthesis. Some HMG-CoA reductase inhibitors have been used to treat hypercholesterolemic individuals. Clinical trials with such compounds have shown that cholesterol levels in hypercholesterolemic patients are greatly reduced. Moreover, reduced serum cholesterol levels have been shown to correlate with improved endothelium-dependent relaxation of atherosclerotic vessels (Treasure, CB et al., N Engl J Med, 1995, 332: 481-487).
. In fact, one of the earliest recognizable benefits after treatment with HMG-CoA reductase inhibitors is the restoration of endothelium-dependent relaxation (supra Anderson, TJ et al., NEngl J Med, 1995, 3
32: 488-493).

The mechanism by which HMG-CoA reductase inhibitors restore endothelial function is mainly in liver HMG-CoA.
It is largely unknown whether inhibition of endothelial HMG-CoA reductase has an additional beneficial effect on endothelial function, although it may be due to inhibition of reductase and subsequent reduction in serum cholesterol levels.

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 have demonstrated reduced NO release in pulmonary arterial blood vessels in patients with pulmonary hypertension (
Giaid, A and Saleh, D, N Engl J Med, 1995, 333: 214-221, Shaul, PW, Am
J Physiol, 1997, 272: L1005-L1012). Furthermore, individuals with pulmonary hypertension exhibit reduced levels of ecNOS expression in pulmonary blood vessels and benefit clinically from inhaled nitric oxide therapy (Roberts, JD et al., N Engl J Med, 1997, 336: 605-610, Kou
youmdjian, C et al., J Clin Invest, 1994, 94: 578-584). Conversely, neonatal lambs treated with mutant mice lacking the ecNOS gene or the ecNOS inhibitor, N-omega-monomethyl-L-arginine (LNMA or N-omega-nitro-L-arginine) showed pulmonary arterial blood pressure and resistance. Develop progressive rise (Steudel, W et al., Circ Res, 1997,
81: 34-41, Fineman, JR et al., J Clin Invest, 1994, 93: 2675-2683). It has also been shown in the prior art that hypoxia causes pulmonary vasoconstriction through 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). Therefore, hypoxia-mediated downregulation of ecNOS may lead to vasoconstrictor and structural changes associated with pulmonary hypertension.

Stroke, often cited as the third leading cause of death in developing countries, is a function of brain function caused by a variety of pathological changes that affect one or several intracranial or extracranial blood vessels. It has been defined as sudden damage. Approximately 80% of all strokes are ischemic strokes resulting from restricted blood flow. Mutant mice lacking the ecNOS gene are hypertensive (Huang, PL et al., Nature, 1995, 377: 239-242, Ste.
udel, W et al., Circ Res, 1997, 81: 34-41), and in response to cuff injury,
Develop more intimal smooth muscle proliferation. Furthermore, occlusion of the middle cerebral artery results in 21% greater infarct size in "ecNOS knockout" mice compared to wild-type mice (Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16: 981-987). . These findings suggest that ecNOS production may play a role in cerebral infarction formation and size. Moreover, since the majority of patients with ischemic stroke have mean or normal cholesterol levels, it is largely unknown what the potential benefits of HMG-CoA reductase inhibitor administration in cerebrovascular events are.

There is a need to identify agents that improve endothelial cell function. There also exists a need to identify agents that can be used in an acute or prophylactic manner to treat conditions that result from low levels of endothelial nitric oxide synthase.

SUMMARY OF THE INVENTION The present invention allows HMG-CoA reductase inhibitors to upregulate endothelial cell nitric oxide synthase (type III) not through interfering with the formation of oxidized LDL. Regarding the discovery. Previously, these reductase inhibitors were thought to function in the cholesterol biosynthetic pathway by lowering serum cholesterol levels by blocking the hepatic conversion of HMG-CoA to L-mevalonate. Surprisingly, HMG-CoA reductase inhibitors may be able to increase nitric oxide synthase activity by directly affecting the endothelium rather than liver HMG-CoA reductase. It has been discovered. Up-regulation of this activity does not depend on reduced cholesterol synthesis and, in particular, on reduced ox-LDL formation. Accordingly, the present invention is useful in restoring endothelial cell nitric oxide synthase activity, or increasing such activity in affected cells or tissues, whenever desired. Tissue is defined to include both cells in the tissue that express endothelial cell nitric oxide synthase, as well as cells in the vasculature that supply the tissue with nutrients.

Nitric oxide synthase activity is involved in many abnormalities, including impotence, heart failure, gastric and esophageal motility disorders, renal disorders such as renal hypertension and progressive renal disease, insulin deficiency, and the like. Individuals with such abnormalities will benefit from increased endothelial cell nitric oxide synthase activity. It was also known that individuals with pulmonary hypertension exhibit reduced levels of nitric oxide synthase expression in the pulmonary blood vessels and benefit clinically from inhalation of nitric oxide. Therefore, the present invention is particularly useful for treating pulmonary hypertension. It has also been demonstrated that hypoxia causes inhibition of endothelial cell nitric oxide synthase activity. Therefore, the present invention is useful for treating a subject having hypoxia-induced abnormalities. Surprisingly, HMG-CoA
It has also been discovered that reductase inhibitors are useful in reducing brain damage that follows stroke.

According to one aspect of the invention, a method of increasing endothelial cell nitric oxide synthase activity in a non-cholesterolemia subject that would benefit from increased endothelial cell nitric oxide synthase activity in tissue. Will be provided. The method provides a HMG-CoA reductase that increases nitric oxide synthase activity in a subject in need of such treatment in an amount effective to increase endothelial cell nitric oxide synthase activity in the tissue of the subject. It involves administering an inhibitor.

In certain embodiments, the use of HMG CoA reductase inhibitors is ruled out if the subject in need of such treatment has an abnormally elevated risk of ischemic stroke. In one important aspect, the HMG-CoA reductase inhibitor does not affect cholesterol levels in the subject. In patients with atherosclerosis, reduced serum cholesterol correlates with improved endothelium-dependent relaxation of atherosclerotic vessels (
Treasure et al., N. Engl. J. Med., 1995, 332: 481-487). HMG-CoA reductase inhibitors have been demonstrated to reduce serum cholesterol within a week, and maximal levels of cholesterol reduction can be achieved months after chronic administration. In contrast, the effects of HMG-CoA reductase inhibitors on ecNOS upregulation occur within days. Thus, the treatment according to the present invention has a significant benefit, even for hypercholesterolemic patients, when administered when presenting a short-term increase in risk of stroke or other embolic events, for example due to surgical intervention. I will provide a.

In certain embodiments, the subject is not hypercholesterolemia or hypertriglyceridemia or both (ie non-hyperlipidemia). In other embodiments, the amount is sufficient to increase endothelial cell nitric oxide synthase activity above the normal baseline levels established by the age control group, described in more detail below.
In certain embodiments, the HMG-CoA reductase inhibitor is administered in an amount that modifies blood LDL cholesterol levels in the subject by less than 10%. The modification may be less than 5%. In certain embodiments, the amount is sufficient to increase endothelial cell nitric oxide synthase activity above the normal baseline levels established by the age-adjusted group, described in more detail below.

The subject can have a condition that is hypoxia-inducible and can be characterized by abnormally low levels of endothelial cell nitric oxide synthase activity. In other embodiments, the subject may have a condition that includes an abnormally low level of endothelial cell nitric oxide synthase activity that is chemically induced. In yet another aspect, the subject may have a condition that is abnormally low in levels of endothelial cell nitric oxide synthase activity that is cytokine-inducible. In certain important aspects, the subject has pulmonary hypertension or an abnormally elevated risk of pulmonary hypertension. In another important aspect, the subject has had an ischemic stroke or has an abnormally elevated risk of an ischemic stroke. In yet another important aspect, the subject has heart failure or progressive renal disease. In yet another important aspect, the subject is chronically exposed to a hypoxic condition.

According to any of the above embodiments, which are preferred, the preferred HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. In a further important aspect, the patient has undergone a thrombotic event or has an abnormally elevated risk of thrombosis. In still other embodiments, the subject has an abnormally elevated risk of atherosclerosis or has atherosclerosis. In another important aspect, the subject has an abnormally elevated risk of developing a myocardial infarction or has experienced a myocardial infarction. In a further important aspect, the subject has an abnormally elevated risk of reperfusion injury. In a preferred embodiment, the subject having an abnormally elevated risk of reperfusion injury is an organ transplant recipient (eg heart, kidney, liver, etc.). In another important aspect, the subject has homocystinuria. In certain other important aspects, the subject has cerebral autosomal dominant arteropathy with subcortical infarction and leukoencephalopathy (CADASIL).
Have a syndrome. In a further important aspect, the subject has a degenerative disorder of the nervous system. In a preferred embodiment, the subject with a degenerative disorder of the nervous system has Alzheimer's disease.

In certain other embodiments, where the subject in need of treatment according to the invention has an abnormally elevated risk of ischemic stroke, the HMG-CoA reductase inhibitor is treated with such subject. Excluded as.

In accordance with any of the foregoing aspects, which may be any, the method further comprises coadministering an endothelial cell nitric oxide synthase substrate, preferably L-arginine, and / or endothelial cell nitric oxide synthase activity. May also be included in the administration of a non-HMG-CoA reductase inhibitor. Preferred such agents are selected from the group consisting of estrogen and angiotensin converting enzyme (ACE) inhibitors. The agent is
It may be administered to a subject having an abnormality, or may be administered prophylactically to a subject having a risk of developing an abnormality, and more preferably an abnormally elevated risk. The inhibitor may also be administered acutely.

In accordance with another aspect of the present invention, in treating a non-hyperlipidemic condition that is favorably affected by increased endothelial cell nitric oxide synthase activity in the tissue, the endothelial cell Methods for increasing nitric oxide synthase activity are provided. Such a condition is exemplified above. The method provides a subject in need of such treatment with HMG-Co in an amount effective to increase endothelial cell nitric oxide synthase activity in tissue of the subject.
It involves administering an A reductase inhibitor. The key conditions are as described above. Also as described above, the method provides a non-HMG-Co that increases the substrate of endothelial cell nitric oxide synthase and / or increases endothelial cell nitric oxide synthase activity.
Administration of an A reductase inhibitor may be accompanied. Preferred compounds are as described above. As mentioned above, the reductase inhibitor may be administered, inter alia, acutely or prophylactically.

In certain embodiments, if the subject in need of such treatment has an abnormally elevated risk of ischemic stroke, the HMG CoA reductase inhibitor is excluded from treating such subject.

According to another aspect of the invention, there is provided a method of reducing brain damage resulting from stroke. The method provides a subject having an abnormally high risk of ischemic stroke with an amount of HMG-Co effective to increase endothelial cell nitric oxide synthase activity in the subject brain.
It involves administering an A reductase inhibitor. As mentioned above, important embodiments include inhibitors selected from the group consisting of simvastatin and lovastatin. Also, as mentioned above, important aspects are non-increasing endothelial cell nitric oxide synthase substrates (preferably L-arginine) and / or endothelial cell nitric oxide synthase activity.
Administering a HMG-CoA reductase inhibitor. Similarly, important aspects include prophylactic and acute administration of the inhibitors.

In certain embodiments, if the subject in need of such treatment has an abnormally elevated risk of ischemic stroke, the HMG CoA reductase inhibitor is excluded from treating such subject.

According to another aspect of the invention, a method of treating pulmonary hypertension is provided. The method involves administering to a subject in need of such treatment a HMG-CoA reductase inhibitor in an amount effective to increase lung endothelial cell nitric oxide synthase activity in the subject. Particularly important aspects are as described above in relation to methods of treating brain injury. Another important aspect is the prophylactic administration of inhibitors to subjects with an abnormally elevated risk of developing pulmonary hypertension, which may affect subjects exposed to chronic hypoxia. Including.

According to another aspect of the invention, a method of treating heart failure is provided. The method 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. As discussed above, important aspects include prophylactic and acute administration of inhibitors. Another important aspect is treating a subject who is non-hyperlipidemic. Preferred compounds and dosing regimens are as described above.

According to yet another aspect of the invention, there is provided a method of 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 subject. Important aspects, as well as preferred compounds and regimens for administration, are:
As related to heart failure, as described above.

According to another aspect of the invention, there is provided a method of increasing blood flow in a subject tissue. 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 subject tissue. In a preferred embodiment, the tissue that increases blood flow includes tissue in the brain. In a particularly preferred embodiment, cerebral blood flow is enhanced. As discussed above, important aspects include prophylactic and acute administration of inhibitors. Preferred compounds and dosing regimens are also as described above. In another important aspect, a subject in a condition treatable by the second agent is co-administered with the second agent in an amount effective to treat the condition, thereby increasing the blood flow. As a result, the method includes enhancing the delivery of the second agent to the subject tissue.

In certain embodiments, the tissue is the brain and the second agent comprises an agent that has a site of action in the brain. In accordance with another aspect of the present invention, there is a screening method for identifying HMG-CoA reductase inhibitors for the treatment of subjects that would benefit from increased endothelial cell nitric oxide synthase activity in tissues. Provided. The method identifies an inhibitor of HMG-CoA reductase that is thought to increase endothelial cell nitric oxide synthase activity, and the inhibitor of HMG-CoA reductase inhibits endothelial cell monocytosis in vivo or in vitro. It involves determining whether it results in increased nitric oxide synthase activity. In certain embodiments, the subject that would benefit from increased endothelial cell nitric oxide synthase activity,
Have an abnormally elevated risk of stroke.

The present invention also involves the use of HMG-CoA reductase inhibitors in the manufacture of a medicament for treating the above mentioned disorders. The important abnormalities and compounds are as described above. The invention further comprises a pharmaceutical preparation comprising an HMG-CoA reductase inhibitor for treating the above mentioned disorders. The preparation may include other agents, such as second agents, ecNOS substrates, ecNOS cofactors, as described above, or directly or indirectly (synergistic, cooperative, additive, etc.) It may be a cocktail of HMG-CoA reductase inhibitors together with a non-HMG-CoA reductase inhibitor that increases the ecNOS activity therein.

In certain embodiments, compositions and pharmaceutical preparations are provided that are cocktails of HMG-CoA reductase inhibitors and L-arginine. In other embodiments, the HMG-CoA reductase inhibitor and L-arginine are in effective amounts to increase blood flow. In a further embodiment, the HMG-CoA reductase inhibitor and L-arginine are in effective amounts to increase blood flow in brain tissue. In a preferred embodiment, administration of the HMG-CoA reductase inhibitor and L-arginine results in increased blood flow. In a particularly preferred embodiment, administration of the HMG-CoA reductase inhibitor and L-arginine results in increased blood flow to the brain. Any of the cocktail compositions described above may also include other cofactors that enhance ecNOS substrate conversion by ecNOS to nitric oxide, with preferred cofactors being NADPH and tetrahydrobiopterin.

The present invention also comprises contacting cells with an effective amount of an HMG-CoA reductase inhibitor, alone or in combination with any of the dosing agents as described above or as a cocktail as described above. Thus, a method of increasing ecNOS activity in cells is also included. Any of the above cocktails may also include a substrate for endothelial cell nitric oxide synthase, the preferred substrate is L-arginine, and / or other cofactors that enhance ecNOS substrate conversion by ecNOS to nitric oxide. Preferred cofactors are NADPH and tetrahydrobiopterin.

These and other aspects of the invention are described in detail below. DETAILED DESCRIPTION OF THE INVENTION The present invention is useful whenever it is desirable to increase endothelial cell nitric oxide synthase (type III isoform) activity in a subject. Nitric oxide synthase is an enzyme that catalyzes the reaction of producing nitric oxide from the substrate L-arginine. As the name implies, endothelial cell nitric oxide synthase refers to the type III isoform of the enzyme found in endothelium.

By “ecNOS activity” is meant the ability of a cell to produce nitric oxide from the substrate L-arginine. Increased ecNOS activity can be achieved by several different methods. For example, increasing the amount of ecNOS protein or increasing the activity of the protein (while maintaining a constant level of protein) can result in an increase in "activity". The increased amount of available protein may result from increased transcription of the ecNOS gene, increased stability of ecNOS mRNA, decreased ecNOS proteolysis.

EcNOS activity in cells or tissues can be measured in a variety of different ways. A direct measurement would be to measure the amount of ecNOS present. Another direct measure would be to measure the conversion of arginine to citrulline by ecNOS or the production of nitric oxide by ecNOS under certain conditions, eg physiological conditions of tissues. ecNOS activity can also be measured more indirectly, for example, by measuring mRNA half-life (upstream index) or by a phenotypic response to the presence of nitric oxide (downstream index). One phenotypic measure used in the art is to detect endothelium-dependent relaxation in response to acetylcholine, whose response is affected by ecNOS activity. The level of nitric oxide present in the sample can be measured using a nitric oxide measuring device. All of the above techniques are known to those of ordinary skill in the art, and some are described in the examples below.

The present invention not only makes it possible to reestablish normal baseline levels of ecNOS activity by causing an increase in ecNOS activity, but it is also possible to increase such activity above normal baseline levels. To Normal baseline levels are age-regulated and have no symptoms that would indicate altered endothelial cell nitric oxide synthase activity (hypoxic conditions, hyperlipidemia and comparable),
This is the amount of activity in the normal control group. Thus, the actual level will depend on the particular age group selected to assay activity and the particular measurement used. Specific examples of various measurements are provided below. In abnormal environments, such as hypoxia and pulmonary hypertension, endothelial cell nitric oxide synthase activity falls below normal levels. Surprisingly, in accordance with the present invention, using reductase inhibitors, it is not only possible to restore normal baseline levels in these abnormal conditions, but also to enhance endothelial cell nitric oxide synthase activity, Endothelial cell nitric oxide synthase activity can be increased much higher than desired above normal baseline levels as desired. Thus, "increased activity" results from treatment with a reductase inhibitor according to the invention, including but not limited to activity and above normal baseline levels that would be sufficient to restore normal baseline levels. Means any increase in endothelial cell nitric oxide synthase activity in the subject, including activity that would be sufficient to increase the activity.

As noted above, nitric oxide synthase activity can be associated with stroke, pulmonary hypertension, thrombosis, atherosclerosis, myocardial infarction, (eg, in an organ transplant recipient).
Includes reperfusion injury, impotence, heart failure, gastric and esophageal motility disorders, renal disorders such as renal hypertension and progressive renal disease, insulin deficiency, hypoxia-induced abnormalities, homocystinuria, neurodegenerative disorders, CADASIL syndrome, etc. , Associated with many anomalies. 1 of the present invention
In one embodiment, the decrease in endothelial cell nitric oxide synthase activity is cytokine-induced. Cytokines are soluble polypeptides produced by a variety of cells that regulate gene activation and cell surface molecule expression. They play an essential role in the development of the immune system and thus in the development of the immune response. But,
In addition to many beneficial properties, cytokines have also been implicated in the mechanisms of development of diverse inflammatory diseases. For example, the cytokines TNF-a and IL-1 are part of the mechanism that causes diseases such as non-cholesterol-induced atherosclerosis, graft arteriosclerosis, rheumatoid arthritis, lupus, scleroderma, and emphysema. it is conceivable that. Subjects with such disorders exhibit lower (and thus "cytokine-induced") levels of endothelial cell nitric oxide synthase activity and may benefit from therapy with agents of the invention.

One important aspect of the present invention is the treatment of ischemic stroke. Ischemic stroke (ischemic cerebral infarction) is an acute neurological injury resulting from diminished blood flow involving the blood vessels of the brain. Ischemic stroke is divided into two broad categories, thrombotic and embolic.

There have been surprising discoveries associated with the treatment of ischemic stroke. In particular, it has been discovered that treatment according to the present invention is able to reduce brain damage following ischemic stroke. The reduction in brain damage as demonstrated in the examples below can be measured by measuring the reduction in infarct size in the treated versus the control group. Similarly, functional tests measuring neurological deficits provided further evidence of reduced brain damage in treated animals versus controls. Cerebral blood flow was also superior in treated animals to controls. Therefore, a positive effect was observed in treated versus control animals in various observed models of brain injury after stroke. All of the above positive results are believed to be due to upregulation of endothelial cell nitric oxide synthase activity, which is believed to be demonstrated in the Examples below.

An important aspect of the present invention is the treatment of subjects with an abnormally elevated risk of ischemic stroke. As used herein, subjects with an abnormally elevated risk of ischemic stroke are a category determined according to conventional medical practice; such subjects are also subject to stroke in conventional medical practice. It can also be identified as having a known risk factor for or having an increased risk of a cerebrovascular event.
Typically, the risk factors associated with heart disease are the same as those associated with stroke. The main risk factors include hypertension, hypercholesterolemia, and smoking. In addition, atrial fibrillation or recent myocardial infarction are important risk factors. As used herein, a subject at abnormally elevated risk of ischemic stroke is also at risk of embolus release, decreased blood pressure or decreased blood flow to the brain, such as carotid endarterectomy, cerebrovascular. Includes individuals undergoing surgical or diagnostic procedures such as imaging, neurosurgical procedures to compress or occlude blood vessels, cardiac catheterization, angioplasty including balloon angioplasty, coronary artery bypass surgery, or similar procedures. Subjects with an abnormally elevated risk of ischemic stroke may also lead to diminished blood flow to the brain, such as atrial fibrillation, ventricular tachycardia, dilated cardiomyopathy and other anticoagulants It also includes an individual having a heart condition such as any of the following. Subjects with an abnormally elevated risk of ischemic stroke also include arteriopathy or cerebral vasculitis,
Also included are individuals with conditions such as, for example, lupus, congenital vascular disorders such as CADASIL syndrome, or migraine, especially long episodes. In certain embodiments, the subject is not hypercholesterolemia or hypertriglyceridemia or both (ie non-hyperlipidemia).

Treatment of stroke may be for patients who have had a stroke or may be prophylactic treatment. Short-term prophylactic treatment reduces the damage due to any ischemic event that may occur as a result of the procedure, and is therefore a surgical or diagnostic procedure at risk of embolus release, decreased blood pressure or decreased blood flow to the brain. Required for subjects with Long-term or chronic prophylactic treatment is required for subjects with cardiac conditions that can lead to diminished blood flow to the brain, or conditions that directly affect the cerebrovascular system. When prophylactic, treatment is for the treatment of subjects at abnormally elevated risk of ischemic stroke, as described above. If the subject has previously undergone an ischemic event and therefore has an abnormally elevated risk of ischemic stroke, prophylactic treatment for these subjects may involve the use of HMG CoA reductase inhibitors. Exclude.
If the subject has had a stroke, treatment may include acute treatment. Acute treatment for stroke subjects refers to the administration of HMG-CoA reductase inhibitors at the onset of abnormal symptoms or at the onset of substantial changes in existing abnormal symptoms.

Another important aspect of the present invention is the treatment of pulmonary hypertension. Pulmonary hypertension is a disease characterized by increased pulmonary arterial blood pressure and pulmonary vascular resistance. Hypoxia, hypocapnia, and the abnormal diffusivity of carbon monoxide are nearly unchanged manifestations of the disease. Furthermore, in accordance with the present invention, pulmonary hypertensive patients also have reduced levels of ecNOS expression in pulmonary blood vessels. Traditionally, the criteria for subjects with or at risk for pulmonary hypertension are Heath and Edwards (Circulation, 1958, 18: 533-547).
Defined based on clinical and histological features according to

The subject may be treated prophylactically to reduce the risk of pulmonary hypertension, or the subject may be treated chronically and / or acutely. If the treatment is prophylactic, the subject being treated is a subject at abnormally elevated risk of pulmonary hypertension. Subjects with an abnormally elevated risk of pulmonary hypertension include those chronically exposed to hypoxic conditions, subjects with persistent vasoconstriction, subjects with multiple pulmonary embolisms, and cardiac hypertrophy subjects. Subjects and / or subjects with a family history of pulmonary hypertension.

Another important aspect of the present invention involves treating hypoxia-induced abnormalities. Hypoxia is defined herein as a decrease in tissue below normal levels of oxygen. Hypoxia can result from a variety of situations, but most often results from impaired lung function. Pulmonary function impairment can include emphysema, smoking, chronic bronchitis, asthma, infectious agents, pneumonia (infectious or chemical), lupus, rheumatoid arthritis, cystic fibrosis and other genetic disorders, obesity, α ₁ -anti It can be caused by trypsin deficiency and its counterparts. It can also result from non-lung damage, such as living at a very high altitude. Hypoxia can result in pulmonary vasoconstriction via inhibition of ecNOS activity.

Another important aspect of the present invention is the treatment of heart failure. Heart failure is a clinical syndrome of diverse etiologies associated by the common features of beating heart failure, and the heart is unable to beat blood equal to the demand of metabolizing tissue, or elevated filling pressure. Characterized by beating only by.

The present invention involves treatment of the aforementioned disorders with HMG-CoA reductase inhibitors. "HMG
-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme A) "is a microsomal enzyme that catalyzes the rate-limiting reaction in cholesterol biosynthesis (HMG-CoA6
Mevalonate). "HMG-CoA reductase inhibitor" inhibits HMG-CoA reductase,
And thus inhibits the synthesis of cholesterol. Described in the art, which have been found to inhibit HMG-CoA reductase, and which are naturally or synthetically derived, forming a category of agents that are useful in practicing the present invention. There are numerous compounds. Traditionally, these agents have been used to treat individuals with hypercholesterolemia. Examples include those commercially available, such as simvastatin (US Pat. No. 4,444,784), lovastatin (US Pat. No. 4,231,938),
Pravastatin sodium (US Pat. No. 4,346,227), fluvastatin (US Pat. No. 4,739,073), atorvastatin (US Pat. No. 5,273,995), cerivastatin, and US Pat. No. 5,622,985, US Pat. No. 5,135,935, US Pat.
356,896, U.S. Patent No. 4,920,109, U.S. Patent No. 5,286,895, U.S. Patent No. 5,262.
, 435, U.S. Patent 5,260,332, U.S. Patent 5,317,031, U.S. Patent 5,283,25.
6, U.S. Patent No. 5,256,689, U.S. Patent No. 5,182,298, U.S. Patent No. 5,369,125, U.S. Patent No. 5,302,604, U.S. Patent No. 5,166,171, U.S. Patent No. 5,202,327,
United States Patent No. 5,276,021, United States Patent No. 5,196,440, United States Patent No. 5,091,386, United States Patent No. 5,091,378, United States Patent No. 4,904,646, United States Patent No. 5,385,932, United States Patent No. 5,250,435, United States Patent No. 5,132,312, United States Patent No. 5,130,306, U.S. Patent No. 5,116,870, U.S. Patent No. 5,112,857, U.S. Patent No. 5,102,911, U.S. Patent No. 5,098,931, U.S. Patent No. 5,081,136, U.S. Patent No. 5,025,000, U.S. Patent No.
5,021,453, U.S. Pat.No. 5,017,716, U.S. Pat.No. 5,001,144, U.S. Pat.No. 5,0
01,128, U.S. Pat.No. 4,997,837, U.S. Pat.No. 4,996,234, U.S. Pat.No. 4,994,
494, U.S. Patent No. 4,992,429, U.S. Patent No. 4,970,231, U.S. Patent No. 4,968,693
No., U.S. Patent No. 4,963,538, U.S. Patent No. 4,957,940, U.S. Patent No. 4,950,675, U.S. Patent No. 4,946,864, U.S. Patent No. 4,946,860, U.S. Patent No. 4,940,800,
U.S. Pat.No. 4,940,727, U.S. Pat.No. 4,939,143, U.S. Pat.No. 4,929,620, U.S. Pat.No. 4,923,861, U.S. Pat.No. 4,906,657, U.S. Pat.No. 4,906,624 and U.S. Pat. And the disclosures of these patents are incorporated herein by reference.

An important aspect of the present invention involves a population that has not previously been treated with HMG-CoA reductase inhibitors. Accordingly, the present invention is directed, in certain aspects, to otherwise HM.
It involves treatment of individuals who do not have symptoms that require treatment with G-CoA reductase inhibitors. The only such clinically recognized abnormality is considered hypercholesterolemia, where reductase inhibitors are administered with the purpose of interfering with cholesterol biosynthesis. Thus, in certain embodiments, the population treated is non-hypercholesterolemic. In other embodiments, the patient has non-triglyceridemia. In still other embodiments, the patient has non-hypercholesterolemia and / or non-hypertriglyceridemia, ie, non-hyperlipidemia.

A non-hypercholesterolemic subject is a subject that does not meet the current standards established for hypercholesterolemic subjects (eg, Harrison's Principles of
See Experimental Medicine, 13th Edition, McGraw-Hill, Inc., New York, "Harrison's"). Hypercholesterolemic and hypertriglyceridemic subjects are associated with an increased incidence of premature coronary heart disease.
Hypercholesterolemic subjects have LDL levels> 160 mg / dl or> 130 mg / dl, and male, family history of premature coronary heart disease, smoking (10 or more daily)
, Hypertension, low HDL (<35 mg / dl), diabetes mellitus, hyperinsulinemia, abdominal obesity,
Have at least two risk factors selected from the group consisting of high lipoprotein (a) and a personal history of cerebrovascular disease or obstructive peripheral vascular disease. Hypertriglyceridemic subjects have triglyceride (TG) levels of> 250 mg / dl. Therefore, hyperlipidemic subjects will have higher cholesterol and triglyceride levels.
For both hypercholesterolemic and hypertriglyceridemic subjects, it is defined as a subject equal to or above the limits set as described above.

Another important aspect of the present invention is the treatment of thrombosis. Thromboembolism is a collective term used for diseases characterized by the formation, development, or presence of thrombi, and blockage of blood vessels by thrombi that are carried by the bloodstream to a thrombotic vessel site. Thromboembolism can reduce blood flow to almost all organs, including the brain and myocardium. Thromboembolism involving the brain is otherwise known as ischemic stroke and is described elsewhere in this application. Thromboembolism involving the heart, otherwise known as myocardial infarction, is also described elsewhere in this application. Harrison's has identified a particular group of patients who are particularly susceptible to thrombosis and embolism. These include: (1) postoperatively fixed patients; (2) patients with chronic congestive heart failure; (3) patients with atherosclerotic vascular disease; (4) patients with malignant tumors; or (5) pregnant Patients included.

An important aspect of the present invention is the treatment of subjects with an abnormally elevated risk of thrombosis (or thromboembolism). As used herein, a subject with an abnormally elevated risk of thrombosis is a category determined according to routine medical practice. Typically, prethrombosis patients can be identified by careful history. According to Harrison's, this diagnosis has three important clues: (1) repeated episodes of thromboembolism without a clear predisposition; (2) a family history of thrombosis; and (3) adolescence and Well documented thromboembolism in early adulthood.

The subject may be treated prophylactically to reduce the risk of a thrombotic episode,
Alternatively, the thrombotic subject may be treated chronically and / or acutely. If the treatment is prophylactic, the subject being treated is a subject at risk of abnormally elevated levels of thrombosis.

Another important aspect of the present invention is the treatment of myocardial infarction. Myocardial infarction is a disease state that occurs with a sudden decrease in coronary blood flow following a pre-narrowed thrombotic occlusion of the coronary arteries due to atherosclerosis. Such damage is caused or promoted by factors such as smoking, hypertension and lipid accumulation.

An important aspect of the present invention is the treatment of subjects with an abnormally elevated risk of myocardial infarction. In the present specification, a subject having an abnormally elevated risk of myocardial infarction,
Patient categories including unstable angina, multiple coronary risk factors (similar to those described for stroke elsewhere in this specification), and those with Printzmetal angina Is. Less common etiological factors include hypercoagulability, coronary embolism, collagen vascular disease, and cocaine abuse.

The subject may be treated prophylactically to reduce the risk of myocardial infarction, or the myocardial infarction subject may be treated chronically and / or acutely. When the treatment is prophylactic, the patient to be treated is a subject with an abnormally elevated risk of myocardial infarction. Subjects with an abnormally elevated risk of myocardial infarction are subjects in the above categories.

Another important aspect of the present invention is the treatment of subjects with an abnormally elevated risk of reperfusion injury. A preferred subject is a patient who is willing or has had a transplant. In accordance with the present invention, increased ecNOS expression and / or activity in blood vessels of transplanted organs is believed to reduce reperfusion injury. Reperfusion injury is a functional, metabolic, or structural change involving necrosis in ischemic tissue that is thought to result from reperfusion of the tissue to the ischemic region. The most common example involves myocardial reperfusion injury. In myocardial reperfusion injury, the ischemic myocardial changes are thought to result from reperfusion into the ischemic region of the heart. Changes can be lethal to myocytes and can include explosive cell swelling and disruption, myocyte membrane disruption, mitochondrial fragmentation, contraction and necrosis, enzyme flux, and edema with calcium overload. is there.

Another important aspect of the invention is the treatment of a homocystinuria subject. Homocystinuria is seven biochemically and clinically distinct disorders, each characterized by increased concentrations of the sulfur-containing amino acid homocysteine in blood and urine. This is due to the loss of the enzyme cystathione synthase, which converts homocysteine and serine into cystathione, the precursor of cysteine. Homocystinuria subjects may also have thrombosis and may have ecNOS expression and / or
Or it may benefit from increased activity.

Another important aspect of the invention is the treatment of subjects with cerebral autosomal dominant arteropathy (CADASIL) syndrome with subcortical infarction and leukoencephalopathy. The disorder is characterized by recurrent stroke with neuropsychiatric symptoms and affects relatively young adults of both sexes. CT scans showed obstructive cerebral vascular infarction in the white matter, which was usually reduced. CADASIL syndrome subjects may also benefit from increased ecNOS expression and / or activity.

Another important aspect of the present invention is the treatment of subjects with neurodegenerative diseases. The term "neurodegenerative disease" is meant to include any pathological condition involving neuronal degeneration and includes Parkinson's disease, Huntington's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS). In a preferred embodiment, the neurodegenerative disease is Alzheimer's disease. Alzheimer's disease is a progressive neurodegenerative disease characterized by loss of function and death of nerve cells in some areas of the brain, leading to loss of cognitive functions such as memory and language. Although the cause of neuronal cell death is unknown, cells are recognized by the appearance of abnormal helical protein filaments (nerve fibril tangles) in neurons and by degeneration in cortical regions of the brain, especially in the frontal and temporal lobes. To be done. Increased cerebral blood flow mediated by increased ecNOS expression and / or activity may also be beneficial to subjects with neurodegenerative diseases.

In accordance with another aspect of the present invention, to identify inhibitors of HMG-CoA reductase inhibitors for the treatment of patients who would benefit from increased endothelial cell nitric oxide synthase activity in tissues. Screening methods are provided. The method identifies an inhibitor of HMG-CoA reductase that is thought to increase endothelial cell nitric oxide synthase activity, and the inhibitor of HMG-CoA reductase inhibits endothelial cell monocytosis in vivo or in vitro. It involves determining whether it results in increased nitric oxide synthase activity. The HMG-CoA reductase inhibitor according to the present invention is a model system in which the inhibitor is a model system in which the inhibitor is any of those described herein, compared to a control.
At least one other HMG-CoA reduction that results in increased ecNOS activity and also as determined in any of the model systems described herein and / or other model systems known in the art. It can be identified by confirming that it inhibits the enzyme-dependent function.

The present invention is also not an HMG-CoA reductase inhibitor, but is capable of acting synergistically or synergistically with such an HMG-CoA reductase inhibitor to increase ecNOS activity. It also includes the administration of certain agents. Thus, an ecNOS substrate that is converted to nitric oxide by ecNOS and a cofactor that enhances such conversion may be co-administered with an HMG-CoA reductase inhibitor according to the present invention. Such ecNOS substrates (eg L-arginine) and cofactors (eg NADPH, tetrahydrobiopterin etc.) may be natural or synthetic, but the preferred substrate is L-arginine.

Similarly, it is not an ecNOS substrate and it is possible to increase ecNOS activity,
There are other agents besides HMG-CoA reductase inhibitors. Agents belonging to these categories are therefore non-HMG-CoA reductase inhibitors, and in the cocktail, rho
It may also be used for co-administration with GTPase inhibitors. Examples of such drug categories are estrogen and ACE inhibitors. Estrogens are a well-defined category of molecules known to those of ordinary skill in the art, and will not be elaborated further herein. All share a high degree of structural similarity. ACE inhibitors have also been well characterized, but they do not always share structural homology.

Angiotensin converting enzyme, or ACE, is an enzyme that catalyzes the conversion of angiotensin I to angiotensin II. ACE inhibitors, which inhibit the activity of ACE,
Included are antibodies against ACE, peptides including amino acids and their derivatives, di- and tripeptides, which interfere with the renin-angiotensin system by reducing or eliminating the formation of the pressor substrate angiotensin II. ACE inhibitors have been used medically to treat hypertension, congestive heart failure, myocardial infarction and renal disease. A class of compounds known to be useful as ACE inhibitors include acyl mercapto and mercapto alkanoyl prolines such as captopril (US Pat. No. 4,105,776) and zefenopril (US Pat. No. 4,316,906).
), Carboxyalkyl dipeptides such as enalapril (US Pat. No. 4,374,
829), lisinopril (US Pat. No. 4,374,829), quinapril (US Pat. No. 4,374,829)
344,949), Ramipril (US Pat. No. 4,587,258), and Perindopril (US Pat.
U.S. Pat. No. 4,508,729), carboxyalkyl dipeptide mimetics such as Cilazapril (U.S. Pat. No. 4,512,924) and Benazapril (U.S. Pat. No. 4,410,520).
), Phosphinylalkanoylproline, such as fosinopril (US Pat. No.
4,337,201) and trandolopril.

The present invention also contemplates co-administration of agents that increase NO production by ecNOS without affecting ecNOS expression, as well as administration of ACE inhibitors or ecNOS substrates and / or ecNOS cofactors. Estrogen upregulates nitric oxide synthase expression, whereas ACE inhibitors do not affect expression, but instead affect the efficiency of nitric oxide synthase action on L-arginine. As such, activity can be increased in a variety of ways. Generally, the activity is increased by increasing the amount of active enzyme present in the cell relative to the amount present in cells not treated with the reductase inhibitor according to the invention. Is increased by.

The reductase inhibitor is administered in an effective amount. In general, the effective amount is any amount that is capable of producing an increase in nitric oxide synthase activity in the desired tissue, and is preferably a preferred representation of the condition, such as reduction, alleviation or elimination of symptoms or abnormalities. Sufficient to cause typological changes.

Generally, an effective amount is the amount of a drug preparation that produces the desired response, either alone or together with further doses. This may include the case of only temporarily delaying the progression of the disease, but more preferably, it permanently stops the progression of the disease or delays the onset of the disease or abnormality, or the disease or abnormality. With preventing the occurrence of. This can be monitored by routine methods.
Generally, doses of active compounds will be from about 0.01 mg / kg per day to 1000 per day.
Will be mg / kg. 50, preferably orally, and in one or several doses per day
It is expected that doses in the range -500 mg / kg will be appropriate.

These amounts, of course, will depend on the particular condition being treated, the severity of the disorder, the age,
Comparable to individual patient parameters including physical condition, size and weight, duration of treatment, nature of concurrent therapy (if any), particular route of administration, and within the knowledge and expertise of the health care professional. Will depend on the factors. A specific mode of administration,
For example, intravenous administration will result in lower doses. If the initial dose applied does not respond adequately in the subject, a higher dose (or an effectively higher dose by a different, more localized route of delivery) may be used, as patient tolerance permits. . Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound. In general, it is preferable to use the maximum dose, ie the highest safe dose according to sound medical judgment. However, it will be appreciated by those of ordinary skill in the art that patients may claim lower or tolerated doses for medical reasons, psychiatric reasons, or for any other reason that may be substantially any. Will be understood.

Reductase inhibitors useful according to the invention may optionally be combined with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein refers to one or more compatible solid or liquid fillers, diluents or encapsulations suitable for administration to humans. Means a substance.
The term "carrier" combines active ingredients to facilitate application,
It means a natural or synthetic organic or inorganic component. The components of the pharmaceutical composition can also be mixed with the molecules of the invention and with each other in such a way that there are no interactions that would substantially impair the desired drug efficacy.

The pharmaceutical composition may include suitable buffering agents including: acetic acid in salt; citric acid in salt; boric acid in salt; and phosphoric acid in salt. The pharmaceutical composition may also optionally comprise suitable preservatives, for example: benzalkonium chloride; chlorobutanol; paraben and thimerosal.

A variety of administration routes are available. The particular mode chosen will, of course, depend on the particular drug chosen, the severity of the disorder to be treated, and the dosage required for therapeutic efficacy. The methods of the present invention are generally practiced using any medically acceptable mode of administration, that is, any mode that produces an effective level of an active compound without causing adverse clinically unacceptable effects. You may. Such modes of administration include oral, rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. The intravenous or intramuscular route is not particularly suitable for long-term therapy and prevention.

The pharmaceutical 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 the carrier which constitutes one or more accessory ingredients. In general, the compositions will associate the active compound (s) with a liquid carrier, a finely divided solid carrier, or both, in a homogeneous and intimate manner and then, if necessary,
Prepared by molding the product.

Compositions suitable for oral administration may be presented as discrete units such as capsules, tablets, lozenges, each containing a predetermined amount of active compound. Other compositions include suspensions in aqueous or non-aqueous liquids, such as syrups, elixirs or emulsions.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of a reductase inhibitor, 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 may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils are conventionally used as solvents or suspending media. For this purpose bland fixed oils, including synthetic mono- or diglycerides, may be used. In addition, fatty acids such as oleic acid may be used in the injectable preparation. Suitable carrier formulations for oral, subcutaneous, intravenous, intramuscular, etc. administration are Remington's Pharmaceutical Scineces, Mack Publishing
It can be found in 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 physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. These include systems based on polymers such as poly (lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, polyanhydrides. Microcapsules of the aforementioned polymers containing drugs are described, for example, in US Pat.
No. 109. Delivery systems also include: lipids including sterols, such as cholesterol, cholesterol esters and fatty acids or neutral fats, such as mono-, di- and triglycerides; hydrogel release systems; sylastic systems; peptide-based systems; wax coatings; conventional Binders and Excipients
Also included are non-polymeric systems that are compressed tablets containing; Specific examples include, but are not limited to: (a) such as those described in U.S. Patent Nos. 4,452,775, 4,675,189 and 5,736,152,
An erosion system comprising an active compound in a matrix form, and (b) US Pat.
Included are diffusion systems in which the active component permeates from the polymer at a controlled rate, as described in US Pat. Nos. 5,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems may be used, some of which are adapted for implantation.

The use of long-term sustained release implants may be desirable. Long-term release means herein that the implant is constructed and arranged to carry therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are known to those of ordinary skill in the art and include some of the release systems described above.

According to another aspect of the invention, a method of increasing blood flow in a subject tissue is provided. 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 subject tissue.

In an important aspect, a subject in a condition treatable by the second agent is co-administered with the second agent in an amount effective to treat the condition, whereby the first agent of the invention is used. Agent (HM
G CoA reductase inhibitor) enhances delivery of the second agent to the subject tissue as a result of increased blood flow. The "second agent" can be any pharmacological compound or diagnostic agent, as desired. A preferable second agent is an agent having a site of action in the brain. These agents include central stimulants, analgesics, anesthetics, adrenergic agonists, anti-adrenergic agonists, amino acids, antagonists, antidotes, anti-anxiety agents, anticholinergics, anticonvulsants. (Ant
icolvunsant), antidepressant drug, antiemetic drug, antiepileptic drug, antihypertensive drug, antifibrinolytic substance, antihyperlipidemic drug, antimigraine drug, antiemetic drug, antitumor drug (brain cancer), anti Antiobessional agents, anti-Parkinson substances, antipsychotics, appetite suppressants, blood glucose regulators, cognitive adjuvants, cognition enhancers, dopaminergic substances, emetic substances, free oxygen radical scavengers, glucocorticoids, low cholesterol Blood sugar, holylipidemic, histamine H2 receptor antagonist, immunosuppressant, inhibitor, memory adjuvant, mental function enhancer, mood regulator, mydriatic, neuromuscular block substance, neuroprotective substance, NMDA antagonist,
Post-stroke and post-head injury remedies, psychotropics, sedatives, sedatives-sleeping pills, serotonin inhibitors, tranquilizers, and cerebral ischemia remedies, calcium channel blockers, free radical scavengers-antioxidants, GABA agonists, Glutamate antagonist, AMPA antagonist, kainate antagonist,
Included are competitive and non-competitive NMDA antagonists, growth factors, opioid antagonists, phosphatidylcholine precursors, serotonin agonists, sodium- and calcium-channel blockers, and potassium channel openers.

In addition to the aforementioned brain-specific categories of agents, examples of other pharmaceutical agent categories that may be used as second agents include: adrenergic agonists; corticosteroids; corticosteroids; alcohol deprivation. Drugs; aldosterone antagonists; amino acids; ammonia detoxifiers; anabolic drugs; central nervous system stimulants; analgesics; androgens; anesthesia, adjuncts to the following: anesthetics; appetite suppressants; antagonists; Acne medications; anti-adrenergic agents; anti-allergic agents; anti-amebic agents;
Antiandrogens; Antianemias; Antianginals; Anxiolytics; Antiarthritics; Antiasthmatics;
Anti-atherosclerotic agents; Anti-bacterial agents; Anti-gallial agents; Anti-galliogenic agents; Anti-cholinergic agents; Anticoagulants; Anticoccidial agents; Anticonvulsants; Antidepressants; Antidiabetic agents; Antidiarrheal agents; Anti Diuretics; Antidotes; Antiemetics; Antiepileptics; Antiestrogens; Antifibrinolytics; Antifungals; Antiglaucomas; Antihemophilia; Antihemorrhagics; Antihistamines; Antihyperlipidemia Antihyperlipoproteinemias; Antihypertensives; Antiinfectives; Topical antiinfectives; Antiinflammatory agents; Antikeratotic agents; Antimalarials; Antimicrobial agents; Antimigraine agents; Antimitotic agents Antifungal drug; Antiemetic drug; Antitumor drug; Antineutropenic drug; Antiobesity drug; Antiparasitic drug; Antiparkinsonian drug; Antiperistaltic drug; Antipneumocystis drug; Antiproliferative drug; Antiprostatic hyperplasia Drugs; Antiprotozoal drugs; Anti-itch drugs; Antipsychotics; Antirheumatic drugs; Antischistosome drugs; Antiseborrheic drugs Anti-secretory drugs; anticonvulsants; antithrombotic drugs; Kosekiyaku; anti-ulcer drugs;
Anti-urinary stone drug; antiviral drug; appetite suppressant; benign prostatic hyperplasia drug; blood glucose regulator; bone resorption inhibitor; bronchodilator; carbonic anhydrase inhibitor; cardiodepressant; cardioprotectant; Cardiovascular agents; Cardiovascular agents; Bile secretagogues; Cholinergic agents; Cholinergic agonists; Cholinesterase deactivators; Coccidial inhibitors; Cognitive adjuvants; Cognitive enhancers; Antidepressants; Diagnostic aids; Diuretics; Dopaminergic agents Substances; ectoparasite eradications; emetics; enzyme inhibitors; estrogens; fibrinolytics; fluorescent agents; free oxygen radical scavengers; gastrointestinal motility effectors; glucocorticoids; gonadotropins; hair growth stimulators;
Hemostatic agents; Histamine H2 receptor antagonists; Hormones; Hypocholesterolemic agents; Hypoglycemic agents; Hypolipidemic agents; Hypotensive agents; Imaging agents; Immunizing agents; Immunomodulators; Immunoregulators; Immunostimulators Immunosuppressants; Adjunctive drugs for immobilization; Inhibitors; Keratolytics; LNRH agonists; Drugs for liver disorders; Lutein; Memory adjuvants; Psychiatric enhancers; Mood regulators; Mucolytics; Mucosal protectants; Eye pupils; Nasal decongestants; Neuromuscular blockers; Neuromuscular protectors; NMDA antagonists; Non-hormonal sterol derivatives; Delivery enhancers; Plasminogen activators; Platelet activator antagonists; Platelet aggregation inhibitors; Stroke Post- and post-head injury treatments; enhancers; progestins; prostaglandins; prostate growth inhibitors; prothyrototropic drugs (prothyrot) Psychoactive substances; Pulmonary interfacial substances; Radioactive substances; Controlling factors; Relaxants; Redistribution agents; Scabies insecticides; Sclerosing agents; Sedatives; Sedative hypnotics; Selective adenosine A1 antagonists; Serotonin antagonists; Serotonin inhibitors Serotonin receptor antagonists; steroids; stimulants; inhibitors; symptomatic multiple sclerosis drugs; synergists; thyroid hormones; thyroid inhibitors; thyroid mimetics; tranquilizers; amyotrophic lateral sclerosis treatments; cerebral ischemia Therapeutic agents; Paget's disease therapeutic agents; unstable angina therapeutic agents; uric acid excretion agents; vasoconstrictors; vasodilators; trauma therapeutic agents; wound healing agents; xanthine oxidase inhibitors.

In another aspect of the invention, a reductase inhibitor is “administered” which means administered substantially simultaneously with another agent. At substantially the same time, the reductase inhibitor is more compatible with other agents (eg, non-HMG-CoA reductase inhibitors, "second agents", etc.)
Is administered to the subject sufficiently close in time to the administration of the two compounds, i.e., to increase ecNOS activity or to deliver the second agent to the tissue via the increased blood flow, It means that it is possible to exert additional or synergistic effects.

[0077]

【Example】

"Upregulation of Endothelial Cell Nitric Oxide Synthase by HMG CoA Reductase Inhibitors" Experimental Procedures All standard culture reagents were obtained from JRH Bioscience (Renexa, KS). Unless otherwise noted, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO). [Α- ³² P] CTP (3000 Ci / mmol) is New England Nucl
Supplied from ear. Purified human LDL was obtained from Calbiochem (San Diego, Calif .; Lot # 730793) and Biomedical Technologies Inc. (Stoughton, Mass.). Endotoxin levels were measured by the chromogenic Limulus migrating cell assay (Biowhittaker Inc., Walkersville, MD). Antibody detection kit (enhanced chemiluminescence) and nylon nucleic acid (Hybond) and protein (PVDF) transfer membranes were purchased from Amersham (Arlington Heights, IL). Simvastatin and Lovastatin are Merck, Sharp
And Dohme, Inc. (West Point, PA). These HMG-CoA reductase inhibitors have been previously described because endothelial cells lack lactonase, which processes simvastatin and lovastatin into active forms (Laufs, U et al., J Biol Chem, 1997, 272: 31725). -31729), chemically activated prior to use.

Cell culture: Human endothelial cells were harvested from the saphenous vein and cultured as described (15). For transfection studies, bovine aortic endothelial cells less than 3 passages were treated with DMEM.
Cultures were performed in growth medium containing (Dulbecco's modified Eagle's medium), 5 mmol / l L-glutamine (Gibco), and 10% fetal bovine serum (Hyclone lot # 1114577). In all experiments, endothelial cells were placed in 10% lipoprotein-deficient serum (Sigma, lot # 26H94031) for 48 hours prior to treatment conditions. In the experiment shown, the endothelial cells were allowed to actinomycin D for 1 h before treatment with ox-LDL and / or simvastatin.
(5 mg / ml). Cell counts, morphology, and cell viability as determined by trypan blue exclusion were maintained for all treatment conditions.

Preparation of LDL : LDL was modified by Chung (Methods Enzymol, 1984, 128: 181-20 with some modifications.
Prepared by intermittent ultracentrifugation according to the method of 9). Fresh plasma from a single donor was anticoagulated with heparin and filtered through a Sephadex G-25 column equilibrated with PBS. The density was 1.21 g / ml with the addition of KBr (0.3265 g / ml plasma)
Adjusted to. An intermittent NaCl / KBr gradient was generated 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, Calif.) In a Beckman Quick-Seal centrifuge tube (5.0 ml). (Capacity) established during. Beck
After ultracentrifugation with a man Near Vertical Tube 90 rotor at 443,000 xg and 7 ° C for 45 minutes (Beckman L8-80M ultracentrifuge), the upper yellow band in the center of the test tube corresponding to LDL was pierced with a needle, And it removed by extracting in a syringe. 100
KBr was removed from LDL by exchanging sterile PBS containing mg / ml polymyxin B, pH 7.4 three times and dialysis.

The purity of the LDL sample was confirmed by SDS / polyacrylamide and cellulose acetate gel electrophoresis. Cholesterol and triglyceride contents were determined as previously described (Liao, JK et al., J Biol Chem, 1995, 270: 319-324). L
DL 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, Mass .; Calbiochem, San Diego, Calif.) Were characterized and used in selected experiments.

Oxidation of LDL : Oxidized LDL was prepared by adding freshly isolated LDL to CuSO ₄ (5) at 37 ° C. for various periods (6-24 hours).
-10 mM). The reaction was performed at 4 ° C in sterile buffer (150 m
It was stopped by dialysis with 3 changes of mol / l NaCl, 0.01% EDTA and 100 mg / ml polymyxin B, pH 7.4). The degree of LDL oxidation was determined as previously described (Yagi, KA, Biochem Med, 1976, 15: 212-21), using a thiobarbituric acid-reactive substance produced using a fluorescence assay for malondialdehyde ( TBARS)
It was estimated by measuring the amount of The degree of LDL modification was expressed as nanomoles of malondialdehyde per mg LDL protein. In this study, 12 and 16 n
Only mild to moderate ox-LDL with TBARS values between mol / mg LDL protein (ie 3-4 nmol / mg LDL cholesterol) was used. All oxidatively modified LDL samples were used within 24 hours of preparation.

Northern blotting: Equal amounts of total RNA (10-20 mg) were separated by 1.2% formaldehyde-agarose gel electrophoresis and transferred to Hybond nylon membranes overnight. 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 radiolabelled with random hexamer priming, [α- ³² P] CTP, and Klenow (Pharmacia). 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 membrane was hybridized to the probe overnight at 45 ° C. All Northern blots were subjected to stringent wash conditions (0.2 x SSC / 0.1% SDS, 6%) prior to autoradiography.
5 ° C). RNA loading was determined by rehybridization with human GAPDH probe.

Western blotting: Cellular proteins were prepared and separated on SDS / PAGE as described (Liao, JK et al., J Biol Chem, 1995, 270: 319-324). Immunoblotting
Murine monoclonal antibody against human ecNOS (1: 400 dilution, Transduction Labo
ratories, Lexington, Kentucky). Immunodetection was performed using sheep anti-mouse secondary antibody (1: 4000 dilution) and enhanced chemiluminescence (ECL) kit (Amersham).
Corp., Arlington Heights, Ill.). Autoradiography was performed at 23 ° C and densitometry quantified appropriate exposure.

Assay for ecNOS activity : ecNOS activity was assayed as previously described (Misko, TP et al. Analytical Biochemistr.
y, 1993, 214: 11-16, Liao, JK et al., J Clin Invest, 1995, 96: 2661-2666), modified nitrite assay. Briefly, endothelial cells were treated with ox-LDL for 24 hours in the presence and absence of simvastatin (0.1 to 1 mM). After treatment, the medium was removed and cells were washed and incubated with phenol red-free medium for 24 hours. After 24 hours, 300: 1 conditioned medium was mixed with 30: 1 freshly prepared 2,3-diaminonaphthalene (1.5 mmol / l DAN in 1 mol / l HCl). The mixture was protected from light and incubated at 20 ° C for 10 minutes. The reaction was stopped with 15: 1 2.8 mol / l NaOH. Fluorescence of 1- (H) -naphthotriazole,
The excitation and emission wavelengths were 365 and 450 nm, respectively. A standard curve was constructed with known amounts of sodium nitrite. Non-specific fluorescence was measured in the presence of LNMA (5 mmol / l).

Nuclear Run-On Assay: Confluent endothelial cells (˜5 × 10 ⁷ cells) grown in LPDS were treated with simvastatin (1 mM) or 95% O ₂ for 24 hours. As described above (Lia
o, JK et al., J Clin Invest, 1995, 96: 2661-2666), isolated nuclei, and in vitr
o Transferred. Equal amounts (1 mg) of purified denatured full-length human ecNOS, human β-tubulin (ATCC # 37855), and linearized pGEM-3z cDNA were applied to a slot blot machine (Sc
Hleicher & Schuell) was used for vacuum transfer on the nitrocellulose membrane. Hybridization of radiolabeled mRNA transcripts to nitrocellulose membranes was performed with 50% formamide, 5x SSC, 2.5x Denhardt's solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 mg / ml salmon sperm. It was performed in a solution containing DNA at 45 ° C. for 48 hours. The membrane was then washed with IX SSC / 0.1% SDS for 72 hours at -80 ° C and 1 hour at 65 ° C before autoradiography.

Transfection assay: For transient transfection, bovine was used rather than human endothelial cells (Graham, FL and Van) due to higher transfection efficiency by calcium phosphate precipitation (12% vs. <4%). der Erb, AJ, Virology, 1973, 52: 456-457). We introduced a human ecNOS promoter construct, F1.LUC, containing a -1.6 kb 5'upstream sequence linked to a luciferase reporter gene, as described by Zhang et al. (J Biol Chem, 1995, 270: 15320-15326). Using. Bovine endothelial cells (60% -70% confluent), 30 mg of the indicated constructs: p.LUC (no promoter), pSV2.LUC.
(SV40 early promoter) or F1.LUC. As an internal control for transfection efficiency, pCMV.bGal plasmid (10 mg) was co-transfected in all experiments. Preliminary results using b-galactosidase staining showed that the cell transfection efficiency was approximately 10% to 14%.

Endothelial cells were placed in lipoprotein-deficient serum 48 hours after transfection and in the presence and absence of simvastatin (1 mM) ox-LDL (50 m
g / ml, TBARS 12.4 nmol / mg) for an additional 24 hours. Luciferase and
b-Galactosidase activity was measured by a chemiluminescence assay (Dual-Light, Tropix, Bedford, MA) using a Berthold L9501 luminometer. The relative promoter assay was calculated as the ratio of luciferase to β-galactosidase activity. Each experiment was performed three times in triplicate.

Data analysis: Band intensities are measured by the National Institutes of Health Image Program (Rasband, W, NIH Imag
e program, v 1.49, National Institutes of Health, Bethesda, 1993). All values are compared to controls and expressed as mean ± SEM in separate experiments. Paired and unpaired Student's t-tests were used to measure any significant changes in densitometry, nitrite production, and promoter activity. Significant differences were considered for P values less than 0.05.

Example 1 Cell Culture Relatively pure (> 95%) human endothelial cell culture was performed using phase contrast microscopy and anti-factor VIII antibody (Gerson, RJ et al., Am J Med, 1989, 887: 28-38) and confirmed by morphological features (ie cubic, cobblestone, contact inhibition). There were no observable adverse effects of ox-LDL or HMG-CoA reductase inhibitors on cell morphology, cell number, immunofluorescent staining, and trypan blue exclusion (> 95%) in all experimental conditions. Higher oxidative modification (ie> 30 nmol / mg TBARS
A higher concentration of (value) ox-LDL (> 100 mg / ml) resulted in vacuolation and some dissociation of endothelial cells after 24 hours. Simvastatin (0.01 to 0.1 mmol
Neither / l) nor lovastatin (10 mmol / l) produced any significant adverse effects on human endothelial cells up to 96 hours. However, higher concentrations of simvastatin (> 15 mmol / l) or lovastatin (> 50 mmol / l) produced cytotoxicity after 36 hours and were therefore not used.

Example 2: Characterization of LDL ADS by SDS / polyacrylamide gel electrophoresis of undenatured or unmodified LDL
A single band (~ 510 kD) corresponding to B-100 was revealed (data not shown)
. Similarly, a single type of low-density lipid (1.
Only one band corresponding to the presence of (02 to 1.06 g / ml density) was revealed. LDL had protein, cholesterol, and triglyceride concentrations of 6.3 ± 0.2, 2.5 ± 0.1, and 0.5 ± 0.1 mg / ml, respectively. In contrast, lipoprotein deficient sera lacked both apo B-100 protein and low density lipid bands and had no detectable levels of cholesterol. There were no detectable levels of endotoxin in the lipoprotein-deficient serum or ox-LDL samples by the chromogenic Limulus migratory cell assay (<0.10 EU / ml).

Furthermore, there were no obvious differences in terms of electrophoretic mobility between our prepared and commercially obtained LDL samples. Native LDL had a TBARS value of 0.3 ± 0.2 nmol / mg, but this value increased to 3.1 ± 0.4 nmol / mg after 72 hours exposure to human saphenous vein endothelial cells in lipoprotein-deficient medium. did. Copper oxide LDL is 4.6 ± 0.5 or
It had a TBARS value in the range of 33.1 ± 5.2 nmol / mg. The degree of ox-LDL used in this study was mild to moderate with TBARS values ranging from 12 to 16 nmol / mg LDL protein (ie 3 to 4 nmol / mg LDL cholesterol).

Example 3 of ox-LDL and HMG-CoA reductase inhibitors for ecNOS protein
Impact We previously found that ox-LDL (50 mg / ml) expressed ecNOS (Liao, JK et al., J Biol Chem, 1
995, 270: 319-324). Ox-LDL compared to untreated cells
Treatment with (50 mg / ml, TBARS 12.2 nmol / mg) caused a 54% ± 6% reduction in ecNOS protein after 48 h (p <0.01, n = 4) (Western blot, 40
mg protein / lane, Figure 1A). There was no difference in the degree of ecNOS down-regulation between our preparation of ox-LDL and commercially available ox-LDL with similar TBARS values. Addition of simvastatin (0.01 mmol / l) did not significantly affect ox-LDL down-regulation of ecNOS (57% ± 8% reduction, p> 0.05, n = 4). However, in the presence of 0.01 mmol / l simvastatin, ox-LDL no longer produced any significant reduction in ecNOS protein levels (4% ± 7% reduction, p <0.0).
1, n = 4). With higher concentrations of simvastatin (1 and 10 mmol / l), ox-L
Not only did DL reverse ecNOS down-regulation, but also a significant increase in ecNOS protein levels above baseline (146% ± 9% and 210% ± 12%, respectively, p <0.
05, n = 4). Simvastatin or lovastatin (not chemically activated
10 mmol / l) had no effect on ecNOS expression.

Treatment with ox-LDL (50 mg / ml, TBARS 12.2 nmol / mg) in a time-dependent manner
ecNOS protein expression was 34% ± 5% after 24, 72 and 96 hours, respectively
, 67% ± 8% and 86 ± 5% (p <0.05 for all values, n = 4) (
(Figure 1B). Simultaneous treatment with simvastatin (0.1 mmol / l) attenuated the decrease in ecNOS protein levels after 24 hours compared to ox-LDL alone (15% ± 2% vs 34% ± 5%).
, P <0.05, n = 4). Longer 72 h and 96 h incubations with simvastatin (0.1 mmol / l) not only reversed the inhibitory effect of ox-LDL on ecNOS expression, but also increased ecNOS protein levels above baseline expression by 110%.
Increased ± 6% and 124% ± 6% (p <0.05, n = 4). Thus, co-treatment with simvastatin increased ecNOS protein expression by 1.3, 3.3, and 8.9-fold after 24, 72, and 96 hours, respectively, compared to ox-LDL alone. These blots are representative of 4 separate experiments.

Example 4: Effect of ox-LDL and HMG-CoA reductase inhibitors on ecNOS mRNA The effect of simvastatin on ecNOS mRNA levels occurred in a time-dependent manner and correlated with the effect on ecNOS protein levels. Northern blot analysis (Northern blot, 20 mg total RNA / lane, Figure 2A) showed ox-LDL (50 mg / ml, T
BARS 15.1 nmol / mg) was 65 ± 5% and 9 after 48 and 72 hours, respectively.
It was shown to produce a time-dependent decrease in ecNOS mRNA levels of 1 ± 4% (p <0.01, n
= 3). Simultaneous treatment with simvastatin (0.1 mmol / l) increased ecNOS mRNA levels by 6.3-fold after 48 hours and 14.5-fold after 72 hours compared to ox-LDL at all time points (all values , P <0.01, n = 3).

To determine if treatment with another HMG-CoA reductase inhibitor had a similar effect as simvastatin, we treated endothelial cells with lovastatin. Again, ox-LDL
Reduced steady-state ecNOS mRNA by 52 ± 5% after 24 hours (p <0.01, n = 3) (Fig.
2B). Treatment with lovastatin (10 mmol / l) not only reversed the inhibitory effect of ox-LDL on ecNOS mRNA, but also caused a 40 ± 9% increase in ecNOS mRNA levels compared to that of untreated cells. It was Co-treatment with lovastatin caused a 3.6-fold increase in ecNOS mRNA after 24 hours compared to ox-LDL alone. However, treatment with lovastatin alone resulted in a 36% increase in ecNOS mRNA levels compared to untreated cells (p <0.05, n = 3). Each experiment was performed three times with comparable results. Loading conditions were normalized using the corresponding ethidium bromide stained 28S band intensities.

Example 5: Effect of ox-LDL and simvastatin on ecNOS activity ecNOS activity was evaluated by measuring LNMA-inhibitable nitrite production from human endothelial cells (Liao, JK et al., J Clin Invest, 1995, 96: 2661-2666). base
The ecNOS activity was 8.8 ± 1.4 nmol / 500,000 cells / 24 hours. ox-LDL (50 mg /
ml, TBARS 16 nmol / mg) for 48 hours, ecNOS-dependent nitrite production 94 ± 3
% (0.6 ± 0.5 nmol / 500,000 cells / 24 hours, p <0.001). Simultaneous treatment with simvastatin (0.1 mmol / l) significantly attenuated this down-regulation, resulting in a 28 ± 3% reduction in ecNOS activity (6.4 ± 0.3 nmol / 500,000 cells / compared to untreated cells). 24 hours, p <0.05). Simultaneous treatment with higher concentrations of simvastatin (1 mmol / l) not only completely reversed the down regulation of ecNOS by ox-LDL, but also increased ecNOS activity by 45 ± 6% compared to baseline. (12.8 ± 2.7 nmol / 500,000
Cells / 24 hours, p <0.05). The experiment was carried out in duplicate in triplicate.

Example 6 Effect of Simvastatin on ecNOS mRNA Stability Actinomycin D (5 mg / ml), a transcription inhibitor, was used to regulate ecNOS mRNA after transcription.
In the presence of. Oxidized LDL (50 mg / ml, TBARS 13.1 nmol / mg) is ecNOS mRN
The half-life of A was shortened (t1 / 2 35 ± 3 hours to 14 ± 2 hours, p <0.05, n = 3). Co-treatment with simvastatin (0.1 mmol / l) prolonged the half-life of ecNOS mRNA by 1.6 fold (t1 / 22 2 ± 3 hours, p <0.05, n = 3). Treatment with simvastatin alone produces ecNO
The half-life of S mRNA was extended 1.3 times from the baseline (t1 / 2 43 ± 4 hours, p <0.05)
, N = 3). The band intensity (relative intensity) of ecNOS mRNA was plotted as a semilogarithmic function of time (t). The data points obtained correspond to the mean ± SEM of three separate experiments.

Example 7: Effect of Simvastatin on ecNOS Gene Transcription To determine if the effect of simvastatin on ecNOS expression occurs at the level of ecNOS gene transcription, we treated endothelium with simvastatin (1 mmol / l) for 24 hours. Nuclear run-on assay was performed using cells. Preliminary experiments with different amounts of radiolabeled RNA transcripts demonstrate that the hybridization is linear and unsaturated under our experimental conditions. The concentration of each ecNOS band
Normalized to the corresponding β-tubulin concentration. The specificity of each band was determined by the lack of hybridization to the non-specific pGEM cDNA vector. In untreated endothelial cells (control), constitutive ecNOS transcriptional activity (relative index 1.0)
was there. Treatment with simvastatin (1 mmmol / l) did not significantly affect ecNOS gene transcription compared to that of untreated cells (relative index 1.2 ± 0.3, p> 0.05).
, N = 4). However, treatment of hyperoxia (95% O ₂ ) endothelial cells significantly increased ecNOS gene expression (relative index 2.5, p <0.05, n = 4). The blot shown is 4
Representative of two separate experiments.

To further confirm the effect of simvastatin on ecNOS gene transcription by different methods, we used the luciferase reporter gene (Zhang, R et al., J Bi.
ol Chem, 1995, 270: 15320-15326), with an ec of -1600 to +22 nucleotides.
The NOS 5'promoter construct (F1.LUC) was used to transfect bovine aortic endothelial cells. This promoter construct has activator protein (AP) -1 and -2, sterol control region-1, retinoblastoma regulatory region, shear stress response region (
SSRE), nuclear factor-1 (NF-1), and the putative cis-acting region of the cAMP response region (CRE). ox-LDL (50 mg / ml, TBARS 14.5 nmol / mg), simvastatin (1 mo
Treatment with l / l) alone or in combination did not significantly affect basal F1 promoter activity. However, laminar fluid shear stress (12 dynes / cm2, 24 hours) was able to induce F1 promoter activity 16-fold after 24 hours, and when the F1 promoter construct was presented to an appropriate stimulus, it functioned. It was shown that it was possible to respond.

Example 8: Effect of simvastatin and lovastatin on ecNOS expression To further characterize the effect of HMG-CoA reductase inhibitors on upregulation of ecNOS expression, we used various time periods (0-84 hours), Simvastatin (0.1 mmol / l
) Treated the endothelial cells. Treatment with simvastatin (0.1 mmol / l) resulted in 4 (6%, 21 (9%, 8%) after 12, 24, 48, 72, and 84 hours, respectively.
0 (8%, 90 (12%, and 95 (16%, increased ecNOS protein sill levels (1
P <0.05, n = 4) at all time points after 2 hours. Higher concentrations of simvastatin also increased ecNOS protein levels, but in significantly less time compared to lower concentrations of simvastatin (Western blot, 40
mg protein / lane).

In a concentration-dependent manner, simvastatin (0.01 to 10 mmol / l, 48 hours) increased 1 (6%, 80 (8%, 190 (10% and 310%, 20%, ecNOS expression, respectively). (P <0.05, n = 4 for concentration (0.1 mmol / l)) (FIG. 3A), thus upregulation of ecNOS expression by simvastatin depends on both concentration and duration of simvastatin treatment. Treatment with lovastatin, lovastatin (0.1 to 10 mmol / l, 48 h) also increased ecNOS expression in a concentration-dependent manner (10, respectively).
(6%, 105 (8% and 180 (11%, p <0.05, n = 3 for concentrations> 0.1 mmol / l))
(Fig. 3B) was significantly less effective than simvastatin at comparable concentrations. Therefore, at the same concentration, simvastatin had a greater effect on ecNOS expression compared to lovastatin. These results are consistent with reported IC50 values for simvastatin and lovastatin (4 nmol / l and 19 nmol / l, respectively) (Van Vliet, AK et al., Biochem Pharmacol, 1996, 52: 1387-1392).
).

Example 9: Effect of L-mevalonate on ecNOS expression To confirm that the effect of simvastatin on ecNOS expression was due to inhibition of endothelial cell HMG CoA reductase, endothelial cells were used alone or in combination. , Ox-LDL (50 mg / ml, TBARS 15.1 nmol / mg), simvastatin (1 mmol / l)
, In the presence of L-mevalonic acid (100 mmol / l). 48 processing with ox-LDL
After hours, ecNOS expression was reduced by 55% ± 6%, which was equivalent to simvastatin (1 mmol / l).
) Was completely reversed and slightly upregulated (150% (8%) higher than basal expression (p <0.05 for both, n = 3).

Compared to endothelial cells treated with ox-LDL and simvastatin, addition of L-mevalonate reduced ecNOS protein by 50% ± 5% (p <0.05, n = 3). further,
Up-regulation of ecNOS expression by simvastatin alone (2.9-fold increase, p <0.05, n = 3)
Was completely reversed by simultaneous treatment with L-mevalonic acid. Treatment with L-mevalonate alone had no discernible effect on basal ecNOS expression (p> 0.05, n
= 3). Similar findings were observed with L-mevalonate and lovastatin. "HMG-CoA Reductase Inhibitors Reduce Cerebral Infarct Size by Up- Regulating Endothelial Cell Nitric Oxide Synthase" Experimental Cell Culture: Human Endothelial Cells II as described above. Type collagenase (Worthington Bi
ochemical Corp., Freehold, NJ) was used to harvest saphenous arteries. Cells less than 3 passages were cultured in medium 199, 20 mM HEPES, 50 mg / ml ECGS (Colla
borative Research Inc., Bedford, Mass.), 100 mg / ml
Heparin sulfate, 5 mM L-glutamine (Gibco), 5% fetal bovine serum (Hyclone, Logan, Utah), and penicillin (100 U / ml) / streptomycin (100 mg)
/ Ml) / fungizone (1.25 mg / ml) in medium containing antibiotic mixture was grown to confluence. For all experiments, endothelial cells were grown to confluence before any treatment conditions. In some experiments, cells were pretreated with actinomycin D (5 mg / ml) 1 hour prior to treatment with HMG-CoA reductase inhibitors.

Exposure of Endothelial Cells to Hypoxia: Confluent endothelial cells grown in 100 mm culture plates were treated with HMG-CoA reductase inhibitors, and then humidified incubation chambers (Billups-Rothenberg, (Delmar, Calif.) Without the culture plate cover. 10 minutes, 20% or 3% O ₂ , 5% before sealing the chamber
Gas was supplied with CO ₂ and equilibrated nitrogen. Chambers have varying durations (0-48 hours)
, Maintained in a 37 ° C incubator, and as previously described (Liao, JK et al.
, J Clin Invest, 1995, 96: 2661-2666), and found that the O ₂ concentration had a fluctuation of less than 2%. Cell confluency and viability were determined by cell counting, morphology, and trypan blue exclusion.

In vitro transcription assay: Confluent endothelial cells (5 × 10 ⁷ cells) were treated with simvastatin (1 mM) for 24 hours in the presence of 20% or 3% O ₂ . As previously described (Liao, JK et al.,
J Clin Invest, 1995, 96: 2661-2666), nuclei were isolated and in vitro transcribed. Equal amount (1 mg) of purified denatured full-length human ecNOS, human β-tubulin (ATCC
# 37855), and the linearized pGEM-3z cDNA in a slot blot machine (Schleicher
& Schuell) was used for vacuum transfer onto the nitrocellulose membrane. Hybridization of radiolabeled mRNA transcripts to nitrocellulose membranes is 50%
45 in 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
It was carried out at 48 ° C for 48 hours. The membrane was then washed with IX SSC / 0.1% SDS for 72 hours at -80 ° C and 1 hour at 65 ° C before autoradiography. The band intensity was analyzed by laser density measurement.

Assay for Nitrite Accumulation: The amount of NO produced by ecNOS was measured by nitrite accumulation in conditioned medium. Nitrite accumulation was converted to 2,3-diaminonaphthalene (1.5 mM DAN in 1 M HCl) and nitrite to 1- (H) -naphthotriazole as previously described (13, 24). It was determined by measuring Non-specific fluorescence was measured in the presence of LNMA (5 mM). Previous studies with nitrite reductase have shown that the conversion of nitrite to nitrate in the medium is approximately 5: 1, and this ratio does not change upon exposure to 20% or 3% O ₂ concentrations. Indicates that

Murine model of cerebral vascular ischemia: Adult male (18-20 g) wild type SV-129 mice (Taconic farm, Germantown, NY) and ecNOS mutant mice (Huang, PL et al., Nature, 1995, 377).
: 239-242), 0.2, 2, or 20 mg of simvastatin or physiological saline (control) per kg of body weight was subcutaneously injected once a day for 14 days. As previously 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), under anesthesia, ischemia was caused by occlusion of the left middle cerebral artery (MCA) with coated nylon monofilament. As stated (Huang, Z et al, J
Cereb Blood Flow Metab, 1996, 16: 981-987, Huang, Z et al., Science, 1994, 2
65: 1883-1885, Hara, H et al., J Cereb Blood Flow Metab, 1997, 1: 515-526),
Arterial blood pressure, heart rate, arterial oxygen tension, and nitric oxide partial pressure were monitored. Filaments were withdrawn after 2 and 4 hours and the mice were sacrificed or as previously described (Huang, Z et al., J Cereb Blood Flow Metab, 1996, 16: 981-987, Hua.
ng, Z et al., Science, 1994, 265: 1883-1885, Hara, H et al., JCereb Blood Flow Met.
ab, 1997, 1: 515-526), using a well-established and standardized observer-blinded protocol for neurological deficit. Motility loss score from 0 (no loss) to 2
The range is (complete loss).

Five coronal 2 mm sections were divided using mouse brain matrix (RBM-200C, Activated Systems, Ann Arbor, Mich., USA). Infarct volume was quantified on a 2% 2,3,5-triphenyltetrazolium chloride stained 2 mm section using an image analysis system (M4, St. Catharines, Ontario, Canada). Serum cholesterol, creatinine and transaminase levels were measured by the Tufts University Veterinary Diagnostic Laboratory (Grafton, MA).

Assay for tissue-derived ecNOS activity: ecNOS activity in mouse aorta and brain was measured by LNMA (5
mM) in the presence and absence of [ ³ H] citrulline to measure [ ³ H] arginine.

Quantitative Reverse Transcription-Polymerase Chain Reaction: Total murine aorta and brain by the guanidinium isothiocyanate method.
RNA was isolated and reverse transcribed using oligo-dT (mRNA preamplification reagent; Gibco BRL) and Taq polymerase (Perkin-Elmer). One tenth of the sDNA was used as a template for the PCR reaction. The following primers were used to amplify the 254-bp fragment of the murine ecNOS cDNA, approximately 0.2 nmol: 5'primer: 5'-GGGCTCCCTC CTTCCGGCTG CCACC.
-3 '(SEQ ID NO: 1) and 3'primer: 5'-GGATCCCTGG AAAAGGCGGT GAGG-3'
(SEQ ID NO: 2) (Hara, H et al., J Cereb Blood Flow Metab, 1997, 1: 515-526.
). For amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 452-
The following primers, 0.1 nmol, were used to amplify the bp fragment: 5'primer: 5'-AC
CACAGTCC ATGCCATCAC-3 '(SEQ ID NO: 3) and 3'primer: 5'-TCCACCACCC
TGTTGCTGTA-3 '(SEQ ID NO: 4). Denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 3 seconds
0 seconds and extension was carried out at 72 ° C. for 60 seconds. Preliminary results show that ecNOS and GAPD
The linear exponential phase for H polymerization was shown to be 30-35 and 20-25 cycles, respectively.

Example 10: Cell Culture Relatively pure (> 98%) human saphenous vein endothelial cell cultures were examined for morphological characteristics using phase-contrast microscopy and immunofluorescence staining with an antibody against Factor VIII. That is, it was confirmed by cubic, cobblestone, contact inhibition). There were no observable adverse effects of HMG-CoA reductase inhibitors, L-mevalonate, or hypoxia on cell morphology.
However, higher concentrations of simvastatin (> 15 mmol / l) or lovastatin (
> 50 mmol / l) produced cytotoxicity after 36 hours and were therefore not used.
Otherwise, cell confluency and viability as measured by trypan blue exclusion were maintained for all treatment conditions described.

Example 11: Effect of HMG-CoA reductase inhibitors on ecNOS activity ecNOS activity was evaluated by measuring 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 conditions was approximately 5: 1, and was similar for hypoxia and normoxia. Basal ecNOS activity at 20% O ₂ is 6.0 ± 3
It was 0.3 nmol / 500,000 cells / 24 hours. Endothelial cells exposed to 3% O ₂ for 24 hours resulted in 75 ± 14% nitrite production (1.5 ± 0.9 nmol / 500,000 cells / 24 hours, p <0.01)
Diminished. Treatment with simvastatin (1 mM) not only completely reversed hypoxia-induced ecNOS down-regulation, but also increased ecNOS activity 3-fold over basal activity (18 ± 5.
0 nmol / 500,000 cells / 24 hours, p <0.05). This upregulation of ecNOS activity was attenuated by the addition of L-mevalonate (400 mM) (9.6 ± 1.3 nmol / 500,000 cells / 24 h, p <0.05). Interestingly, simvastatin (1 mM) alone up-regulated nitrite production 5-fold (30 ± 6.5 nmol / 500,000 cells / 24 h, p <0.01), which was induced by L-mevalonate (400 mM). , Completely blocked (8.6 ± 2.9 nmol / 500,000
Cells / 24 hours, p <0.05). Similar findings were observed with lovastatin, but at a concentration 10 times higher than that of simvastatin.

[0113] Example 12: In effect concentration-dependent manner of HMG-CoA reductase inhibitor substance for ecNOS protein and mRNA levels, the treatment with simvastatin (0.01 to 10 mM, 48 hours), the ecNOS expression, respectively, 1 (6%, 80 (8%, 190 (10% and 310 (20%, increased (at 0.1 mM concentration, p <0.05, n = 4). Treatment with simvastatin (0.1 mM) Protein levels after 12 hours, 24 hours, 48 hours, 72 hours, and 84 hours, respectively, 4 (6%, 21 (9%, 80 (8%, 90 (12%, and 95 (16%, hour Increased in a dependent manner (p <0.05, n = 4 at all time points after 12 h), another HMG-CoA reductase inhibitor, lovastatin, was also time- and concentration-dependent in a time- and concentration-dependent manner. Lovastatin has a higher IC50 value for HMG-CoA reductase compared to simvastatin Thus, at equimolar concentrations, it is 10 times less potent than simvastatin in upregulating ecNOS protein levels.

We have previously shown that hypoxia downregulates ecNOS protein expression (L
iao, JK et al., J Clin Invest, 1995, 96: 2661-2666). Exposure to hypoxia (3% O ₂ ) was 46 ± 4 after 24 and 48 hours, respectively, compared to normoxia (20% O ₂ ).
% And 75 ± 3% decrease in ecNOS protein levels (p <0.01, n = 3).
In a concentration-dependent manner, treatment with simvastatin resulted in a progressive reversal of hypoxia-mediated downregulation of ecNOS protein levels after 48 hours. Higher concentrations (1 and 10 mM
) Simvastatin up-regulated ecNOS protein levels to basal levels of 159 ± 13% and 223 ± 21% (p <0.05, n = 3). Co-treatment with L-mevalonate (400 mM) completely blocked the simvastatin-induced increase in ecNOS protein levels after 48 h (35 ± 2.4%). However, treatment with L-mevalonate alone did not cause any significant effect on basal ecNOS protein levels in untreated cells exposed to hypoxia (25 ± 3.9%, p> 0.05, n = 3). ). Furthermore, chemically unactivated simvastatin had no effect on ecNOS expression. These results indicate that the simvastatin- and lovastatin-mediated increase in ecNOS protein expression is mediated by inhibition of endothelial HMG-CoA reductase.

To determine whether changes in ecNOS protein levels were due to changes in ecNOS steady-state mRNA levels, we determined that in the presence or absence of simvastatin (1 mM) and lovastatin (10 mM), Northern blotting was performed on endothelial cells exposed to normoxia and hypoxia. Simvastatin alone, ecNO
S mRNA levels were increased to 340 ± 24% (p <0.01, n = 3). Exposure of endothelial cells to hypoxia reduced ecNOS mRNA levels by 70% ± 2% and 88 ± 4% after 24 and 48 hours, respectively, compared to GAPDH mRNA levels. Simultaneous treatment with simvastatin not only completely reversed the hypoxia-mediated decrease in ecNOS mRNA levels, but
ecNOS mRNA levels were basal at 195 ± 1 after 24 and 48 hours, respectively.
2% and 530 ± 30% (p <0.01, n = 3). Similarly, lovastatin (1
0 mM), ecNOS message alone, under hypoxia, 350 ± 27% and 41
It was increased to 0 ± 21% (p <0.01, n = 3). Neither simvastatin nor lovastatin caused any significant changes in G-protein and b-actin mRNA levels under normoxic or hypoxic conditions. These results indicate that the effects of HMG-CoA reductase inhibitors are relatively selective with respect to their effects on ecNOS mRNA expression.

Example 13: Effect of HMG-CoA reductase inhibitors on ecNOS mRNA half-life The half-life of ecNOS mRNA was determined in the presence of actinomycin D (5 mg / ml). Hypoxia reduced the half-life of ecNOS mRNA from 28 ± 4 hours to 13 ± 3 hours. Treatment with simvastatin (1 mM) increased ecNOS half-life under normoxic and hypoxic conditions to 46 ± 4 hours and 38 ± 4 hours, respectively (p <0.05, n for both).
= 3). These results suggest that HMG-CoA reductase inhibitors prevent hypoxia-mediated reduction of ecNOS expression by stabilizing ecNOS mRNA.

Example 14: Effect of HMG-CoA Reductase Inhibitors on ecNOS Gene Transcription A nuclear run-on assay showed that hypoxia caused a 85 ± 8% reduction in ecNOS gene transcription (p <0.01, n = 3). Treatment with simvastatin (1 mM) is ecNOS
There was no significant effect on the hypoxia-mediated reduction of gene transcription (ecNO
83 ± 6% reduction in S gene transcription, p> 0.05 compared to hypoxia alone). Moreover, simvastatin alone produced a minimal increase in ecNOS gene transcription under normoxic conditions (20 ± 5% increase in ecNOS gene transcription, p <0.05 compared to normoxic control).
).

Preliminary studies with different amounts of radiolabeled RNA transcripts demonstrate that the hybridization is linear and non-saturating under our experimental conditions. The concentration of each ecNOS band was normalized to the corresponding β-tubulin band concentration (relative intensity). To exclude the possibility that hypoxia or simvastatin caused changes in β-tubulin gene transcription, another gene, GAPDH, was included in each of the nuclear run-on blots. normalize ecNOS gene transcription to GAPDH gene transcription,
A similar relative index was obtained. The specificity of each band was determined by the lack of hybridization to a non-specific pGEM cDNA vector.

Example 15: Effect of HMG-CoA reductase inhibitors on mouse physiology To determine whether up-regulation of ecNOS by HMG-CoA reductase inhibitors occurs in vivo, SV-129 wild type and ecNOS. Knockout mice were treated subcutaneously with 2 mg / kg simvastatin or saline for 14 days. Wild type and ec
Mean arterial blood pressure in NOS mutant mice was previously reported (Huang, PL et al., Nature, 19
95, 377: 239-242). The ecNOS mutant is relatively hypertensive. After 14 days of simvastatin treatment, there was no significant change in mean arterial blood pressure in wild-type mice (81 ±
7 mmHg vs 93 ± 10 mmHg, p> 0.05, n = 8). There were also no significant group differences in heart rate, arterial blood gas, and temporal muscle temperature before ischemia or after reperfusion. Furthermore, there were no significant differences in serum cholesterol (control: 147 ± 10 vs. simvastatin 161 ± 5.2 mg / dl), creatinine and transaminase levels after treatment with simvastatin compared to control values.

[0120] Example 16: Effect of HMG-CoA reductase enzyme inhibitor for ecNOS expression and function in mouse aorta simvastatin treatment (2 mg / kg, sc, 14 days) and ecNOS activity in the aorta of saline injected mice , LNMA-inhibitable conversion of arginine to citrulline (C ¹⁴ ) was determined. EcNOS activity in aorta from simvastatin treated mice was significantly higher than in control group (0.39 ± 0
.09 vs. 0.18 ± 0.04 U / mg protein, n = 8, p <0.05).

EcNOS mRNA expression in the aorta of simvastatin treated and untreated mice was examined by quantitative RT-PCR. There was a significant, dose-dependent, 3-fold increase in ecNOS message in simvastatin treated mice compared to that in GAPDH (n = 3). These findings indicate that simvastatin upregulates ecNOS expression in vivo.

Example 17: Effect of HMG-CoA reductase inhibitor on cerebral ischemia in mice Endothelium-derived NO protects against ischemic cerebral injury (Huang, Z et al., J Cereb Bloo).
d Flow Metab, 1996, 16: 981-987). We therefore sought whether the observed in vivo upregulation of ecNOS by simvastatin had a beneficial effect on cerebral infarct size. After 14 days treatment with 2 mg / kg simvastatin, ischemia was caused by occluding the left middle cerebral artery for 2 hours. After 22 hours of reperfusion, mice were tested for neurological deficit using a well-established and standardized observer-blinded protocol. The neurological motility deficits score was almost 2-fold improved (0.8 ± 0.2) in simvastatin-treated mice (n = 18) compared to those in controls (n = 12).
V 1.7 ± 0.2, p <0.01).

Simvastatin treated wild type mice (n = 18) had 25% smaller cerebral infarct size compared to untreated animals (73.8 ± 8.5 mm3 vs 100.7 ± 7.3 mm3, n = 12, p <0.05).
(Figure 4A). This effect was concentration dependent (0.2, 2, 20 mg / kg simvastatin), lasted up to 3 days, and also occurred at higher relative concentrations, but with lovastatin treatment. Furthermore, simvastatin increases cerebral blood flow by 23% and 35% respectively from baseline at concentrations of 2 mg / kg and 20 mg / kg (n = 8, p <0.05 for both). These findings suggest that simvastatin reduces cerebral infarct size and neurological deficits.

Finally, to demonstrate that the reduction in cerebral infarct size by simvastatin was due to ecNOS upregulation, ecNOS mutant mice lacking the ecNOS gene were treated with simvastatin (2 mg / kg, 14 days) and Cerebral ischemia was applied in the absence. There was no significant difference between cerebral infarct size in simvastatin treated and untreated ecNOS mutant mice (n = 6, p <0.05) (FIG. 4B). These findings indicate that upregulation of ecNOS mediates the beneficial effects of HMG-CoA reductase inhibitors on cerebral infarct size.

[0125] Example 18: Compared to the impact GAPDH mRNA levels of HMG-CoA reductase inhibitor for ecNOS expression in mouse brain by quantitative RT-PCR, ischemic and contralateral side of mouse brain (non-ischemic) I examined the hemisphere. Simvastatin treated mice (n = 3) (2 mg / kg, 14 days) showed 1.5 ecNOS expression in the ipsilateral (I) hemisphere compared to the contralateral (C) non-infarct side.
Or a two-fold increase. In contrast, in naive mice, there was no difference in ecNOS expression between infarcted and non-infarcted hemispheres. These findings indicate that simvastatin selectively increases ecNOS expression in ischemic and hypoxic infarct zones,
It suggests that it may reduce cerebral infarction size.

Example 19: Effect of L-arginine on cerebral blood flow L -arginine infusion at 300 mg / kg, iv was used in several preliminary experiments.
After infusion, it caused a moderate (10%) and diverse increase in regional cerebral blood flow (rCBF) (n = 4, data not shown). In this experiment, 450 mg / kg or physiological saline was used.
Wild type mouse, endothelial cell nitric oxide synthase deficient mutant mouse (eNOS null),
Mice receiving chronic daily administration of simvastatin (2 mg / kg) were infused at a constant rate of 100 microliters / kg / min for 15 minutes. Local cerebral blood flow (r
CBF) was monitored in a group of urethane anesthetized and ventilated mice by laser Doppler flow measurement. Additional physiological variables were also monitored in mice, including mean arterial blood pressure (MABP), heart rate, blood pH, PaO ₂ and PaCO ₂ .

Results Physiological variables during laser Doppler flow measurement in urethane anesthesia ventilated wild type, simvastatin treated and eNOS null mice infused with L-arginine or saline are shown in Table 1. The number of mice in each group is shown in parentheses. Values are reported as mean ± SEM. ^* Is statistically significantly different compared to eNOS null mice (p <0.05)
# Indicates one-way ANOVA followed by Scheffe's test to be statistically significantly different (p <0.05) compared to baseline. MABP indicates mean arterial blood pressure; sim indicates that mice were chronically administered simvastatin.

There were no intragroup differences in mean arterial blood pressure and heart rate during the observation period, but these values were elevated in eNOS null mice, as previously reported. PaCO ₂ values did not differ in or among all groups, but pH values decreased after L-arginine infusion.

RCBF response to L-arginine: FIG. 5 shows a constant rate of 100 microliters / kg / min of L-arginine (450 mg / kg) or saline infusion for 15 minutes followed by 40 minutes of wild-type. FIG. 6 is a bar graph showing the local CBF change of type and eNOS null mouse. The number of mice in each group is shown in parentheses. Error bars indicate standard error of the mean (SEM), and asterisk ( ^* ) is one-way AN
Shows statistically significant differences (p <0.05) compared to baseline controls by OVA followed by Fisher's protected least squares difference test.

L-arginine injection (450 mg / kg, iv) increased rCBF in the parietal cortex of wild-type mice, as shown in Figure 1 (Figure 1). It increased rCBF after injection begins at 5-1 0 minutes and achieved statistical significance in 10-15 minutes. The maximum value reached in 20-25 minutes reached 26% above, after which the value decreased to the control level. In contrast, 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 of wild-type mice did not significantly increase rCBF.

RCBF Response to Simvastatin Added to L-Arginine: FIG. 6 is a bar graph showing local CBF changes in Simvastatin treated mice for 40 minutes after injection of the same dose of L-arginine or saline. The number of mice in each group is shown in parentheses; sim indicates simvastatin. Error bars indicate SEM and asterisks ( ^* ) indicate statistically significant differences (p <0.05) compared to baseline controls by Fisher's protected least squares difference test following one-way ANOVA.

After chronic daily administration of simvastatin alone, baseline rCBF increased by 25%.
L-arginine infusion significantly increased rCBF above baseline simvastatin, but not saline infusion. A significant increase was observed in the 10-15 minute period. Maximum increase was observed at 15-20 minutes and was 29-31% higher than baseline.
These increases lasted an additional 20 minutes, much longer than after L-arginine treatment alone. The maximum response to L-arginine in the presence of simvastatin is
It did not increase statistically. However, the response to L-arginine was more persistent in simvastatin treated mice. In the 30-40 minute period, increased blood flow is due to untreated
Greater in simvastatin treated mice compared to troll (p <0.05).

[0133]

[Table 1]

[0134]

[0135]   All references disclosed herein are fully incorporated by reference.

[0136]

[Sequence list]

[Brief description of drawings]

FIG. 1 Western blot showing the effect of oxidized (ox) LDL on ecNOS protein levels in the presence and absence of simvastatin. FIG. 1A shows the effect of increasing concentrations of simvastatin on ecNOS protein levels. FIG. 1B shows the effect of increasing concentrations of simvastatin on ecNOS protein levels in a time-dependent manner.

FIG. 2: ecNOS mRN in the presence or absence of HMG-CoA reductase inhibitor
Northern blot showing the effect of ox-LDL on A levels. FIG. 2A shows the effect of simvastatin on ecNOS mRNA levels. FIG. 2B shows the effect of lovastatin on ecNOS mRNA levels.

FIG. 3: Simvastatin (vs ecNOS protein levels after 48 hours
Western blot showing the concentration-dependent effects of Figure 3A) and lovastatin (Figure 3B).

FIG. 4. Cerebral infarct volume as a percentage of control after 2 hours of fibrous middle cerebral vein occlusion and 22 hours of reperfusion with or without simvastatin treatment. FIG. 4A shows cerebral infarction in wild type SV-129 mice. FIG. 4B shows cerebral infarction in ecNOS-deficient mice.

FIG. 5: Wild type and eNOS 40 min after L-arginine or saline injection.
A bar graph showing local CBF changes in null mice.

FIG. 6 is a bar graph showing regional CBF changes in mice treated with simvastatin 40 minutes after injection of the same dose of L-arginine or saline.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/00 A61P 1/00 3/00 3/00 3/04 3/04 5/50 5/50 7 / 02 7/02 9/10 9/10 101 101 9/12 9/12 11/00 11/00 11/06 11/06 13/02 13/02 13/12 13/12 15/10 15/10 17 / 00 17/00 25/28 25/28 29/00 101 29/00 101 31/04 31/04 37/06 37/06 43/00 111 43/00 111 C12Q 1/02 C12Q 1/02 1/26 ZNA 1/26 ZNA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD) , SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB , BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Entreth, Matthias Federal Republic of Germany Day-10437 Greimstraße, Berlin 60 (72) Inventor Mosco Wids, Michael A. 02178 Massachusetts, USA Belmont, Prospect Street 257 F-term (reference) 4B063 QA01 QA18 QQ20 QR02 QR72 QR77 QS24 QS28 4C084 AA17 AA19 AA20 AA22 AA23 AA24 MA02 NA14 ZA36 ZA45 NA42 ZA36 ZA36 ZA70 ZA89 ZA91 ZB08 ZB15 ZB35 ZC19 ZC20 ZC21 ZC35 4C086 AA01 AA02 BA07 CB09 EA16 MA01 MA02 MA03 MA04 NA14 ZA36 ZA42 ZA45 ZA59 ZA66 MA59 ZA66 MA42 MA45 MA42 MA45 MA45 MA42 MA45 MA45 MA45 MA45 MA45 MA32 MA45 ZA70 ZA89 ZA91 ZB08 ZB15 ZB35 ZC19 ZC20 ZC21 ZC35

Claims (216)

[Claims] 1. An endothelial cell nitric oxide synthase (Nitric Oxide Synth) in a tissue.
A method for increasing endothelial cell nitric oxide synthase activity in a non-hypercholesterolemic subject that may benefit from increased activity, such as: non-hypercholesterolemia requiring such treatment. In a subject having the disorder, an amount of an amount effective to increase endothelial cell nitric oxide synthase activity in the tissue of the subject.
Administering an HMG-CoA reductase inhibitor. 2. The method of claim 1, wherein the subject is a non-hypertriglyceridemic subject. 3. The method of claim 1, wherein the subject is a non-hyperlipidemic subject. 4. The amount of blood LDL cholesterol level in a subject is 10
The method of claim 1, wherein the amount is less than the amount that is changed. 5. The method of claim 1, wherein the amount is sufficient to increase endothelial cell nitric oxide synthase activity above normal baseline levels. 6. The method of claim 1, wherein the subject has a condition that includes abnormally low levels of chemically induced endothelial cell nitric oxide synthase activity. 7. The subject has an abnormally elevated risk of pulmonary hypertension.
The method described in 1. 8. The method of claim 1, wherein the subject has pulmonary hypertension. 9. The method of claim 1 having an abnormally elevated risk of ischemic stroke. 10. The method of claim 1, wherein the subject has experienced ischemic stroke. 11. The subject, wherein the subject is chronically exposed to hypoxic conditions.
The method described in. 12. The subject has an abnormally elevated risk of thrombosis.
The method described in. 13. The method of claim 1, wherein the subject has thrombosis. 14. The subject has an abnormally elevated risk of arteriosclerosis.
The method described in 1. 15. The method of claim 1, wherein the subject has arteriosclerosis. 16. The subject has a risk of abnormally elevated myocardial infarction.
The method described in 1. 17. The method of claim 1, wherein the subject has undergone myocardial infarction. 18. The method of claim 1, wherein the subject has an abnormally elevated risk of reperfusion injury. 19. The method of claim 18, wherein the subject is a transplant recipient. 20. The method of claim 1, wherein the subject has homocystinuria. 21. The method of claim 1, wherein the subject has a neurodegenerative disease. 22. The method of claim 21, wherein the neurodegenerative disease is Alzheimer's disease. 23. The method of claim 1, wherein the subject has CADASIL syndrome. 24. A blood cholesterol level of HMG-CoA reductase inhibitor is 5%.
2. The method according to claim 1, which is administered in an amount that is not sufficient for the above changes. 25. A blood cholesterol level of HMG-CoA reductase inhibitor is increased to 15
2. The method of claim 1, wherein the method is administered in an amount that is not sufficient to cause a change of more than 1%. 26. The method of any one of claims 1-25, further comprising administering an endothelial cell nitric oxide synthase substrate. 27. The method of claim 26, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 28. The method of claim 26, further comprising administering an endothelial cell nitric oxide synthase cofactor. 29. The method of claim 28, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 30. The method of any one of claims 1-25, wherein the HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. 31. The HMG-CoA reductase inhibitor is lovastatin.
The method described in. 32. The method of claim 30, further comprising administering an endothelial cell nitric oxide synthase substrate. 33. The method of claim 32, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 34. The method of claim 32, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 35. The method of claim 34, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 36. Further comprising co-administering at least one different HMG-CoA reductase inhibitor in an amount sufficient to increase endothelial cell nitric oxide synthase activity in said tissue of the subject, The method according to any one of claims 1 to 25. 37. The method of claim 36, further comprising administering an endothelial cell nitric oxide synthase substrate. 38. The method of claim 37, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 39. The method of claim 37, further comprising administering an endothelial cell nitric oxide synthase cofactor. 40. The method of claim 39, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 41. The method further comprising co-administering at least one different HMG-CoA reductase inhibitor that increases endothelial cell nitric oxide synthase activity.
The method according to any one of 25. 42. The method of claim 41, further comprising administering an endothelial cell nitric oxide synthase substrate. 43. The method of claim 42, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 44. The method of claim 42, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 45. The method of claim 44, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 46. Increase endothelial cell nitric oxide synthase activity in a subject,
A method for treating a non-hyperlipidemic condition that is favorably affected by an increase in endothelial cell nitric oxide synthase activity in a tissue, comprising: HMG-for a subject in need of such treatment. Administering a CoA reductase inhibitor in an amount sufficient to increase endothelial cell nitric oxide synthase activity in the tissue of the subject. 47. The method of claim 46, wherein the amount is less than the amount that changes the blood LDL cholesterol level in the subject by 10%. 48. The method of claim 46, wherein the subject has non-hypercholesterolemia. 49. The method of claim 46, wherein the subject has non-hyperlipidemia. 50. The method of claim 46, wherein the amount is sufficient to increase endothelial cell nitric oxide synthase activity above normal baseline levels. 51. The method of claim 46, wherein the subject has a condition with abnormally low levels of chemically induced endothelial cell nitric oxide synthase activity. 52. The method of claim 46, wherein the subject has an abnormally elevated risk of pulmonary hypertension. 53. The method of claim 46, wherein the subject has pulmonary hypertension. 54. The method of claim 46, wherein the subject has an abnormally elevated risk of ischemic stroke. 55. The method of claim 46, wherein the subject is experiencing ischemic stroke. 56. The subject wherein the subject is chronically exposed to a hypoxic condition.
The method described in. 57. The method of claim 46, wherein the subject has an abnormally elevated risk of thrombosis.
The method described in. 58. The method of claim 46, wherein the subject has thrombosis. 59. The subject is at risk of abnormally elevated arteriosclerosis.
The method described in 46. 60. The method of claim 46, wherein the subject has arteriosclerosis. 61. The subject has a risk of abnormally elevated myocardial infarction.
The method described in 46. 62. The method of claim 46, wherein the subject has had myocardial infarction. 63. The method of claim 46, wherein the subject has an abnormally elevated risk of reperfusion injury. 64. The method of claim 63, wherein the subject is a transplant recipient. 65. The method of claim 46, wherein the subject has homocystinuria. 66. The method of claim 46, wherein the subject has a neurodegenerative disease. 67. The method of claim 66, wherein the neurodegenerative disease is Alzheimer's disease. 68. The method of claim 46, wherein the subject has CADASIL syndrome. 69. A blood cholesterol level of HMG-CoA reductase inhibitor is adjusted to 10
47. The method of claim 46, wherein the method is administered in an amount that is not sufficient to cause a change of more than 1%. 70. If the subject in need of such treatment has an abnormally elevated risk of ischemic stroke, the HMG-CoA reductase inhibitor is fasudil (fasudil).
47. The method of claim 46, which is not an udil). 71. The method of any one of claims 46-70, further comprising administering an endothelial cell nitric oxide synthase substrate. 72. The method of claim 71, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 73. The method of claim 71, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 74. The method of claim 73, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 75. The method of any one of claims 46-70, wherein the HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. 76. The HMG-CoA reductase inhibitor is lovastatin.
The method described in. 77. The method of claim 75, further comprising administering an endothelial cell nitric oxide synthase substrate. 78. The method of claim 77, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 79. The method of claim 77, further comprising coadministering an endothelial cell nitric oxide synthase cofactor. 80. The method of claim 79, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 81. further comprising administering at least one different HMG-CoA reductase inhibitor in an amount sufficient to increase endothelial cell nitric oxide synthase activity within the tissue of the subject, 71. The method according to any one of claims 46 to 70. 82. The method of claim 81, further comprising administering an endothelial cell nitric oxide synthase substrate. 83. The method of claim 82, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 84. The method of claim 82, further comprising administering an endothelial cell nitric oxide synthase cofactor. 85. The method of claim 84, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 86. The method of any one of claims 46-70, further comprising administering a non-HMG-CoA reductase inhibitor that increases endothelial cell nitric oxide synthase activity. 87. The method of claim 86, further comprising administering an endothelial cell nitric oxide synthase substrate. 88. The method of claim 87, wherein the endothelial cell nitric oxide synthase substrate is L-arginine. 89. The method of claim 87, further comprising administering an endothelial cell nitric oxide synthase cofactor. 90. The method of claim 89, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 91. A method of reducing brain damage resulting from stroke, comprising: endothelial cell nitric oxide synthesis in brain tissue of a subject, as opposed to a subject having an abnormally high risk of ischemic stroke. Effective amount of HMG-Co to increase enzyme activity
Administering an A reductase inhibitor. 92. An HMG-CoA reductase inhibitor, which increases blood cholesterol level to 1
92. The method of claim 91, wherein the method is administered in an amount that is not sufficient to change 0% or more. 93. The method of claim 91, wherein the subject has non-hypercholesterolemia. 94. The method of claim 91, wherein the subject has non-hyperlipidemia. 95. The method of claim 91, wherein the HMG-CoA reductase inhibitor is administered prophylactically. 96. The method of claim 91, wherein the HMG-CoA reductase inhibitor is administered acutely. 97. The method of any one of claims 91-96, wherein the HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. 98. The HMG-CoA reductase inhibitor is lovastatin.
The method described in. 99. The method of any one of claims 91-96, further comprising administering a substrate of endothelial cell nitric oxide synthase. 100. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 99. 101. The method of claim 99, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 102. The method of claim 101, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 103. The method of claim 9, further comprising co-administering at least one different HMG-CoA reductase inhibitor that increases endothelial cell nitric oxide synthase activity.
The method according to any one of 1 to 96. 104. The method of claim 103, further comprising co-administering a substrate for endothelial cell nitric oxide synthase. 105. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 104. 106. The method of claim 104, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 107. The method of claim 106, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 108. further comprising coadministering to the tissue of the subject an amount of at least one different HMG-CoA reductase inhibitor effective to increase endothelial cell nitric oxide synthase activity. The method according to any one of items 91 to 96. 109. The method of claim 108, further comprising administering a substrate of endothelial cell nitric oxide synthase. 110. The endothelial cell nitric oxide synthase substrate is L-arginine
110. The method of claim 109. 111. The method of claim 109, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 112. The method of claim 111, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 113. A method of treating pulmonary hypertension: for a subject in need of such treatment, effective to increase endothelial cell nitric oxide synthase activity in the lung tissue of the subject. Administering a different amount of HMG-CoA reductase inhibitor, provided that the HMG-CoA reductase inhibitor is not HMG-CoA reductase inhibitor. 114. The method of claim 113, wherein the subject has non-hypercholesterolemia. 115. The method of claim 113, wherein the subject has non-hyperlipidemia. 116. The method according to claim 113, wherein the rho GTPase function inhibitor is administered prophylactically to a subject at risk of abnormally elevated progressive pulmonary hypertension. 117. The method according to claim 113, wherein the HMG-CoA reductase inhibitor is acutely administered to a subject having pulmonary hypertension. 118. The method of claim 118, wherein the subject is chronically exposed to a hypoxic condition.
The method described in 113. 119. The method of any one of claims 113-118, wherein the HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. 120. The HMG-CoA reductase inhibitor is lovastatin.
The method described in 19. 121. The method of any one of claims 113-118, further comprising coadministering a substrate for endothelial cell nitric oxide synthase. 122. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 121. 123. The method of claim 121, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 124. The method of claim 123, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 125. The method further comprising co-administering at least one different HMG-CoA reductase inhibitor that increases endothelial cell nitric oxide synthase activity.
The method according to any one of 13 to 118. 126. further comprising co-administering to the tissue of the subject an amount of at least one different HMG-CoA reductase inhibitor effective to increase endothelial cell nitric oxide synthase activity. Item 113. The method according to any one of items 113 to 118. 127. The method of claim 125, further comprising co-administering a substrate for endothelial cell nitric oxide synthase. 128. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 127. 129. The method of claim 127, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 130. The method of claim 129, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 131. A method for treating heart failure, which is effective to increase endothelial cell nitric oxide synthase activity in a subject's heart tissue for a subject in need of such treatment. Administering the HMG-CoA reductase inhibitor in an amount, wherein the HMG-CoA reductase inhibitor is not a rho GTPase function inhibitor. 132. The method of claim 131, wherein the subject has non-hypercholesterolemia. 133. The method of claim 131, wherein the subject has non-hyperlipidemia. 134. For a subject at risk of abnormally elevated heart failure,
132. The method of claim 131, wherein the HMG-CoA reductase inhibitor is administered prophylactically. 135. The method of claim 131, wherein the HMG-CoA reductase inhibitor is acutely administered to a subject having heart failure. 136. The method of any one of claims 131-135, wherein the HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. 137. The HMG-CoA reductase inhibitor is lovastatin.
The method described in 36. 138. The method of claim 136, further comprising administering a substrate of endothelial cell nitric oxide synthase. 139. The endothelial cell nitric oxide synthase substrate is L-arginine,
139. The method of claim 138. 140. The method of claim 138, further comprising coadministering an endothelial cell nitric oxide synthase cofactor. 141. The method of claim 140, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 142. further comprising co-administering to the tissue of the subject an amount of at least one different HMG-CoA reductase inhibitor effective to increase endothelial cell nitric oxide synthase activity. Item 131. The method according to any one of items 131 to 135. 143. The method of claim 142, further comprising co-administering an endothelial cell nitric oxide synthase substrate. 144. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 143. 145. The method of claim 143, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 146. The method of claim 145, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 147. The method of any one of claims 147. 131-135 further comprising administering the endothelial cell nitric oxide synthase substrate, 148. The endothelial cell nitric oxide synthase substrate is L-arginine
The method of claim 147. 149. The method of claim 147, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 150. The method of claim 149, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 151. The method further comprising co-administering at least one different HMG-CoA reductase inhibitor that increases endothelial cell nitric oxide synthase activity.
The method according to any one of 31-135. 152. The method of claim 151, further comprising administering an endothelial cell nitric oxide synthase substrate. 153. The endothelial cell nitric oxide synthase substrate is L-arginine,
153. The method of claim 152. 154. The method of claim 152, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 155. The method of claim 154, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 156. A method of treating progressive renal disease, for increasing endothelial cell nitric oxide synthase activity in renal tissue of a subject in need of such treatment. Administering an effective amount of an HMG-CoA reductase inhibitor, provided that it is not an HMG-CoA reductase inhibitor rho GTPase function inhibitor. 157. The method of claim 156, wherein the subject has non-hypercholesterolemia. 158. The method of claim 156, wherein the subject has non-hyperlipidemia. 159. The HMG-CoA reductase inhibitor is administered prophylactically.
The method described in. 160. The method of claim 156, wherein the HMG-CoA reductase inhibitor is administered acutely. 161. The method of any one of claims 156-160, wherein the HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. 162. The HMG-CoA reductase inhibitor is lovastatin.
The method described in 61. 163. The method of any one of claims 156-160, further comprising co-administering a substrate for endothelial cell nitric oxide synthase. 164. The method further comprising coadministering at least one different HMG-CoA reductase inhibitor endothelial cell increasing nitric oxide synthase activity.
The method according to any one of 156 to 160. 165. further comprising co-administering to the tissue of the subject an amount of at least one different HMG-CoA reductase inhibitor effective to increase endothelial cell nitric oxide synthase activity. Item 16. The method according to any one of items 156 to 160. 166. The method of claim 164, further comprising co-administering a substrate of endothelial cell nitric oxide synthase. 167. A method of increasing blood flow in a tissue of a subject, which method enhances endothelial cell nitric oxide synthase activity in the tissue of the subject for such a subject in need of such treatment. Administering an effective amount of a HMG-CoA reductase inhibitor. 168. The method of claim 167, wherein blood flow is increased in brain tissue. 169. The method of claim 167, wherein the subject has non-hypercholesterolemia. 170. The method of claim 167, wherein the subject has non-hyperlipidemia. 171. The method of claim 168, wherein the subject has non-hypercholesterolemia. 172. The method of claim 168, wherein the subject has non-hyperlipidemia. 173. The HMG-CoA reductase inhibitor is administered prophylactically, 167.
The method described in. 174. The method of claim 167, wherein the HMG-CoA reductase inhibitor is administered acutely. 175. The method of any one of claims 167-174, wherein the HMG-CoA reductase inhibitor is selected from the group consisting of simvastatin and lovastatin. 176. The HMG-CoA reductase inhibitor is lovastatin.
The method described in 75. 177. The method of any one of claims 167-174, further comprising coadministering a substrate of endothelial cell nitric oxide synthase. 178. The endothelial cell nitric oxide synthase substrate is L-arginine
The method of claim 177. 179. The method of claim 177, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 180. The method of claim 179, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 181. The method further comprising co-administering at least one different HMG-CoA reductase inhibitor that increases endothelial cell nitric oxide synthase activity.
The method according to any one of 67 to 174. 182. further comprising co-administering to the tissue of the subject an amount of at least one different HMG-CoA reductase inhibitor effective to increase endothelial cell nitric oxide synthase activity. The method according to any one of paragraphs 167-174. 183. The method of claim 181, further comprising administering a substrate of endothelial cell nitric oxide synthase. 184. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 183. 185. The method of claim 183, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 186. The method of claim 185, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 187. With respect to a subject having a condition treatable by the second agent,
Further comprising administering an effective amount of a second agent to treat the condition, thereby enhancing delivery of the second agent to the tissue of the subject as a result of increased blood flow,
The method according to any one of claims 167-174. 188. The method of claim 187, further comprising co-administering a substrate for endothelial cell nitric oxide synthase. 189. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 188. 190. The method of claim 188, further comprising administering an endothelial cell nitric oxide synthase cofactor. 191. The method of claim 190, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 192. For a subject having a condition treatable by the second agent,
167. further comprising administering a second agent in an amount effective to treat the condition, thereby enhancing delivery of the second agent to the brain of the subject as a result of increased blood flow. The method of any one of-174. 193. The method of any one of claims 167-174, further wherein the tissue is the brain and the second agent comprises the site of action in the brain. 194. The second agent is a central stimulant (analeptic), an analgesic, an anesthetic, an adrenergic substance, an anti-adrenergic substance, an amino acid, an antagonist, an antidote, an anti-anxiety substance, Anticholinergics, anticonvulsants (anticolvunsant), antidepressants, antiemetics, antiepileptics, antihypertensives, antifibrinolytics, antihyperlipidemics, antimigraine, antiemetics, Antitumor substance (brain cancer), antiobessional agent, antiparkinsonian substance, antipsychotic drug, appetite suppressant, blood glucose regulator, cognitive adjuvant, cognitive enhancer, dopaminergic substance, emetogenic Substance Emetic substance, free oxygen radical scavenger, glucocorticoid, hypocholesterolemic agent, holylipidemic, histamine H2
Receptor antagonists, immunosuppressants, inhibitors, memory adjuvants, mental function enhancers, mood regulators, mydriatics, neuromuscular block substances, neuroprotective substances, NMDA antagonists, post-stroke and post-head trauma agents, Psychotropics, sedatives, sedatives-hypnotics, serotonin inhibitors, tranquilizers, and cerebral ischemia treatments, calcium channel blockers, free radical scavengers-
Antioxidants, GABA agonists, glutamate antagonists, AMPA antagonists, kainate antagonists, competitive and non-competitive NMDA antagonists, growth factors, opioid antagonists, phosphatidylcholine precursors, serotonin agonists, sodium- and calcium-channel blockers, and potassium 192. The method of claim 192, selected from the group consisting of channel openers. 195. An agent having a site of action in the brain is a central stimulant, an analgesic,
Anesthetics, adrenergic substances, anti-adrenergic substances, amino acids, antagonists, antidotes, anti-anxiety substances, anticholinergics, anticonvulsants (anticol)
vunsant), antidepressant, antiemetic, antiepileptic, antihypertensive, antifibrinolytic, antihyperlipidemic, antimigraine, antiemetic, antitumor (brain cancer), anti Antiobessional agents, anti-Parkinson's drugs, antipsychotics, appetite suppressants, blood glucose regulators, cognitive adjuvants, cognition enhancers, dopamine agonists, emetics, free oxygen radical scavengers, glucocorticoids, low cholesterol Phosphemia, holylipidemic, histamine H2 receptor antagonist, immunosuppressant, inhibitor, memory adjuvant, mental function enhancer, mood regulator,
Mydriatics, neuromuscular block substances, neuroprotective substances, NMDA antagonists, post stroke and post head trauma agents, psychotropics, sedatives, sedatives-hypnotics, serotonin inhibitors, tranquilizers, and cerebral ischemia , Calcium channel blockers, free radical scavengers-antioxidants, GABA agonists, glutamate antagonists, AMPA antagonists, kainate antagonists, competitive and non-competitive NMDA antagonists, growth factors, opioid antagonists, phosphatidylcholine precursors, serotonin 195. The method of claim 193, selected from the group consisting of agonists, sodium- and calcium-channel blockers, and potassium channel openers. 196. The second agent is a central stimulant, an analgesic, an anesthetic, an adrenergic substance, an anti-adrenergic substance, an amino acid, an antagonist, an antidote, an anti-drug.
Anxiety agents, anticholinergics, anticolvunsant, antidepressants,
Anti-emetic drug, anti-epileptic drug, anti-hypertensive drug, anti-fibrinolytic substance, anti-hyperlipidemic drug,
Anti-migraine drug, anti-emetic drug, anti-neoplastic substance (brain cancer), anti-obesity drug (antiobessional age)
nt), anti-Parkinson's drug, anti-psychotic drug, appetite suppressant, blood glucose regulator,
Cognitive adjuvant, cognition enhancer, dopaminergic substance, emetic substance, free oxygen radical scavenger, glucocorticoid, hypocholesterolemic agent, ho
lipidemic, histamine H2 receptor antagonists, immunosuppressants, inhibitors, memory adjuvants, psychological enhancers, mood regulators, mydriatics, neuromuscular block substances, neuroprotective substances, NMDA antagonists, post-stroke agents and head trauma Post-treatments, psychotropics, sedatives, sedatives-hypnotics, serotonin inhibitors, tranquilizers, and cerebral ischemic treatments, calcium channel blockers, free radical scavengers-antioxidants, GABA agonists, glutamate antagonists, AMPA antagonists , Kainate antagonists, competitive and non-competitive NMDA antagonists, growth factors, opioid antagonists, phosphatidylcholine precursors, serotonin agonists, sodium- and calcium-channel blockers, and potassium It is selected from the group consisting of catcher down channel opener A method according to claim 187. 197. The method of claim 192, further comprising co-administering a substrate of endothelial cell nitric oxide synthase. 198. The endothelial cell nitric oxide synthase substrate is L-arginine
The method of claim 197. 199. The method of claim 197, further comprising co-administering an endothelial cell nitric oxide synthase cofactor. 200. The method of claim 199, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 201. The method of claim 193, further comprising administering a substrate of endothelial cell nitric oxide synthase. 202. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 201. 203. The method of claim 201, further comprising administering an endothelial cell nitric oxide synthase cofactor. 204. The method of claim 203, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin. 205. A screening method for identifying an HMG-CoA reductase inhibitor for treating a subject who may benefit from increased endothelial cell nitric oxide synthase activity in a tissue, comprising: (a ) Identifying HMG-CoA reductase inhibitors suspected to increase endothelial cell nitric oxide synthase activity, and (b) in vivo or in vitro, the HMG-CoA reductase inhibitors are Determining whether to cause an increase in nitric oxide synthase activity. 206. Does the subject who can benefit from increased endothelial cell nitric oxide synthase activity in the tissue have an abnormally elevated risk of ischemic stroke,
209. The method of claim 205, which has also had a stroke and the increased endothelial cell nitric oxide synthase activity is increased in the brain. 207. A composition comprising an HMG-CoA reductase inhibitor and L-arginine. 208. The composition of claim 207, which is a pharmaceutical composition. 209. The composition of claim 207, wherein the HMG-CoA reductase inhibitor and L-arginine are in effective amounts to increase blood flow. 210. The composition of claim 207, wherein the HMG-CoA reductase inhibitor and L-arginine are in effective amounts to increase blood flow. 211. Administration of the composition results in increased blood flow.
The composition of claim 207. 212. The composition of claim 207, wherein administration of the composition results in increased blood flow to the brain. 213. The method of claim 182, further comprising coadministering a substrate of endothelial cell nitric oxide synthase. 214. The endothelial cell nitric oxide synthase substrate is L-arginine,
The method of claim 213. 215. The method of claim 213, further comprising administering an endothelial cell nitric oxide synthase cofactor. 216. The method of claim 215, wherein the endothelial cell nitric oxide synthase cofactor is NADPH or tetrahydrobiopterin.