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Title:  Upregulation of type III endothelial cell nitric oxide synthase by agents that disrupt actin cytoskeletal organization

United States Patent:  6,423,751

Inventors:  Liao; James K. (Weston, MA)

Assignee:  The Brigham and Women's Hospital, Inc. (Boston, MA)

Appl. No.:  115387

Filed:  July 14, 1998

Abstract

A use for agents that disrupt actin cytoskeletal organization is provided. In the instant invention, agents that disrupt actin cytoskeletal organization are found to upregulate endothelial cell Nitric Oxide Synthase activity. As a result, agents that disrupt actin cytoskeletal organization are useful in treating or preventing conditions that result from the abnormally low expression and/or activity of endothelial cell Nitric Oxide Synthase. Such conditions include pulmonary hypertension, ischemic stroke, impotence, heart failure, hypoxia-induced conditions, insulin deficiency, progressive renal disease, gastric or esophageal motility syndrome, etc. Subjects thought to benefit mostly from such treatments include nonhyperlipidemics and nonhypercholesterolemics, but not necessarily exclude hyperlipidemics and hypercholesterolemics.

SUMMARY OF THE INVENTION

The invention involves the discovery that agents which disrupt actin cytoskeletal organization can upregulate endothelial cell Nitric Oxide Synthase (Type III) expression. The invention, therefore, is useful whenever it is desirable to restore endothelial cell Nitric Oxide Synthase activity or increase such activity in a cell, tissue or subject, provided the cell or the tissue expresses endothelial cell Nitric Oxide Synthase.

Nitric Oxide Synthase activity is involved in many conditions, including impotence, heart failure, gastric and esophageal motility disorders, kidney disorders such as kidney hypertension and progressive renal disease, insulin deficiency, etc. Individuals with such conditions would benefit from increased endothelial cell Nitric Oxide Synthase activity. It also was known that individuals with pulmonary hypertension demonstrate reduced levels of Nitric Oxide Synthase expression in their pulmonary vessels and benefit clinically from inhalation of Nitric Oxide. The invention therefore is particularly useful for treating pulmonary hypertension. It also has been demonstrated that hypoxia causes an inhibition of endothelial cell Nitric Oxide Synthase activity. The invention therefore is useful for treating subjects with hypoxia-induced conditions. It also has been discovered, surprisingly, that agents which disrupt actin cytoskeletal organization are useful for reducing brain injury that occurs following a stroke.

According to one aspect of the invention, a method is provided for increasing endothelial cell Nitric Oxide Synthase activity in a subject who would benefit from increased endothelial cell Nitric Oxide Synthase activity in a tissue. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount(s) effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. In one important embodiment agents that disrupt actin cytoskeletal organization do not affect cholesterol levels in a subject. In certain embodiments, however, agents that disrupt actin cytoskeletal organization as well as increasing endothelial cell Nitric Oxide Synthase activity in the tissue of a subject can also affect cholesterol levels in the subject. In certain embodiments, the subject is nonhyperlipidimic. In other embodiments the amount is sufficient to increase endothelial cell Nitric Oxide Synthase activity above normal baseline levels established by age-controlled groups, described in greater detail below.

The subject can have a condition characterized by an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is hypoxia-induced. In other embodiments the subject can have a condition comprising an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is chemically induced. In still other embodiments the subject can have a condition comprising an abnormally low level of endothelial cell Nitric Oxide Synthase activity which is cytokine induced. In certain important embodiments, the subject has pulmonary hypertension or an abnormally elevated risk of pulmonary hypertension. In other important embodiments, the subject has experienced an ischemic stroke or has an abnormally elevated risk of an ischemic stroke. In still other important embodiments, the subject has heart failure or progressive renal disease. In yet other important embodiments, the subject is chronically exposed to hypoxic conditions.

According to any of the foregoing embodiments, the preferred agent that disrupts actin cytoskeletal organization is selected from the group consisting of a myosin light chain kinase inhibitor, a myosin light chain phosphatase, a protein kinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, and cytochalasin D. In some embodiments the myosin light chain kinase inhibitor is selected from the group consisting of 2,3-butanedione 2-monoxime, 1-(5-iodonaphthalene-1-sulphonyl)-1-hexahydro-1,4-diazepine hydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride. Likewise, in any of the foregoing embodiments, the method can further comprise co-administering an endothelial cell Nitric Oxide Synthase substrate and/or co-administering an agent other than an agent that disrupts actin cytoskeletal organization that also increases endothelial cell Nitric Oxide Synthase activity, and/or co-administering at least one different agent that disrupts actin cytoskeletal organization. A preferred agent other than an agent that disrupts actin cytoskeletal organization is selected from the group consisting of estrogens and angiotensin-converting enzyme (ACE) inhibitors. The agents may be administered to a subject who has a condition or prophylactically to a subject who has a risk, and more preferably, an abnormally elevated risk, of developing a condition. The inhibitors also may be administered acutely.

According to another aspect of the invention, a method is provided for increasing endothelial cell Nitric Oxide Synthase activity in a subject to treat a condition favorably affected by an increase in endothelial cell Nitric Oxide Synthase activity in a tissue. Such conditions are exemplified above. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. In important embodiments, agents that disrupt actin cytoskeletal organization do not affect cholesterol levels in a subject. In certain embodiments, however, agents that disrupt actin cytoskeletal organization as well as increase endothelial cell Nitric Oxide Synthase activity in the tissue of a subject can also affect cholesterol levels in the subject. In certain embodiments, the subject is nonhyperlipidimic. Important conditions are as described above. Also as described above, the method can involve co-administration of substrates of endothelial cell Nitric Oxide Synthase and/or co-administering an agent other than an agent that disrupts actin cytoskeletal organization that also increases endothelial cell Nitric Oxide Synthase activity, and/or co-administering at least one different agent that disrupts actin cytoskeletal organization. Preferred compounds are as described above. As above, the agents that disrupt actin cytoskeletal organization with or without the co-administered compounds can be administered, inter alia, acutely or prophylactically.

According to another aspect of the invention, a method is provided for reducing brain injury resulting from stroke. The method involves administering to a subject having an abnormally high risk of an ischemic stroke an agent that disrupts actin cytoskeletal organization in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the brain of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. As above, important embodiments include the agent being selected from the group consisting of a myosin light chain kinase inhibitor, a myosin light chain phosphatase, a protein kinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, and cytochalasin D. As above, in some embodiments a myosin light chain kinase inhibitor is selected from the group consisting of 2,3-butanedione 2-monoxime, 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride, and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydro-chloride. Also as above, important embodiments include co-administering a substrate of endothelial cell Nitric Oxide Synthase and/or co-administering an agent other than an agent that disrupts actin cytoskeletal organization that also increases endothelial cell Nitric Oxide Synthase activity, and/or co-administering at least one different agent that disrupts actin cytoskeletal organization. Likewise, important embodiments include prophylactic and acute administration of the agent(s).

According to another aspect of the invention, a method is provided for treating pulmonary hypertension. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase pulmonary endothelial cell Nitric Oxide Synthase activity in the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. Particularly important embodiments are as described above in connection with the methods for treating brain injury. Another important embodiment is administering the agent prophylactically to a subject who has an abnormally elevated risk of developing pulmonary hypertension, including subjects that are chronically exposed to hypoxic conditions.

According to another aspect of the invention, a method for treating heart failure is provided. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase vascular endothelial cell Nitric Oxide Synthase activity in the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. As discussed above, important embodiments include prophylactic and acute administration of the agent(s). Preferred compounds and co-administration schemes are as described above.

According to yet another aspect of the invention, a method is provided for treating progressive renal disease. The method involves administering to a subject in need of such treatment an agent that disrupts actin cytoskeletal organization in an amount effective to increase renal endothelial cell Nitric Oxide Synthase activity in the kidney of the subject, provided that the agent that disrupts actin cytoskeletal organization is not a rho GTPase function inhibitor. Important embodiments and preferred compounds and schemes of co-administration are as described above in connection with heart failure.

According to another aspect of the invention, a method for increasing blood flow in a tissue of a subject is provided. The method involves administering to a subject in need of such treatment a first agent that disrupts actin cytoskeletal organization in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the tissue of the subject, provided that the first agent is not an agent selected from the group consisting of a rho GTPase function inhibitor and fasudil. In certain embodiments the first agent is not a myosin light chain kinase inhibitor. In other embodiments the first agent is selected from the group consisting of a myosin light chain phosphatase, a protein kinase N inhibitor, a phospatidylinositol 4-phosphate 5-kinase inhibitor, and cytochalasin D. Other important embodiments include co-administering a second agent to the subject with a condition treatable by the second agent in an amount effective to treat the condition, whereby the delivery of the second agent to a tissue of the subject is enhanced as a result of the increased blood flow. In certain embodiments where a second agent is administered, the condition treatable by the second agent does not involve the brain tissue.

The invention also involves the use of agents that disrupt actin cytoskeletal organization in the manufacture of medicaments for treating the above-noted conditions. Important conditions, compounds, etc. are as described above. The invention further involves pharmaceutical preparations that are cocktails of agents that disrupt actin cytoskeletal organization according to the invention [non-rho GTPase function inhibitor(s)]. In certain embodiments, however, the cocktails can include a rho GTPase function inhibitor(s) that disrupts actin cytoskeletal organization together with the non-rho GTPase function inhibitor agent of the invention. The invention also involves pharmaceutical preparations that are cocktails of agents that disrupt actin cytoskeletal organization together with agents other than agents that disrupt actin cytoskeletal organization that also increase ecNOS activity in a cell.

The invention also involves methods for increasing ecNOS activity in a cell by contacting the cell with an effective amount of an agent that disrupts actin cytoskeletal organization (excluding rho GTPase function inhibitors), alone, or together with any of the agents co-administered as described above, or as a cocktail as described above.

In any of the foregoing aspects of the invention the agent can be a non-fasudil agent that disrupts actin cytoskeletal organization.

Claim 1 of 9 Claims

What is claimed is:

1. A method for reducing brain injury resulting from a stroke comprising:

administering to a subject having experienced an ischemic stroke or having an abnormally high risk of an ischemic stroke, an agent that disrupts actin cytoskeletal organization in an amount effective to increase endothelial cell Nitric Oxide Synthase activity in the brain tissue of the subject, provided that the agent that disrupts actin cytoskeletal organization is not an agent selected from the group consisting of a rho GTPase function inhibitor, a cytochalasin, and an isoquinoline sulphonyl compound.
 


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