A method for treating type 1 diabetes in a human in need of such treatment, comprising administering to the human a biologically effective amount of at least one inhibitor of inducible nitric oxide synthase or a pro-inflammatory cytokine, wherein the inhibitor is an inhibitor of HMG-CoA reductase or a pharmaceutically acceptable salt thereof, and the inhibitor specifically inhibits the activity of HMG-CoA reductase.

Description of the Invention

SUMMARY OF THE INVENTION

The invention generally provides methods of treating nitric oxide (NO) cytotoxicity comprising providing a biologically effective amount of an inducible nitric oxide synthase (iNOS) and/or proinflammatory cytokine induction suppressor and/or inhibitor. The invention provides a solution to the cytotoxicity induced or fostered by the presence of NO and/or proinflammatory cytokines which is observed in individuals suffering from autoimmune or inflammatory diseases, including stroke, neurodegenerative diseases, demyelinating conditions (e.g., multiple sclerosis, experimental allergic encephalopathy, X-adrenoleukodystrophy), brain trauma, ischemia-reperfusion, Alzheimer's disease, aging, Landry-Guillain-Barre-Strohl syndrome, rheumatoid arthritis, endotoxic shock, myocardial infarction, tissue injury or HIV-mediated NO neurotoxicity.

The invention first provides a method for suppressing the induction of inducible nitric oxide synthase and/or proinflammatory cytokines in a cell comprising contacting said cell with an effective amount of at least one induction suppressor and/or inhibitor of inducible nitric oxide synthase. Preferred cells throughout the various embodiments of the invention are lymphocytes, macrophages, endothelial cells, astrocytes, masengial cells, myocytes, Kuffer cells, epithelial cells, microglia, oligodendrocytes and neurons. Proinflammatory cytokines that are preferred include TNF-.alpha., IL-1.beta., IL-2, IL-6, IL-8 and IFN-.gamma.. As used herein certain embodiments "induction" may mean an increase in the overall rate of gene transcription and/or translation. Induction may also mean that the rate of gene message or protein product destruction is decreased, producing a net increase in the amount of a message or translated protein. As used herein certain embodiments, the phrase "inhibition of nitric oxide cytotoxicity" denotes any measurable decrease in the production of NO. Inhibition of nitric oxide cytotoxicity includes inhibition of iNOS activity, production of iNOS protein, production or translation of iNOS mRNA, inhibition of LPS- or cytokine-induced NF-k.beta. activation in a cell. As used herein certain embodiments, "inhibitors" refers to such compounds or agents that produce any measurable decrease in the activity, production, or secretion of a protein or biological compound, or the translation of mRNA, in, or in the case of secretion, from, a cell. Proteins and biological compounds that are specifically contemplated in the invention include iNOS and proinflammatory cytokines. As used herein certain embodiments, a "enhancer" or "stimulator" refers to such compounds or agents that produce any measurable increase in the activity, production, or secretion of a protein or biological compound, or the translation of mRNA, in, or in the case of secretion, from, a cell. As used herein certain embodiments, an "inducer" refers to such compounds or agents that produce any measurable increase in the content, production, translation, or secretion of a protein or biological compound, or the translation of mRNA, in, or in the case of secretion, from, a cell. As used herein certain embodiments, "a suppressor" refers to an agent or compound that produces any measurable reduction in the induction of a gene. Thus, a "suppressor" is a type of "inhibitor", that acts reduce the net rate of transcription or translation of a target gene.

In preferred aspects of the invention, the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines may be selected from the group including, but not limited to, lovastatin, mevastatin, FPT inhibitor II, forskolin, rolipram, phenylacetate (NaPA), N-acetyl cysteine (NAC), pyrolidine dithiocarbamate (PDTC), 4-phenylbutyrate (4PBA), 5-aminoimmidazole-4-carboxamide ribonucleoside (AICAR), theophylline, papaverine, cAMP, 8-bromo-cAMP, (S)-cAMP, and salts, analogs, or derivatives thereof.

In some embodiments, the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines may be an inhibitor of the Ras/Raf/MAP kinase pathway. In certain embodiments the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines may be an inhibitor of NF-kB, such as for example an inhibitor of NF-kB activation, and/or a suppressor of its induction. In certain preferred embodiments the inhibitor of NF-kB activation includes, but is not limited to, lovastatin, NaPA, metastatin, 4-phenylbutyrate, FPT inhibitor II, AICAR and salts, analogs, or derivatives thereof. In some embodiments, the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines may be an inhibitor of mevalonate synthesis. In certain embodiments the inhibitor of mevalonate synthesis may be an inhibitor of the farnasylation of a protein. In certain preferred embodiments the inhibitor of mevalonate synthesis may be an inhibitor of HMG-CoA reductase and/or suppressor of its induction, including but not limited to, lovastatin or AICAR and salts, analogs, or derivatives thereof. In certain preferred embodiments the inhibitor of HMG-CoA reductase is a stimulator of AMP-activated protein kinase, including but not limited to, AICAR and salts, analogs, or derivatives thereof In certain embodiments, the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines may be a stimulator of AMP-activated protein kinase. In certain other preferred embodiments the inhibitor of of inducible nitric oxide synthase and/or proinflammatory cytokines may be an inhibitor of mevalonate pyrophosphate decarboxylase and/or suppressor of its induction, including but not limited to, phenylacetic acid, 4-phenylbutyrate and salts, analogs, or derivatives thereof. In certain preferred embodiments the inhibitor of mevalonate synthesis may be lovastatin, mevastatin, NaPA, AICAR, 4-phenylbutyrate and salts, analogs, or derivatives thereof. In certain aspects embodiments the inhibitor of of inducible nitric oxide synthase and/or proinflammatory cytokines is an inhibitor of farnesyl pyrophosphate. Preferred inhibitors of farnesyl pyrophosphate include, but are not limited to 4-phenylbutyrate or NaPA.

In other embodiments the suppressor of inducible nitric oxide synthase and/or proinflammatory cytokines is an antioxidant. In preferred embodiments the antioxidant may be, but is not limited to, N-acetyl cysteine, PDTC, and salts, analogs, or derivatives thereof.

In certain other embodiments the inducible nitric oxide synthase and/or proinflammatory cytokines induction suppressor and/or inhibitor is an enhancer of intracellular cAMP, inhibitor of the Ras/Raf/MAP kinase pathway, and/or inhibitor of NF-kB, NF-kB activation and/or suppressor of NF-kB induction. In a preferred embodiment, the inhibitor of the Ras/Raf/MAP kinase pathway includes, but is not limited to, AICAR and salts, analogs, or derivatives thereof. The enhancer of intracellular cAMP may be an inhibitor of cAMP phosphodiesterase and/or suppressor of its induction. In preferred aspects of the invention, the inhibitor of cAMP phosphodiesterase may be, but is not limited to rolipram and salts, analogs, or derivatives thereof. In certain other aspects of the invention, the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines is cAMP and salts, analogs, or derivatives thereof. Derivatives of cAMP include, but are not limited to, 8-bromo-cAMP or (S)-cAMP. In other aspects of the invention, the enhancer of intracellular cAMP may be, but is not limited to, forskolin, rolipram, 8-bromo-cAMP, theophylline, papaverine, cAMP and salts, analogs, or derivatives thereof. In certain embodiments, the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines may be a enhancer of protein kinase A. In other aspects of the invention, the enhancer of protein kinase A may include, but is not limited to, forskolin, rolipram, 8-bromo-cAMP, (S)-cAMP, cAMP and salts, analogs, or derivatives thereof may be, but is not limited to, forskolin, rolipram, 8-bromo-cAMP, theophylline, papaverine, cAMP and salts, analogs, or derivatives thereof.

In yet another aspect of the invention, the induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines may be a Ras farnesyl protein transferase inhibitor and/or induction suppressor, an inhibitor of the farnasylation of Ras, and/or an activator of G-proteins. In a preferred embodiment, the Ras farnesyl protein transferase inhibitor and/or induction suppressor includes, but is not limited to, a FPT inhibitor and salts, analogs, or derivatives thereof. In a preferred embodiment, the inhibitor of the farnasylation of Ras, includes, but is not limited to, a FPT inhibitor II and salts, analogs, or derivatives thereof.

In one embodiment of the invention, the inducible nitric oxide synthase and/or proinflammatory cytokines inhibitor and/or induction suppressor is selected from the group consisting of lovastatin, mevastatin, FPT inhibitor II, forskolin, rolipram, phenylacetate (NaPA), N-acetyl cysteine (NAC), PDTC, 4-phenylbutyrate (4PBA), 5-aminoimmidazole4-carboxamide ribonucleoside (AICAR), theophylline, papaverine, cAMP, 8-bromo-cAMP, (S)-cAMP, and salts, analogs, or derivatives thereof. In a further embodiment of the invention, combinations of two or more inhibitors and/or induction suppressors are preferred for use in the methods described herein.

A "salt" is understood herein certain embodiments to mean a compound formed by the interaction of an acid and a base, the hydrogen atoms of the acid being replaced by the positive ion of the base. Salts, within the scope of this invention, include both the organic and inorganic types and include, but are not limited to, the salts formed with ammonia, organic amines, alkali metal hydroxides, alkali metal carbonates, alkali metal bicarbonates, alkali metal hydrides, alkali metal alkoxides, alkaline earth metal hydroxides, alkaline earth metal carbonates, alkaline earth metal hydrides and alkaline earth metal alkoxides. Representative examples of bases that form such base salts include ammonia, primary amines such as n-propylamine, n-butylamine, aniline, cyclohexylamine, benzylamine, p-toluidine, ethanolamine and glucamine; secondary amines such as diethylamine, diethanolamine, N-methylglucamine, N-methylaniline, morpholine, pyrrolidine and piperidine; tertiary amines such as triethylamine, triethanolamine, N,N-dimethylaniline, N-ethylpiperidine and N-methylmorpholine; hydroxides such as sodium hydroxide; alkoxides such as sodium ethoxide and potassium methoxide; hydrides such as calcium hydride and sodium hydride; and carbonates such as potassium carbonate and sodium carbonate. Preferred salts are those of sodium, potassium, ammonium, ethanolamine, diethanolamine and triethanolamine. Particularly preferred are the sodium salts.

As used herein, "derivatives" refers to chemically modified inhibitors or stimulators that still retain the desired effects on property(s) of iNOS or pro inflammatory gene, protein, and/or activity induction or suppression. Derivatives may also retain other desired properties described herein, such as suppressing the accumulation of very long chain fatty acids, defined herein as fatty acids with more than 22 carbon atoms. Such derivatives may have the addition, removal, or substitution of one or more chemical moieties on the parent molecule. Such moieties may include, but are not limited to, an element such as a hydrogen or a halide, or a molecular group such as a methyl group. Such a derivative may be prepared by any method known to those of skill in the art. The properties of such derivatives may be assayed for their desired properties by any means described herein or known to those of skill in the art.

As used herein, "analogs" include structural equivalents or mimetics, described further in the detailed description.

In administering the inducible nitric oxide synthase and/or proinflammatory cytokines inhibitors and/or induction suppressors to a mammal, preferably a human, pig, cats, dogs, rodent, or cattle including but not limited to, sheep, goats and cows, the inhibitor is formulated in a pharmaceutically acceptable vehicle. The induction suppressor and/or inhibitor may be administered to a patient in a dose therapeutic to treat a diseases, conditions and disorders where there is an advantage in inhibiting the nitric oxide synthase enzyme and/or the production of proinflammatory cytokines.

A "patient", as used herein, may be an animal. Preferred animals are mammals, including but not limited to humans, pigs, cats, dogs, rodents, or cattle including but not limited to, sheep, goats and cows. Preferred patients are humans.

The induction suppressors, also known as "suppressing agents", and/or inhibitors of iNOS and/or proinflammatory cytokines, in pure form or in a pharmaceutically acceptable carrier, will find benefit in treating conditions and disorders, described below, where there is an advantage in inhibiting and/or suppression the induction of proinflammatory cytokines and/or the inducible isoform of nitric oxide synthase enzyme. These induction suppressors and/or inhibitors may also be used to treat conditions and disorders created, induced, enhanced and/or aggravated by the contact of a cell with bacterial endotoxin (LPS).

For example, the suppressing agents and/or inhibitors may be used to treat circulatory shock including its various aspects such as vascular and myocardial dysfunction, metabolic failure including the inhibition of mitochondrial enzymes and cytochrome P450-mediated drug metabolism, and multiple organ dysfunction syndrome including adult respiratory distress syndrome. Hypotension and/or circulatory shock may be a result of gram-negative and gram positive sepsis (a.k.a. septic shock), toxic shock, trauma, hemorrhage, burn injury, anaphylaxis, cytokine immunotherapy, liver failure, kidney failure or systemic inflammatory response syndrome. Suppressing agents and/or inhibitors also may be beneficial for patients receiving therapy, including cancer therapy, with cytokines such as TNF-.alpha., IL-1.beta., IL-2, IL-6, IL-8 and/or IFN-.gamma., or therapy with cytokine-inducing agents, or as an adjuvant to short term immunosuppression in transplant therapy. In addition, the suppressing agents and/or inhibitors may be useful to inhibit NO synthesis in patients suffering from inflammatory conditions in which an excess of NO contributes to the pathophysiology of the condition, such as adult respiratory distress syndrome (ARDS) and myocarditis, for example.

There is also evidence that an NO synthase enzyme and/or proinflammatory cytokines may be involved in the pathophysiology of autoimmune and/or inflammatory conditions such as arthritis, rheumatoid arthritis and systemic lupus erythematosus (SLE) and in insulin-dependent diabetes, mellitus type 1 diabetes, and therefore, the suppressing agents may prove helpful in treating these conditions.

Furthermore, it is now clear that there are a number of additional inflammatory and noninflammatory diseases and/conditions that are associated with NO overproduction. Examples of such physiological disorders include: inflammatory bowel diseases such as ileitis, ulcerative colitis and Crohn's disease; inflammatory lung disorders such as asthma, bronchitis, oxidant-induced lung injury and chronic obstructive airway disease; inflammatory disorders of the eye including corneal dystrophy, ocular hypertension, trachoma, onchocerciasis, retinitis, uveitis, sympathetic ophthalmitis and endophthalmitis; chronic inflammatory disorders of the gum including periodontitis; chronic inflammatory disorders of the joints including arthritis, septic arthritis and osteoarthritis, tuberculosis, leprosy, glomerulonephritis sarcoid, and nephrosis; disorders of the skin including sclerodermatitis, sunburn, psoriasis and eczema; inflammatory diseases of the central nervous system, including amyotrophic lateral sclerosis, chronic demyelinating diseases such as multiple sclerosis, dementia including AIDS-related neurodegeneration and Alzheimer's disease, encephalomyelitis and viral or autoimmune encephalitis; autoimmune diseases including immune-complex vasculitis, systemic lupus and erythematosis; and disease of the heart including ischemic heart disease, heart failure and cardiomyopathy. Additional disease that may benefit from the use of suppressing agents include adrenal insufficiency; hypercholesterolemia; atherosclerosis; bone disease associated with increased bone resorption, e.g., osteoporosis, pre-eclampsia, eclampsia, uremic complications; chronic liver failure, noninflammatory diseases of the central nervous system (CNS) including stroke and cerebral ischemia; and other disorders associated with inflammation and undersirable production of nitric oxide and/or proinflamatory cytokines such as cystic fibrosis, tuberculosis, cachexia, ischeimia/reperfusion, hemodialysis related conditions, glomerulonephritis, restenosis, inflammatory sequelae of viral infections, hypoxia, hyperbaric oxygen convulsions and toxicity, dementia, Sydenham's chorea, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, epilepsy, Korsakoff's disease, imbecility related to cerebral vessel disorder, NO mediated cerebral trauma and related sequelae, ischemic brain edema (stroke), pain, migraine, emesis, immune complex disease, as immunosuppressive agents, acute allograft rejection, infections caused by invasive microorganisms which produce NO and for preventing or reversing tolerance to opiates and diazepines, aging, and various forms of cancer. All these nitric oxide and/or proinflammatory cytokine and/or endotoxin induced, mediated, enhanced, and/or aggravated diseases and disorders are contemplated as being treatable in a cell by contacting the cell with at least one suppressing agent and/or inhibitor of iNOS and/or proinflammatory cytokines. A patient with may also be treated by administering at least one suppressing agent and/or inhibitor of iNOS and/or proinflammatory cytokines. When administered to a patient, the at least one suppressing agent and/or inhibitor is formulated in a pharmaceutically acceptable vehicle.

In another aspect the present invention provides a method of identifying, or screening for, a candidate inducible nitric oxide synthase and/or proinflammatory cytokines inhibitor and/or induction suppressor, comprising preparing a cell capable of producing inducible nitric oxide synthase and/or proinflammatory cytokines activity and testing the candidate inhibitor and/or induction suppressor for the ability to inhibit the inducible nitric oxide synthase and/or proinflammatory cytokines activity, wherein the inhibition is indicative of a candidate inducible nitric oxide synthase and/or proinflammatory cytokines inhibitor and/or induction suppressor. These candidate inhibitor and/or induction suppressor are known herein as "candidate substances". A further aspect of this method is to identify an iNOS specific inhibitor and/or induction suppressor that does not inhibit or suppress one or more proinflammatory cytokines. Another aspect of this invention is to identify an inhibitor and/or induction suppressor that does not inhibit or suppress iNOS, but does inhibit or suppress one or more proinflammatory cytokines.

This method of identifying a candidate inducible nitric oxide synthase and/or proinflammatory cytokines induction suppressor and/or inhibitor comprising the steps of a) obtaining a cell comprising at least the capability of producing inducible nitric oxide synthase and/or proinflammatory cytokines activity; b) obtaining a candidate inducible nitric oxide synthase and/or proinflammatory cytokines induction suppressor and/or inhibitor; c) contacting the cell with the candidate inducible nitric oxide synthase and/or proinflammatory cytokines induction suppressor and/or inhibitor under conditions normally inducing, enhancing, and/or stimulating iNOS and/or proinflammatory cytokines; and d) determining the ability of the candidate inducible nitric oxide synthase and/or proinflammatory cytokines induction suppressor and/or inhibitor to inhibit the formation of nitric oxide in the presence of inducible nitric oxide synthase, wherein the inhibition of the formation of nitric oxide in the presence of inducible nitric oxide synthase is indicative of a candidate inducible nitric oxide synthase induction suppressor and/or inhibitor. In an aspect of the invention, decreased content or production of at least one proinflammatory cytokine by a cell is indicative of a candidate proinflammatory cytokine induction suppressor. In another aspect of the invention, decreased bioactivity of at least one proinflammatory cytokine is indicative of a candidate proinflammatory cytokine inhibitor and/or induction suppressor. In further aspects of this method, an induction suppressor and/or inhibitor is further identified by detecting the amount of iNOS and/or proinflammatory cytokine mRNA message and/or protein content and/or biological activity. In additional aspects of the invention, an induction suppressor and/or inhibitor is further identified by comparing the amount of iNOS and/or proinflammatory cytokine mRNA message and/or protein content and/or biological activity to another cell under conditions normally inducing, enhancing, and/or stimulating iNOS and/or proinflammatory cytokines in the absense of the candidate inhibitor and/or induction suppressor. The preferred conditions inducing, enhancing, and/or stimulating iNOS and/or proinflammatory cytokines is contacting a cell with endotoxin and/or at least one cytokine and/or at least one inducer or stimulator of at least one proinflammatory cytokine. Preferred cytokines are proinflammatory cytokines.

In preferred embodiments, a candidate induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines is selected from agents that have certain traits or modes of action common to those of the suppressors and/or inhibitors identified herein. Preferred candidate substances would either inhibit the Ras/Raf/MAP kinase pathway, inhibit and/or suppress the induction and/or activation of NF-kB, inhibit mevalonate synthesis, be an enhancer of protein kinase A, and/or inhibit the farnasylation of proteins, including but not limited to Ras. In certain embodiments the inhibitor of mevalonate synthesis may be an inhibitor of HMG-CoA reductase or suppressor of its induction. In certain aspects the inhibitor of HMG-CoA reductase is a stimulator of AMP-activated protein kinase. In certain other embodiments the inhibitor of of inducible nitric oxide synthase and/or proinflammatory cytokines may be an inhibitor of mevalonate pyrophosphate decarboxylase or suppressor of its induction. In other embodiments the candidate substance is an antioxidant. In other embodiments the candidate substance is an enhancer of intracellular cAMP. The enhancer of intracellular cAMP may be an inhibitor of cAMP phosphodiesterase and/or suppressor of its induction. In other embodiments the candidate substance is a farnesyl protein transferase inhibitor and/or induction suppressor.

Proinflammatory cytokine and/or iNOS RNA message, protein content, or activity can be detected by any method described herein or known to those of skill in the art (see for example, Sambrook et al., 1989), and include but are not limited to Northern analysis of iNOS and/or inflammatory cytokine message, PCR.TM. amplification of target message, immunodetection techniques including Western analysis of iNOS and/or proinflammatory cytokine content or production, and chemical or biological activity assays for iNOS or cytokine activity.

Candidate inhibitors and/or induction suppressors identified by the method of the invention are preferably purified. When administered to a mammal, the purified candidate inducible nitric oxide synthase inhibitor and/or induction suppressor is formulated in a pharmaceutically acceptable vehicle.

In another preferred embodiment, the invention provides a method of inhibiting nitric oxide cytotoxicity comprising contacting a cell capable of producing nitric oxide with a biologically effective amount of at least one inducible nitric oxide synthase induction suppressor and/or inhibitor identified by the screening assay of the invention. In preferred embodiments, the cell is in a patient.

In another preferred embodiment, the invention provides a method of inhibiting proinflammatory cytokine or endotoxin treated, induced or aggravated conditions and disorders, where there is an advantage in inhibiting and/or suppression the induction of proinflammatory cytokines. In certain embodiments, the method comprises contacting a cell with a biologically effective amount of at least one induction suppressor and/or inhibitor of: at least one proinflammatory cytokine and/or iNOS. In certain aspects of the invention, the at least one induction suppressor and/or inhibitor is identified by the screening assay of the invention. In preferred embodiments, the cell is in a patient.

The invention also provides a method of suppressing the accumulation of very long chain fatty acids in a cell, by contacting the cell with a biologically effective amount of at least induction suppressor and/or inhibitor of: inducible nitric oxide synthase and/or at least one proinflammatory cytokine. In certain aspects of the invention, the at least one induction suppressor and/or inhibitor is identified by the screening assay of the invention. In preferred embodiments, the cell is in a patient. Such methods have use in inflammatory conditions including, but not limited to, demylenating diseases or neural trauma, and particularly in treating patients with X-ALD. In certain aspects of the invention, lignoceric acid .beta.-oxidation is stimulated. In other aspects of the invention, the ratios of C.sub.26:0/C.sub.22:0 or C.sub.24:0/C.sub.22:0 fatty acids are lowered.

The invention provides a method of treating a nitric oxide and/or cytokine mediated disorder in a cell, by contacting the cell with a biologically effective amount of at least one induction suppressor and/or inhibitor of: inducible nitric oxide synthase and/or at least one proinflammatory cytokine. In certain aspects of the invention, the at least one induction suppressor and/or inhibitor is identified by the screening assay of the invention. In preferred embodiments, the cell is in a patient. In preferred aspects, the disorder is X-ALD, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis, lupus, septic shock, stroke, ischemia/reperfusion, rheumatoid arthritis, osteoarthritis or aging. In other preferred aspects, the nitric oxide or cytokine mediated disorder is myelinolytic inflammation, a demyelinating condition or an inflammatory demyelinating disease, or a neuroinflammatory disease. The inflammatory disease is preferably X-ALD, multiple sclerosis, Landry-Guillain-Barre-Strohl syndrome, Alzheimer's disease and/or aging.

In another preferred embodiment, the invention provides a method of treating septic shock comprising contacting a cell capable of producing excess nitric oxide and/or at least one proinflammatory cytokine under conditions of septic shock with a biologically effective amount of an inducible nitric oxide synthase and/or proinflammatory cytokine induction suppressor and/or inhibitor. In certain aspects of the invention, the induction suppressor and/or inhibitor is identified by the screening assay of the invention. In preferred aspects of the invention, the cell is in a patient. Methods of treating septic shock with inhibitors of nitric oxide synthase activity are described in U.S. Pat. Nos. 5,028,627 and 5,296,466, each incorporated herein by reference in entirety.

The present invention is further directed to methods for inducing or suppressing apoptosis in the cells and/or tissues of individuals suffering from degenerative disorders characterized by inappropriate cell proliferation or inappropriate cell death, or in some cases, both. The method comprises contacting a cell capable of producing excess nitric oxide under conditions of degenerative disorders with a biologically effective amount of an inducible nitric oxide synthase and/or proinflammatory cytokines induction suppressor and/or inhibitor. In preferred aspects of the invention, the cell is in a patient. In certain aspects of the invention, the cytokines induction suppressor and/or inhibitor identified by the screening assay of the invention. Inappropriate cell proliferation will include the statistically significant increase in cell number as compared to the proliferation of that particular cell type in the normal population. Also included are disorders whereby a cell is present and/or persists in an inappropriate location, e.g., the presence of fibroblasts in lung tissue after acute lung injury, and cancer cells which exhibit the properties of invasion and metastasis and are highly anaplastic. Such cells include but are not limited to, cancer cells including, for example, tumor cells. Inappropriate cell death will include a statistically significant decrease in cell number as compared to the presence of that particular cell type in the normal population. Such underrepresentation may be due to a particular degenerative disorder, including, for example, viral infections such as AIDS (HIV), which results in the inappropriate death of T-cells, and autoimmune diseases which are characterized by inappropriate cell death. Autoimmune diseases are disorders caused by an immune response directed against self antigens. Such diseases are characterized by the presence of circulating autoantibodies or cell-mediated immunity against autoantigens in conjunction with inflammatory lesions caused by immunologically competent cells or immune complexes in tissues containing the autoantigens. Such diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis. Standard reference works setting forth the heneral principles of immunology include Stites and Terr, 1991 and Abbas et al., 1991.

The invention particularly relates to the use of at least one iNOS and/or pro-inflammatory cytokine induction suppressor and/or inhibitors, preferably reductants such as NAC or other thiol compounds to reduce NO-mediated cytotoxicity as well as ceramide-mediated apoptosis in neuroinflammatory diseases and degenerative disorders. Suppressing agents in this class would be particularly preferred in treating diseases characterized by excessive or inappropriate cell death, including, for example, neuro-degenerative diseases and injury resulting from ischemia. Degenerative disorders characterized by inappropriate cell proliferation include, for example, inflammatory conditions, cancer, including lymphomas, such as prostate hyperplasia, genotypic tumors, etc. Degenerative disorders characterized by inappropriate cell death include, for example, autoimmune diseases, acquired immunodeficiency disease (AIDS), cell death due to radiation therapy or chemotherapy, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Landry-Guillain-Barre-Strohl syndrome, multiple sclerosis, etc. In certain aspects of the invention, the at least one induction suppressor and/or inhibitor is identified by the screening assay of the invention.

The invention further provides a method for enhancing the production of an inducible nitric oxide synthatase or a proinflammatory cytokine in a cell comprising providing a biologically effective amount of a inducible nitric oxide synthatase and/or proinflammatory cytokine stimulator. In certain aspects of the invention, the at least one induction stimulator and/or enhancer is identified by the screening assay of the invention. A stimulator in this aspect of the invention is preferably an induction stimulator. Preferred stimulators include a PKA inhibitor or enhancer of intracellular cAMP. PKA inhibitors may include, but are not limited to, H-89, myristoylated PKI, (R)-cAMP and salts, analogs, or derivatives thereof. The enhancers of intracellular cAMP may also be selected from the group comprising forskolin, 8-bromo-cAMP and rolipram. In other preferred aspects of the invention, the enhancer of intracellular cAMP is an inhibitor of cAMP phosphodiesterase. A preferred inhibitor of cAMP phosphodiesterase is rolipram. In other aspects of this method, a biologically effective amount of LPS and/or one or more proinflammatory cytokine is administered to stimulate iNOS and/or proinflammatory cytokines' induction or activity. Preferred proinflammatory cytokine that are administered include TNF-.alpha., IL-1.beta., IL-2, IL-6, IL-8 and/or IFN-.gamma..

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention discloses the novel uses of compounds which inhibit the induction of iNOS and/or proinflammatory cytokines and the production of NO and/or proinflammatory cytokines by cells, including lymphocytes, macrophages, endothelial cells, astrocytes and microglia in response to inflammatory cytokines for the therapeutic treatment of disease affecting the vascular and nervous systems. The therapeutic uses described herein utilizing these compounds provide protection against NO toxicity to including lymphocytes, macrophages, endothelial cells, astrocytes microglia, oligodendrocytes and neurons in neuroinflammatory disease, stroke, ischemia-reperfusion and tissue injury and HIV-mediated NO neurotoxicity for which there is no effective treatment presently available.

The present disclosure further describes the discovery of a novel role of the mevalonate pathway in controlling the expression of iNOS and different cytokines in lymphocytes, macrophages, endothelial cells, astrocytes and microglia. This discovery provides the basis for novel screening assays of previously unknown inhibitors of iNOS and the production of NO. An understanding of the cellular mechanisms involved in the induction of iNOS and cytokines allows identification of novel targets for the therapeutic intervention of NO-mediated, proinflammatory cytokine and/or endotoxin-mediated pathophysiology in inflammatory diseases.

The inventor demonstrates herein that LPS- and cytokine-induced production of NO can be blocked by antioxidants. Therefore maintenance of the thiol/oxidant balance appears to be crucial for protection against proinflammatory cytokine production and, at least, in NO cytotoxicity. The inventor has discovered that the use of reductants, such as N-acetyl cysteine (NAC) or other thiol compounds, is beneficial in restoring cellular redox and in inhibiting the production of proinflammatory cytokines and in reducing cytotoxic levels of NO. N-acetyl cysteine blocks the induction of TNF-.alpha. and iNOS and is a nontoxic drug that enters the cell readily and serves both as a scavenger of reactive oxygen species and a precursor of glutathione, the major intracellular thiol (Smilkstein et al., 1988; Aruoma et al., 1989). Therefore, the use of reductants such as NAC or other thiol compounds, may be beneficial in restoring cellular redox and in inhibition of production of proinflammatory cytokines and in reducing cytotoxic levels of NO.

The inventor investigated the cellular regulation of the induction of iNOS and cytokines by lovastatin and NaPA in rat primary lymphocytes, macrophages, endothelial cells, astrocytes and microglia. This investigation disclosed the first evidence that the induction of inducible nitric oxide synthase (iNOS) and cytokine (for example TNF-.alpha., IL-1.beta. and IL-6) gene expression are uniquely sensitive to the drugs lovastatin and the sodium salt of phenylacetic acid (NaPA) in astrocytes, glial cells and macrophages. The reversal of lovastatin-mediated inhibition of iNOS induction by mevalonate and FPP, and reversal of the inhibitory effect of NaPA by FPP, and inhibition of Ras farnesyl protein transferase by an inhibitor (FPT inhibitor II) demonstrated that the farnesylation reaction is a key step in the regulation of LPS-mediated induction of iNOS and production of NO and cytokines.

The inventor have discovered that lovastatin and NaPA, alone or in combination represent therapeutic agents directed against cytokine- and nitric oxide-mediated brain disorders, particularly in stroke, trauma, Alzheimer's Disease and in demyelinating conditions such as multiple sclerosis and X-adrenoleukodystrophy (X-ALD).

The inventor's results demonstrate that the inhibition iNOS expression by lovastatin, NaPA and FPT inhibitor II may be due to the inhibition of NF-k.beta. activation. Previous studies of Law et al. (1992) demonstrating the inhibition of NF-k.beta. activation by mevinolin and 5'-methylthioadenosine indicated a role of protein farnesylation and carboxyl methylation reactions in the activation of NF-k.beta.. The Ras protooncogene proteins function by binding to cytoplasmic surface of plasma membrane. Since mevalonate availability regulates the post-translational isoprenylation of many intracellular signaling proteins including Ras p21 (Goldstein et al., 1990), the observed inhibition of NF-k.beta. activation and induction of iNOS by lovastatin and NaPA appears to be due to decreased, or a lack of, isoprenylation of Ras that in turn leads to the lack of or abnormal signal transmission from receptor tyrosine kinase to Ras/Raf/MAP kinase cascade, activation of NF-k.beta. and induction of iNOS.

The inventor has also investigated the effect of other antioxidants on the induction of NO by LPS and/or cytokine-stimulated macrophages, C6 glioma cell lymphocytes, endothelial cells, astrocytes and microglia. These results clearly show that antioxidants (N-acetyl cysteine (NAC) and pyrolidine dithiocarbamate (PDTC)) inhibit the LPS- and cytokine-induced production of NO, iNOS activity, production of iNOS protein and iNOS mRNA indicating a role of reactive oxygen species (e.g., H.sub.2O.sub.2, O.sub.2 and OH) in iNOS induction. Superoxide (O.sub.2.sup.-) and hydroxyl radical (OH) are reported to be involved in the production of NO in brain cerebellum (Mittal, 1993) where the hydroxyl radical was indicated to hydroxylate L-arginine during its conversion to citrulline and NO (Mittal, 1993). The inventor has discovered through the inhibition of iNOS activity and induction of iNOS protein and mRNA in LPS- and cytokine-activated macrophages by NAC that reactive oxygen species (ROS) modulate the intracellular signal pathways for the induction of iNOS biogenesis.

Several lines of evidence disclosed herein clearly support the conclusion that inhibitors of HMG-CoA reductase (for example, lovastatin or mevastatin) and mevalonate pyrophosphate decarboxylase (NaPA) have an inhibitory effect on the induction of inflammatory mediators (iNOS, TNF-.alpha., IL-1.beta. and IL-6) in rat astrocytes, microglia and macrophages demonstrating the involvement of mevalonate metabolite(s), farnesyl pyrophosphate, in the induction of inflammatory mediators. This conclusion was based on the following observations. First, the LPS-induced expression of iNOS, TNF-.alpha., IL-1.beta. and IL-6, and activation of NF-k.beta. was inhibited by lovastatin and NaPA. Second, inhibitory effects of lovastatin and NaPA on LPS-mediated induction of iNOS and cytokines was not reversed by cholesterol and ubiquinone, end products of the mevalonate pathway, indicating that this inhibitory effect of lovastatin was not due to depletion of end products of mevalonate pathway. Third, the reversal of inhibitory effects of lovastatin by mevalonate and FPP and the reversal of inhibitory effects of NaPA by FPP, but not by mevalonate, indicates a role of farnesylation in LPS-mediated induction of iNOS. Fourth, the inhibition of LPS-induced activation of NF-k.beta. and induction of iNOS by FPT inhibitor II, an inhibitor of Ras farnesyl protein transferase, demonstrates that farnesylation of Ras is required for signal transduction in the LPS-induced expression of iNOS. Since the iNOS, TNF-.alpha., IL-1.beta. and IL-6 have been implicated in the pathogenesis of demyelinating and neurodegenerative diseases (Mitrovic et al., 1994; Bo et al., 1994; Merrill et al., 1993), these results provide an important mechanism whereby inhibitors of HMG-CoA reductase and mevalonate pyrophosphate decarboxylase can ameliorate neural injury.

Therapy For X-Adreno Leukodystrophy

Since X-ALD is a metabolic disorder of the very long chain fatty acids (VLCFA) that eventually leads to an inflammatory bilateral demyelination with marked activation of microglia and astrocytes and accumulation of proinflammatory cytokines (TNF-.alpha. and IL-1.beta.) and extracellular matrix proteins (Powers et al., 1992; McGuinness et al., 1995), the inventor developed a therapy that should normalize the VLCFA and inhibit the induction of proinflammatory cytokines by astrocytes and microglia. Example 4 described herein demonstrates that the compounds that increase the intracellular levels of cAMP and the activity of protein kinase A (PKA) normalize the levels of VLCFA possibly by increasing the peroxisomal activity for .beta.-oxidation of VLCFA. Moreover, the same compounds also inhibit the induction of TNF-.alpha. and IL-1.beta. in lipopolysaccharide (LPS) stimulated astrocytes and microglia. These observations demonstrate the therapeutic potential of compounds that increase the activity of PKA in correction of the metabolic defect and inhibition of the neuroinflammatory disease process in X-ALD.

The inventor provides evidence that in X-ALD cultured skin fibroblasts, up regulation of PKA activity increased the .beta.-oxidation of lignoceric acid, decreased the chain elongation of fatty acids and lowered cellular content of VLCFA to the normal level, despite the status (mutation or deletion) of the ALD gene. The detailed mechanism leading to the normalization of VLCFA in X-ALD is not known at the present, but is likely to involve cAMP-dependent protein kinase A. This conclusion is based on the following observations. First, cAMP analogs and rolipram, an inhibitor of cAMP phosphodiesterase, stimulated transport and .beta.-oxidation of lignoceric acid and decreased the chain elongation of fatty acids in X-ALD as well as control skin fibroblasts whereas H-89 and myristoylated PKI, specific inhibitors of PKA, inhibited transport and .beta.-oxidation of lignoceric acid, stimulated chain elongation of fatty acids and blocked the observed effects in normalization of VLCFA by cAMP analogs. Second, a long-term treatment of fibroblasts of X-ALD with cAMP analogs and rolipram although had no effect on protein and mRNA for X-ALD gene but lowered the accumulation of VLCFA to the control level that is also blocked by inhibitors of PKA. These results clearly indicate that increasing cAMP level in fibroblasts of X-ALD normalizes the VLCFA pathogen by a mechanism that is dependent on the activity of PKA but independent of the involvement of the ALD gene product.

Previous studies (Singh et al., 1984; Hashmi et al., 1986; Lageweg et al., 1991; Lazo et al., 1988; Lazo et al., 1989) have shown that VLCFA (lignoceric and cerotic acids) are preferentially .beta.-oxidized in peroxisomes. The increased transport of lignoceric acid into cAMP-treated cells indicates that the observed increase in .beta.-oxidation of lignoceric acid is due to higher availability of lignoceric acid in these cells. However, the increase in .beta.-oxidation of lignoceric acid in cell-free extracts or permealized X-ALD cells, or cell homogenates demonstrate that normalization of VLCFA is due to increased activity of fatty acid .beta.-oxidation system. In the cell, fatty acids are .beta.-oxidized in mitochondria and peroxisomes (Singh, 1997). The lack of effect of etomoxir, an inhibitor of mitochondrial carnitine palmitoyl transferase-I (Mannaerts et al., 1979), on the cAMP-stimulated oxidation indicates that the higher lignoceric acid oxidation activity observed in cAMP-stimulated cells was due to increase in the activity of peroxisomal .beta.-oxidation system. These observations provide the first evidence that peroxisomal .beta.-oxidation of fatty acids is regulated by intracellular second messenger (cAMP).

The pathogenetic mechanism of X-ALD is poorly understood. The constant "hallmark" of X-ALD is an excessive accumulation of VLCFA with subsequent involvement of CNS with induction of proinflammatory cytokines (TNF-.alpha. and IL-1.beta.) and extracellular matrix proteins by reactive astrocytes and microglia and demyelination/inflammatory dysmyelination and loss of oligodendrocytes (Powers et al., 1992; McGuinness et al., 1995; Powers, 1995). The documentation of immunoreactive TNF-.alpha. and IL-1.beta. in astrocytes and microglia of X-ALD brain indicated the involvement of these cytokines in immunopathology of X-ALD and aligned X-ALD with multiple sclerosis (MS), the most common immune-mediated demyelinating disease of the CNS in man. However, apart from traditionally higher expression of cytokines by microglia than in astrocytes of MS and other neurodegenerative disorders, the expression of TNF-.alpha. and IL-1.beta. is more prominent in astrocytes than microglia of X-ALD brain (Powers et al., 1992).

At present it is not known how the inherited metabolic abnormality of accumulation of VLCFA subsequently triggers a neuroinflammatory response in X-ALD brain. Since the metabolic defect appears prior to the detection of neuroinflammatory disease, the assumption is that these VLCFA, by themselves or as a constituent of complex lipid, act as a trigger for the inflammatory response that in turn becomes the basis for the observed demyelination and loss of oligodendrocytes in X-ALD. The data presented here indicate that cAMP may also inhibit the induction of proinflammatory cytokines in reactive astrocytes and microglia. The treatments of rat brain primary astrocytes or microglia with forskolin or rolipram inhibit the LPS-induced induction of TNF-.alpha. and IL-1.beta..

Previously it has been shown that cAMP derivatives and rolipram inhibit the cytokine-induced expression of inducible nitric oxide synthase and production of NO in astrocytes. The inventor's studies indicate that proinflammatory cytokines down regulate the peroxisomal function in the metabolism of VLCFA thereby aggravating the inherited metabolic abnormality by accumulating 4-times higher VLCFA and around the plaque than in normal looking X-ALD brain and these alterations by proinflammatory cytokines are mediated by NO toxicity (Khan et al., 1997). The inhibition of induction of cytokines as well as induction of iNOS by compounds that increase the activity of PKA (e.g., cAMP and rolipram) in astrocytes and microglia indicate that these compounds should be beneficial in terms of blocking the induction of proinflammatory cytokines in X-ALD

These results provide the basis of a therapy to normalize the metabolic abnormality and block the neuroinflammatory process by inhibiting the induction of proinflammatory cytokines. The studies described in Example 4 clearly demonstrate that the compounds (e.g. forskolin, 8-Br-cAMP, rolipram) that increase cAMP and activate PKA meet both of these conditions. Moreover, recent reports showing the prevention of progression of autoimmune encephalomyelitis in mice (Sommer et al., 1995) as well as in marmoset by rolipram indicate that rolipram does cross the blood brain barrier and inhibit the cytokine-induced neuropathologies in these animal models.

The studies described in Example 5 demonstrate that lovastatin and sodium phenylacetate (NaPA), inhibitors of mevalonate pathway, normalize the levels of VLCFA in skin fibroblasts of X-ALD by increasing the peroxisomal activity for .beta.-oxidation of VLCFA. In light of the fact that these compounds also inhibit the induction of proinflammatory cytokines and nitric oxide synthase in astrocytes and microglia, the inventor deduced that these drugs may have therapeutic potential in correction of the metabolic defect and inhibition of the neuroinflammatory disease process in X-ALD.

The inventor found that PD 98059, an inhibitor of MAP kinase (MEK), the kinase responsible for the activation of MAP kinase, inhibits the LPS-induced activation of NF-kB and the induction of iNOS in astrocytes indicating the possible involvement of the MAP kinase pathway in the induction of iNOS. MAP kinases exhibit dual-specificity, regulating both serine (Ser)/threonine (Thr) phosphorylation and Tyr autophosphorylation (Blenis, 1993; Rossomando et al., 1994; Her et al., 1993). In addition, MAP kinases themselves require concurrent Thr and Tyr phosphorylation for activation, and are, in turn, substrates for MEK (Blenis, 1993; Rossomando et al., 1994; Her et al., 1993). MEK is also a dual specificity kinase whose activation requires Ser/Thr phosphorylation (Blenis, 1993; Rossomando et al., 1994; Her et al., 1993). The inventor deduced form these observations that cellular regulation of this signaling pathway may utilize Ser/Thr phosphatases to modulate the phosphorylation state of critical phosphoproteins.

Since phosphoprotein phosphatases (PP) 1 and PP 2A are the two most abundant Ser/Thr phosphatases in the cell, the study presented in Example 6 was undertaken to investigate the cellular regulation of the induction of iNOS by PP 1 and PP 2A in rat primary astrocytes and macrophages. The results clearly demonstrate that calyculin A, microcystin, cantharidin and okadaic acid, inhibitors of PP 1 and PP 2A, stimulate the LPS- and cytokine-mediated expression of iNOS and production of NO in astrocytes and C.sub.6 glial cells while the same inhibitors inhibit the LPS- and cytokine-mediated expression of iNOS and production of NO in macrophages and RAW 264.7 cells. Consistent with this observation, okadaic acid stimulates the iNOS promoter-derived chloramphenicol acetyl transferase (CAT) activity in LPS-treated astrocytes but inhibits the iNOS promoter-derived CAT activity in LPS-treated macrophages. This differential regulation of the induction of iNOS in astrocytes and macrophages by inhibitors of PP 1/2A indicates that, although PP 1/2A functions as a physiological inhibitor of the induction of iNOS in astrocytes, the induction of iNOS in macrophages requires the involvement of PP 1/2A. However, in spite of this differential regulation of the induction of iNOS in astrocytes and macrophages, inhibitors of PP 1/2A stimulate the activation of NF-kB and the production of TNF-.alpha. in both astrocytes and macrophages.

Transient modulation of protein phosphorylation and dephosphorylation is a major mechanism of intracellular signal transduction pathways triggered by different cytokines. Therefore, the inventor hypothesized that inhibition of protein phosphatase 1 and 2A (PP 1 and 2A) activities will influence cytokine induced signal transduction pathways for the induction of iNOS. The signaling events in cytokine-mediated induction of iNOS in astrocytes and macrophages are not well understood. An understanding of the cellular mechanisms involved in the induction of iNOS should identify novel targets for therapeutic intervention in NO-mediated neuroinflammatory diseases. Several lines of evidence presented in Example 6 support the conclusion that inhibition of PP 1/2A activity differentially modulates the LPS- and cytokine-induced expression of iNOS and production of NO in rat primary astrocytes and macrophages. The conclusion is based on the following observations. First, treatment of astrocytes and macrophages with LPS and/or cytokines induced the expression of iNOS and production of NO, and inhibitors of PP 2B (cypermethrin, deltamethrin and fenvalerate) had no effects on the LPS- and cytokine-mediated induction of iNOS and production of NO. Second, compounds (calyculin A, microcystin, okadaic acid and cantharidin) that inhibit PP 1/2A stimulated the LPS- and cytokine-mediated production of NO as well as expression of iNOS protein and mRNA in astrocytes and C.sub.6 glial cells. However, in contrast, these inhibitors inhibited the LPS- and cytokine-mediated production of NO and expression of iNOS in rat resident macrophages and RAW 264.7 cells. Third, the inhibitors of PP 1/2A stimulated iNOS promoter-derived chloramphenicol acetyl transferase (CAT) activity in LPS-treated astrocytes but inhibited iNOS promoter-derived CAT activity in LPS-treated macrophages. These results indicate that the signaling events required for the induction of iNOS in astrocytes differ from those required for the induction of iNOS in macrophages.

Cytokines (INF-.alpha., IL-1.beta. or IFN-.gamma.) and LPS bind to their respective receptors and induce iNOS expression via activation of NF-kB (Xie et al., 1994, Kwon et al., 1995). The nuclear expression and biological function of the NF-kB transcription factor are tightly regulated through its cytoplasmic retention by the ankyrin-rich inhibitor IkB.alpha. (Beg et al., 1992). Activation of NF-kB by various cellular stimuli involves the proteolytic degradation of IkB.alpha. and the concomitant nuclear translocation of the liberated NF-kB heterodimer. Although the biochemical mechanism underlying the degradation of IkB.alpha. remains unclear, it appears that degradation of IkB.alpha. induced by various mitogens and cytokines occurs in association with the transient phosphorylation of IkB.alpha. on serines 32 and 36. Further the inventor has found that the 90 kDa ribosomal S6 kinase (a downstream candidate of the well characterized Ras-Raf-MEK-MAP kinase pathway), but not p70 S6 kinase or MAP kinase, phosphorylates the N-terminal regulatory domain of IkB.alpha. on serine 32. However, in vivo, only phorbol 12-myristate 13-acetate produced rapid activation of p90 RSK, other potent NF-kB inducers including TNF-.alpha. and the Tax transactivator of human T-cell lymphotrophic virus, type I, failed to activate p90 RSK indicating that more than a single IkB.alpha. kinase exists within the cell and that these IkB.alpha. kinases are differentially activated by different NF-kB inducers. By phosphorylation, IkB.alpha. which is still bound to NF-kB has apparently turned into a high affinity substrate for an ubiquitin-conjugating enzyme. Following this phosphorylation-controlled ubiquitination, IkB.alpha. is rapidly and completely degraded by the 20 S or 26 S proteosome.

Okadaic acid and other inhibitors of PP 1/2A have also been shown to induce the activation of NF-kB in monocytes, Jurkat T cells and Hela cells (Menon et al., 1993; Suzuke et al., 1994) due to the phosphorylation of IkB.alpha. at protein phosphatase 2A-sensitive phosphorylation sites which are different than cytokine-induced phosphorylation sites (Sun et al., 1995). However, according to Baeuerle and colleagues (Schmidt et al., 1995), okadaic acid-mediated activation of NF-kB in Hela cells requires the induction of oxidative stress. Identification of binding site of NF-kB in the promoter region of iNOS gene and the activation of NF-kB during cytokine-induced iNOS expression establishes the role of NF-kB activation in the induction of iNOS (Xie et al., 1994; Kwon et al., 1995). In contrast to the ability of okadaic acid on the activation of NF-kB in other cell types (Menon et al., 1993; Suzuke et al., 1994), okadaic acid by itself was unable to induce the activation of NF-kB in rat primary astrocytes. However, okadaic acid markedly stimulated LPS- or cytokine-mediated activation of NF-kB in astrocytes. Increase in the activation of NF-kB in LPS-stimulated astrocytes by okadaic acid paralleled the increase in induction of iNOS indicating that stimulation of iNOS expression in LPS-activated rat primary astrocytes by inhibitors of PP 1/2A is probably mediated via enhanced activation of NF-kB. However, consistent with the effect of okadaic acid on the activation of NF-kB in other cell types (Menon et al., 1993; Suzuke et al., 1994), okadaic acid by itself induced the activation of NF-kB in macrophages but this activation of NF-kB by okadaic acid did not result in the induction of iNOS indicating that activation of NF-kB by okadaic acid is not sufficient for the induction of iNOS in macrophages. Although similar to astrocytes, okadaic acid stimulated the LPS-mediated activation of NF-kB in rat peritoneal macrophages, yet in sharp contrast to the effect of okadaic acid on the induction of iNOS in astrocytes, the stimulation of NF-kB activation by okadaic acid in LPS-treated macrophages did not parallel with the expression of iNOS. Instead, consistent with a previous report, okadaic acid and other inhibitors of PP 1/2A markedly inhibited LPS- and cytokine-induced expression of iNOS in macrophages. However, the basis for this differential regulation of induction of iNOS in astrocytes and macrophages by inhibitors of PP 1/2A is not understood at the present time.

Earlier, the inventor observed that cAMP-dependent protein kinase A (PKA) also differentially modulates the induction of iNOS in astrocytes and macrophages. Inhibition of the activation of NF kB and the induction of iNOS with the increase in PKA activity, and stimulation of the activation of NF-kB and the induction of iNOS with the decrease in PP 1/2A activities in astrocytes indicate that both PKA (a serine-threonine protein kinase) and PP 1/2A (serine-threonine phosphoprotein phosphatases) function as inhibitory signals for the induction of iNOS in astrocytes modulating different steps of the signal transduction pathways. In contrast, in macrophages, inhibitors of PKA inhibited the LPS-mediated activation of NF-kB and induction of iNOS, and inhibitors of PP 1/2A stimulated the LPS-mediated activation of NF-kB but inhibited the induction of iNOS indicating that both PKA and PP 1/2A are necessary components of the LPS-mediated signaling pathways for the induction of iNOS. However, the molecular basis for the differential regulation of activation of NF-kB and expression of iNOS gene by inhibitors of PP 1/2A in rat peritoneal macrophages is not known. In light of the fact that NF-kB is necessary but not sufficient for the expression of iNOS gene and that many of the signal transduction events are cell type specific, the apparent stimulation of NF-kB and inhibition of iNOS gene expression by inhibitors of PP 1/2A clearly delineate that apart from the activation of NF-kB some other signaling pathway(s) sensitive to PP 1/2A is/are responsible for the expression of iNOS gene in macrophages.

The inventor examined the possible involvement of ROS in cytokine-mediated activation of sphingomyelin breakdown and ceramide formation and found that intracellular GSH plays a crucial role in the breakdown of SM to ceramide, in that low GSH levels are required for ceramide generation and high GSH levels inhibit production of ceramide. Inhibition of cytokine-mediated breakdown of SM to ceramide by antioxidants like N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) and induction of ceramide production by oxidants or pro-oxidants like hydrogen peroxide, aminotriazole, diamide and L-buthione (S,R)-sulfoximine clearly delineate a novel function of ROS and GSH in regulation of the first step of sphingomyelin signal transduction pathway. Moreover, decreased levels of GSH and increased levels of ceramide correlate with the DNA fragmentation in rat primary oligodendrocytes as well as in the banked human brains from patients with neuroinflammatory diseases (e.g. multiple sclerosis and X=adrenoleukodystrophy).

Changes in the cellular redox state toward either prooxidant or antioxidant conditions have profound effects on cellular functions. Several lines of evidence presented herein indicate that the first step of cytokine-induced sphingomyelin signal transduction pathway (i.e. breakdown of sphingomyelin to ceramide and phosphocholine) is redox sensitive. First, cytokines like TNF-.alpha. and IL-1.beta. decreased intracellular GSH and induced the degradation of sphingomyelin to ceramide in rat primary astrocytes, oligodendrocytes, microglia and rat C.sub.6 glial cells, and pretreatment of the cells with antioxidants like NAC restored the levels of GSH and blocked the degradation of sphingomyelin to ceramide. Second, depletion of endogenous glutathione by diamide or buthione sulfoximine alone induces the degradation of sphingomyelin to ceramide which is blocked by NAC. Third, the increase in intracellular H.sub.2O.sub.2 by the addition of exogenous H.sub.2O.sub.2 or by the inhibition of endogenous catalase by aminotriazole induced the, degradation of sphingomyelin to ceramide which is also blocked by NAC. Fourth, besides NAC, pyrrolidine dithiocarbamate (PDTC), an amioxidant but not the precursor of GSH (Laight et al., 1997), also inhibited the TNF-.alpha. and IL-1.beta.-induced hydrolysis of sphingomyelin to ceramide.

Several studies support a role for hydrolysis of sphingomyelin as a stress-activated signaling mechanism in which ceramide plays a role in growth suppression and apoptosis in various cell types including glial and neuronal cells (Brugg et al., 1996; Wiesner and Dawson, 1996). Ceramide activates the proteases of the interleukin converting enzyme (ICE) family, (especially prICE/YAMA/CPP32), the protease responsible for cleavage of poly-ADP-ribose polymerase (PARP) (Martin et al., 1995) and that the activation of prICE by ceramide and induction of apoptosis are inhibited by overexpression of Bcl-2 (Zhang et al., 1996). Addition of exogenous ceramides or sphingomyelinase to cells induces stress activated protein kinase (SAPK)-dependent transcriptional activity through the activation of c-jun (Latinis and Koretzky, 1996) and a dominant negative mutant of SEK1, the protein kinase responsible for phosphorylation and activation of SAPK, interferes with ceramide-induced apoptosis (Verheij et al., 1996). These observations also indicate that both Bcl-2 and SAPK function downstream of ceramide in the apoptotic pathway.

The inventor has found that DNA fragmentation and increase in ceramide and decrease in GSH in primary oligodendrocytes and banked human brains with X-ALD and MS clearly indicate that intracellular redox (level of GSH) is an important regulator of apoptosis via controlling the generation of ceramide. This conclusion is based on following observations. First, treatment of oligodendrocytes with TNF-.alpha. decreased intracellular level of GSH, increased degradation of SM to ceramide and induced DNA fragmentation, however, pretreatment of oligodendrocytes with NAC blocked the TNF-.alpha.-mediated decrease in GSH level, increase in ceramide level and increase in DNA fragmentation. Second, treatment of oligodendrocytes only with diamide, a thiol-depleting agent, decreased intracellular level of GSH, increased level of ceramide and induced DNA fragmentation which are prevented by pretreatment of NAC, a thiol-replenishing agent. Third, the inventor found increased fragmentation of DNA in brains from patients with X-ALD and MS where the levels of GSH and ceramide were lower and higher respectively compared to those found in control human brains. These observations clearly indicate that maintenance of the thiol/oxidant balance is crucial for protection against cytokine-mediated ceramide production and thereby against ceramide-induced cytotoxicity.

Recent observation demonstrated that ceramide potentiates the cytokine-mediated induction of inducible nitric oxide synthase (iNOS) in astrocytes and C.sub.6 glial cells. Although ceramide by itself did not induce the expression of iNOS and production of NO, it markedly stimulated the cytokine-induced expression of iNOS and production of NO indicating that sphingomyelin-derived ceramide generation may be an important factor in cytokine-mediated cytotoxicity in neurons and oligodendrocytes in neuroinflammatory diseases. The N-acetyl cysteine (NAC), which has been used to block the cytokine-induced ceramide production in this study and to inhibit cytokine-mediated induction of iNOS is a nontoxic pharmaceutical drug that enters the cell readily and serves both as a scavenger of ROS and a precursor of GSH, the major intracellular thiol (Smilkstein et al., 1988). Therefore, the use of reductants such as NAC or other thiol compounds, may be beneficial in restoring cellular redox and in inhibition of cytokine-mediated induction of iNOS and breakdown of sphingomyelin thus reducing NO-mediated cytotoxicity as well as ceramide-mediated apoptosis in neuroinflammatory diseases.

Inhibitors, Enhancers and Screening Assays

In still further embodiments, the present invention provides methods for identifying new iNOS and/or proinflammatory cytokine inhibitory compounds, which may be termed as "candidate substances." It is contemplated that such screening techniques will prove useful in the general identification of any compound that will serve the purpose of inhibiting iNOS and/or proinflammatory cytokines, and in preferred embodiments, will provide candidate therapeutic compounds. The present invention also provides methods for identifying new iNOS and/or proinflammatory cytokine stimulatory or enhancing compounds.

It is further contemplated that useful compounds in this regard will in no way be limited to proteinaceous or peptidyl compounds. In fact, it may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assays will be non-peptidyl in nature and, e.g., which will serve to inhibit or enhance iNOS and/or proinflammatory cytokine activity or transcription through a tight binding or other chemical interaction. Candidate substances may be obtained from libraries of synthetic chemicals, or from natural samples, such as rain forest and marine samples.

In preferred embodiments, a candidate induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines is selected from agents that have certain traits or modes of action common to those of the suppressors and/or inhibitors identified herein. Preferred candidate substances would either inhibit the Ras/Raf/MAP kinase pathway, inhibit and/or suppress the induction and/or activation of NF-kB, inhibit mevalonate synthesis, be an enhancer of protein kinase A, and/or inhibit the farnasylation of proteins, including but not limited to Ras. In certain embodiments the inhibitor of mevalonate synthesis may be an inhibitor of HMG-CoA reductase or suppressor of its induction. In certain aspects the inhibitor of HMG-CoA reductase is a stimulator of AMP-activated protein kinase. In certain other embodiments the inhibitor of of inducible nitric oxide synthase and/or proinflammatory cytokines may be an inhibitor of mevalonate pyrophosphate decarboxylase or suppressor of its induction. In other embodiments the candidate substance is an antioxidant. In other embodiments the candidate substance is an enhancer of intracellular cAMP. The enhancer of intracellular cAMP may be an inhibitor of cAMP phosphodiesterase and/or suppressor of its induction. In other embodiments the candidate substance is a farnesyl protein transferase inhibitor and/or induction suppressor.

In other preferred embodiments, a candidate induction suppressor and/or inhibitor of inducible nitric oxide synthase and/or proinflammatory cytokines is selected from agents that have certain traits or modes of action common to those of the stimulators and/or enhancers identified herein. For example, a preferred candidate stimulators or enhancers would include a PKA inhibitor.

In other embodiments, the present invention provides methods for identifying new iNOS and/or proinflammatory cytokine inhibitory or stimulatory compounds. To determine whether a candidate substance has inhibitory, suppressor, stimulator, or enhancer activity for iNOS, and/or proinflammatory cytokines, assays may be employed to detect or measure the change in the message, content, and/or activity of iNOS, proinflammatory cytokines such as TNF-.alpha., IL-1.beta., IL-2, IL-6, IL-8 and/or IFN-.gamma., proteins involved in second messenger pathways, or transcription factors such as NF-k.beta..
 

Claim 1 of 36 Claims

1. A method for treating type 1 diabetes in a human in need of such treatment, comprising administering to the human a biologically effective amount of at least one inhibitor of inducible nitric oxide synthase, wherein the inhibitor is an inhibitor of HMG-CoA reductase or a pharmaceutically acceptable salt thereof, and the inhibitor specifically inhibits the activity of HMG-CoA reductase.

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