Inhibitors of nitric oxide synthase and use thereof to prevent glutamate ...

INHIBITORS OF NITRIC OXIDE SYNTHASE AND
USE THEREOF TO PREVENT GLUTAMATE
NEUROTOXICITY

This invention was made with government support.
The government retains certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to the use of inhibitors of nitric 10 oxide synthase to prevent glutamate neurotoxicity.

BACKGROUND OF THE INVENTION

Nitric Oxide (NO) was originally identified in vascular endothelial cells (Palmer et al. (1987) Nature 327: 15 524-526 and Palmer et al. (1988) Nature 333: 664-666) and has been identified as being identical to endothelium-derived relaxing factor (Moncada et al. (1989) Biochem. Pharmacol. 38: 1709-1715; Furchgott (1990) Acta Physiol. Scand. 139: 257-270 and Ignarro (1990) 20 Annu. Rev. Pharmacol. Toxicol. 30: 535-560). Besides endothelial cells, NO formation has been demonstrated in macrophages (Hibbs et al. (1987) Science 235: 473-476 and Marietta et al. (1988) Biochemistry 27: 8706-8711), neutrophils (McCall et al. (1989) Biochem. 25 J. 262: 293-297; Salvemini et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6328-6332 and Wright et al. (1989) Biochem. Biophys. Res. Commun. 160: 813-819), some tumor cells (Amber et al. (1988) J. Leuk. Biol. 44: 58-65), adrenal glands (Palacios et al. (1989) Biochem. 30 Biophys. Res. Commun. 165: 802-809), Kupffer cells (Billiar et al. (1989) J. Exp. Med. 169: 1467-1472) and in brain tissue (Garthwaite et al. (1988) Nature 336: 385-388; Knowles et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5159-5162 and Bredt and Snyder (1989) Proc. 35 Natl. Acad. Sci. USA 86: 9030-9033).

Endothelium derived NO relaxes the smooth muscles of blood vessels (Palmer et al. (1987) Nature 327: 524-526 and Ignarro et al. (1987) Proc. Natl. Acad. Sci. USA 84: 9265-9269) and inhibits platelet adhesion 40 (Radomski et al. (1987) Biochem. Biophys. Res. Commun. 148: 1482-1489). NO production by cocultures of Kupffer cells and hepatocytes mediates inhibition of hepatocyte protein synthesis (Billiar et al. (1989) J. Exp. Med. 169: 1467-1472). NO is responsible for mediating 45 the cytotoxic effects of macrophages and neutrophils (Hibbs et al. (1987) J. Immunol. 138: 550-556). NO has also been shown to be a major neuronal messenger- in the brain (Bredt and Snyder (1989) Proc. Natl. Acad. Sci USA 86:9030-9033). The meditation of functions of 50 tissues as diverse as the brain, endothelium and blood cells indicates a wide-spread role for NO as a messenger molecule.

NO is formed by nitric oxide synthetase (NOS) from L-arginine with stoichiometric formation of L-citrul- 55 line. Studies have shown that a guanidino nitrogen of L-arginine is used to form NO (Iyengar et al. (1987) Proc. Natl. Acad. Sci USA 84: 6369-6373; Palmer et al. (1988) Nature 333: 664-666 and Marietta et al. (1988) Biochemistry 27: 8706-8711). 60

The formation of NO appears to involve the same or a similar enzyme in brain and endothelial cells but a different enzyme in macrophages. The brain-endothelium enzyme has been found to require calcium and calmodulin for activity (Bredt and Snyder (1990) Proc. 65 Natl. Acad. Sci. USA 87: 682-685). The macrophage enzyme does not require calcium-calmodulin but does require tetrahydrobiopterin for activity (Tayeh and

Marietta (1989) J. Biol. Chem. 264: 19654-19658; Soo Kwon et al. (1989) J. Biol. Chem. 264: 20496-20501).

The brain (i.e., calmodulin-dependent) NOS enzyme (EC 1.14.23.-) has been purified to homogeneity from rat brain, revealing a 150,000 kD protein (Bredt and Snyder (1990) Proc. Natl. Acad. Sci. USA 87: 682-685). The purification and molecular cloning of calmodulindependent NOS, as well as the preparation of antibodies immunoreactive with calmodulin-dependent NOS, is described in U.S. application Ser. No. 642,002, the disclosure of which is hereby incorporated by reference.

In addition to the differences between NOS activities in brain and endothelial cells as compared to macrophages, the regulation of NOS expression appears to differ as well. The synthesis of NO does not occur in macrophages unless they have been exposed to endotoxin (e.g., bacterial lipopolysaccharide) or cytokine (e.g., interferon-,y, -/3 or -a, tissue necrosis factor-a or -13). However, in the brain and vascular endothelium, NOS is present without exposure to inducing agents (Knowles et al. (1990) Biochem, J. 270: 833-836). The arginine derivative L-N^-nitroarginine (N02Arg) has been described as being a competitive inhibitor of NOS (Moore (1990) Br. J. Pharmacol. 99: 408-412).

NO has been demonstrated to mediate neuronal relaxation of intestines (Bult et al. (1990) Nature 345: 346-347; Gillespie et al. (1989) Br. J. Pharmacol. 98: 1080-1082 and Ramagopal and Leighton (1989) Eur. J. Pharmacol. 174: 297-299) and to mediate stimulation by glutamate of cGMP formation (Bredt and Snyder

(1989) Proc. Natl. Acad. Sci. USA 86: 9030-9033). Glutamate, the major excitatory neurotransmitter in the brain, acts through several receptor subtypes, some of which stimulate the formation of cGMP (Ferrendelli et al. (1974) J. Neurochem. 22: 535-540). Glutamate, acting at N-methyl-D-aspartate (NMDA) subtype of receptors, is responsible for neurotoxic damage in vascular strokes. Selective antagonists of NMDA glutamate receptors prevent neuronal cell death in animal models of hypoxic-ischemic brain injury (Choi (1990) J. Neurosci. 10: 2493-2501). Glutamate neurotoxicity has also been implicated in neurodegenerative disorders such as Alzheimer and Huntington diseases (Choi (1990) J. Neurosci. 10: 2493-2501 and Meldrum and Garthwaite

(1990) Trends in Pharmacol. Sci. 11: 379-387).

An effective method of preventing, treating or ameliorating diseases caused by glutamate neurotoxicity is needed in the art.

SUMMARY OF THE INVENTION

It is an object of the invention is to provide a method of treating diseases caused by glutamate neurotoxicity.

It is another object of the invention to provide a method of preventing or treating vascular stroke.

Another object of the invention is to provide a method of treating a neurodegenerative disease, such as Huntington's disease, Alzheimer's disease and Parkinson's disease.

These and other objects of the invention are provided by one or more of the embodiments described below.

In one embodiment of the invention, a method of preventing or treating diseases caused by glutamate neurotoxicity is provided, which method comprises administering to a mammal in need thereof a therapeutically effective amount of an NOS inhibitor.

In another embodiment of the invention, a method of preventing or treating vascular stroke in a mammal, in particular a human patient, is provided, which comprises administering a therapeutically effective amount of an NOS inhibitor.

In still another embodiment of the invention, a method of preventing or treating a mammal for a neurodegenerative disease caused by glutamate neurotoxic- 5 ity, in particular preventing or treating a neurodegenerative disease such as Huntington's disease, Alzheimer's disease and Parkinson's disease in a human patient, is provided, which method comprises administering a therapeutically effective amount of an NOS inhibitor. 10

It has been discovered that NO mediates glutamate neurotoxicity and that inhibitors of NOS can be used to prevent neuronal cell death by preventing neurotoxicity mediated through glutamate receptors. Since glutamate neurotoxicity is implicated in vascular stroke and neu- IS rodegenerative disorders, such as Alzheimer's disease and Huntington's disease, NOS inhibitors can be used therapeutically in such conditions.

FIG. 1 shows the irreversible inhibition of cerebellar NOS by NO^Arg in vitro.

FIGS. 2A and 2B show the time dependent inhibition of cerebellar NOS by NOaArg in cerebellar and activated macrophage homogenates, respectively. 25

FIG. 3 shows the irreversible inhibition of NOS by N02Arg in vivo.

FIGS. 4A-4P are photomicrographs of cortical cell cultures 24 hours after treatment for NMDA toxicity with various agents. 30

FIG. 5 shows cGMP formation in cortical cell cultures.

FIG. 6 shows the concentration-response relationship of NOS inhibitors in inhibiting NMDA neurotoxicity.

FIG. 7 shows the reversal of N02Arg protection 35 from NMDA cytotoxicity by L-arginine, homoarginine and D-arginine.

FIG. 8 compares cytotoxicity following exposure to sodium nitroprusside and cGMP formation following exposure to reduced hemoglobin. 40

DETAILED DESCRIPTION OF THE
INVENTION

It has been discovered that NOS inhibitors may be used to prevent neuronal cell death. It has now been 45 found that NO mediates glutamate neurotoxicity and that NOS inhibitors may be used therapeutically to prevent neurotoxicity.

NOS inhibitors may be used to prevent, treat, arrest, or ameliorate the progression of any disease condition 50 caused by glutamate neurotoxicity. Such conditions include vascular strokes and neurodegenerative diseases, such as Alzheimer's, Huntington's and Parkinson's diseases, as well as other disease states. For example, following the symptoms of a stroke, a patient is 55 administered an NOS inhibitor to block damage to the brain. Patients with symptoms of Alzheimer's or Huntington's disease are treated with NOS inhibitors to halt the progression of the disease. The symptoms of these disease states are known by one skilled in this art. 60

Inhibitors of NOS are compounds which compete for the substrate binding site of NOS or other sites on the enzyme, and include both reversible and irreversible inhibitors. The present invention contemplates the use of any physiologically acceptable inhibitor which inhib- 65 its NOS activity. The effectiveness of a compound, and its relative potency as an NOS inhibitor, can be tested and routinely determined by measuring inhibition of

NOS activity by monitoring the conversion of arginine to citrulline by NOS in, for example, cerebellar homogenates. A reduction in citrulline formation indicates inhibitory activity of the compound. The percent reduction in citrulline formation, compared to the amount of citrulline formed in the absence of the compound being tested, indicates the potency of the compound as an NOS inhibitor.

Both L-N<"-nitroarginine (N02Arg) and L-N°>monomethylarginine (MeArg), two NOS inhibitors, have been found to prevent neurotoxicity in proportion to their relative potencies as NOS inhibitors. In addition to N02Arg and MeArg, other inhibitors of NOS have been developed. Inhibitors have been prepared from arginine, nitroarginine and guanidinoalkanoic acids, wherein the guanidino group, the amino group, the carboxyl group and the backbone have been systematically altered. Various types of structural alterations affect the selectivity of inhibitors toward brain or macrophage enzyme.

Among the nitroarginine analogs tested, nitroarginine was the most potent inhibitor. In general, the introduction of the nitro group on the guanidine moiety appears to result in selective inhibition of the brain enzyme. Moreover, it was found that the nature of substituents on the a-amino group seemed to dictate the inhibition potency of the compounds. Whereas a free a-amino group offers good inhibitors with 5 to 200 fold selectivity toward brain, the substitution of the amino group with a bulky protective group, such as benzyloxy, results in total loss of activity. A small substituent, such as a formyl group, appears to be favorable.

In the guanidinoalkanoic acid series, it has been found that 6-guanidinohexanoic acid (6-GHA) and 5guanidinopentanoic acid (5-GPA) are potent inhibitors and show good selectivity toward the macrophage enzyme but were inactive against the brain NOS. The nitro analogs of 6-GHA and 5-GPA were found to be generally inactive, as were guanidinoalkanoic acid analogs made rigid by the cyclization of the guanidino group.

The dosage and length of treatment depends on the disease state being treated. The duration of treatment may be a day, a week or longer and may, as in the case of a chronic progressive illness, such as Alzheimer's, last over the entire lifetime of the patient. The inhibitors are administered in a therapeutically effective amount, a typical human dosage of N02Arg ranging from about 0.01 mg/kg of body weight of N02Arg to about 10 mg/kg of N02Arg, in single or divided doses. The dosage will vary depending on the NOS inhibitor to be used and its relative potency. Dosage and length of treatment are readily determinable by the skilled practitioner based on the condition and stage of disease.

In therapeutic use, NOS inhibitors may be administered by any route whereby drugs are conventionally administered. Such routes of administration include intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally and intraventricularly, as well as orally.

Typical preparations for administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water alcoholic/aqueous and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishes,