| PATENT ASSIGNEE'S COUNTRY | USA |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 17.10.2000 |
| PATENT TITLE |
Methods of inhibiting neurodegerative diseases |
| PATENT ABSTRACT |
Methods for inhibiting a nitric oxide-mediated pathological condition and a neurodegenerative disease such as Parkinson's disease, Huntington's disease, Alzheimers's disease, and amyotrophic lateral sclerosis in a human patient, comprising administering an effective amount of a nitroindazole capable of inhibiting a neuronal nitric oxide synthase. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | December 14, 1998 |
| PATENT GOVERNMENT INTERESTS | This invention was made in part with Government funding and the Government may therefore have certain rights in the invention. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method of treating a neurodegenerative disease in a human patient, the method comprising administering to the patient a therapeutically effective amount of a compound that selectively inhibits a neuronal nitric oxide synthase. 2. The method of claim 1, wherein said neurodegenerative disease is Parkinson's Disease. 3. The method of claim 1, wherein said neurodegenerative disease is Huntington's Disease. 4. The method of claim 1, wherein said neurodegenerative disease is Alzheimer's Disease. 5. The method of claim 1, wherein said neurodegenerative disease is amyotrophic lateral sclerosis. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION This invention relates to the treatment of neurodegenerative diseases. In human and nonhuman primates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical, neuropathologic changes analogous to those observed in idiopathic Parkinson's disease. The neurotoxic effects of MPTP are thought to be initiated by 1-methyl-4-phenylpyridinium (MPP+), which is a metabolite formed by the monoamine oxidase B-mediated (MAO-B) oxidation of MPTP (for review, see Tipton and Singer (1993) J. Neurochem. 61:1191-1206). MPP+ is selectively taken up by high-affinity dopamine and noradrenaline uptake systems and is subsequently accumulated within mitochondria of dopaminergic neurons. There it disrupts oxidative phosphorylation by inhibiting complex I of the mitochondrial electron transport chain (Gluck et al. (1994) J. Biol. Chem. 269:3167-3174). The interruption of oxidative phosphorylation results in decreased levels of ATP (Chan et al. (1991) J. Neurochem. 57:348-351), which may lead to partial neuronal depolarization and secondary activation of voltage-dependent NMDA receptors, resulting in excitotoxic neuronal cell death (Beal (1992) Ann. Neurol. 31:119-130). Although excitotoxic neuronal damage has been linked to Ca.sup.2+ influx, the subsequent crucial steps that lead to cell death remain unknown. The entry of calcium through N-methyl-D-aspartate (NMDA) receptor channels into cells stimulates nitric oxide synthase (NOS) activity by binding to calmodulin, a cofactor for NOS (Bredt and Snyder (1990) Proc. Natl. Acad. Sci. USA 87:682-685). Studies in dissociated cell cultures showed that NOS inhibitors blocked NMDA-induced cell death (Dawson et al. (1991) Proc. Natl. Acad. Sci. USA 88:6368-6371). NO.sup..cndot. may react with superoxide (O.sub.2.sup..cndot.) to generate peroxynitrite (Beckman et al. (1990) Proc. Natl. Acad. Sci. USA 87:1621-1624). Peroxynitrite has been identified as a potent oxidant (Beckman et al. (1992) Arch. Biochem. Biophys. 298:438-445; Ischiropoulos et al. (1992) Arch. Biochem. Biophys. 298:431-437), mediating the nitration of tyrosine and producing hydroxyl radicals (Beckman et al. (1990) supra; Crow et al. (1994) Free Radic. Biol. Med. 16:331-338; van der Vliet et al. (1994) FEBS Lett. 339:89-92). NOS has been implicated as having a role in focal ischemia (Huang et al. (1994) Science 265:1883-1885). Recently, improved inhibitors of NOS have been described. 7-nitroindazole (7-NI) has been reported to be a potent and selective inhibitor of neuronal NOS in vitro and in vivo (Babbedge et al., (1993) Br. J. Pharmacol. 110:225-228; Moore et al. (1993) Br. J. Pharmacol. 110:219-224). Although in vitro studies suggest that 7-NI inhibits both endothelial and neuronal NOS, in vivo studies showed no effect on blood pressure and no effects on endothelium-dependent blood vessel relaxation and acetylcholine-induced vasodepressor effects (Babbedge et al. (1993) supra; Moore et al. (1993) supra, Wolff and Gribin (1994) Arch. Biochem. Biophys. 311:300-306). 7-NI has been shown to be efficacious against focal ischemic lesions in vivo (Yoshida et al. (1994) J. Cereb. Blood Flow Metab. 14:924-929). SUMMARY OF THE INVENTION The invention features treating neurodegenerative diseases by administration of a therapeutically effective amount of inhibitor of neuronal nitric oxide synthase, e.g., a nitroindazole such as 7-NI. By the term "neurodegenerative disease" is meant any pathological state involving neuronal degeneration, including Parkinson's Disease, Huntington's Disease, Alzheimer's Disease, and amyotrophic lateral sclerosis (ALS). By the term "therapeutically effective amount" as used herein means an amount sufficient to effect sufficient in vivo inhibition of a neuronal nitric oxide synthase to bring about clinical improvement in a human patient. A therapeutically effective amount of nitroindazole may be prepared for administration to a patient in need thereof in a number of ways known to the art, including parental, intranasal, and oral formulations. Nitroindazole may also be formulated for implants. The concentration of nitroindazole in a physiologically acceptable formulation will vary depending on a number of factors, including the dosage to be administered, the route of administration, and the specific neurodegenerative condition being treated and its severity. The preferred dosage of nitroindazole will be determined by variables generally considered by the healthcare provider in determining dosages. Typical dosage of nitroindazole to be administered is from about 0.0001 mg nitroindazole/kg body weight to about 10 mg nitroindazole/kg body weight. Frequency of administration may vary from as frequently as daily to as infrequently as once every 1-2 weeks, or once every 6-8 weeks. Treatment will generally be continued as long as necessary to maintain inhibition of neuronal nitric oxide synthase and neuronal injury. The inventors herein provide evidence that nitric oxide plays a role in in vivo neurotoxicity. Accordingly, in one embodiment, the invention features a method for treating nitric oxide-mediated pathological conditions by administration of a therapeutically effective amount of neuronal nitric oxide synthase inhibitor. By "nitric oxide-mediated pathological condition" is meant a degenerative condition resulting at least in part from in vivo production of neuronal nitric oxide. Other features and advantages of the invention will be apparent from the following preferred embodiments thereof, and from the claims. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) controls or MPTP-treated animals receiving 0, 25 or 50 mg/kg 7-nitroindazole (7-NI), as described in Example 1 (mean.+-.SEM; n=11-12). #=p<0.05, ##=p<0.01, and ###=p<0.001, *=p<0.05, **=p<0.001, ***=p<0.001 (ANOVA followed by Fisher PLSD post hoc test) (ANOVA). FIG. 2 shows the effect of MPTP treatment on the formation of 3-nitrotyrosine in mouse striatum and mice additionally treat with 7-NI (mean.+-.SEM; n=11-12). **=p<0.001, #=p<0.005 (ANOVA). FIG. 3 shows the effect of pretreatment with 7-NI on striatal lesions produced by NMDA, AMPA and KA. *=p<0.05; n=9-10 (Student's t-test). FIG. 4 shows the effects of pretreatment with 7-NI on striatal lesions produced by 3 .mu.mol malonate. **=p<0.01, ***=p<0.001; n=10 (ANOVA). FIG. 5 shows the effects of pretreatment with 7-NI on ATP and lactate concentrations. *=p<0.05, **=p<0.01, ***=p<0.001 (paired Student's t-test), #=p<0.05, ##=p<0.01 (unpaired Student's t-test). FIG. 6 shows the T.sub.2 -weighted images and striatal proton spectra in a rat 1.5 h after striatal injection of 3 .mu.mol malonate without (upper panel) and with pretreatment of 50 mg/kg of 7-NI. NAA=N-acetylaspartate, PPM=parts per million. FIG. 7 shows the effects of 7-NI on striatal lesions produced by injection of 10 mg/kg of 3-NP every 12 h. In addition animals received either 25 mg/kg 7-NI or vehicle at the same time points. Animals were sacrificed at the 4th and 5th day. The table gives the number of animals showing a striatal lesion per group (n=12, .chi..sup.2 =0.0005). For calculation of lesion volume the lesions in both hemispheres were combined. ***=p<0.001 (Student's t-test). FIG. 8 shows the digitized photomicrographs of Nissl/NADPH-diaphorase stained sections through the caudate/putamen at the level of the anterior commissure of 3-nitropropionic acid (3-NP) treated rats. In A, a lesion (pale zone) is present in a rat treated with 3-NP alone. A higher magnification of the lesion is presented in C. There is marked neuronal loss and gliosis with NADP-diaphorase neuronal sparing (arrow heads). In contrast, the rat concomitantly treated with 3-NP and 7-nitroindazole (7-NI), the 7-NI provided complete protection (B and D). FIG. 9 shows the digitized photomicrograph of .sup.8 hydroxydeoxyguanosine immunoreactivity around lesion site of FIG. 8A. Activity is present within neurons at this locus. FIG. 10 shows the production of 2,3 and 2,5 DHBA and 3-nitrotyrosine by systemic treatment with 3-NP and effects of 7-NI (n=10). #=p<0.05 (ANOVA). FIG. 11A shows the mean firing rate under three conditions, spontaneous, after injection of 7-NI (50 mg/kg i.p.) and after MK-801 (4 mg/kg i.p.). Four animals received 7-NI and three of these also received MK-801. The spike counts were averaged. ***=p<0.001 (ANOVA). FIG. 11B shows the actual firing rate of neurons in the striatum from one of the test animals. The baseline spontaneous activity was recorded between 0 and 16 minutes. The first gap in the graph represents 60 min which elapsed from the time the animal was injected with 7-NI (black arrow head). During this gap no change in activity occurred. The second gap indicates the 30 minutes that passed after administering the MK-801 (white arrow). FIG. 12 is a schematic representation of the NMDA receptor mediated cascade of cell death and potential steps of therapeutic intervention. DETAILED DESCRIPTION The inventors have shown, for the first time, that inhibition of a neuronal isoform of NOS offers protection against neurotoxicity in vivo, and that blocking the activity of neuronal NOS with an NOS inhibitor provides protection against neurotoxicity in murine models of Parkinson's and Huntington's Diseases. Example 1 below describes the inhibition of neuronal NOS by 7-nitroindazole (7-NI) resulting in in vivo protection against MPTP-induced neurotoxicity. These results show for the first time that treatment with 7-NI dose-dependently protects against MPTP-induced dopamine depletions in vitro. 7-NI was effective in two different dosing regimens of MPTP that produce varying degrees of dopamine depletion. At 50 mg/kg of 7-NI there was almost complete protection in both paradigms. Similar effects were seen with MPTP-induced depletions of both homovallinic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC). These neuroprotective effects do not appear to be due to an inhibition of MAO-B activity, because 7-NI had no significant inhibitory effect on this enzyme either in vitro and in vivo. 7-NI also had no influence on dopamine uptake in vitro. Nor are the observed effects due to temperature; 7-NI produced only small changes in body temperature and showed the same degree of protection when the temperature of mice was maintained at 37.5.degree. C. These results therefore indicate that NO.sup..cndot. plays a critical role in MPTP-induced neurotoxicity. One mechanism by which NO.sup..cndot. may mediate toxicity is by interacting with superoxide radical to form peroxynitrite (ONOO.sup.--). Consistent with this hypothesis, MPTP neurotoxicity in mice resulted in a significant increase in the concentration of 3-nitrotyrosine, which was attenuated by treatment with 7-NI. These results also show the involvement of peroxynitrite in neurotoxicity. In Example 2, the ability of 7-NI to block striatal lesions produced by direct acting and secondary excitotoxins was examined. Direct acting excitotoxins include N-methyl-D-aspartate (NMDA), kainic acid, and .alpha.-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Secondary excitotoxic striatal lesions were produced by the reversible succinate dehydrogenase inhibitors malonate and the irreversible inhibitor 3-nitropropionic (3-NP). It has previously been found that both malonate and 3-NP produce striatal lesions by a secondary excitotoxic mechanism as a consequence of impaired energy metabolism (Beal et al. (1993) J. Neurochem. 61:1147-1150; Greene et al. (1993) J. Neurochem. 61:1151-1154; Henshaw et al. (1994) Brain Res. 647:161-166). The results described in Example 2 show that 7-NI significantly attenuated lesions produced by intrastriatal injections of NMDA, but not kainic acid or AMPA. 7-NI attenuated malonate lesions and resulted in nearly complete protection against striatal lesions produced by systemic administration of 3-NP. This is the first evidence that inhibition of neuronal NOS can attenuate striatal lesions produced by either intrastriatal administration of malonate or systemic administration of 3-nitropropionic acid (3-NP). 7-NI was much more effective against these secondary excitotoxic lesions than it was against the direct acting excitotoxin NMDA. To evaluate the mechanism of neuroprotection, we investigated the in vivo effects of 7-NI on spontaneous striatal electrophysiological activity in rat striatum. In contrast to the NMDA antagonist MK-801, which inhibited electrophysiological activity, 7-NI had no significant effects, suggesting that it is not acting at NMDA receptors. The ability of 7-NI to attenuate ATP depletions and increases in lactate concentrations produced by intrastriatal administration of malonate was examined. 7-NI protected both against malonate-induced decreases in ATP, and increases in lactate, as assessed by chemical shift magnetic resonance spectroscopy. This result contrasts with those of a free radical spin trap which exerts neuroprotective effects but had no effect on malonate-induced depletions of ATP (Schulz et al. (1995) 64:in press). Similarly, MK-801 which has neuroprotective effects against 3-NP-induced neurotoxicity in vitro had no effect on ATP depletions (Riepe et al. (1994) NeuroReport 5:2130-2132). The effect of 7-NI on 2,3 dihydroxybenzoic acid (DHBA) and 3-nitrotyrosine were also investigated. Hydroxyl radical generation results in an increase in the conversion of salicylate to 2,3 DHBA (Floyd et al. (1984) J. Biochem. Biophys. Methods 10:221-235; Hall et al. (1993) J. Neurochem. 60:588-594). As is shown in Example 2, 3-NP produced an increase in 2,3 DHBA/salicylate, which is attenuated by pretreatment with 7-NI. 3-NP also induced increases in 3-nitrotyrosine, which was also attenuated by prior treatment with 7-NI. These results suggest that peroxynitrite may play a role in 3-NP-induced neurotoxicity in vivo. A mechanism of NMDA receptor mediated cascade of cell death and potential steps of therapeutic intervention are illustrated in FIG. 12. The upper panel of FIG. 12 shows how mitochondria provide ATP that fuels a multitude of ion pumps which produce and maintain voltage and ion gradients across neuronal membranes, thereby creating a resting potential of -80 mV. The cytoplasmic Ca.sup.2+ concentration is maintained several order of magnitude lower than outside the cell by means of ATPases that actively move Ca.sup.2+ out of the cell or into intracellular storage organelles such as the endoplasmic reticulum (ER). The lower panel of FIG. 12 illustrates how excitotoxicity may occur as a consequence of a defect in energy metabolism. This mechanism may be due to membrane depolarization due to ATP depletion, followed by relief of the voltage dependent Mg.sup.2+ block of the NMDA receptor, leading to an ion influx, especially the inward movement of Na.sup.+ and Ca.sup.2+. The intracellular Ca.sup.2+ concentration increases dramatically leading to an activation of Ca.sup.2+ dependent enzymes, including neuronal NOS which produces NO.sup..cndot.. NO.sup..cndot. may interact with superoxide radicals (O.sub.2.sup..cndot.) to form peroxynitrite (ONOO.sup.--).sup.8. The formation of peroxynitrite does not require transition metals, and once formed it can diffuse over several cell diameters where it can oxidize lipids, proteins and DNA. It also can produce nitronium ions which then nitrate tyrosine residues. Peroxynitrite can also be protonated to form ONOOH which may then decompose to OH.sup..cndot.. NO.sup..cndot. can inhibit cytochrome oxidase in vitro and de-energizes mitochondria, thereby leading to an additional decline in mitochondrial function. Increased mitochondrial Ca.sup.2+ concentrations also lead to an increase in OH.sup..cndot. (Dykens (1994) J. Neurochem. 63:584-591). The block of the electron flux through the electron transport chain produces O.sub.2.sup..cndot.. We provide herein evidence that NO.sup..cndot. plays a role in NMDA, malonate and 3-NP neurotoxicity in vivo. This evidence provides an important new insight for the treatment of neurodegenerative disease such as Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases which are mediated by NO.sup..cndot.. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the instant invention to its fullest extent. Accordingly, the following experimental examples are illustrative and by no means intended to limit the scope of the claimed invention. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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