USOO6369052B1 (12) United States Patent (10) Patent No.: US 6,369,052 B1 Kellar et al. (45) Date of Patent: Apr. 9, 2002 (54) COMBINATION OF HUPERZINE AND Ayer et al., 1989," Alkaloids of Lycopodium selago. On the NCOTINC COMPOUNDS ASA identity of Selagine with huperzine A and the Structure of a NEUROPROTECTIVE AGENT related alkaloid”, Can. J. Chem. 67: 1538–1540. Bai et al., 1993, "Traditional Chinese medicines and New (75) Inventors: Kenneth J. Kellar, Bethesda, MD drug development', Pure and Appl. Chem. 65:1103-1112. (US); Alan P. Kozikowski, Princeton, Choi et al., 1989, “Aspartate Neurotoxicity on Cultured NJ (US) Cortical Neurons', J. of Neuroscience Res. 23:116-121. Choi et al., 1993, "Calcium and Excitotoxic Neuronal (73) Assignee: Georgetown University, Washington, Injury', annals NY Acdemy of Sciences 162-170. DC (US) Coyle et al., 1984,. “Alzheimers Disease: A disorder of cortical cholinergic innervation”, Science 219:1184-1190. (*) Notice: Subject to any disclaimer, the term of this Decker et al., 1994, “(S)-3-Methyl-5-(1-Methyl-2- Pyr patent is extended or adjusted under 35 rolidinyl) Isoxazole (ABT 418): A novel cholinergic ligand U.S.C. 154(b) by 0 days. with cognition enhancing and anxiolytic activities: II. In vivo characterization”, J. of Pharmacology and Exp. Ther (21) Appl. No.: 09/910,819 270:319-328. (22) Filed: Jul. 23, 2001 Decket et al., 1992, “Effects of nicotine on spatial memory deficits in rats with septal lesions”, Brain Res. 572:281-285. Gordon et al., 1997, "Huperzine-A(HUP-A) Interacts with Related U.S. Application Data the NMDA Receptor Ion Channel”, FASEB Journal, Core (60) Provisional application No. 60/223,605, filed on Aug. 7, Abstracts, 11(9):268. 2000, and provisional application No. 60/241,670, filed on Harsing et al., 1992, “Dopamine Efflux from Striatum After Oct. 18, 2000. Chronic Nicotine: Evidence for Autoreceptor Desensitiza tion”, J. of Neurochem. 59(1):48–54. (51) Int. 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Neurology 5,104,880 A 4/1992 Kozikowski 5,106,979 A 4/1992 Kozikowski et al. 5798):719–731. 5,177,082 A 1/1993 Yu et al. Linville et al., 1993, “Nicotinic Agonists Modulate Basal 5,547,960 A 8/1996 Kozikowski et al. Forebrain Control of Cortical Cerebral Blood Flow in Anes 5,663,344 A 9/1997 Kozikowski et al. thetized Rats', J. of Pharmacology and Exp. Ther. 5,869.672 A 2/1999 Kozikowski et al. 267:441-447. 5,929,084 A 7/1999 Zhu et al. (List continued on next page.) OTHER PUBLICATIONS Primary Examiner Raymond Henley, III Akaike et al., 1994, “Nicotine induced protection of cultured cortical neurons against N-methyl-D-aspartate receptor (74) Attorney, Agent, or Firm-Pennie & Edmonds LLP mediated glutamate cytotoxicity', Brain Res. 644:181-187. (57) ABSTRACT Akaike et al., 1994, "Regulation by neuroprotective factors of NMDA receptor mediated nitric oxide synthesis in the The present invention provides compositions and methods brain and retina”, Prog Brain Res. 103:391-403. for treating, preventing, or reversing neuronal dysfunction Arneric et al., 1994, “Nicotinic agonists in Alzheimers including cognitive decline, Such as cognitive decline asso disease: does the molecular diversity of nicotine receptors ciated with aging and minimal cognitive impairment; Severe offer the opportunity for developing CNS-selective cholin neurodegenerative disorders, Such as Alzheimer's disease; ergic channel activators?”, Recent Advances in the Treat and neuronal dysfunction associated with loSS of motor ment of Neurodegenerative Disorders and Cognitive Dys skills, Such as Parkinson's disease and amyotrophic lateral function. Sclerosis. The compositions and methods of the invention Arneric et al., 1994, "(S)-3-Methyl-5-(1-Methyl-2- Pyr can also treat or prevent neuronal dysfunction resulting from rolidinyl) Isoxazole (ABT 418): A Novel Cholinergic CNS injury, Such as Stroke, Spinal-cord injury, and Ligand with Cogniton-Enhancing and Anxiolytic Activi peripheral-nerve injury. The compositions of the invention ties”, J. of Pharmacology and Exp. Ther. 270:310-318. comprises a huperzine compound and a nicotinic compound. Ayer et al., 1989, “Some new Lycopodium alkaloids”, Can. J. Chem. 67; 1077–1086. 58 Claims, 3 Drawing Sheets US 6,369,052 B1 Page 2 OTHER PUBLICATIONS Sanberg et al., 1993, “Nicotine Potentiation of Haloperidol Liu et al., 1986, “The Structures of Huperzine A and B two Induced Catalepsy: Striatal Mechanisms”, Pharmacology new alkaloids exhibiting marked anticholinesterase activ Biochem. And Behavior 46:303-307. ity”, Can. J. Chem. 6 4:837-839. Sjak-Shie et al., 1991, “Some Novel Actions of Nicotine in Marin et al., 1994, “Nicotine protects cultured striatal neu Nucleus Basalis Lesioned Rats and in Hippocampal Slices”, rones against N-methily-D-aspartate receptor-mediated Cholinergic Basis for Alzheimer Therapy 379-385. neurotoxicity”, NeuroReport 5:1977-1980. Mattson et al., 1988, “Neurotransmitters in the regulation of Sullivan et al., 1995, “Cholinergenic Channel Activators: neuronal cytoarchitecture', Brain Res. Reviews Novel Opportunities for the Treatment of CNS Disorders”, 13:179-212. Proc. West. Pharmacol. Soc. 38:127-130. Meldrum and Garthwaite, 1990, “Excitatory amino acid neurotoxicity and neurodegenerative disease”, TIPS Ved et al., 1997, "Huperzine A, a potential therapeutic agent 11:379-387. for dementia, reduces neuronal cell death caused by Sahaklan et al., 154, “The Effects of Nicotine on Attention, glutamate”, Medicinal Chem. 8(43):963–968. Information Processing, and Short Term Memory in Patients Wesnes et al., 1984, “Effects of Scopolamine and nicotine on with Demential of the Alzheimer Type”, British J. of Psy human rapid information processing perfromance', Psy chiatry 154:797–800. chopharmacology 82:147-150. U.S. Patent Apr. 9, 2002 Sheet 1 of 3 US 6,369,052 B1 3 4. S. t C2 ces ce -S. S. U v. 2 C N ver 1. -- v. CD- L CS S LL 2 E. N 1. l Ol O C S : O O O O O O O Se do d r N JOXO WOWN O % O 9 S is < Y - 5a CD - 2, l U 4. 5 C2 2. C O O O O O O S2 OO O tr OXO WONN O % U.S. Patent Apr. 9, 2002 Sheet 2 of 3 US 6,369,052 B1 O O d se Z R O C2 s O 2 Le S. on 2 CN S. 2 Se S C D l O LL t N n 1 1 Ll 55 C C C O O O Co O O O O C O on O CO CO sh- N ALIOIXOL 3Olddd OIOIAWV-8 40% O O S. s. g Cn s s 3 CD g A. Ll R O C 2. O O O O O O O O O CN O O d s on ALIOXO1 3OLld3d OIOJWV-8 40% U.S. Patent Apr. 9, 2002 Sheet 3 of 3 US 6,369,052 B1 O O C U s 2 s O O Se O is s S. 9. O S2O is4 no 5 tL Us 95 asCD 55N - E. Ll 1 1 U Ol Cl O O C O O O O O O O O 3 o O r on OXO WOWN O % US 6,369,052 B1 1 2 COMBINATION OF HUPERZINE AND binding to N-methyl-D-aspartate (“NMDA') receptors, NCOTINC COMPOUNDS ASA which play a crucial role in glutamate-induced neurodegen NEUROPROTECTIVE AGENT eration (Mattson, 1988, Brain Res. 13:174; Choi et al., 1989, J. Neurosci. Res. 23:116; Hartley & Choi, 1989, J. Phar 5 macol. Exp. Ther. 250:752; Meldrum & Garthwaire, 1990, This application claims the benefit of U.S. Provisional Trends Pharmacol. Sci. 11:379). For example, symptoms of Application No. 60/223,605, filed Aug. 7, 2000, hereby neuronal damage caused by organophosphate (“OP”) toxic incorporated herein by reference in its entirety and U.S. ity (i.e. tremors, seizures or convulsions) have been partially Provisional Application No. 60/241,670, filed Oct. 18, 2000, attributed to the release of large quantities of glutamate hereby incorporated herein by reference in its entirety. during OP intoxication. The released glutamate causes acti This invention was made in part under Department of vation of NMDA receptors and increases Ca' influx that in Army Grant No. DAMD 17-99-2-9907. turn causes persistent elevation in Ca" concentrations, 1. FIELD OF THE INVENTION eventually resulting in neuronal death (Ved et al., 1997, The present invention relates to compositions comprising Neuroreport 8:963; Choi, 1994, Ann. NY Acad. Sci. huperzine compounds and nicotinic compounds and to 15 747:162). Nicotine protects cultured cortical neurons against methods of using huperzine compounds and nicotinic com NMDA receptor-mediated, glutamate-induced cell death pounds in combination to provide a neuroprotective effect. (Marin et al., 1994, Neuropharmacology and Neurotoxicol ogy 5:1977; Kaneko et al., 1997, Brain Research 765:135.