USOO5888996A United States Patent (19) 11 Patent Number: 5,888,996 Farb (45) Date of Patent: Mar. 30, 1999 54 INHIBITION OF NMDA RECEPTOR Gyermek, L., et al., “Structure-Activity Relationship of ACTIVITY AND MODULATION OF Some Steroidal Hypnotic Agents,” Steroids. CCX, GLUTAMATE-MEDIATED SYNAPTC 11:117–125 (1968). ACTIVITY Wu, F.-S., et al., “Pregnenolone Sulfate: A Positive Allos teric Modulator at the N-Methyl-D-aspartate Receptor,” 75 Inventor: David H. Farb, Cambridge, Mass. Molecular Pharmacology, 40:333-336 (1991). 73 Assignee: Trustees of Boston University, Boston, Park-Chung, M., et al., “3C-Hydroxy-5B-pregnan-20-one Mass. Sulfate: A Negative Modulator of the NMDA-Induced Cur rent in Cultured Neurons,” Molecular Pharmacology, 21 Appl. No.: 559,442 46:146-150 (1994). Wieland, S., et al., “Anxiolytic Activity of the Progesterone 22 Filed: Nov. 15, 1995 Metabolite 5C-pregnan-3C-ol-20-one,” Brain Research, Related U.S. Application Data 565:263-268 (1991). Belelli, D., et al., “Anticonvulsant Profile of the Progester 63 Continuation-in-part of Ser. No. 507,757, Jul. 26, 1995, one Metabolite 5C-pregnan-3C-ol-20-one.” European abandoned. Journal of Pharmacology, 166:325–329 (1989). (51) Int. Cl." ..................................................... A61K 31/56 Lan, N. C., et al., “Neurocactive Steroid Actions at the 52 U.S. Cl. .......................... 514/182; 514/177; 514/178; GABA Receptor.” Hormones and Behavior; 28:537-544 514/179 (1994). 58 Field of Search ..................................... 514/182, 177, 514/178, 179 Primary Examiner Rebecca Cook Attorney, Agent, or Firm-Hamilton, Brook, Smith & 56) References Cited Reynolds, P.C. U.S. PATENT DOCUMENTS 57 ABSTRACT 5,212,167 5/1993 Farb ........................................ 514/178 5,366,968 11/1994 Farb ........................................ 514/178 The present invention relates to a method of inhibiting N-methyl-D-aspartate (NMDA) glutamate receptor FOREIGN PATENT DOCUMENTS mediated ion channel activity (NMDA receptor activity), WO 93/04687 4/1991 WIPO. comprising contacting a neuronal cell (e.g., hippocampal WO 93/05786 4/1993 WIPO. neuron, Spinal cord cell) with an effective amount (e.g., 1 to WO 94/27608 12/1994 WIPO. 500 uM) of a derivative of pregnenolone sulfate. Derivatives of pregnenolone sulfate that inhibit NMDA receptor activity OTHER PUBLICATIONS include pregnenolone Sulfate in which the A ring includes at CA 122:23982 Irwinet et al., 1994. least one double bond or is fully unsaturated, the double CA 121:125584 Park-Chung et al., 1994. bond at the C5-C6 position is reduced, the moiety at the C3, CA 119:86563 Bowlby, 1993. C10, C11 or C13 position is modified, alone or in combi CA 119:20512 Farb, Mar. 18, 1993. nation. It further relates to pregnenolone Sulfate derivatives CA 117:185114 Malone, 1992. which have modifications at other positions (e.g., C5, C7, Wong, M., and Moss, R.L., “Patch-Clamp Analysis of C10, C16, C17, C18, C19, C20, C21), alone or in Direct Steroidal Modulation of Glutamate Receptor-Chan combination, and are inhibitors of NMDA recepteor activity. nels”, Jour: of Neuroendocrinology, 6:347–355, (1994). The pregnenolone Sulfate derivatives differ from preg Bowlby, M.R., “Pregnenolone Sulfate Potentiation of nenolone Sulfate at least one position. The present invention N-Methyl-D-aspartate Receptor Channels in Hippocampal also relates to a method of modulating or altering (e.g., Neurons”, Mol. Pharmacol., 43(5):813–819, (1993). potentiating; inhibiting) excitatory glutamate-mediated Syn Irwin, R. P., et al., “Steroid Potentiation and Inhibition of aptic activity comprising contacting neurons with preg N-Methyl-D-Aspartate Receptor-Mediated Intracellular nenolone Sulfate and derivatives of pregnenolone Sulfate. Ca++ Responses: Structure-Activity Studies,” J. Pharm. and Experimental Therapeutics, 271(2):677-682 (1994). 17 Claims, 5 Drawing Sheets U.S. Patent Mar. 30, 1999 Sheet 1 of 5 5,888,996 CH3 CH3 C=O CO OSO Osc-O Pregnanolone CH Sulfate ?2 CH3 CH2 C=O o2 so Pregnanalone hemisu CCindte O 3SO 3CI-Hydroxy-5B-pregnan-2O-One Sulfate (pregnanolone Sulfate) 5B3CIS CH CH3 C C=O Osc O CH2 OSO CH2 3 H oCo 3B-Hydroxy-5B-pregnan-20- 17 B-estradio-3- One Sulfote he misuCCinCte 5B3 BS O2SO3 CH3 AndrosterOne Sulfote - CH C=O '3 HO C= O O3SO OSO | B-OH-pregnanolone 3 H Sulfate 3CI-Hydroxy-5CI-pregnan-2O One Sufote 5C3O. FG. U.S. Patent Mar. 30, 1999 Sheet 2 of 5 5,888,996 A NMDA 5B3CISNMDAt 5B3CIS NMDA . OOpA OS B K 5B3OSK+5B3CIS K i. 4OOJA OS C AMPA 5B3CIS AMPA+5B3CIS AMPA 25OJA OS FG. 2 U.S. Patent Mar. 30, 1999 Sheet 3 of 5 5,888,996 - Of O-6 IO-5 O-4 O-3 O-2 (5B3CIS) M FG. 3 U.S. Patent Mar. 30, 1999 Sheet 4 of 5 5,888,996 KX 5B3CIS (OOM 5B3CHS IOOM E2 HS2OOM Spino Cord rot hippocompo Electrophysiology 3Ou MNMDA FG. 4 U.S. Patent Mar. 30, 1999 Sheet 5 of 5 5,888,996 OO |: Chronic ShOrt term 25 JM NMDA 3OJM NMDA 24 hrs 5 min NMDA excitotoxicity F.G. 5 5,888,996 1 2 INHIBITION OF NMDA RECEPTOR include pregnenolone Sulfate derivatives in which the A ring ACTIVITY AND MODULATION OF includes at least one double bond or is fully unsaturated, GLUTAMATE-MEDIATED SYNAPTC pregnenolone sulfate derivatives in which the double bond at ACTIVITY the C5-C6 position is reduced, pregnenolone Sulfate in which the moiety at the C3, C10, C11 or C13 position is RELATED APPLICATIONS modified, alone or in combinations thereof. It further relates This application is a Continuation-in-Part application of to pregnenolone Sulfate derivatives which have modifica U.S. application Ser. No. 08/507,757, filed Jul. 26, 1995, tions at other positions (e.g., C5, C7, C10, C16, C17, C18, abandoned, entitled “Inhibition of NMDA Receptor Activ C19, C20, C21), alone or in combination, and are inhibitors ity” by David H. Farb, which claims priority to U.S. Pro of NMDA receptor activity. The pregnenolone sulfate visional Application, 60/001,439, filed on Jul. 24, 1995, derivatives differ from pregnenolone Sulfate at least one entitled “3C-Hydroxy-5B-Pregnan-20-One Sulfate: A Nega position. tive Modulator of the NMDA-Induced Current in Cultured In one embodiment the present invention relates to a method for inhibiting NMDA glutamate receptor mediated Neurons” by David H. Farb and claims the benefit thereof. 15 ion-channel activity comprising contacting a neuronal cell FUNDING with an effective amount of a derivative of pregnenolone Sulfate wherein the derivative is Selected from the group Work described herein was funded by grants from the consisting of 3C-hydroxy-5B-pregnan-20-one Sulfate National Institutes of Mental Health (Grant MH-49469). (5 B3CS), 3B-hydroxy-5E-pregnan-20-one sulfate (5 B3 BS), The United States government has certain rights in the 3C-hydroxy-5C.-pregnan-20-one sulfate (50.3C.S), invention. 3.C.-hydroxy-5C.-pregnan-20-one he misuccinate (5f.3C. he misuccinate), 17 B-estradiol hemisuccinate, 11 f-OH BACKGROUND pregnenolone Sulfate and androsterone Sulfate. L-Glutamate is thought to be the major excitatory neu The present invention also relates to a method of inhib rotransmitter in the vertebrate central nervous System and is 25 iting toxic effects associated with activation of the NMDA known to activate at least three major, pharmacologically receptor in neurons (e.g., hippocampal neurons, Spinal cord distinct classes of glutamate-gated ion channels: N-methyl cells), comprising contacting the neurons with a derivative D-aspartate (NMDA), C.-amino-3-hydroxy-5-methyl-4- of pregnenolone Sulfate Selected from the group consisting isoxazole-propionate (AMPA), and kainate receptors. These of: 533Cs, 5B3 Bs, 5C3Cs, 5B3C. hemisuccinate, 17B three inotropic receptors are named according to their Selec estradiol hemisuccinate, 11B-OH-pregnenolone Sulfate and tive agonists. androsterone Sulfate. NMDA receptors have attracted particular attention Thus, the ability to selectively inhibit the NMDA receptor because of their importance in normal brain function and in acroSS nerve cell membranes offers the means for pharma pathophysiological conditions Such as epilepsy and cerebral cological intervention in various glutamate-induced condi ischemia (Rothman, S. M. and Olney, J. W., Trends 35 tions Such as excitotoxicity, epilepsy, cerebral ischemia and Neurosci., 10:299-302 (1987)). The NMDA receptor Stroke. appears to be essential for the induction of long-term poten The present invention also relates to a method of modul tiation (Collingridge, G. L. and Bliss, T. V. P., Trends lating or altering (e.g., potentiating; inhibiting) excitatory Neurosci., 10:288-293 (1987)), a proposed underlying 40 glutamate-mediated Synaptic activity comprising contacting mechanism for learning and memory (Madison, D. V., et al., neuronal cells with pregnenolone Sulfate or a derivative of Annu. Rev. Neurosci., 14:379-397 (1991)), ischemic cell pregnenolone Sulfate. In one embodiment, the invention death, epilepsy, and other neurological disorders (Simon, R. relates to a method of potentiating excitatory glutamate P., et al., Science 226:850-852 (1984); Choi, D. W., J. mediated Synaptic activity comprising contacting neuronal Neurosci. 10:2493-2501 (1990) such as hypoxic neuronal cells with pregnenolone Sulfate or a derivative of preg damage (Simon, R., et al., Science, 226:850-852 (1984)), 45 nenolone Sulfate (e.g., dehydroepiandrosterone Sulfate). In Schizophrenia (Carlsson, M., et al., Trends NeuroSci., another embodiment, the invention relates to a method of 13:272-276 (1990); Watchel, H., et al., Trends Pharmacol. inhibiting excitatory glutamate-mediated Synaptic activity Sci., 11:219–220 (1990)) and excitotoxicity (Onley, J., et al., comprising contacting neuronal cells with a derivative of Brain Res., 221:207-210 (1981)). The integral channel of 50 pregnenolone Sulfate (e.g. 5(33CS). the NMDA receptor is permeable to Na+, K+, and Ca".