(12) United States Patent (10) Patent No.: US 6,265,147 B1 Mobley Et Al
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USOO6265147B1 (12) United States Patent (10) Patent No.: US 6,265,147 B1 Mobley et al. (45) Date of Patent: Jul. 24, 2001 (54) METHOD OF SCREENING FOR Carmeci, Charles, et al., “Identification of a Gene (GPR30) NEUROPROTECTIVE AGENTS with Homology to the G-Protein-Coupled Receptor Super family ASSociated with Estrogen Receptor Expression in (75) Inventors: William C. Mobley, Palo Alto; Ronald Breast Cancer,” Genomics (1997) vol. 45:607-617. J. Weigel, Woodside; Chengbiao Wu, Owman, Christer, et al., “Cloning of Human cDNA Encod San Jose; Har Hiu Dawn Lam, ing a Novel Hepathelix Receptor Expressed in Burkitt's Stanford, all of CA (US) Lymphoma and Widely Distributed in Brain abd Peripheral Tissues,” Biochemical and Biophysical Research Cimmuni (73) Assignee: The Board of Trustees of the Leland cations (1996) vol. 228:285–292. Stanford Junior University, Palo Alto, Singh, Meharvan, et al., “Estrogen-Induced Activation of CA (US) Mitogen-Activated Protein Kinase in Cerebral Cortical Explants: Convergence of Estrogen and Neurotrophin Sig (*) Notice: Subject to any disclaimer, the term of this naling Pathways,” Journal of Neuroscience (Feb. 15, 1999) patent is extended or adjusted under 35 vol. 19(4): 1179–1188. U.S.C. 154(b) by 0 days. Toran-Allerand, C. Dominique, “The Estrogen/Neurotro phin Connection During Neural Development: Is Co-Lo (21) Appl. No.: 09/452,531 calization of Estrogen Receptors with the Neurotrophins and Their Receptors Biologically Revelant?”, Dev: Neurosci. (22) Filed: Dec. 1, 1999 (1996) vol. 18:36–48. (51) Int. Cl." ................................................... C12O 1/00 Genbank Accession Number Y08162. (52) U.S. Cl. ............................... 435/4; 435/69.1; 514/169 * cited by examiner (58) Field of Search ......................... 435/4, 69.1, 514/12, 514/169, 182 Primary Examiner Ralph Gitomer (74) Attorney, Agent, or Firm-Pamela Sherwood; (56) References Cited Bozicevic, Field & Francis LLP U.S. PATENT DOCUMENTS (57) ABSTRACT 5,135,956 * 8/1992 Borg et al. ........................... 514/724 Neurological dysfunction is prevented or treated by the 5,554,601 * 9/1996 Simplins et al....... ... 514/182 administration of ligands that activate the GPR30 receptor. 5,723,291 3/1998 Kushner et al. ......................... 435/6 Ligands include, but are not limited to, estrogens and 5,843.934 * 12/1998 Simpkins .......... ... 514/182 Structurally related molecules. In a preferred embodiment, OTHER PUBLICATIONS the GPR30 ligand is an orally available drug that can cross into the brain from blood. Of particular interest are ligands Carmeci C. Identification of a Gene (GPR30)... Genomics that do not activate other estrogen receptors, and therefore 45:607-617, 1997.* do not have the classical estrogenic effects attributable to Hermann C. Quantitative Analysis of the Complex Between these receptorS. p21 and the Ras-Binding Domain of the Human Raf-1 Protein Kinase. J Biol Chem 270(7)2901–2905, Feb. 1995.* 4 Claims, 5 Drawing Sheets U.S. Patent Jul. 24, 2001 Sheet 1 of 5 US 6,265,147 B1 - Er x -3. - 3. CR U.S. Patent Jul. 24, 2001 Sheet 3 of 5 US 6,265,147 B1 - RE 3 U.S. Patent Jul. 24, 2001 Sheet 4 of 5 US 6,265,147 B1 4. U.S. Patent Jul. 24, 2001 Sheet 5 of 5 US 6,265,147 B1 US 6,265,147 B1 1 2 METHOD OF SCREENING FOR for memory and learning in postmenopausal women. Indeed, NEUROPROTECTIVE AGENTS these data have Suggested that estrogen's neurotrophic actions could benefit the function and Survival of neurons in Neurodegenerative diseases are characterized by the dyS both women and men and that estrogen, or an estrogen function and death of neurons, leading to the loSS of neu derivative that is active on estrogen receptors, could be used rologic functions mediated by the brain, Spinal cord and the to treat patients with disorders in which there is dysfunction peripheral nervous System. These disorders have a major and death of neurons. impact on Society. For example, approximately 4 to 5 Estrogen elicits a Selective enhancement of the growth million Americans are afflicted with the chronic neurode and differentiation of axons and dendrites (neurites) in the generative disease known as Alzheimer's disease. Other developing brain. Widespread colocalization of the receptors examples of chronic neurodegenerative diseases include for estrogen and neurotrophic factors in a number of neu diabetic peripheral neuropathy, multiple Sclerosis, amyo ronal populations, including neurons of the basal forebrain, trophic lateral Sclerosis, Huntington's disease and Parkin cerebral cortex, Sensory ganglia, and PC12 cells, has been Son's disease. Normal brain aging is also associated with correlated with the differential and reciprocal transcriptional loSS of normal neuronal function and may entail the deple 15 regulation of these receptors by their ligands. Even more tion of certain neurons. Not all neurodegenerative diseases important, estrogen and neurotrophic factor receptor coex are chronic. Stroke is an acute neurodegenerative disease. pression leads to convergence or cross-coupling of their Sudden loSS of neurons may also characterize the brains of Signaling pathways, particularly at the level of the mitogen patients with epilepsy and those that Suffer hypoglycemic activated protein (MAP) kinase cascade. The ability of insults and traumatic injury of the brain, peripheral nerves or estrogen to regulate a broad array of cytoskeletal and Spinal cord. growth-associated genes involved in neurite growth and Though the mechanisms responsible for the dysfunction differentiation is of great interest in the development of and death of neurons in neurodegenerative disorders are not pharmaceutical agents and methodologies for the treatment well understood, a common theme is that loSS of neurons of neurological disorders. results in both the loss of normal functions and the onset of 25 Relevant Literature adverse behavioral Symptoms. For example, patients with Estrogen elicits a Selective enhancement of the growth Alzheimer's disease demonstrate memory loSS and cognitive and differentiation of axons and dendrites (neurites) in the deficits as well as bizarre and Sometimes aggressive behav developing CNS. There is widespread colocalization of iors. Therapeutic agents that have been developed to retard estrogen and neurotrophin receptors within developing fore loSS of neuronal activity and Survival are largely ineffective. brain neurons and reciprocal transcriptional regulation of Some have toxic side effects that limit their usefulness. these receptors by their ligands (Toran-Allerand (1996) Dev Other promising therapies, Such as neurotrophic factors, are Neurosci 18(1-2):36–48). Estradiol elicited rapid tyrosine prevented from reaching their target site because of their phosphorylation/activation of mitogen-activated protein inability to cross the blood-brain barrier. The blood-brain (MAP) kinases. This extracellular signal-regulated protein barrier is a complex of Structural and enzymatic components 35 kinase activation was inhibited successfully by the MEK1 that retards the passage of both large and charged Small inhibitor PD98059, but not by the estrogen receptor (ER) molecules, thereby limiting the access of Such molecules to antagonist ICI 182,780, and did not appear to result from cells in the brain. estradiol-induced activation of trk (Singh et al. (1999) J Senile dementia of the Alzheimer's type is a debilitating Neurosci 19(4): 1179–88). neurodegenerative disease, mainly afflicting the elderly; it is 40 U.S. Pat. No. 5,843,934, issued Dec. 1, 1998, a method is characterized by a progressive intellectual and personality described for conferring a cytoprotective effect on a popu decline, as well as a loSS of memory, perception, reasoning, lation of cells by administering an estrogen compound orientation and judgment. One highly reproducible feature having insubstantial SeX related activity. of the disease is dysfunction and loSS of Selected populations of neurons in the brain. Among these are cholinergic neurons 45 SUMMARY OF THE INVENTION of the basal forebrain, whose normal function contributes Compositions and methods are provided for the treatment importantly to attention, learning and memory. There is an of neurological dysfunction, by the administration of ligands observed decline in the function of cholinergic Systems, and that activate the GPR30 receptor. Ligands include, but are a Severe depletion of these cholinergic neurons. not limited to, estrogens and structurally related molecules. It is increasingly apparent that estrogen has an important 50 Conditions that benefit from protection of neurons include role in regulating neuronal Survival and function. There is brain trauma, Stroke, multiple Sclerosis, neurodevelopmental perhaps no better demonstration of this than the finding that disorders and neurodegenerative disorders including, but not administration of estrogen to postmenopausal women Sig limited to, Alzheimer's disease, Parkinson's disease, and nificantly decreases the incidence of Alzheimer's disease. amyotrophic lateral Sclerosis. These Striking data have encouraged new interest in the 55 biological functions of estrogen and, in particular, of its In a preferred embodiment, the GPR30 ligand is an orally actions on neurons. available drug that can cross into the brain from blood. Of In recent years it has become apparent that estrogen particular interest are estrogen derivatives that do not acti enhances the differentiation of neurons, including the out Vate other estrogen receptors, and therefore