Method of screening for neuroprotective agents

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...

Reexamination Certificate

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C435S069100, C514S169000

Reexamination Certificate

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06265147

ABSTRACT:

Neurodegenerative diseases are characterized by the dysfunction and death of neurons, leading to the loss of neurologic functions mediated by the brain, spinal cord and the peripheral nervous system. These disorders have a major impact on society. For example, approximately 4 to 5 million Americans are afflicted with the chronic neurodegenerative disease known as Alzheimer's disease. Other examples of chronic neurodegenerative diseases include diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease and Parkinson's disease. Normal brain aging is also associated with loss of normal neuronal function and may entail the depletion of certain neurons. Not all neurodegenerative diseases are chronic. Stroke is an acute neurodegenerative disease. Sudden loss of neurons may also characterize the brains of patients with epilepsy and those that suffer hypoglycemic insults and traumatic injury of the brain, peripheral nerves or spinal cord.
Though the mechanisms responsible for the dysfunction and death of neurons in neurodegenerative disorders are not well understood, a common theme is that loss of neurons results in both the loss of normal functions and the onset of adverse behavioral symptoms. For example, patients with Alzheimer's disease demonstrate memory loss and cognitive deficits as well as bizarre and sometimes aggressive behaviors. Therapeutic agents that have been developed to retard loss of neuronal activity and survival are largely ineffective. Some have toxic side effects that limit their usefulness. Other promising therapies, such as neurotrophic factors, are prevented from reaching their target site because of their inability to cross the blood-brain barrier. The blood-brain barrier is a complex of structural and enzymatic components that retards the passage of both large and charged small molecules, thereby limiting the access of such molecules to cells in the brain.
Senile dementia of the Alzheimer's type is a debilitating neurodegenerative disease, mainly afflicting the elderly; it is characterized by a progressive intellectual and personality decline, as well as a loss of memory, perception, reasoning, orientation and judgment. One highly reproducible feature of the disease is dysfunction and loss of selected populations of neurons in the brain. Among these are cholinergic neurons of the basal forebrain, whose normal function contributes importantly to attention, learning and memory. There is an observed decline in the function of cholinergic systems, and a severe depletion of these cholinergic neurons.
It is increasingly apparent that estrogen has an important role in regulating neuronal survival and function. There is perhaps no better demonstration of this than the finding that administration of estrogen to postmenopausal women significantly decreases the incidence of Alzheimer's disease. These striking data have encouraged new interest in the biological functions of estrogen and, in particular, of its actions on neurons.
In recent years it has become apparent that estrogen enhances the differentiation of neurons, including the outgrowth of processes from CNS neurons; estrogen binds to specific receptors on neurons in the brain and regulates the levels of classical neurotrophic factors, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BNDF), and their receptors. These findings are evidence that estrogen is itself a neurotrophic factor, and they argue strongly that estrogen's neurotrophic actions are responsible for enhancing the survival and function of neurons important for memory and learning in postmenopausal women. Indeed, these data have suggested that estrogen's neurotrophic actions could benefit the function and survival of neurons in both women and men and that estrogen, or an estrogen derivative that is active on estrogen receptors, could be used to treat patients with disorders in which there is dysfunction and death of neurons.
Estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing brain. Widespread colocalization of the receptors for estrogen and neurotrophic factors in a number of neuronal populations, including neurons of the basal forebrain, cerebral cortex, sensory ganglia, and PC12 cells, has been correlated with the differential and reciprocal transcriptional regulation of these receptors by their ligands. Even more important, estrogen and neurotrophic factor receptor coexpression leads to convergence or cross-coupling of their signaling pathways, particularly at the level of the mitogen-activated protein (MAP) kinase cascade. The ability of estrogen to regulate a broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation is of great interest in the development of pharmaceutical agents and methodologies for the treatment of neurological disorders.
Relevant Literature
Estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing CNS. There is widespread colocalization of estrogen and neurotrophin receptors within developing forebrain neurons and reciprocal transcriptional regulation of these receptors by their ligands (Toran-Allerand (1996)
Dev Neurosci
18(1-2):36-48). Estradiol elicited rapid tyrosine phosphorylation/activation of mitogen-activated protein (MAP) kinases. This extracellular signal-regulated protein kinase activation was inhibited successfully by the MEK1 inhibitor PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780, and did not appear to result from estradiol-induced activation of trk (Singh et al. (1999)
J Neurosci
19(4):1179-88).
U.S. Pat. No. 5,843,934, issued Dec. 1, 1998, a method is described for conferring a cytoprotective effect on a population of cells by administering an estrogen compound having insubstantial sex related activity.
SUMMARY OF THE INVENTION
Compositions and methods are provided for the treatment of neurological dysfunction, by the administration of ligands that activate the GPR30 receptor. Ligands include, but are not limited to, estrogens and structurally related molecules. Conditions that benefit from protection of neurons include brain trauma, stroke, multiple sclerosis, neurodevelopmental disorders and neurodegenerative disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.
In a preferred embodiment, the GPR30 ligand is an orally available drug that can cross into the brain from blood. Of particular interest are estrogen derivatives that do not activate other estrogen receptors, and therefore do not have the classical estrogenic effects attributable to these receptors.


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Carmeci, Charles, et al., “Identification of a Gene (GPR30) with Homology to the G-Protein-Coupled Receptor Superfamily Associated with Estrogen Receptor Expression in Breast Cancer,”Genomics(1997) vol. 45:607-617.
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Singh, Meharvan, et al., “Estrogen-Induced Activation of Mitogen-Activated Protein Kinase in Cerebral Cortical Explants: Convergence of Estrogen and Neurotrophin Signaling Pathways,”Journal of Neuroscience(Feb. 15, 1999) vol. 19(4):1179-1188.
Toran-Allerand, C. Dominique, “The Estrogen/Neurotrophin Connection During Neural Development: Is Co-Localization of Estrogen Receptors w

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