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Protein kinase c epsilon as modulator of anxiety, alcohol consumption and self-administration of drugs of abuseRelated Patent Categories: Multicellular Living Organisms And Unmodified Parts Thereof And Related Processes, Nonhuman Animal, Transgenic Nonhuman Animal (e.g., Mollusks, Etc.), Mammal, MouseProtein kinase c epsilon as modulator of anxiety, alcohol consumption and self-administration of drugs of abuse description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070039064, Protein kinase c epsilon as modulator of anxiety, alcohol consumption and self-administration of drugs of abuse. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is related to U.S. Provisional Application No. 60/091,755, filed Jul. 6, 1998, and U.S. Provisional Application No. 60/125,995, filed Mar. 24, 1999. I. FIELD OF THE INVENTION [0002] The present invention relates to: cells and non-human animals deficient for the protein kinase C isozyme .epsilon. (PKC.epsilon.); the use of PKC.epsilon. as a target for drugs; the use of modulators of PKC.epsilon. in methods of reducing anxiety, modulating alcohol consumption and self-administration of other drugs of abuse, altering the effects of alcohol, and treating conditions associated with insufficient activity of the GABA.sub.A receptor; and the identification of individuals with enhanced susceptibility to alcoholism or other forms of addiction. II. BACKGROUND OF THE INVENTION [0003] Anxiety is very common sensation that, if severe or persistent, can be quite disabling. Anxiety-related disorders are so prevalent that benzodiazepines, the most frequently prescribed anxiolytic agents, regularly appear in lists of the top 20 or 25 most frequently prescribed drugs. Given the undesirable side effects of benzodiazepines and other anxiety-reducing drugs, there is a need for new treatments for anxiety. [0004] Alcoholism is the most common form of drug abuse and a major public health problem worldwide. Nevertheless, few drugs exist that modify alcohol intake and the genetic factors that influence alcohol's effects on brain and behavioral processes remain largely uncharacterized. Thus, there is a need for diagnostic tests that can identify individuals with a predisposition to becoming alcoholics and a need for treatments that can alter alcohol consumption. [0005] The Lewin Group estimated the economic cost to U.S. society in 1992 due to alcohol and drug abuse to be $246 billion, $148 billion of which was attributed to alcohol abuse and alcoholism and $98 billion of which stemmed from drug abuse and dependence (H. Harwood et al., The Economic Costs of Alcohol and Drug Abuse in the United States, 1992, NIH Publication Number 98-4327 (September 1998)). When adjusted for inflation and population growth, the alcohol estimates for 1992 are very similar to cost estimates produced over the past 20 years, and the drug estimates demonstrate a steady and strong pattern of increase. The current estimates are significantly greater than the most recent detailed estimates developed for 1985 for alcohol and for drugs (Rice et al. 1990)--42 percent higher for alcohol and 50 percent greater for drugs over and above increases due to population growth and inflation. [0006] Protein kinase C (PKC) is a multigene family of phospholipid-dependent, serine-threonine kinases central to many signal transduction pathways. So far, ten members, i.e., isozymes, of the PKC family have been described, which are encoded by nine different genes. The ten isozymes are designated as the .alpha.-, .beta.I, .beta.II, .gamma.-, .delta.-, .epsilon.-, .xi.-, .eta.`-, `-, and .theta.-isozymes. Nishizuka, 1992, Science 258:607-614; Selbie et al., 1993, J. Bio. Chem. 268:24296-24302. Based on sequence homology and biochemical properties, the PKC gene family has been divided in three groups. A first group, i.e., the .alpha., .beta.1, .beta.2, and .gamma. isozyme, designated as "conventional" PKCs, are regulated by calcium, diacylglycerol and phorbol esters. A second group, i.e., the .delta., .epsilon., .theta. and .eta. isozymes, designated as "novel" PKCs, are calcium-independent, but diacylglycerol and phorbol ester-sensitive. Finally, a third group, i.e., the .xi., and isozymes, designated as "atypical" PKCs, are insensitive to calcium, diacylglycerol, and PMA. In addition, two related phospholipid-dependent kinases, PKC.mu. and protein kinase D, share sequence homology in their regulatory domains to novel PKCs and may constitute a new subgroup. Johannes et al., 1994, J. Biol. Chem. 269:6140-6148; Valverde et al., 1994, Proc. Natl. Acad. Sci. USA 91:8572-8576. [0007] A number of studies with tumor promoting phorbol esters suggest that PKC modulates neural differentiation. For example, phorbol esters induce neural tissue from ectoderm in Xenopus embryos (Otte et al., 1988, Nature 334:618-620) and elicit neurite outgrowth from chick sensory ganglia (Mehta et al., 1993, J. Neurochem. 60:972-98 1, Hsu et al., 1984, Cancer Res. 44:4607-4614), chick ciliary ganglion neurons (Bixby, 1989, Neuron 3:287-297), several human neuroblastoma cell lines (Pahlman et al., 1983, Cell Diff. 12:165-170; Spinelli et al., 1982, Cancer Res. 42:5067-5073), and rat PC12 cells (Roivainen et al., 1993, Brain Res. 624:85-93; Hall et al. 1988, J Biol. Chem. 263:4460-4466). Studies using purified isozymes, kinase-defective mutants, and transgenic or mutant cell lines have implicated PKC.alpha., -.beta., -.delta., -.epsilon., and -.xi. in the differentiation of nonneural cells (Berra et al., 1993, Cell 74:555-563; Goodnight et al., 1994, Adv. Cancer Res. 64:159-209; Gruber et al., 1992, J. Biol. Chem. 267:13356-13360; Macfarlane and Manzel, 1994, J. Biol. Chem. 269:4327-4331; Powell et al., 1992, Proc. Natl. Acad. USA 89:146-151). Overexpression of PKC.alpha. or -.beta. in Xenopus embryos enhances neural induction (Otte and Moon, 1992, Cell 68:1021-1029), but little else is known about the identity of specific PKC isozymes that regulate neural differentiation. [0008] Recent evidence suggests that PKC.epsilon. plays a role in neural differentiation and plasticity. PKC.epsilon. is expressed predominantly in the nervous system and is particularly abundant in the hippocampus, olfactory tubercle, and layers I and II of cerebral cortex (Saito et al., 1993, Brain Res. 607:241-248). Within immunoreactive neurons, it is localized to the Golgi apparatus and to axons and presynaptic nerve terminals (Saito et al., supra). PKC.epsilon. is activated by growth factors that stimulate neural differentiation such as insulin (Heidereich et al., 1990, J. Biol. Chem. 265:15076-15082) and NGF (Ohmichi et al., 1993, Biochem J. 295:767-772). In addition, in developing chick brain, it is the major isozyme found in nondividing, differentiating neurons (Mangoura et al., 1993, J. Neurosci. Res. 35:488-498). [0009] Further evidence for involvement of PKC.epsilon. in neural differentiation has come from studies with PC12 cells. PC12 cells are derived from neural crest and, when treated with NGF or fibroblast growth factors, undergo dramatic biochemical and morphological differentiation, developing several characteristics of mature sympathetic neurons. Greene et al., 1991, in: Culturing Nerve Cells (Banker, G. and Goslin, K eds) pp. 207-226, MIT Press, Cambridge, Mass. PKC-activating phorbol esters enhance NGF-induced activation of ERK1 and ERK2 mitogen-activated protein (MAP) kinases and neurite outgrowth in PC12 cells, suggesting that PKC modulates responses to NGF (Rolvainen et al., 1993, supra; Hall et al.; 1988, supra; Rolvainen et al., 1995, Proc. Natl. Acad. Sci. USA 92:1891-1895). Studies with ethanol-treated PC12 cells suggested that PKC.epsilon. is responsible for this effect. Like phorbol esters, ethanol increases NGF-induced MAP kinase activation and neurite outgrowth through a PKC-dependent mechanism (Roivainen et al., 1993, supra; Roivainen et al., 1995, supra). Ethanol promotes PKC-mediated phosphorylation in PC12 cells by increasing levels of messenger RNA and protein for two PKC isozymes, PKC.delta. and PKC.epsilon. (Messing et al., 1991, J. Biol. Chem., 266:23428-23432; Roivainen et al., 1994, Toward a Molecular Basis of Alcohol Use and Abuse, pp. 29-38). Recent data demonstrate that overexpression of PKC.epsilon., but not of PKC.delta., enhances NGF-induced MAP kinase activation and neurite outgrowth (Hundle et al., 1995, J. Biol. Chem. 270:30134-30140). These findings establish PKC.epsilon. as a positive modulator of neurite growth. They also suggest that PKC.epsilon. mediates the neurite-promoting effect of ethanol and phorbol esters in PC12 cells. [0010] A recent study suggests that PKC.epsilon. specifically mediates enhancement of MAP kinase activation and neurite growth by phorbol esters and ethanol in PC12 cells. PKC activation is generally associated with enzyme translocation to lipid containing structures in particulate fractions of cells. Specifically, studies with PC12 cell lines that stably express the fragments .epsilon.V1 or .delta.V1, which are derived from the first variable domains of PKC.epsilon. or PKC.delta., showed that each fragment selectively inhibited phorbol ester-induced translocation of its corresponding isozyme, indicating that these fragments can function as isozyme-selective translocation inhibitors. NGF-induced MAP kinase phosphorylation and neurite outgrowth are not enhanced by phorbol esters or ethanol in cells expressing .epsilon.V1, but they are increased by these agents in cells expressing .delta.V1 and in cells transfected with empty vector. [0011] It has been demonstrated that chronic exposure to ethanol increases total PKC activity, high affinity phorbol ester binding and PKC-mediated phosphorylation in PC12 cells (Messing et al., 1991, J. Biol. Chem. 266:23428-23432), which is associated with a selective increase in immunoreactivity and mRNA levels for two PKC isozymes, PKC.delta. and PKC.epsilon. (Roivainen et al., 1994, Protein kinase C and adaptations to ethanol, in: Toward a Molecular Basis of Alcohol Use and Abuse. Jansson B., Jorvall H., Rydberg U., Terenius L., and Vallee B. L., eds. Birkhduser Verlag, Basel, 1994. pp. 29-38). Ethanol does not increase diacylglycerol formation in PC12 cells or alter PKC activity in an in vitro assay using a mixture of PKC isozymes partially purified from rat brain. These findings suggest that chronic exposure to ethanol increases PKC activity by increasing expression of PKC.delta. and PKC.epsilon.. Further, it has been demonstrated that PKC.epsilon. is involved in two ethanol-induced processes in PC12 cells; first, it has been shown that ethanol potentiates NGF-induced activation of mitogen-activated protein kinases and neurite outgrowth in PC12 cells by a PKC.epsilon.-dependent mechanism (Roivainen et al., 1993, Brain Res. 624:85-93, Roivainen et al., 1995, Proc. Natl. Acad. Sci USA 92:1891-1895; Messing et al., 1991, Brain Res. 565:301-311); second, evidence suggesting that ethanol increases the number of N-type voltage gated Ca.sup.2+ channels in PC12 cells and rodent brain by a PKC.epsilon.-dependent process (Messing et al., Alcoholism Clinical and Experimental Research 22: Abstract S26:2 (1998)). Since both neural plasticity (Robinson and Kolb, 1998, J. Neurosci. 17:8491-8497) and increases in the activity of Ca.sup.2+ channels (Messing and Diamond, 1997, Molecular biology of alcohol dependence, in: The Molecular and Genetic Basis of Neurological Disease. Rosenberg R., Prusiner S., DiMauro S., and Barchi R., eds. Butterworth-Heinemann, Boston, pp. 1109-1126) may contribute to drug dependence, PKC.epsilon. may have a behavior-modulating effect. [0012] GABA (gamma amino butyric acid) is the major inhibitory neurotransmitter in the brain and GABA.sub.A receptors are receptor-gated chloride channels. Upon binding GABA, these channels open, allowing chloride to pass in or out of the cell. This tends to hold the membrane potential of the cell at negative values close to the resting membrane potential, thereby preventing the generation of an action potential. Benzodiazepines are a class of drugs commonly used to reduce anxiety. Benzodiazepines bind with high affinity to GABA.sub.A receptors in the central nervous system. DeLorey and Olsen, 1992, J. Biol. Chem. 267:16747-16750. Pentobarbital and benzodiazepines such as diazepam allosterically regulate the GABA.sub.A receptor channel, increasing the Cl.sup.- channel open time or the probably of channel opening in response to GABA (A. Guidotti, M. G. Corda, B. C. Wise, F. Vaccarino, E. Costa, Neuropharmacology 22, 1471-9 (1983)). GABA-dependent neurotransmission is thereby enhanced. In contrast, muscimol binds competitively to the GABA recognition site on GABA.sub.A receptors and can elevate Cl.sup.- conductance independently of endogenous GABA. [0013] Previous studies have provided conflicting reports regarding PKC regulation of GABA.sub.A receptors. GABA.sub.A receptors are heteropentameric complexes of related subunits, several of which contain consensus sequences for PKC phosphorylation. Moss, 1992, J. Biol. Chem. 267:14470-14476. The .gamma.2 subunit of the GABA.sub.A receptor exists in two forms produced by alternate splicing of mRNA, and some studies suggest that the long splice variant (.gamma..sub.2L), which contains a unique consensus site for PKC phosphorylation, is specifically required for ethanol sensitivity of GABA.sub.A receptors (Wafford et al., 1990, Science 249:291-293; K. A. Wafford, et al., Neuron 7, 27-33 (1991); K. A. Wafford, P. J. Whiting, Febs Letters 313, 113-7 (1992)). However, others have failed to observe this requirement (W. Marszalec, Y. Kurata, B. J. Hamilton, D. B. Carter, T. Narahashi, Journal of Pharmacology and Experimental Therapeutics 269, 157-63 (1994); E. Sigel, R. Baur, P. Malherbe, FEBS Letters 324, 140-142 (1993); D. W. Sapp, H. H. Yeh, Journal of Pharmacology and Experimental Therapeutics 284, 768-76 (1998)), and mice lacking .gamma..sub.2L show normal behavioral and electrophysiological responses to ethanol (G. E. Homanics, J. J. Quinlan, R. M. Mihalek, L. L. Firestone, Frontiers in Bioscience 3, D548-53 (1998)). Phorbol ester treatment of mouse cerebellar microsacs or of Xenopus oocytes and human kidney cells expressing GABA.sub.A receptor subunits inhibits receptor activation by GABA or muscimol (B. J. Krishek, et al., Neuron 12, 1081-95 (1994); N. J. Leidenheimer, R. A. Harris, Advances in Biochemical Psychopharmacology 47, 269-79 (1992); S. Kellenberger, P. Malherbe, E. Sigel, The Journal of Biological Chemistry 267, 25660-25663 (1992)). In contrast, an active catalytic domain of PKC enhances GABA-stimulated currents when expressed in fibroblasts or microinjected into CA1 hippocampal pyramidal neurons (F. Poisbeau, M. C. Cheney, M. D. Browning, I. Mody, Journal of Neuroscience 19, 674-83 (1999); Y. F. Lin, M. D. Browning, E. M. Dudek, R. L. Macdonald, Neuron 13, 1421-1431 (1994)). [0014] As discussed above, prior to the present invention, little was known about the role of PKC.epsilon. in vivo in alcoholism, anxiety, drug abuse or GABA.sub.A receptor function. III. SUMMARY OF THE INVENTION [0015] To study the role of PKC.epsilon. in vivo in alcoholism, anxiety, drug abuse, GABA.sub.A receptor function, and other processes, the inventors have used gene targeting by homologous recombination to create mutant mice that lack PKC.epsilon.. [0016] The present invention relates, inter alia, to: 1) the production of PKC.epsilon. deficient cells and non-human animals; 2) the identification and the use of the PKC isozyme .epsilon. (PKC.epsilon.) as a target for the modulation of anxiety in a mammal; 3) the use of modulators of PKC.epsilon. to modulate alcohol consumption and self-administration of other drugs of abuse and the effects of alcohol and other drug consumption; 4) the use of inhibitors of PKC.epsilon., either alone or in conjunction with allosteric agonists of GABA.sub.A receptors, to treat conditions, such as anxiety, addiction, withdrawal syndrome, skeletal muscle spasms, convulsive seizures, and epilepsy, that are amenable to treatment by allosteric agonists of GABA.sub.A receptors; and 5) a diagnostic method for identifying individuals at risk for becoming alcoholics or abusers of other drugs. [0017] The present invention is based, in part, on the inventors' discovery that PKC.epsilon..sup.-/- mice have less fear and anxiety than wild-type mice. This suggests that PKC.epsilon. is a target for the development of anxiety-reducing drugs. Furthermore, the invention is based, in part, on the inventors' discovery that PKC.epsilon..sup.-/- mice sleep twice as long as wild-type mice when injected intraperitoneally with drugs that act at GABA.sub.A receptors, such as ethanol, pentobarbital or benzodiazepines. This result suggests that PKC.epsilon..sup.-/- mice are hypersensitive to the sedative-hypnotic effects of compounds acting at GABA.sub.A receptors. Thus; inhibition of PKC.epsilon. augments GABA.sub.A receptor-mediated signaling, and based on the fact that GABA.sub.A agonists are anxiolytics, it can be concluded that PKC.epsilon. inhibitors are potent suppressors of anxiety. This conclusion is supported by the observation that PKC.epsilon..sup.-/- mice have reduced basal levels of stress-associated hormones and accelerated reduction of hormone levels in the wake of an event that increases such levels. [0018] In one specific aspect, the present invention is directed to animal cells that are PKC.epsilon. deficient due to a disruption in the PKC.epsilon. coding nucleic acid sequences. An additional aspect of the present invention is the use of a genetically modified PKC.epsilon. deficient cell to generate PKC.epsilon.-deficient non-human transgenic embryos and animals. Other aspects of the present invention are the PKC.epsilon.-deficient non-human, preferably mouse, transgenic embryos and animals, and offspring that comprise a targeted disruption in the PKC.epsilon. gene, and hence produce less than wild-type levels of PKC.epsilon. activity. The PKC.epsilon. deficient non-human transgenic animals of the present invention may be heterozygous or homozygous for the mutated PKC.epsilon. allele. [0019] The present invention is also directed to assays for identifying anxiolytic compounds. The assays of the invention comprise identification of a compound that inhibits the enzymatic activity of PKC.epsilon., and isolation of such compound. In another specific aspect, the present invention is directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound inhibiting the enzymatic activity of PKC.epsilon. and a pharmaceutically acceptable carrier. In addition, the present invention is directed to the treatment of anxiety by administration of such pharmaceutical compositions. [0020] Other aspects of the present invention are methods of modulating consumption of a drug of abuse and/or the effects of such drug by administering a modulator of PKC.epsilon.. Administration of an inhibitor of PKC.epsilon. would thereby reduce consumption of alcohol, barbituates, nicotine, opiates, or psychostimulants. Increased consumption of such drugs would result from embodiments of the method that involve the administration of enhancers of PKC.epsilon.. 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