United States Patent (19) 11 Patent Number: 6,007,986 Sadée (45) Date of Patent: *Dec
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US006007986A United States Patent (19) 11 Patent Number: 6,007,986 Sadée (45) Date of Patent: *Dec. 28, 1999 54 METHODS FOR ANTI-ADDICTIVE Yu et al., “Efficacy and Tolerance of Narcotic Analgesics at NARCOTIC ANALGESIC ACTIVITY the u Opioid Receptor in Differentiated Human Neuroblas SCREENING toma Cells,” J. Pharmacol. Exp. Ther, 245, pp. 350-355 (1988). 75 Inventor: Wolfgang Sadée, Ross, Calif. Yu et al., “Differentiation of Human Neuroblastoma Cells: Marked Potentiation of Prostaglandin E-Stimulated Accu 73 Assignee: The Regents of the University of mulation of cAMP by Retinoic Acid,” J. Neurochem., 51, pp. California, Oakland, Calif. 1892–1899 (1988). * Notice: This patent issued on a continued pros Yu et al., “Regulation of Cyclic AMP by the u-Opioid ecution application filed under 37 CFR Receptor in Human Neuroblastoma SH-SY5Y Cells,” J. 1.53(d), and is subject to the twenty year Neurochem., 55:4, pp. 1390–1396 (1990). patent term provisions of 35 U.S.C. Schlitz et al., “Reverse Intrinsic Activity of Antagonists of G 154(a)(2). Protein-Coupled Receptors,” TiPS, 13 (Oct. 1993), pp. 376-379. 21 Appl. No.: 08/703,637 Chen et al., “Moleculare Cloning and Functional Expression of a u-Opioid Receptor from Rat Brain,” Molecular Phar 22 Filed: Aug. 27, 1996 macology, 44, pp. 8-12 (1993). Wang et al., “Constitutive it Opioid Receptor Activation as Related U.S. Application Data a Regulatory Mechanism Underlying Narcotic Tolerance 63 Continuation-in-part of application No. 08/261,500, Jun. 16, and Dependence,” Life Sciences, 54:20, pp. 339–350 (1994). 1994, abandoned, which is a continuation-in-part of appli Smith et al., “Problems and Approaches in Studying Mem cation No. 08/081,612, Jun. 23, 1993, abandoned. brane Opioid Receptors,” Molecular Approaches to Drug 51) Int. Cl. ............................... C12O 1/42; C12O 1/68; Abuse Research Volume I, edited by Lee et al., U.S. Dept. of GO1N 33/53 Health & Human Services, pp. 69-84 (1991). 52 U.S. Cl. .............................. 435/6; 435/7.2; 435/7.21; Hawkins et al., “HHH-D-Phe-Cys-Try-D-Trp-Orn 435/21 Thr-Pen-Thr-NH. (HICTOP), A Potent and Highly 58 Field of Search ..................................... 435/72, 7.21, Selective Peptide for Mu Opioid Receptors in Rat Brain,” J. 435/6, 21 Pharmacol. & Exp. Ther, 24.8:1, pp. 73–80 (1989). Abdelhamid et al., “Characteristics of u and ö Opioid 56) References Cited Binding Sites in Striatal Slices of Morphine-Tolerant and -Dependent Mice,” Eur: J. Pharmacol., 198, pp. U.S. PATENT DOCUMENTS 157-163 (1991). 5,061,715 10/1991 Sunkara et al. ......................... 514/314 (List continued on next page.) OTHER PUBLICATIONS Biedler et al., “Morphology and Growth, Tumorigenicity, Primary Examiner Anthony C. Caputa and Cytogenetics of Human Neuroblastoma Cells in Con Assistant Examiner Robert C. Hayes tinuous Culture,” Cancer Res., 33, pp. 2643–2652 (1973). Attorney, Agent, or Firm Majestic, Parsons, Siebert & Costa et al., “Drug Efficacy at Guanine Nucleotide-Binding HSue Regulatory Protein-Linked Receptors: Thermodynamics 57 ABSTRACT Interpretation of Negative Antagonism . .” Molec. Phar macol., 41, pp. 549–560 (1992). The present invention provides assays to measure the regu lation of the narcotic analgesic addictive State. Practice of Frey et al., “Ali-Opioid Receptor in 7315c Tumor Tissue the invention permits classification of test compounds for Mediates Inhibition of Immunoreactive Prolactin Release their effects on an activated opioid it receptor State. When and Adenylate Cyclase Activity,” Endocrin., 115, pp. opioid it receptor cells are treated with a test composition 1797–1804 (1984). under investigation, then in one embodiment the propensity Kogan et al., “Elevated Basal Firing Rates and Enhanced of the test composition to elicit a spontaneous cAMP over Responses to 8-Bromo-cAMP in Locus coeruleus Neurons shoot and an inverse agonist induced cAMP overshoot is in Brain Slices from Opiate Dependent Rats,” Eur: J. Phar determined and Serves as a Surrogate measure of addiction macol., 211, pp. 47-53 (1992). liability. The inverse agonist induced cAMP overshoot sig Nestler, “Molecular Mechanisms of Drug Addiction,” J. nifies the presence of what is designated as the constitutively Neurosci. 12:7, pp. 2439-2450 (1992). active State for the opioid it receptors. The use of these Rasmussen et al., “Opiate Withdrawal and the Locus coer assays has led to the identification of compounds that have uleus: Behavioral, Elactrophysiological, and Biochemical the desired effects on the constitutive activation of the opioid Correlates,” J. Neurosci., 10, pp. 2308-2317 (1990). tl receptors. The therapeutic potential of these compounds Sibinga et al., “Opioid Peptides and Opioid Receptors in include treating patients who are addicted to a narcotic Cells of the Immune System,” Ann. Rev. Immunol. , 6, pp. analgesic or who have taken an overdose of a narcotic 219–249 (1988). analgesic, or whose pain is being relieved with a narcotic Yu et al., “A Human Neuroblastoma Cell Line Expresses it analgesic. and ö Opioid Receptor Sites,” J. Biol. Chem., 261, pp. 1065-1070 (1986). 5 Claims, 3 Drawing Sheets 6,007,986 Page 2 OTHER PUBLICATIONS gang-Goethe University, held in Fort Lauderdale, Florida, Sharma et al., “Dual Regulation of Adenylate Cyclase Nov. 9-12, 1994. Accounts for Narcotic Dependence and Tolerance,” Proc. Baker, Mitzi, “New Hypothesis on How Narcotics Produce Antl. Acad. Sci. USA, 72:8, pp. 3092-3096 (1975). Tolerance, Dependency, Synapse (newspaper of the Uni Sadée et al., “Constitutive Activation of the u-Opioid versity of California, San Francisco), 39:15 (Jan. 12, 1995), Receptor: A Novel Paradigm of Receptor Regulation in pp. 1 and 5. Narcotic Analgesia, Tolerance, and Dependence,” Analge sia, 1:1, pp. 11-14 (1994). Högger et al., “Activating and Inactivating Mutations in Lameh et al., Abstract of “Agonist Induced Conversion of N-and C-terminal i3 Loop Junctions of Muscarinic Acetyl the Hml Muscarinic Cholinergic Receptor to a Constitu choline Hm1 Receptors,” J. Biol. Chem..., 270:13, (1995), pp. tively Active State: A Novel Papadigm of Receptor Regu 7405-741O. lation,” p. 47 of the program for Subtypes of Muscarinic Wang et al., “Agonist Induced Constitutive Activation of the Receptors. The Sixth International Symposium, Sponsored lu Opioid Receptor by Phosphorylation' Regulatory Pep by Boston University School of Medicine and Johann Wolf tides, 54:1, (1994), pp. 323-324. U.S. Patent 6,007,986 U.S. Patent Dec. 28, 1999 Sheet 2 of 3 6,007,986 6000 5000 4000 CAMP Naloxone (pmoles/mg 3000 protein / 15 min) 2OOO 1000 FIG.2A CONTROL MORPHINE MORPHINE (No Pretreatment) Pretreatment Pretreatment 15000 12500 10000 CAMP Naloxone (pmoles / mg 7500 protein / 15 min) 5000 2500 FIG.2B CONTROL MORPHINE MORPHINE (No Pretreatment) Pretreatment Pretreatment 6,007,986 1 2 METHODS FOR ANT-ADDICTIVE matic upregulation of the cAMP Second messenger System NARCOTIC ANALGESIC ACTIVITY at every major Step between receptor and physiological SCREENING response, which leads to a dependent State. See, for example, Nestler at 2440. This is a continuation-in-part of Ser. No. 08/261,500, To date, assays of the u opioid receptor System have been filed Jun. 16, 1994, now abandoned, which was a unable to detect any major changes of that System during continuation-in-part of Ser. No. 08/081,612, filed Jun. 23, narcotic addiction. Thus, much of the current work has 1993, now abandoned, all of common assignment herewith. focused on events downstream of the receptor, Such as This invention was made with Government support long-term gene regulation, in attempting to account for the under Grant No. DA 04166, awarded by the U.S. Depart dependent State. Because the dependence liability of nar ment of Health and Human Services. The Government has cotic drugs. Severely limits their clinical utility as potent certain rights in this invention. analgesics and exerts a heavy toll on Society through illicit narcotic drug use, a Screen for agents that could prevent or FIELD OF THE INVENTION reverse the narcotic dependent State or might facilitate The present invention generally relates to Screening for 15 gradual withdrawal would greatly enhance the clinical utility the pharmacological activities of narcotic drugs, and more of narcotic analgesics and could serve as an effective phar particularly relates to assays useful in classifying the nar macological weapon in the fight against illicit drug use. cotic activity of compounds and treatments using anti addictive agents determinable from Such assayS. SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION A novel mechanism is proposed where opioid it receptor exposure to agonist leads to constitutive activation of those Endogenous opiate receptors were discovered in the receptors. It is through this mechanism that the addictive 1970s, and have been intensely studied in seeking the State to narcotic analgesics is regulated. mechanisms by which particular drugs lead to addiction. 25 One aspect of the present invention is to provide a means However, in his 1992 review of molecular mechanisms of for assaying or measuring the regulation of the addictive drug addition, Nestler noted that Such mechanisms have State, in the Search for compounds that prevent or reverse remained elusive. J. Neurosci, 12 (7), pp. 2439-2450 constitutive it receptor activation, and to classify test com (1992). pounds for their effects on the constitutively active it recep A number of different opioid receptor types have been tor State. identified. Among the known receptor types is the opioid ul Other aspects of the present invention are methods for receptor. treating patients who are addicted to a narcotic analgesic, or Narcotic analgesics act at the opioid u receptor to produce who have taken an Overdose of a narcotic analgesic, or analgesia. However, continued use of narcotic analgesics whose pain is being relieved with a narcotic analgesic. typically leads to habituation or addiction, and use of one 35 Therapeutic methods in accordance with the invention nor leads to cross-tolerance/dependence for the others.