Charles R. Schuster, Ph.D. Wayne State University IMMUNOLOGICAL APPROACHES TO THE TREATMENT OF DRUG DEPENDENCE: LESSONS FROM THE PAST Biography From 1986 to 1992, Dr. Schuster served as the Director of the National Institute on Drug Abuse. In 1992, Dr. Schuster returned to his research career as a Research Scientist at the Addiction Research Center of NIDA. In 1995, Dr. Schuster joined the Department of Psychiatry and Behavioral Neurosciences at Wayne State University School of Medicine as the Director of the Clinical Research Division on Substance Abuse. He is the editor of the APA journal Experimental and Clinical Psychopharmacology. Prior to joining NIDA, Dr. Schuster was the Director of the University of Chicago's Drug Abuse Research Center, and Professor of Psychiatry, Pharmacology, and Behavioral Science. He has authored or co-authored over 150 scientific journal articles, as well as numerous book chapters and several books. He is a Fellow of the American Association for the Advancement of Science, the American College of Neuropsychopharmacology, and the American Psychological Association, and a member of the Institute of Medicine. S. Michael Owens, Ph.D. University of Arkansas for Medical Sciences RATIONAL DESIGN AND DEVELOPMENT OF PROTEIN-BASED THERAPIES FOR DRUG ABUSE Focus of Presentation Recent advances in immunotherapy, enzymology, and large-scale production of biological reagents suggest these novel approaches could be beneficial in the treatment of drug abuse. While the potential benefits for a new generation of biological modifiers for treating drug abuse is exciting, the therapeutic principles involved in successful therapy are poorly understood. This presentation will use experimental data and theoretical considerations to show that protein-based therapies such as antibodies and enzymes follow many of the same basic principles as more conventional therapeutics. Donald W. Landry, M.D., Ph.D. Columbia University ANTI-COCAINE CATALYTIC ANTIBODIES Biography Donald W. Landry completed his Ph.D. in organic chemistry under Nobel laureate Robert Burns Woodward at Harvard University in 1979 and then obtained the M.D. degree from Columbia University in 1983. After completing his Residency in Internal Medicine at the Massachusetts General Hospital, he returned to Columbia University as an NIH Physician-Scientist, 1985-90. In 1991, he established a laboratory at Columbia University to investigate medical applications of artificial enzymes. He is presently an Associate Professor of Medicine at Columbia University College of Physicians and Surgeons. Focus of Presentation Cocaine can be degraded by hydrolysis of the benzoyl ester group to yield inactive products, and a catalyst for this reaction could reduce serum cocaine concentrations, deprive the cocaine abuser of the behavioral reinforcing effect of the drug, and thus favor extinction of the addiction. Catalytic antibodies are programmable artificial enzymes obtained by immunization with a transition-state analog of the substrate in the midst of the desired chemical transformation. We have synthesized several analogs of cocaine hydrolysis, immunized mice, and generated antibodies that hydrolyze cocaine. One of these enzymes has achieved the activity necessary for preclinical studies, and we have demonstrated its ability to block lethal doses of cocaine in rat. We have begun a rational effort to improve on the activity of these first artificial enzymes to degrade cocaine through the synthesis of novel transition-state analogs and the mutagenesis of our most active antibody. Presentation Conclusions Anti-cocaine catalytic antibodies can degrade cocaine to inactive products in vivo. Catalytic antibodies with only moderate Km and Kcat parameters are effective in animal models of cocaine overdose (and addiction). Cocaine abuse is an appropriate target for a therapeutic strategy based on antibody catalysis. Gail D. Winger, Ph.D. University of Michigan THE EFFECTS OF ANTI-COCAINE ANTIBODIES ON CARDIOVASCULAR, LOCOMOTOR, REINFORCING, AND LETHAL EFFECTS OF COCAINE IN RODENTS Biography Gail Winger, Ph.D., is a Research Scientist in the Department of Pharmacology, University of Michigan. Dr. Winger received her Ph.D. (Psychology) from the University of Michigan, Ann Arbor, in 1971. She then joined the Downstate Medical Center, SUNY, as a Clinical Assistant Professor in the Department of Psychiatry. Prior to assuming her current position, Dr. Winger served as Assistant Research Scientist (1975-1989) and Associate Research Scientist (1989-1996), Department of Psychology, University of Michigan. Dr. Winger is widely published in the areas of psychology and psychopharmacology, and is an ad hoc member of study sections for the National Institute on Drug Abuse (NIDA) and the National Institute on Alcohol Abuse and Alcoholism (NIAAA). John Cashman, Ph.D. Human BioMolecular Research Institute DEVELOPMENT OF CATALYTIC ANTIBODIES FOR COCAINE Biography John Cashman received B.A. degrees in Chemistry and Biology from the University of California, Santa Barbara in 1977. He then went on to receive M.S. and Ph.D. degrees in Medicinal Chemistry from the University of Kansas in 1980 and 1982, respectively. From 1982-1984 he was a postdoctoral fellow in the laboratory of Professor E.J. Corey at Harvard University. In 1984 he took a position as Assistant Professor of Chemistry and Pharmaceutical Chemistry at the University of California, San Francisco. In 1992 he became the Associate Director of IGEN Research Institute in Seattle, WA. In 1994 he moved to Seattle Biomedical Research Institute as Senior Scientist before becoming Director of a new Seattle non-profit research institute named the Human BioMolecular Research Institute in 1998. Focus of Presentation Significant progress has been made in the development of pharmacotherapies for cocaine abuse and addiction. However, additional alternative approaches must be developed. Catalytic antibodies, in principle, represent an unlimited source of novel catalysts that could be developed where there is no or poor biological precedent. Because human butyrylcholinesterase is a sluggish catalyst that only slowly detoxicates (-)-cocaine, efficient cocaine esterolytic catalytic antibodies hold significant promise as therapeutic agents. The approach taken was to procure monoclonal catalytic antibodies against a stable phosphonate transition state analog of (-)-cocaine hydrolysis. A number of monoclonal antibodies were obtained and the most catalytically active ones were examined in some detail. The results suggested that efficient catalysts can be procured and that further evaluation may demonstrate therapeutic utility. Kim D. Janda, Ph.D. The Scripps Research Institute BASICS OF ANTIBODY CATALYSIS and A LITTLE CHEMISTRY AND MOLECULAR BIOLOGY IN REGARDS TO IMMUNOPHARMACOLOGICAL PROTOCOLS IN THE ABATEMENT OF COCAINE ABUSE AND ADDICTION Biography Kim D. Janda was born in Cleveland, Ohio, in 1958. He received a B.S. degree in Clinical Chemistry from the University of South Florida (1980), an M.S. degree (1983), and a Ph.D. degree (1984) both in Organic Chemistry from the University of Arizona. He has been at The Scripps Research Institute since 1985 and currently is the Ely R. Callaway Professor of Chemistry in the Department of Chemistry and Molecular Biology. His research interests include catalytic antibodies, enzyme inhibition, combinatorial chemistry, and immunopharmacotherapy. Focus of Presentations A general prospectus on catalytic antibodies will be presented. The lecture will encompass hapten design strategies, hybridoma selection for catalysts, and kinetic analysis of isolated catalysts. Several examples of catalytic antibodies isolated from the investigator's laboratory will be discussed as case studies. New strategies will be detailed that allow procurement of more efficient antibody catalysts. Within this context "reactive immunization" and "direct selection" approaches for catalytic antibodies will be discussed. In the second presentation, a discussion will be given on the chemistry that has evolved in our immunological strategies for the treatment of cocaine abuse and addiction. In addition, "phage display" techniques will be presented in regards to our humanization of anti-cocaine antibodies for passive immunization therapy. Presentation Conclusions Catalytic antibodies are tailored catalysts to the designer's needs. Catalytic antibodies can possess exquisite specifications and chemical reactivity. Direct selection and reactive immunization are the tickets for the isolation of more efficient catalytic antibodies. A combination of chemistry, immunology, and molecular biology can be a powerful multifaceted approach to the treatment of cocaine abuse and addiction. M. Rocio A. Carrera The Scripps Research Institute IMMUNIZATION AS A THERAPEUTIC STRATEGY AGAINST COCAINE ABUSE Biography M. Rocio A. Carrera was born in Mexico were she received a degree in Clinical Psychology from the Center of Technical and Social Education in 1985. She later obtained her bachelor's degree from the University of California, San Diego in 1990. She received her master's degree at the same institution in 1991, were she is presently a doctoral candidate in the Department of Psychology. Ms. Carrera conducted her predoctoral research at the Scripps Research Institute in the division of neuropsychopharmacology in the laboratory of Dr. George F. Koob. She is currently working in the department of chemistry with Dr. Kim Janda, where she is extending the studies involving the anti-cocaine vaccines synthesized in this laboratory. Focus of Presentation Cocaine abuse continues to be prevalent, and effective therapies for its treatment remain elusive. In the last several years, immunopharmacotherapy has been proposed as a promising means to treat this illness. By using the organism's natural immune response, an anti-cocaine vaccine promotes the production of cocaine-specific antibodies that sequester the ingested cocaine molecules before their passage into the brain where they exert their reinforcing and thus addictive effects. A series of studies demonstrating the effectiveness of anti-cocaine immunization in blocking the psychostimulant and reinforcing properties of cocaine in animals models will be reviewed. Presentation Conclusions Active immunization with cocaine conjugate GNC-KLHJ suppressed the psychoactive (locomotor activity and stereotyped behavior) effects of cocaine in rats. This effect was enhanced across further cocaine challenges. Moreover, after cocaine injection, brain cocaine levels were found to be up to 77% lower in immunized rats compared to controls. This immune-mediated response was also effective in preventing reinstatement of cocaine self-administration upon noncontingent cocaine infusions in an animal model of cocaine self-administration relapse. Cocaine-induced locomotor activity was dramatically suppressed by passive immunization with murine mAb GNC92h3, and active immunization with the new conjugate GND-KLH. Immunization against cocaine offers a means of blocking the actions of the drug by preventing it from entering the central nervous system and may offer a nontoxic, substance-specific strategy that should not affect normal neurochemical physiology, presenting a solid scientific approach for cocaine abuse treatment. Cocaine addiction is a complex phenomenon with social, psychological, and physiological components. Therefore, in a clinical setting, immunotherapy could be a potentially useful tool if offered in concert with psycho- and pharmacotherapeutic interventions in order to optimize relapse prevention. Barbara S. Fox, Ph.D. ImmuLogic Pharmaceutical Corporation THERAPEUTIC VACCINE FOR THE TREATMENT OF COCAINE ADDICTION Biography Barbara S. Fox received her Ph.D. in biochemistry from MIT in 1983 and carried out post-doctoral training in cellular immunology at the NIH. She moved to the Department of Medicine, Division of Rheumatology at the University of Maryland School of Medicine in 1987 where she rose to the rank of Associate Professor. In 1993, she joined ImmuLogic Pharmaceutical Corporation and led the Discovery Research Program from 1994 to 1996. Dr. Fox has been Vice President of Immunology since 1996 and has been responsible for moving the cocaine vaccine from early product concept into the human clinical testing. Focus of Presentation Cocaine addiction is a significant medical and public health issue in the United States, yet there are currently no approved medications for the treatment of cocaine dependence. As a novel approach to the treatment of cocaine addiction, ImmuLogic Pharmaceutical Corporation is developing a therapeutic cocaine vaccine. The vaccine is designed to elicit high titer cocaine-specific antibodies following active immunization of cocaine-addicted individuals. These antibodies will bind to cocaine in the bloodstream and inhibit both the amount and rate at which cocaine crosses into the brain, thereby reducing the reinforcing potential of the drug. The vaccine is intended to be administered as an aid to maintenance of abstinence as part of a comprehensive behavioral cocaine dependency treatment program. Presentation Conclusions A cocaine vaccine has been produced which induces cocaine-specific antibodies in immunized animals. The antibodies induced by the vaccine inhibit the entry of cocaine into the brain. The cocaine vaccine is ready to begin human testing. Kathleen M. Kantak, Ph.D. Boston University EVALUATION OF ANTI-COCAINE ANTIBODIES AND A COCAINE VACCINE IN A RAT SELF-ADMINISTRATION MODEL Biography Dr. Kantak received her Ph.D. in Biopsychology from Syracuse University in 1977. Following post-doctoral training in Behavioral Neurochemistry from the University of Wisconsin School of Medicine and in Psychopharmacology from Tufts University, Dr. Kantak joined the faculty of the Department of Psychology at Boston University in 1982. Since 1982 her work has focused on the effects of magnesium and other NMDA antagonists on the behavioral effects of cocaine, and more recently, she has turned her attention to evaluating anti-cocaine antibodies and a cocaine vaccine in collaboration with ImmuLogic Pharmaceutical Corporation. Dr. Kantak is on the Editorial Advisory Board for Pharmacology, Biochemistry, and Behavior and is an elected member of ASPET, CPDD, and other scientific societies. Focus of Presentation Previous pre-clinical studies with anti-cocaine antibodies left open several issues critical in assessing the feasibility of a vaccine for the treatment of cocaine addiction in human subjects. First, it was unclear how much antibody was needed to block the reinforcing properties of cocaine. Second, it was unknown whether an individual could easily overcome the effects of immunization simply by increasing the self-administered dose of cocaine. The data presented in this talk address both of these issues. The vaccine used here was similar to the one we described previously (Fox et al., 1996), but uses a different carrier protein which is more appropriate for use in humans. Presentation Conclusions Based on passive administration studies, serum antibody levels sustained above 0.05 mg/ml are more effective at reducing cocaine self-administration behavior when cocaine is rapidly delivered. The cocaine vaccine IPC-14,551 induces a sufficient level of antibody to disrupt the reinstatement of drug-seeking and drug-consummatory behavior under conditions of rapid drug delivery. Antagonism of cocaine self-administration after immunization is also evident across a range of doses of cocaine and is only apparent in animals whose serum antibody levels exceed 0.05 mg/ml. The effects of immunization are not easily surmountable in that even 6- to 10-fold increases in the usual dose fail to reinstate drug-seeking and drug-consummatory behavior to baseline levels. The cocaine vaccine IPC-14,551 may be useful to guard against relapse in human cocaine addicts seeking treatment. Andrew B. Norman, Ph.D. University of Cincinnati School of Medicine TOWARDS A PASSIVE IMMUNOTHERAPY FOR COCAINE ADDICTION Biography 1977-1980 - B.Sc. (with honors) in Pharmacology from King's College, London 1980-1983 - Ph.D. in Pharmacology from King's College, London 1983-1986 - Postdoctoral fellow at Department of Neuroscience, University of California, San Diego (Advisor, Dr. Ian Creese) 1986-present - Department of Psychiatry, University of Cincinnati College of Medicine 1991: Awarded tenure 1995-present - Scientific Director, Cincinnati NIDA/VA Medications Development Research Unit Author on 82 published papers/reviews and 105 published abstracts. Major research interests: 1. Neuropharmacology of addiction 2. Medications development Focus of Presentation Passive immunotherapy of cocaine addiction with a well-characterized antibody would ensure maximum safety and predictable efficacy. Furthermore, protection is immediate in onset, does not require an intact host immune system, and is reversible. We have characterized a mouse monoclonal anti-cocaine antibody with a Kd of 6 nM and an approximate 50-1,000-fold selectivity for cocaine over its major metabolites. These properties are hypothesized to be suitable for effective immunotherapy. Preliminary data suggest that the mouse monoclonal antibody increases the dose of cocaine required to initiate cocaine self-administration and abolishes the self-administration of low unit-doses of cocaine in a novel quantitative animal model of relapse. If sufficient cocaine is infused into the rats, normal self-administration can be reinstated. A human anti-cocaine antibody is currently under development that should permit passive immunotherapy of human cocaine addicts. Presentation Conclusions Monoclonal antibodies can be generated which have properties expected to confer effective passive immunity to cocaine. A monoclonal anti-cocaine antibody dose-dependently increased the amount of cocaine necessary to initiate cocaine self-administration. The antibody also abolished cocaine self-administration at low unit doses of cocaine. The monoclonal antibody did not effect the mechanisms underlying cocaine self-administration as self-administration could be reinstated once the antibody was saturated. New technologies are available that will permit the development of human anti-cocaine antibodies which should be suitable for passive immunotherapy in human cocaine addicts. Marion Kasaian, Ph.D. ImmuLogic Pharmaceutical Corporation DEVELOPMENT OF A VACCINE FOR TREATMENT OF NICOTINE DEPENDENCE Biography Dr. Kasaian received her B.S. in biochemistry from Brown University in 1981 and Ph.D. in biochemistry from Case Western Reserve University in 1987. After completing postdoctoral fellowships in viral and molecular immunology, she joined ImmuLogic Pharmaceutical Corporation in 1992. At ImmuLogic, she has investigated the immunochemistry of allergens as well as the effector functions of allergen-specific IgE, and has contributed to studies of autoantigen recognition in autoimmune diseases. She currently heads a project to develop a novel vaccine approach to induce specific antibodies for treatment of nicotine dependence. Focus of Presentation ImmuLogic is adapting vaccine technology to the treatment of substance abuse and drug dependency. This talk will review development of a novel therapeutic strategy for reducing nicotine dependence. A vaccine was produced that induces generation of drug-specific antibodies. In a mouse model, the antibodies were shown to bind nicotine in the blood stream and decrease drug uptake into the brain. By preventing nicotine-activated brain reward mechanisms, the vaccine should help to reduce the reinforcing properties of the drug. For smokers attempting to quit the habit, this would provide an additional treatment option to help ensure maintenance of long-term abstinence. Presentation Conclusions An effective immunogen was produced by conjugating nicotine to a protein carrier. Immunization of mice with the nicotine vaccine generated antibodies highly specific for nicotine. Immune mice displayed an altered pharmacokinetic distribution of a 3H-nicotine challenge. Antibody bioactivity correlated with the ability to bind free nicotine. Antibody effects on nicotine self-administration will be tested in a rat model. Mariangela Segre, D.Sc. University of Illinois IS A COCAINE ANTI-IDIOTYPIC VACCINE FEASIBLE? Biography Dr. Segre's research on immunoregulation, especially of the humoral response, includes studies on the age-related sequential deterioration of various immune parameters in long-lived strains of mice, tolerance and autoimmunity. She is currently working on a longitudinal study of the immunological competence of aging dogs and the effect of caloric restriction on their immune system. Other recent work examines the relevance of the idiotypic network on the regulation of antibody production, particularly on the elicitation of polyreactive antibodies and epibodies in conventional responses to a hapten coupled to different carriers. She is also involved in the search for an immunologic biomonitor for polychlorinated biphenyl environmental contamination. Focus of Presentation This project examines the feasibility of using anti-idiotypic antibodies that mimic the configuration of the cocaine molecule to immunize experimental animals, thereby reducing the level of cocaine reaching the brain after challenge. Several cocaine-specific monoclonal antibodies were produced and used to elicit anti-idiotypic antibodies (Ab2). Among the different species of Ab2, only those which carry the internal image of the antigen were selected and in turn used to immunize Balb/c mice. Cocaine-specific antibodies were present in the blood of Ab2-immunized mice and led to a statistically significant reduction of cocaine levels in the brains of immunized animals following cocaine injection. Presentation Conclusions A cocaine anti-idiotipic vaccine is indeed feasible. An accurate selection of the anti-idiotypic antibody (Ab2) molecule is essential. An anti-idiotypic vaccine should incorporate a mixture of different molecules of Ab2. Paul Pentel, M.D. University of Minnesota EFFECTS OF ACTIVE IMMUNIZATION ON NICOTINE DISTRIBUTION IN RATS Biography My training is in internal medicine and clinical pharmacology. For many years the focus of our laboratory was the mechanisms and treatment of tricyclic antidepressant toxicity. A particular interest was pharmacokinetic interventions for overdose, including the use of drug-specific antibodies or antibody fragments as antidotes. Our work has more recently been directed toward drug abuse. Studies of immunization for nicotine abuse focus on understanding the pharmacokinetic mechanisms underlying the relationship between drug distribution and effect. Our laboratory also participates in clinical trials of treatments for cocaine and nicotine abuse as a part of the University of Minnesota's NIDA Medications Development Center. Focus of Presentation A vaccine that elicits nicotine-specific antibodies has been studied in rats. Rapid injection of nicotine in the jugular vein was used to simulate nicotine absorption from cigarette smoking. After a single 0.03 mg/kg dose of nicotine (equivalent to 2 cigarettes), actively immunized animals had higher arterial and venous plasma nicotine concentrations than controls, increased protein binding of nicotine in serum, and a reduced free (unbound) nicotine concentration in serum. Brain nicotine concentration was substantially reduced as early as 30 sec, and the extent of reduction was a function of the serum antibody titer. Presentation Conclusions Suitable immunogens can elicit nicotine-specific antibodies in rats with high titers and reasonable affinity. Immunized rats bind a significant fraction of a clinically relevant nicotine dose in serum. Immunization reduces nicotine distribution to brain by binding nicotine in serum and reducing the free (unbound) nicotine concentration. S. Michael Owens, Ph.D. University of Arkansas for Medical Sciences USE OF A DRUG CLASS-SPECIFIC MONOCLONAL FAB TO TREAT PHENCYCLIDINE ABUSE AND OVERDOSE IN A RAT MODEL Focus of Presentation The development of treatment strategies for drug intoxication has been hindered in part by the lack of clinically useful antagonists. Consequently, the major goal of these studies was to demonstrate that monoclonal antibody Fab fragments could be used as safe and effective drug class-specific pharmacokinetic antagonists for reversing central nervous system drug toxicity. These results indicate antibody-based therapies can be developed to treat toxicity caused by classes of drugs, as well as by individual drugs. These data also show that urinary elimination of Fab is dose-dependent, and that adequate urine output is required for maximal Fab and drug elimination. David A. Gorelick, M.D., Ph.D. National Institute on Drug Abuse ENHANCING COCAINE METABOLISM WITH BUTYRYLCHOLINESTERASE AS AN APPROACH TO COCAINE ADDICTION TREATMENT Biography David A. Gorelick, M.D., Ph.D., is Chief of the Pharmacotherapy Section in the Intramural Research Program of the National Institute on Drug Abuse (NIDA) in Baltimore, MD, and Adjunct Professor of Psychiatry at the University of Maryland School of Medicine. Dr. Gorelick received his M.D. and Ph.D. (pharmacology) from the Albert Einstein College of Medicine, Bronx, NY, in 1976, and did his medical internship and psychiatric residency at the University of California, Los Angeles. After completion of training in 1980, he joined the faculty of the UCLA Department of Psychiatry and the medical staff of the West LA VA Medical Center, where he directed several substance abuse treatment programs. Dr. Gorelick came to NIDA in 1989. His research interests focus on the clinical pharmacology of substance abuse and its pharmacological treatment. Dr. Gorelick is Board certified in psychiatry with added qualifications in addiction psychiatry, and certified in addiction medicine by the American Society of Addiction Medicine. Focus of Presentation Current pharmacotherapy of cocaine addiction has not been broadly successful. An alternative approach is to enhance cocaine metabolism by administration of the cocaine-metabolizing enzyme butyrylcholinesterase (BChE). Studies in rodents suggest that enzyme pretreatment can substantially reduce the physiological and behavioral effects of a cocaine challenge, at doses that themselves have no behavioral or toxic effects. Potential advantages include several days of increased enzyme activity after a single BChE injection, a benign pattern of cocaine metabolites, and no dependence for efficacy on an intact immune system. Research in humans is needed to evaluate the safety and efficacy of this treatment approach. Presentation Conclusions: Pharmacotherapy of cocaine addiction has not been widely successful. Hydrolysis by butyrylcholinesterase is a major cocaine metabolic pathway in primates. Hydrolysis produces pharmacologically benign cocaine metabolites. Butyrylcholinesterase treatment blunts the acute physiological and behavioral effects of cocaine. Gilberto N. Carmona National Institute on Drug Abuse ATTENUATION OF COCAINE'S ACTIVITY BY BUTYRYLCHOLINESTERASE (BChE) Focus of Presentation Previous studies have implicated butyrylcholinesterase (BChE; E.C. 3.1.1.8) as the primary plasma enzyme for metabolizing cocaine. In rats tested in a locomotor activity chamber following 17.0 mg/kg cocaine IP, HS-BChE pretreatment produced a significant attenuation in cocaine-induced locomotor activity. When added to plasma, BChE reduced the cocaine half-life from over 5 hours to less than 5 min. BChE altered the cocaine metabolic pattern such that the relatively non-toxic metabolite ecgonine methyl ester was produced, rather then benzoylecgonine. Thus, systemic administration of BChE can increase metabolism of cocaine sufficiently to alter a behavioral effect of cocaine, and should be investigated as a potential cocaine abuse treatment. Kazuo Matsubara, Ph.D. Asahikawa Medical College Hospital EFFECT OF JTP-4819 ON COCAINE METABOLISM Biography Education 1978 - Kyoto University, B.Pharm. 1987 - Shimane Medical University, Ph.D. Personal Experience 1979 - Research Assistant of Dept. of Legal Med., Shimane Medical Univ. 1987 - Assistant Professor of Dept. of Legal Med., Shimane Medical Univ. 1990-1991 - Visiting Assistant Professor of Dept. of Molecular and Cellular Biochem., Loyola Univ., Chicago 1991 - Associate Professor of Dept. of Legal Med., Shimane Medical Univ. 1997-present - Professor of Hospital Pharmacy & Pharmacology, Asahikawa Medical College Recent bibliography (1998) 1. K. Matsubara et al., Structural significance of azaheterocyclic amines related to Parkinson's disease for dopamine transporter. Eur. J. Pharmacol. 1998 (in press) 2. K. Matsubara et al., Endogenously occurring beta-carboline induces parkinsonism in non-primate animals: a possible causative pro-toxin in idiopathic Parkinson's disease. J. Neurochem., 70, 727-735, 1998. 3. K. Shimizu, K. Matsubara et al., Reduced dorsal hippocampal glutamate release significantly correlates with the spatial memory deficits produced by benzodiazepines and ethanol. Neuroscience, 83, 701-706, 1998. Focus of Presentation JTP-4819, (-)-(2S)-1-benzylaminocarbonyl-[(2S)-2-glycolonylpyrrolidinyl]-2-pyrrolineca rboxamide, is a specific prolyl endopeptidase inhibitor. This compound also increased the activity of plasma cholinesterase in experimental animals and humans. Thus, we investigated the effect of JTP-4819 on cocaine metabolism. The sub-chronic treatment of JTP-4819 decreased the unchanged cocaine level in the serum of mice after its administration, suggesting that this substance facilitated the cocaine metabolism. The treatment of JTP-4819 did not affect the spontaneous activity of mice. Interestingly, the strikingly increased locomotion activity induced by cocaine was attenuated by the pre-treatment of JTP-4819. These results indicate that JTP-4819 would dilute cocaine toxicity via the enhanced cocaine metabolism. Presentation Conclusions JTP-4819 is a specific prolyl endopeptidase inhibitor. This compound increased plasma cholinesterase activity. The treatment of JTP-4819 facilitated cocaine metabolism in mice. The increased locomotion activity induced by cocaine was attenuated by JTP-4819. These results indicate that JTP-4819 would attenuate cocaine toxicity. Clarence A. Broomfield, Ph.D. U.S. Army Medical Research Institute of Chemical Defense THE DEVELOPMENT OF XENOBIOTIC SCAVENGERS BY PROTEIN ENGINEERING Biography Clarence A. Broomfield was born on 18 September 1930 in Mount Morris, Michigan. Bachelor of Science in Chemistry, The University of Michigan, 1953 (Regents-Alumni Scholar). Ph.D. in Chemistry, with a concentration in Biochemistry, Michigan State University, 1958 (National Science Foundation Fellow). National Institutes of Health Postdoctoral Fellow, Cornell University in the laboratory of Professor Harold Scheraga, 1958-1962. Since 1962, Research Chemist and Research Biochemist in the U.S. Army Medical Research Institute of Chemical Defense (MRICD) and its precursor organizations. Dr. Broomfield is a member of the American Society of Biochemists and Molecular Biologists, the American Chemical Society, The New York Academy of Sciences, and Sigma Xi. Focus of Presentation This will be a summary of our recent efforts to produce, by site-directed mutagenesis of human butyrylcholinesterase, an enzyme that can be administered to humans that will catalyze the hydrolysis of nerve agents in the blood stream. Animal experiments have shown that it is possible to protect against several lethal doses of nerve agents by pretreatment with catalytic scavengers if the catalytic activity is high enough. Since the drugs of abuse are also xenobiotics, suggestions will be made to relate our work on protection against nerve agents to possible approaches for treating addiction and overdose. Presentation Conclusions Specific catalytic activity can now be generated by mutation of existing enzymes or catalytic antibodies. Use of scavenger molecules should be considered in the treatment of any toxic substance. Perhaps the most efficacious route of administration of scavengers will prove to be form of gene therapy. Oksana Lockridge, Ph.D. University of Nebraska Medical Center AN IMPROVED COCAINE HYDROLASE: THE A328Y MUTANT OF HUMAN BUTYRYLCHOLINESTERASE IS FOURFOLD MORE EFFICIENT Biography Ph.D. from Northwestern University, Evanston, IL 1971 (advisor Laszlo Lorand) Post-Doctoral, University of Michigan, Ann Arbor, MI (advisor Vincent Massey) Research Scientist at University of Michigan until 1990 Associate Professor, University of Nebraska Medical Center, 1990 to present Have worked on butyrylcholinesterase for 24 years. Focus of Presentation Our goal is to modify human butyrylcholinesterase to make it a better (-)-cocaine hydrolase. Butyrylcholinesterase is responsible for detoxicating cocaine by hydrolyzing it to benzoic acid and ecgonine methyl ester. Though butyrylcholinesterase is very important in this role, it performs it at the slow rate of 4 per minute. We would like to increase this rate so it approaches the rate at which it hydrolyzes the pharmacologically inactive (+)-cocaine, that is, to 10,000 per minute. We are modifying amino acids near the active site of human butyrylcholinesterase. A second approach is to study the rate of cocaine hydrolysis by butyrylcholinesterase from animals to see if multiple amino acid mutations have created a faster cocaine hydrolase. Presentation Conclusions The A328Y mutant of human butyrylcholinesterase is fourfold more efficient at hydrolyzing (-)-cocaine than is wild-type butyrylcholinesterase. Horse butyrylcholinesterase hydrolyzes (-)-cocaine with a twofold higher kcat but its binding affinity is lower so that it is not a better cocaine hydrolase than human butyrylcholinesterase. People who have the atypical variant (D70G) of human butyrylcholinesterase are at risk of suffering severe or fatal complications from cocaine use. Their butyrylcholinesterase is unable to bind cocaine (Km value is tenfold higher) and therefore a standard dose is a tenfold overdose. An improved cocaine hydrolase, the A328Y mutant of human butyrylcholinesterase, is fourfold more efficient. John Cashman, Ph.D. Human BioMolecular Research Institute NOVEL COCAINE ESTERASES Biography John Cashman received B.A. degrees in Chemistry and Biology from the University of California, Santa Barbara in 1977. He then went on to receive M.S. and Ph.D. degrees in Medicinal Chemistry from the University of Kansas in 1980 and 1982, respectively. From 1982-1984 he was a postdoctoral fellow in the laboratory of Professor E.J. Corey at Harvard University. In 1984 he took a position as Assistant Professor of Chemistry and Pharmaceutical Chemistry at the University of California, San Francisco. In 1992 he became the Associate Director of IGEN Research Institute in Seattle, WA. In 1994 he moved to Seattle Biomedical Research Institute as Senior Scientist before becoming Director of a new Seattle non-profit research institute named the Human BioMolecular Research Institute in 1998. Focus of Presentation Overuse and addiction to the psychomotor stimulant (-)-cocaine represents a significant public health problem in the United States with enormous economic and social consequences. Cocaine is largely detoxicated in humans by the action of esterases. However, the major detoxication catalyst, human butyrylcholinesterase (hBuChE), is rather sluggish at hydrolyzing naturally occurring (-)-cocaine. In contrast, the unnatural (+)-cocaine isomer is hydrolyzed approximately 2000-fold faster and is essentially nontoxic. To explore this apparent stereochemical difference in hBuChE esterase action, phosphonothiolate inhibitors corresponding to the transition state for (+)- and (-)-cocaine hydrolysis were synthesized and the separated stereoisomers (about phosphorous) were used to probe human and mouse BuChE active site topology. The results of kinetic investigations suggested specific amino acids to modify to increase the hydrolytic activity of BuChE against (-)-cocaine. Mouse BuChE was used as a model system. Site-directed mutagenesis studies ultimately provided a double mutant with considerable (-)-cocaine hydrolytic activity. It is possible that protein engineering of hBuChE may provide a therapeutic to combat cocaine addiction and abuse. Buddy Pouw, M.D., M.P.H.* University of Oklahoma College of Pharmacy NOVEL APPROACH FOR DEVELOPING CATALYTIC ANTIBODIES AGAINST COCAINE Biography Born in 1942, I received my medical degree from the University of Airlangga, Indonesia, in 1967. In 1980, I received an award from W.H.O. to get a Master's Degree from the School of Public Health, University of Michigan. I run my private practice (1967-1990), and since 1974 I had conducted several health services researches. I came to the United States in 1990, became a citizen in 1996, and started my career as a member of the research staff at Immuno-Mycologics, Inc., a company that produces diagnostic agents for deep mycosis. In 1993, I joined the College of Pharmacy at the University of Oklahoma as a Postdoctoral Research Fellow, and later as an Associate in Research I was involved in studies on developing catalytic antibodies against cocaine. Recently I have been involved in a study on NMDA antagonists in an attempt to block the acute toxicity of cocaine (convulsion and lethality). Focus of Presentation Introducing a new approach in developing catalytic antibodies against cocaine is a novel strategy that uses a Transition State Analog (TSA) of cocaine couple to a carboxylesterase at its active site. By introducing the active site of the enzyme (that hydrolyzed cocaine) to the immune system, we believe that catalytic antibodies against cocaine will be fruitfully generated. Anita D. Wentworth, Ph.D.* The Scripps Research Institute ANTIBODY CATALYSIS: IMPROVING CATALYTIC PROFICIENCY USING ALTERNATIVE HAPTEN DESIGNS Biography Anita D. Wentworth, Ph.D., nee Datta, was born in Blackburn, England, in 1972. She studied Chemistry at the University of Sheffield from where she received first her B.Sc. (Hon.) in 1993 and then her Ph.D. in 1996. Her Ph.D. thesis, entitled "Antibody Catalysis of Carbamate Ester Hydrolysis" was completed under the direction of Professor G. Mike Blackburn. Currently, Anita is working as a Research Associate in the laboratory of Professor Kim D. Janda at the Scripps Research Institute with continued interests in the field of catalytic antibodies while also gaining experience in liquid-phase organic chemistry. *Poster Presenter [Index] [Back to Program List]