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Peripheral Blockers as Treatments for Substance Abuse and Dependence


Charles R. Schuster, Ph.D.
Wayne State University


In this presentation some of the early work carried out at the Drug Abuse Research Center of the University of Chicago to investigate an immunological approach to the treatment of heroin dependence will be reviewed. In this research, a method for preparing morphine-6-hemisuccinate and conjugating it to bovine serum albumin was developed. Studies of the competitive inhibition of 14C-morphine binding to Anti-M-6-HS-BSA by the prior addition of increasing concentration of unlabelled opioids showed the selectivity of the antibodies for heroin and structurally related compounds. In vitro studies showed that preincubation of morphine with Anti-M-6-HS-BSA globulin prevented morphine from inhibiting electrically-induced contractions of the guinea pig ileum. Further, Anti-M-6-HS-BSA reversed morphine's inhibitory effect on ileum contractions. Subsequent studies demonstrated that active or passive immunization of rhesus monkeys trained to self-administer both cocaine and heroin specifically blocked the reinforcing effects of heroin. Saturation of the antibody pool by very high doses of heroin caused the reinstatement of heroin self-administration. This line of research was discontinued for two reasons. First, the immunological approach to the treatment of drug dependence is limited by the specificity of the antibodies. Although immunized animals stopped self-administering heroin there is no doubt that they would have
self-administered fentanyl and other opioids whose chemical structure would make them non-reactive to the Anti-M-6-HS-BSA. Secondly, at the time this research was conducted, naltrexone was developed and seemed to be a more useful treatment intervention for opiate dependence since it antagonized all opiates regardless of their chemical structure. Compliance problems with naltrexone have unfortunately, gravely limited its usefulness for the treatment of opiate dependence and would undoubtedly be a problem for any other type of "blocker" therapy.

This early experience points to several implications for the current renewal of interest in immunological approaches for the treatment of drug dependence. First, compliance problems must be dealt with. Evidence will be presented that voucher-based reinforcement procedures are useful in increasing compliance with naltrexone administration and would no doubt enhance compliance for any other type of therapy. Drug substitution may be a more difficult problem. Cathinone, methamphetamine, and a host of other psychomotor stimulants are potentially available on the illicit market and would no doubt be substituted at least by some immunized cocaine abusers. On the other hand, immunization for those who are motivated to become cocaine abstinent might be very helpful. Since there are no currently available effective pharmacological treatments for cocaine dependence, peripheral blockers should be developed and evaluated for their efficacy and usefulness.

S. Michael Owens, Ph.D.
University of Arkansas for Medical Sciences


Knowledge gained from basic research into the neurobiology of drug abuse has led to major discoveries in medicine. Nevertheless, the development of medical strategies for treating the complex array of neurological problems associated with drug abuse has been frustratingly slow. This is partially because the central nervous system toxicity and/or addiction, which results from the use of drugs of abuse, is difficult to treat medically. Furthermore, successful therapy has been hindered by the lack of useful chemical-based agonist or antagonists for many of these drugs and by the extensive distribution of these chemicals out of the blood stream. 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 is exciting, the therapeutic principles involved in successful therapy are poorly understood. In addition, drug abusers usually self-administer large doses of drugs, which can easily exceed the therapeutic capacity of antibody-based therapy (after active and passive immunizations) or passive administration of enzymes. Therefore, the use of these medications must be carefully matched to the appropriate drug abuse-related medical condition.

This presentation will present 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. Indeed a full understanding of the pharmacokinetic and pharmacodynamic relationships will provide the framework for current and future development of these biological modifiers which act as pharmacokinetic antagonists for the treatment of drug abuse. Finally, methods for large-scale production of biological modifiers will be discussed. (Supported by NIDA grants DA07610 and KO2 DA0110.)

Gail D. Winger, Richard Briscoe, Lee Koetzner, Camilo Cabrera, and James H. Woods
University of Michigan


We have evaluated three different interventions that should reduce the rate of access of cocaine to the brain. Several procedures that reflect the behavioral effects of cocaine in rodents were used to determine the effectiveness of these interventions. One intervention was to actively immunize mice against cocaine through administration of a cocaine-BSA antigen (Fox et al., 1996). Using an ELISA assay, we were able to measure antibody titers to this immunogen 10 days following a single administration of the cocaine-protein complex in an alum adjuvant. A booster administration of the same amount of antigen did not greatly increase the titer level in the serum. The effects of cocaine in these mice and in control animals were evaluated in a locomotor activity apparatus. There was no difference between the ability of cocaine (15 and 30 mg/kg) to stimulate locomotor activity in mice that had been immunized and those that had not.

The ability of the catalytic antibody 15A10 (Yang et al., 1996) to modify the effects of cocaine in rats and mice has also been studied. Mab 15A10 did not alter the amount of cocaine, given intravenously as a constant infusion (2 mg/kg/min), required to produce convulsions and death in mice. This may have been because large doses of cocaine (approximately 140 mg/kg) were necessary to produce lethality in mice, and these doses may have overwhelmed the antibody. We therefore sought for measures that were more sensitive to cocaine administration. Consistent blood pressure increases were observed following intravenous (through a jugular canula) administration of doses as small as 0.056 mg/kg cocaine to awake, freely moving mice. These blood pressure increases lasted approximately 20 sec. The catalytic antibody (100 mg/kg) did not alter the effects of cocaine on these acute blood pressure changes. This may have been due to the rapid onset and short duration of action of cocaine in this preparation. Much larger doses of cocaine (100 mg/kg) were required to produce chronic blood pressure changes when the drug was administered i.p. Nevertheless, these blood pressure changes were significantly reduced, although not completely eliminated, by 100 mg/kg Mab 15A10. Administration of 15 mg/kg BuChE, however, was able to prevent completely the effects of 100 mg/kg cocaine (i.p.) on blood pressure increases and lethality in mice.

In the rat, 9-12 mg of Mab 15A10 were able to block the reinforcing effects of i.v. delivered cocaine 0.3 mg/kg/inj. In the presence of these doses of Mab 15A10, behavior maintained by cocaine had the appearance of behavior maintained by saline. Self-administration of bupropion, another dopamine uptake blocker, was not modified by Mab 15A10, nor was behavior maintained by sweetened condensed milk.

Our data indicate that, not surprisingly, the ability of anti-cocaine antibodies to reduce the behavioral and cardiovascular effects of cocaine may depend critically on the amount of cocaine required to produce the effect and the rate of cocaine administration.

Fox, B.S., et al., Nature Med. 2, 1129-1132, 1996

Yang, G., et al., J. Am. Chem. Soc. 118, 5581-5586, 1996

John Cashman, Ph.D.
Human BioMolecular Research Institute


Catalytic antibodies offer a new method to design a catalyst where previously no such catalytic activity exists or where the catalyst that is present is sluggish or inefficient. In principle, elaboration of a highly efficient catalyst and humanization of the catalytic antibody could provide an unprecedented approach to development of selective human therapeutics. An example of a therapeutic human catalyst that is urgently needed is the need to develop an efficient anti-cocaine (-)-cocaine detoxication catalyst. Such an agent would be useful to decrease addiction liability if used in conjunction with psychological and medical counseling. The goal of our research was to develop a highly efficient mouse anti-cocaine catalytic antibody and to convert it into an antibody that would be suitable for human therapy by modern recombinant methods. Related objectives were to develop active immunization strategies and to develop high-affinity anti-cocaine antibodies. The approach that was taken was to immunize mice with a stable transition state analog for cocaine hydrolysis, procure the most catalytically active antibody with a high throughput rapid screen for activity, prepare monoclonal antibodies of the most active catalysts and characterize the monoclonal antibodies obtained. We compared the catalytic efficiency of the most active anti-cocaine catalytic antibodies with a number of other cocaine esterases. The most active anti-cocaine catalytic antibodies more closely resembled pig liver esterase and to a less extent, butyrylcholinesterase. It is likely that the anti-cocaine catalytic antibodies active site has a broader active site binding domain and provides less steric interference to (-)-cocaine hydrolysis than does human butyrylcholinesterase.

Supported by NIH grants DA08531 and DA00269.

Kim D. Janda, Ph.D.
The Scripps Research Institute


Since the first reports of antibody catalysis, a defining tenet for catalyst generation has been the mimicry and incorporation of a reaction's transition-state stereoelectronics into hapten design. Success depends upon accurate assumptions of mechanistic details, the extent to which the hapten is a model of the transition-state, and how the immune system responds to the antigen. With these thoughts in mind, a general discussion of antibody catalysis will be presented. Hapten design strategies will be detailed in regards to a transition-state analogue, bait and switch, and reactive immunization approaches. Hybridoma selection will be described as well as kinetic analysis and the criteria used in judging the antibody preparation "contaminate free." Several antibody catalysts from the investigator's group will be described including cationic cyclization reactions, Diels-Alder reactions, the kinetic resolution of pharmaceutically relevant drugs, and phosphodiester hydrolysis. New strategies will be detailed that describe the "direct selection" of catalytic antibodies. Within this context, disulfide bond interchange, primary amide, and glycosidic bond cleavage will be presented as successful case studies emerging from our laboratories. A summary of what we can expect from catalytic antibodies in the future will round out the lecture.


Our efforts have been programmed to specifically delineate aspects of the chemistry, immunochemistry, and immunology necessary for the implementation of immunopharmacological protocols in the abatement of cocaine abuse. Our work encompasses the synthesis of haptens and reagents, monoclonal antibodies that bind cocaine with high affinity and specificity, catalytic antibodies that degrade cocaine, development of noncatalytic and catalytic vaccine designs, human anti-cocaine monoclonal antibodies derived from combinatorial libraries, and sophisticated animal behavioral paradigms for testing all therapies. I will briefly highlight the synthesis of two haptens that furnish both polyclonal and monoclonal antibodies highly specific for cocaine rather than cocaine metabolites. Our selection of these structures accounts for hydrogen bonding interactions, shape selectivity, and enhanced stability of the immunoconjugates. One antibody elicited from our novel cocaine haptens will be described in detail in terms of its binding specificity. Finally, "phage display" techniques will be discussed in the context of reconstructing this monoclonal mouse antibody specific to cocaine into a human single chain antibody with similar specificity and affinity. The goal of this latter effort is to make a human antibody clone that can be prepared in an economically efficient manner for passive immunization therapy.

M. Rocio A. Carrera
The Scripps Research Institute


Cocaine is a powerfully addictive psychostimulant that produces euphorigenic and reinforcing effects. In order for cocaine to exert its rewarding effects and thus be a potential drug of abuse, it must penetrate the central nervous system (CNS) by crossing the blood-brain barrier. The reinforcing properties of cocaine and other psychomotor stimulants have been linked to the activation of central dopaminergic neurons as well as other neurochemical systems, making it difficult for pharmacotherapeutical interventions to target cocaine abuse successfully and without side effect problems. An alternative strategy has involved immunological reagents and the immune system, whereby the presence of specific anticocaine antibodies act as peripheral blockers of the cocaine thus impeding its passage into the CNS. We previously demonstrated that active immunization with a new stable cocaine conjugate (GNC-KLH) suppressed locomotor activity and stereotyped behavior in rats induced by cocaine but not by amphetamine. Also, this effect was enhanced with consequent cocaine challenges. Moreover, brain levels of cocaine were lower in immunized rats after cocaine injection. Recently, we have extended these data by testing the effects of immunization with GNC-KLH in an animal model of cocaine self-administration relapse in the rat. Animals previously trained to self-administer cocaine were immunized while subjected to a period of extinction by replacing cocaine with saline. When exposed to cocaine again, priming-induced reinstatement was achieved in control but not in immunized rats. Taken together, these results indicate that active immunization with GNC-KLH effectively blocks the psychoactive as well as the reinforcing effects of cocaine. In the context of relapse prevention, immunotherapy may offer a nontoxic, substance-specific approach for cocaine abuse treatment.

Barbara S. Fox, Ph.D.
ImmuLogic Pharmaceutical Corporation


A cocaine vaccine has been developed by conjugating a cocaine derivative to an immunogenic carrier protein. Immunization of mice or rats with the vaccine induces a high titer antibody response against cocaine. The antibodies that are induced bind to cocaine, and its pharmacologically active metabolites and derivatives (norcocaine and cocaethylene), but not to its inactive metabolites (benzoylecgonine and ecgonine methyl ester). When [3H]cocaine is administered intravenously to immunized mice, the majority of the cocaine in the plasma is antibody-bound. As a result of antibody binding, the levels of cocaine in the plasma are increased in immunized animals while the levels of cocaine in the brain are decreased. The inhibition of brain uptake of cocaine can be seen across a range of cocaine doses and following either intravenous or intranasal administration of cocaine. Cocaine dosing of immunized animals does not detectably decrease the anti-cocaine antibody titer and the presence of the antibody has only a modest effect on the metabolism and half-life of cocaine in the circulation. As discussed by Dr. Kathleen Kantak in the following talk, the vaccine was also able to significantly reduce cocaine self-administration in rats. Together, these data suggest that the cocaine vaccine will provide a powerful new tool for the treatment of cocaine addiction. An IND application for ImmuLogic's cocaine vaccine was cleared by the FDA in late 1997 and the drug is ready to begin evaluation in human clinical trials.

Kathleen M. Kantak1, Stephanie L. Collins1, Elizabeth G. Lipman1, Julian Bond2, Kate Giovanoni2, and Barbara S. Fox2
1Laboratory of Behavioral Neuroscience, Department of Psychology, Boston University, Boston, Massachusetts; and 2ImmuLogic Pharmaceutical Corporation, Waltham, Massachusetts


Previous pre-clinical studies with an anti-cocaine monoclonal antibody left open several issues critical in assessing the feasibility of a vaccine for cocaine addiction treatment in human subjects, namely, how much antibody was needed to block cocaine reinforcement and whether the effects of immunization were easily surmountable. Two experiments were conducted using a second-order schedule of drug delivery in rats. A passive transfer experiment with control and 4 or 12 mg anti-cocaine monoclonal antibody treatments showed that antagonism of 1 mg/kg cocaine was dependent on antibody dose. In animals whose serum antibody levels were sustained above 0.05 mg/ml, there was a sufficient amount of antibody to blunt the effects of the first and second cocaine infusions and to disrupt the response rate as well as the intake and pattern of subsequent infusions for several days. In an active immunization experiment, the cocaine vaccine IPC-14,551 induced average serum antibody levels of 0.08 mg/ml and blocked the reinstatement of behavior by 1 mg/kg cocaine. The vaccine also lengthened the inter-infusion-interval and engendered a progressive increase in the latency to the first infusion. Antagonism of cocaine self-administration after immunization was evident across a range of doses of cocaine and was only apparent in animals whose serum antibody levels exceeded 0.05 mg/ml. Furthermore, there was no evidence that the antagonism was surmountable within the dose range examined (up to 10 mg/kg). A therapeutic vaccine for cocaine relapse prevention would open a new avenue for the treatment of this serious addiction.

Marion Kasaian, Ph.D.
ImmuLogic Pharmaceutical Corporation


Approximately 16 million people attempt to quit smoking each year, but fewer than 1.5 million are able to achieve long-term abstinence, even with the help of pharmacologic interventions such as nicotine replacement therapy and anti-depressant drugs. The aim of our vaccine approach is to elicit nicotine-specific antibodies in treated subjects, lessening the reinforcing properties of nicotine by blocking its entry into the brain. Used in conjunction with behavioral counseling and other therapies, the vaccine should help to reduce nicotine dependence in those motivated to quit. To produce the vaccine, nicotine was conjugated to an immunogenic protein carrier. Mice immunized with the nicotine-protein conjugate on alum generated antibodies that bound both to the conjugate and to nicotine free in solution. A broad range of biological molecules with structural similarity to nicotine was screened for binding to anti-nicotine sera, and no detectable cross-reactivity was found. The presence of high levels of nicotine-specific antibody in immune mice resulted in an alteration of the pharmacokinetic distribution of nicotine following a 3H-nicotine challenge. As compared to naive controls, the immune mice had decreased levels of 3H-nicotine in the brain and increased retention in the plasma one minute after an i.v. bolus of the drug. The magnitude of this in vivo effect was related to the levels of 3H-nicotine captured by the antiserum in vitro. The observation that anti-nicotine antibody reduces drug uptake into the brain suggests that antibodies may have the capacity to alter the psychoactive effects underlying nicotine dependence and reinforcement. Future studies will examine the influence of anti-nicotine antibody on drug-seeking behavior in rats trained to self-administer nicotine.

Mariangela Segre, D.Sc.
University of Illinois


In order to circumvent the problems associated with the instability of the cocaine molecule, we propose the use of an antibody molecule that mimics the configuration of cocaine as the antigen. In numerous cases anti-idiotypic vaccines have been used successfully to provide protective immunity. As Jerne's network theory describes, by virtue of its dual nature, the Ab molecule recognizes the antigen through its combining site (paratope) and is recognized by other Abs through its idiotopes. Antibodies specific to the antigen (Ab1), elicit anti-idiotypic antibodies (Ab2) which in turn elicit Ab3 and so on. The Ab2s which recognize the paratope of the Ab1 contain the internal image of the antigen and therefore can be used in place of the antigen. Cocaine specific Ab1 antibodies were elicited using two cocaine conjugates presenting opposite sides of the molecule to the immune system. The Ab2 antibodies produced from these Ab1 were linked to KLH and were used for the vaccination of male BALB/c mice. Mice were challenged with 5 mg/kg of cocaine ip and sacrificed at 10 minutes. After solid phase extraction the level of cocaine in the resulting extracts of brain tissue were analyzed by HPLC. We found a significant decrease in the level of cocaine in the brain of mice vaccinated with Ab2-KLH, when compared with control mice. Through these experiments we have established the feasibility of an anti-idiotypic cocaine vaccine. A more thorough examination of the metabolism of cocaine in vaccinated mice, as well as behavioral studies, must be performed to determine if this vaccination could be a practical treatment for cocaine addiction.

Y. Hieda, D.E. Keyler, J. VanDeVoort, J.K. Kane, C.A. Ross, D. Raphael, R.S. Niedbala, P.R. Pentel
University of Minnesota


The ability of active immunization to alter nicotine distribution was studied in rats. Animals were immunized with 6-(carboxymethylureido)-(Å)-nicotine (CMUNic) linked to keyhole limpet hemocyanin (KLH). Antibody titers determined by ELISA using CMUNic coupled to albumin as the coating antigen were >1:10,000. Antibody binding was not inhibited by the nicotine metabolites cotinine or nicotine-N-oxide. Antibody affinity for nicotine measured by soluble RIA was 2.4Å1.6 x 107 M-1.

Anesthetized rats immunized with either CMUNic-KLH or KLH alone received nicotine 0.03 mg/kg (equivalent to 2 cigarettes in a human) via the jugular vein over 10 sec. This dosing regimen was shown to mimic the arterial-venous nicotine concentration gradient typical of nicotine delivered by cigarette smoking in humans.

Both arterial and venous serum nicotine concentrations were higher in immunized animals than in controls, even at the first (7.5 sec) sampling time. Brain nicotine concentration was reduced in immunized rats over the entire 6 min sampling period immediately following nicotine dosing (mean reduction 38%). A reduction was found at the earliest sampling time (30 sec) and was maximal at 1 minute (48%). Immunization increased the serum protein binding of nicotine from 10.9 to 91.2%, and reduced the unbound nicotine concentration in venous serum from 13.4 to 10.0 ng/ml. The reduction in brain nicotine concentration correlated with the serum antibody titer and the percent of nicotine bound in serum.

These data demonstrate that nicotine-specific antibodies produced by active immunization rapidly bind nicotine in arterial blood, reduce the unbound nicotine concentration, and reduce the early distribution of nicotine to brain. Effects were observed using a clinically relevant nicotine dose and route of administration.

S. Michael Owens, Ph.D.
University of Arkansas for Medical Sciences


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. Changes in drug-induced locomotor effects in a rat model were used to assess the ability of the anti-phencyclidine Fab to reverse the behavioral effects of phencyclidine (PCP) and other potent arylcyclohexylamines. In addition to reversing the locomotor effects of PCP, the anti-PCP Fab completely reversed the locomotor effects induced by two other potent analogs, 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and N-ethyl-1-phenylcyclohexylamine (PCE). For example, the average distance traveled (and duration of effects) with saline or anti-PCP Fab treatment for TCP was 541 vs. 107 m (98 vs. 21 min) and for PCE was 691 vs. 125 m (138 vs. 27 min) (P < .05 in all cases). These results indicate antibody-based therapies can be developed to treat toxicity caused by classes of drugs, as well as by individual drugs. To minimize potential toxicity, and to maximize therapeutic effectiveness, the renal elimination of Fab and PCP were studied. The hypothesis was that increasing urine output would lead to increases in the elimination of intact Fab and PCP in the urine. The total amount of Fab appearing in the urine was 59.9*9.0% of the Fab dose. To study the effect of Fab dose on Fab elimination, rats (n=3-4) received PCP (0.1, 0.3, 1.0 and 3.0 mg/kg) followed 10 min later by a mole equivalent Fab dose (21, 62, 206 and 617 mg/kg). Elimination of Fab following the 21 mg/kg dose (45.3*14.2%) was found to be significantly lower than that of the 62 mg/kg (71.2*5.5%) and 617 mg/kg (65.4*7.6%) doses (P < .05), but not the 206 mg/kg dose (58.2*4.3%). These data suggest that urinary elimination of Fab is dose-dependent, and that adequate urine output is required for maximal Fab elimination. Thus fluid loading can play an important role in Fab elimination and help to maximize therapeutic effectiveness. (Supported by NIDA grants DA07610, KO2 DA0110 to S.M.O., and F31 DA05795 to J.W.P.)

David A. Gorelick, M.D., Ph.D.
National Institute on Drug Abuse


Current pharmacodynamic approaches to pharmacotherapy for cocaine addiction have not been broadly successful. An alternative pharmacokinetic approach would be to enhance cocaine metabolism by administration of butyrylcholinesterase (E.C.; BChE), a major cocaine-metabolizing enzyme in primates. Exogenous BChE administration has been used to successfully treat patients with organophosphate poisoning or exaggerated responses to succinylcholine, both of which are also metabolized by BChE. The application of this approach to human cocaine addiction is supported by the following findings: 1) inverse correlation between plasma BChE activity and severity of acute cocaine-associated medical events; 2) decrease of cocaine half-life in vitro with addition of human BChE; 3) decrease in the acute behavioral and physiological effects of a cocaine challenge in rodents after BChE treatment, at enzyme doses that themselves have no behavioral or toxic effects. The in vitro kinetics of human BChE with cocaine as substrate (Km = 11.9 M, Vmax = 1.17 M/min, specific activity = 69.7 nM/min/mg enzyme) are consistent with meaningful metabolism of self-administered cocaine. BChE administration offers potential advantages over other pharmacokinetic approaches to cocaine addiction treatment: 1) administration of an existing, presumably safe, endogenous compound, 2) no dependence on a functioning immune system, 3) generation of inactive or beneficial metabolites. Potential disadvantages are the need for repeated administration (although enzyme half-life may be several days) and limited enzyme contact with smoked cocaine (since little exogenous enzyme will enter the CNS). Clinical research is needed to further explore the usefulness of this treatment approach.

Gilberto N. Carmona
National Institute on Drug Abuse


Pharmacological approaches to cocaine abuse and toxicity have not been very successful, due in part to the lack of drug-receptor specificity. A more fruitful approach for the treatment of cocaine abuse and toxicity may be to provide treatment that is aimed at altering the metabolic profile of cocaine in a way that further reduces the overall effects of cocaine both in the central and peripheral nervous systems. Previous studies have implicated butyrylcholinesterase (BChE; E.C. as the primary plasma enzyme for metabolizing cocaine. Systemically administered BChE has been shown to afford protection against both the acute and chronic physiological and behavioral effects of organophosphate poisons in rats and monkeys. In our experiment, male Sprague-Dawley rats surgically prepared with jugular catheters were given an IV injection of saline or HS-BChE prior to being placed in locomotor activity chambers. Neither saline nor HS-BChE had an effect on activity during the 30-minute habituation period. After the 30-minute habituation session, the rates were removed from the chambers and then given IP injection (1.0 mg/kg) of either saline or cocaine (17.0 mg/kg) and immediately placed back into their respective chambers for an additional 120 minutes. HS-BChE pretreatment produced a significant attenuation in cocaine-induced locomotor activity. When added to rat plasma in vitro, BChE reduced the cocaine half-life from over 5 hours to less than 5 minutes. Thus, systemic administration of BChE can increase metabolism of cocaine sufficiently to alter a behavioral effect of cocaine, and may be useful as an adjunct to cocaine abuse treatment.

Kazuo Matsubara, Ph.D.
Asahikawa Medical College Hospital


JTP-4819, (-)-(2S)-1-benzylaminocarbonyl-[(2S)-2-glycolonylpyrrolidinyl]-2-pyrroline carboxamide, is a specific prolyl endopeptidase inhibitor. This drug has been developed as a new therapeutic agent for Alzheimer's disease. JTP-4819 also increased plasma cholinesterase and acetylcholinesterase activities in experimental animals and humans, although in brain, these enzyme activities were unchanged. Plasma cholinesterase activity has been shown to be a critical factor in cocaine detoxification. Thus, we investigated the effect of JTP-4819 on cocaine metabolism. JTP-4819 (100 mg/kg) was orally administered to male ICR mice for 5 days. Control mice received saline instead of JTP-4819. Five minutes after the last injection, 20 mg/kg of cocaine was intraperitoneally injected in the mice. Cocaine and its metabolites were determined using GC/MS. Treatment with JTP-4819 decreased serum unchanged cocaine and benzoylecgonine levels in mice when cocaine levels were relatively high in control mice, suggesting that this substance facilitated cocaine metabolism. Norcocaine and ecgonine methylester concentrations were not affected by treatment with JTP-4819. We also evaluated JTP-4819 treatment on locomotor activity induced by cocaine. Mice pre-treated with JTP-4819 were placed in an activity cage with two series of infrared beams (low and high levels) using a Scanet MV-10 apparatus. Treatment with JTP-4819 did not affect spontaneous activity of mice. Interestingly, the strikingly increased locomotion and rearing activities induced by cocaine were attenuated by pre-treatment with JTP-4819. These results indicate that JTP-4819 would dilute cocaine toxicity via enhanced cocaine metabolism.

Oksana Lockridge1, Wei Hua Xie1, C.V. Altamirano1, C.F. Bartels, R.J. Speirs2, J.R. Cashman2
1Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center and 2Human BioMolecular Research Institute


Butyrylcholinesterase (BChE) has a major role in cocaine detoxication. The rate at which human BChE hydrolyzes cocaine is slow, with a kcat of 3.9 min-1 and Km of 14 M. Our goal was to improve cocaine hydrolase activity by mutating residues near the active site. The mutant A328Y had a kcat of 10.2 min-1 and Km of 9 M for a fourfold improvement in catalytic efficiency (kcat/Km). Since benzoylcholine (kcat 15,000 min-1) and cocaine form the same acyl-enzyme intermediate but are hydrolyzed at 4000-fold different rates, it was concluded that a step leading to formation of the acyl-enzyme intermediate was rate-limiting. BChE purified from plasma of cat, horse, and chicken was tested for cocaine hydrolase activity. Compared to human BChE, horse BChE had a twofold higher kcat but a lower binding affinity, cat BChE was similar to human, while chicken BChE had only 10% of the catalytic efficiency. Naturally occurring genetic variants of human BChE were tested for cocaine hydrolase activity. The J and K variants (E497V and A539T) had kcat and Km values similar to wild-type, but because these variants are reduced to 66% and 33% of normal levels in human blood, people with these variants may be at risk for cocaine toxicity. The atypical variant (D70G) had a tenfold lower binding affinity for cocaine, suggesting that persons with the atypical variant of BChE may experience severe or fatal cocaine intoxication when administered a dose of cocaine that is not harmful to others.

Supported by the Nebraska Affiliate of the American Heart Association Grant 9707841S (to O.L.), AASERT Award DAAG55-07-1-0244 from the U.S. Army Research Office (to O.L.) NIH grants DA08531 and DA00269 (to J.R.C.), DA011707 (to O.L.), and U.S. Army Medical Research and Materiel Command Grant DAMD17-97-1-7349 (to O.L.).

John Cashman, Ph.D.
Human BioMolecular Research Institute


Development of pharmacotherapeutic agents that antagonize or partially agonize receptors or transporters to combat the addiction liability of cocaine has made considerable progress. However, new approaches that rely on modern developments in structural and molecular biology are needed to expedite drug discovery and create new therapeutics to address diseases of drug abuse. The goal of our work is to provide fundamental biochemical information that could be useful in the discovery of human cocaine detoxication therapies. It was known that the major cocaine esterase in human serum, butyrylcholinesterase, is relatively slow at catalyzing the hydrolysis of (-)-cocaine. On the other hand, human butyrylcholinesterase is an efficient (+)-cocaine esterase. Thus, butyrylcholinesterase possesses all of the mechanistic imperative to hydrolyze (-)-cocaine, but it is likely that one or more active site residue blocks enzyme action. The approach taken was to investigate a closely related enzyme, acetylcholinesterase, that was known to be virtually inactive as a cocaine hydrolytic catalyst with the idea that engineering an increase in acetylcholinesterase would provide significant insight into improving butyrylcholinesterase cocaine esterase activity. The experimental approach was divided into 4 parts: 1) Design, synthesis and testing of stereochemically defined mechanism-based inhibitors of cholinesterases, 2) Refinement of the inhibitor data by molecular modeling of the enzyme, 3) Evaluation of the model by investigating site-directed mutagenized acetylcholinesterases and 4) Application of the information obtained to design improved human cocaine esterases.

Supported by NIH grants DA08531 and DA00269.

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