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Anticocaine catalytic antibodies
Journal of Immunological Methods 269 (2002) 299 – 310 www.elsevier.com/locate/jim
Anticocaine catalytic antibodies Shi Xian Deng, Paloma de Prada, Donald W. Landry * Division of Clinical Pharmacology and Experimental Therapeutics, Department of Medicine, Columbia University, Box 84, 630 W 168th Street, New York, NY 10032, USA Received 27 March 2002; accepted 10 April 2002
Abstract Cocaine mediates its reinforcing and toxic actions through a ‘‘loss of function’’ effect at multiple receptors. The difficulties inherent in blocking a pleiotropic blocker pose a great obstacle for the classical receptor – antagonist approach and have contributed to the failure (to date) to devise specific treatments for cocaine overdose and addiction. As an alternative, we have embarked on an investigation of catalytic antibodies, a programmable class of artificial enzyme, as ‘‘peripheral blockers’’— agents designed to bind and degrade cocaine in the circulation before it partitions into the central nervous system to exert reinforcing or toxic effects. We synthesized transition-state analogs of cocaine’s hydrolysis at its benzoyl ester, immunized mice, prepared hybridomas and developed the first anticocaine catalytic antibodies with the capacity to degrade cocaine to nonreinforcing, nontoxic products. We subsequently identified several families of anticocaine catalytic antibodies and found that the most potent antibody possessed sufficient activity to block cocaine-induced reinforcement, organ dysfunction and sudden death in rodent models of addiction, toxicity and overdose, respectively. With the potential to promote cessation of use, prolong abstinence and provide a treatment for acute overdose, the artificial enzyme approach comprehensively responds to the problem of cocaine. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cocaine; Addiction; Toxicity
1. Introduction The problem of cocaine has been an intractable one for the organic chemist. The crux of the problem stems from the fact that cocaine itself is a blocker, and analogs of cocaine that can displace it from its binding site, still block function just as cocaine would. The difficulties inherent in blocking
* Corresponding author. Tel.: +1-212-305-1890; fax: +1-212305-3475. E-mail address: [email protected] (D.W. Landry).
a blocker led us to an alternative approach based on peripheral blockade rather than central blockade. Regardless of the route by which an addictive drug enters the body, the drug must pass through the blood to reach the brain. An agent that intercepted a drug in the peripheral circulation before it partitions into the central nervous system could circumvent limitations on central blocks that act directly within the central nervous system. For peripheral blocking agents, we chose catalytic antibodies, biocompatible artificial enzymes that are programmable to particular molecular targets and have the potential for an extended plasma half-life.
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 2 3 7 - 5
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2. Background For over a thousand years, Peruvian Indians chewed Coca leaves, a practice that increased their energy and enabled them to work with increased vigor at high altitude. However, it was not until the 1850s when German chemists purified the active constituent of coca leaves that cocaine arose as a potent drug of abuse. Cocaine was initially popularized by Sigmund Freud as a treatment for opium addiction and he did succeed in curing opiate addicts but in their place appeared a legion of cocaine addicts. Enthusiasm for cocaine culminated in the cocaine pandemic of the 1890s. Cocaine fell acutely from favor but appreciation for its potential faded and widespread addiction recrudesced again in the 1920s. Stimulant abuse emerged once more in the 1950s with amphetamine as the stimulant of choice, and again in the 1960s with the rise of metamphetamine. The fifth and current stimulant epidemic began in the 1980s with the reemergence of cocaine. Renewed use of cocaine was driven in part by an enormous increase in supply and in part by a new method of administration, the smoking of crack cocaine. Over the last 20 years, 60 million Americans have been exposed to cocaine, with approximately 6 million regular users and 2 –3 million hard-core addicts (Office of the National Drug Control Policy, 1996). The clinical characteristics of cocaine underscore its potential for abuse. First, there is a magnification of pleasure. The euphoria induced by cocaine is dosedependent. A consequence of heavy use is progressive social isolation and a transition to binges. Periods of abstinence also have well-defined clinical characteristics. After heavy use, there is a crash, a period of hypersomnolence and dysphoria, usually mild, lasting 12– 96 h and followed by a period of withdrawal marked by anergia (decreased energy) and anhedonia (decreased capacity to experience pleasure). The symptoms of cocaine withdrawal differ markedly from those of heroin or alcohol withdrawal, both of which can be life threatening. In the case of cocaine, withdrawal is principally experienced as craving, and is not associated with vascular collapse. In alcohol withdrawal, an addict can at any given moment indicate the progress of withdrawal, initially intense and diminishing over time. For cocaine addiction, craving is a function of perceived availability of the
drug. Thus, a cocaine addict may feel no desire for the drug, but the sudden introduction of a social cue can precipitate craving. For example, seeing a person who supplies the drug or with whom the drug was used can be a potential precipitant. One consequence of this physiology is that an agent that suddenly blocked the actions of cocaine would not have the deleterious consequences, e.g., vascular collapse anticipated for an analogous agent that suddenly blocked the actions of alcohol or heroine. At the same time, an agent capable of blocking the actions of cocaine could through a diminution in the perceived availability of the drug actually diminish the incidence or magnitude of craving. At the moment, no treatment for cocaine addiction exists and relapse is the rule. Up to 90% of cocaine addicts in treatment use cocaine while in treatment. Motivated individuals who enlist in treatment programs relapse repeatedly, but if they persist, as many as 50% can be in remission at the end of 1 year. With abstinence, cravings gradually diminish in frequency and intensity, the process of extinction. A treatment for cocaine addiction that could be applied over a period of 4– 6 months would have an excellent chance of fostering remission. The potential utility of such an agent can be appreciated by considering the experience with methadone in the treatment of heroin addiction. Without methadone, the success rate for heroin treatment programs is approximately 20– 30%, but rises to 70– 80% when methadone maintenance is included. The pattern of initial stimulant use and the transition to heavy abuse is well documented for cocaine (Denison et al., 1998). After a first exposure, a typical pattern is one of stable intermittent use. Then after some period of time, usually years and perhaps as many as 5, there is a sudden conversion to heavy use and hard-core addiction. This transition is often associated with an increase in the supply of the drug. But in this latest stimulant epidemic, the transition has been accelerated due to an improved delivery system, the smoking of crack. To appreciate the role of delivery and route of administration on cocaine addiction, we need to consider the elementary chemistry of cocaine. Cocaine hydrochloride is the form of cocaine found in coca leaves. The hydrochloride salt is soluble in water and it is the form that is snorted in the course of nasal administration and injected in intravenous
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administration. If cocaine hydrochloride is dissolved in water, treated with a base such as bicarbonate and extracted with an organic solvent, then evaporation of the separated organic phase yields a plate-like residue that cracks when tapped, i.e. ‘‘crack’’ cocaine or the free base of cocaine. The important property unique to the free base is its potential for volatilization on heating. In contrast to cocaine hydrochloride, which decomposes upon heating, the free base is efficiently volatilized by smoking. Because the surface area of the lungs is similar to that of a tennis court, the absorption of the smoked crack cocaine can be extremely rapid and of equivalent efficiency to an intravenous injection of cocaine hydrochloride. Intravenous administration is at once more potent that nasal but also an impediment to widespread use of a drug and, thus, the smoking of crack cocaine is especially pernicious and greatly expands the potential pool of addicts with access to a most addictive formulation (Verebey and Gold, 1988). The difference in these various forms of administration can be seen in the pharmacokinetic curves depicting plasma concentrations versus time. The oral administration of cocaine hydrochloride results in a slow rise to a peak level of the drug at f 60 min. The same dose of cocaine hydrochloride administered nasally results in a more rapid rise and a peak level achieved at f 30 min because cocaine hydrochloride constricts the blood vessels of the nasal mucosa, it actually retards its own absorption. In contrast, the intravenous injection of cocaine hydrochloride results in rapid rise to a peak level and a higher peak than that of oral or intranasal administration at the same dose. The smoking of crack cocaine at this dose results in a rapid rise to a peak level that is virtually identical to intravenous administration. The relationship of pharmacokinetics to physiologic effect has been studied and the concordance between a peak plasma level due to an intravenous injection and a peak effect, for example, an increase in heart rate, is well documented (Verebey and Gold, 1988). This increase in heart rate is mediated by the central nervous system and is not a direct effect on the heart. Qualitatively similar relationships have been established for cocaine peak plasma concentrations and responses to common cocaine use, such as perceived euphoria. Interestingly, the decline in physiologic response, for example the increase in heart rate,
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is more rapid than the decline in the plasma concentration, and this discordance reflects the tachyphylaxis that occurs in the course of a single administration of the drug. Parameters important for physiological and behavioral effects include the peak serum level, but also the rate of rise to the peak and the baseline from which the rise occurs. It is the rapid rise to peak serum concentration upon the smoking of crack that results in a rapid rise in the central nervous system concentration and profound reinforcing effects. Cocaine is thought to mediate reinforcement of self-administration—the hallmark of addiction—by increasing neurotransmission in the mesolimbocortical reward pathway. Cocaine’s actions have been localized to the nucleus accumbens of the striatum and, even more specifically, to a dopaminergic synapse in the nucleus acumbens (Leshner and Koob, 1999). This dopaminergic reward pathway evolved in preconscious life to be activated by behaviors conducive to survival, such as finding food or a mate. After an appropriate latency period, this pathway would provide a drive for the repetition of that beneficial behavior. Cocaine, by activating this pathway, causes that same drive for repetition but now the repetition is entrained to drug seeking and drug taking behavior. In addition to this drive to repeat, there is, for conscious life, the subjective experience of euphoria. The specific site of action of cocaine within the synapse is known to be the dopamine reuptake transporter. In dopaminergic neurotransmission, dopamine is released from a presynaptic neuron and its binding to postsynaptic dopamine receptors causes continued neurotransmission. Dopaminergic neurotransmission is turned off by the reuptake of dopamine into the presynaptic nerve terminal, a process driven by the cotransport of sodium and chloride. Cocaine’s amplification of dopaminergic neurotransmission occurs because it blocks the dopamine reuptake transporter. In some cases, this blockade appears competitive with dopamine; in others, it appears competitive with sodium binding and transport (Gantenberg and Hageman, 1992). Regardless of the molecular details, the competitive nature of this inhibition has important consequences that can be appreciated by considering the opiate receptor. Heroin binds to opiate receptors that are also present in the reward pathway thereby causing activation of neurotransmission that is the basis of heroin’s addictive effect. By modifying the
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heroin structure, organic chemists can produce a drug such as naltrexone, which binds tightly but lacks the full array of binding interactions that cause activation; the result is a heroin blocker for the treatment of overdose. Other modifications in opiate structures can provide methadone for the treatment of addiction. But consider the dopamine reuptake transporter; in this case, dopamine is the natural agonist and cocaine is the antagonist. Analogs of cocaine produced by the organic chemist capable of displacing cocaine from its binding site, will nonetheless inhibit dopamine transport. While this mapping of cocaine binding to either dopamine or sodium binding sites is not point for point, the difficulties inherent in blocking a competitive blocker have impeded the development of useful cocaine antagonists and partial agonists. As an alternative to the classic central approach to antidrug therapeutics, we considered the possibility of peripheral blockade, i.e., the use of a binder to intercept a toxic substance before it has an opportunity to reach its receptor. Antibodies—evolved for surveillance in the circulation and for binding of foreign proteins—were first investigated as peripheral blockers by Schuster et al. (Bonese et al., 1974) in rhesus monkey self-administering heroin. Rhesus monkeys addicted to heroin—and also to cocaine as a control addiction—were immunized with an opiate attached to a carrier protein. When an animal with a high titer of opiate-neutralizing antibodies was reintroduced into the self-administration protocol, cocaine self-administration was unaffected but heroin selfadministration was markedly blunted. When the serum titers of antiopiate declined, self-administration resumed. The conclusions of this trial were that, first, the binding of drug by antibody was sufficiently rapid to intercept intravenously administered drug before it partitioned into the central nervous system to exert a reinforcing effect. Second, the relevant antibody concentration appeared to be circulating antibody because the titers of antibody in cerebral spinal fluid were very low. And third, antibody is depleted by the act of binding and at lower titers self-administration resumes. A solution to the problem of stoichiometric binding would be a binder that not only bound but also destroyed what it bound to. The utility of catalytic degradation was underscored in a study of PETlabeled 11C-cocaine binding to baboon brain by
Gatley et al. (1990, 1994, 1998). Comparing the binding of ( ) cocaine, the natural form of cocaine, to its mirror image, the (+) enantiomer, Gatley expected to see specific uptake for ( ) cocaine in the striatum and only nonspecific uptake with (+) cocaine, with the difference between those two defining the specific binding sites. However, no trace of (+) cocaine was observed in the baboon brain, despite the fact that its acid –base properties, lipophilicity and CNS partition coefficient are identical to those of ( ) cocaine. Assays of ( ) and (+) cocaine in baboon blood showed a well-defined peak for ( ) cocaine but no discernable peak for (+) cocaine when the resolution was in minutes. A circulating esterase, butyrylcholinesterase (BuChE), was ultimately shown to be responsible for the degradation of (+) cocaine in seconds. Butyrylcholinesterase metabolizes ( ) cocaine, the active form of cocaine with a half-life of 10 –20 min but by chance, metabolizes (+) cocaine a 1000-fold more rapidly, with a half-life of approximately 5 s. These findings demonstrate that a circulating cocaine esterase can metabolize intravenously administered cocaine with sufficient rapidity that it never partitions into the brain (Stewart et al., 1977, 1979; Xie et al., 1999). BuChE has a plasma half-life of days, and so while a potentially useful treatment for acute cocaine overdose, its utility for treating addiction was likely to be limited, since protection lasting for weeks is essential for a population with a high propensity to recidivism. For this reason, we considered catalytic antibodies as a class of biocompatible artificial enzymes with programmable specificity and the potential for prolonged half-life. The basic principles underlying antibody catalysis can be appreciated by considering the hydrolysis of an ester. Hydrolysis of a triagonal planar ester proceeds through a tetrahedral intermediate to yield a carboxylic acid and an alcohol. According to transition state theory, the highest point on the energy diagram, the transition state, resembles the intermediate closest to it in energy, in this case the tetrahedral intermediate. Most simply, a natural enzyme is a structure that stabilizes the transition state of a chemical reaction over the ground state starting material. In the case of ester hydrolysis, an enzyme would possess a binding pocket that stabilizes the charged tetrahedral transition state over the uncharged triagonal planar ground state. This stabilization lowers the energy to the transition
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state and lowered energy results in rate acceleration. As proposed by Jencks (1969), and realized by Richard Lerner (Tramontano et al., 1986), and independently by Peter Schultz (Pollack et al., 1986), the stable analog of an evanescent transition state could elicit antibodies that favor binding of the transition state structure over the ground state structure. These antibodies placed in the presence of substrate, stabilize the transition state, accelerate the reaction and function as a catalyst. The application of catalytic antibodies to the problem of cocaine requires that the transformation of cocaine yield inactive products. The large epitope provided by the benzoyl ester, rendered this site a superior target, and the products of benzoyl hydrolysis, ecgonine methyl ester and benzoic acid, are nontoxic and nonreinforcing. A second consideration—that the desired chemical transformation fall within the purview known for antibody-based catalysts—was also satisfied. Among the over 50 transformations known for antibody catalysis, ester hydrolysis is one of the most facile. Thus, both the general chemistry and specific target supported the antibody-catalyzed degradation of cocaine.
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3. Methodology Substitution of the uncharged triagonal planar benzoyl ester carbonyl with a charged tetrahedral phosphonate monoester provided a high fidelity transition state analog (TSA) of cocaine benzoyl ester hydrolysis (Fig. 1). Because antibodies are not elicited by small molecules alone, a tether was constructed for attachment to carrier protein. Three tether sites were easily accessible, the first at the carbomethoxyl group (TSA1), the second at the 4V position of the phenyl group (TSA2) and the third at the tropane methyl group (TSA3). We favored attachment at the carbomethoxyl group because the tropane nitrogen and the phenyl group remain unencumbered for strong binding interactions. However, each tether site exposes different epitopes to the immune system, thus, we constructed all three. We envisioned the attachment to carrier protein through an activated ester and protection of the phosphonate monoester as a diester, until the penultimate step in the synthesis. Using our unique tetrazole catalysis method (Zhao and Landry, 1992; Yang et al., 1996), we prepared TSA1, TSA2
Fig. 1. Hydrolysis of cocaine at the benzoyl ester and the corresponding phosphonate monoester analogs.
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Fig. 2. Representative synthesis of one of the TSAs—TSA1.
and TSA3 (Fig. 2). The core phosphonate monoester structure was identical in each, and only the tether sites varied (Fig. 1). The nine-atom tether contained a 14 C label to permit easy assessment of the degree of incorporation into carrier protein (Fig. 2). Mice were immunized with each analog conjugated to BSA, and high-titer antisera were elicited by each. Monoclonal antibodies were prepared by standard protocols, and hybridomas secreting analog-specific antibodies were selected by an enzymelinked immunosorbent assay (ELISA). All IgG antianalog antibodies were prepared in ascites or cell culture, and purified by protein A affinity column chromatography (Landry et al., 1993; Yang et al., 1996). The phosphonate monoester was an effective immunogen and an assay was required to determine which of the antianalog antibodies was also an anticocaine esterase. We sought a sensitive assay that would not alter the chemical structure of the molecule and devised an assay based on the hydrolysis of cocaine radiolabel on the benzoyl group. After the acidification of the reaction, the benzoic acid product appears in the organic phase, and unreacted 14C-cocaine remains in the aqueous phase. Using this assay we identified 3B9 and 6A12 as the first artificial cocaine hydrolysases (Landry et al., 1993). Compared to controls, both antibodies released radiolabeled benzoic acid above background, and the release of benzoic acid was inhibited by free transition state analog (free TSA). Using analogs TSA1, TSA2 and TSA3, a total of nine
catalytic antibodies with saturation kinetics and the first order rate constant (Kcat) were obtained from the 107 antianalog antibodies elicited (Yang et al., 1996). TSA1 yielded six out of 50 antianalog antibodies; TSA2 yielded 1 out of 8 and TSA3 yielded 2 out of 49. All catalysts were inhibited by free TSA at 50 AM but not by an inhibitor of serum esterases, eserine, at 1 mM. The most potent at pH 8 was 15A10, with a Km of 220 AM and a Kcat 2.3 per min.
4. Results When we cloned and sequenced the complementarity determining regions (CDRs) of the light chains and heavy chains of the nine catalytic antibodies, we identified several structural classes. A comparison of the CDRs of the antibodies showed four discrete nonoverlapping families that were elicited specifically by TSA1 (3B9 – 6A12 – 2A10 and 9A3 – 19G8 – 15A10) and by TSA3 (8G4G and 8G4E). TSA2 yielded one antibody 12H1 highly homologous to the 3B9 family from TSA1 and without homology to the antibodies derived from TSA3. These structural families overlapped in part with two broad groups defined by competitive ELISA (Yang et al., 1996). Competitive ELISA defined a group consisting of Mabs 3B9, 6A12, 2A10, and 12H1 that displayed high affinity for TSA1, moderate affinity for TSA2, and very low affinity for TSA3. All members of this group could have been elicited by TSA1; the one
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antibody derived from TSA2, Mab 12H1, bound TSA1 with even greater affinity than TSA2. Nonetheless, most if not all members of the group could likely have been elicited by TSA2 since the range of affinities for TSA2 overlapped with the range of affinities for the TSAs that elicited each antibody. In contrast, the very low affinity of TSA3 for every member of this group suggests that TSA3 could not yield any member of the group. Thus, a strategy to obtain catalytic antibodies against cocaine based only on a TSA tethered at the tropane nitrogen would have failed to identify this group of antibodies (Yang et al., 1996). The second group defined by competitive ELISA consisted of five catalytic antibodies from three structural families: 9A3 – 19G8 – 15A10 derived from TSA1; 8G4G and 8G4E from TSA3. These five antibodies displayed equally high affinity for TSA1 and TSA3, and in principle either TSA1 or TSA3 could have elicited every catalytic antibody in this group. None of these antibodies could have been obtained with TSA2 and, thus, three of the four structural families would not have been identified with this conjugate. TSA1 elicited the most active catalytic antibody, Mab 15A10. Moreover, on the basis of the high affinity for TSA1 for all nine catalytic antibodies, TSA1 could plausibly have elicited every antibody described. This result was unexpected but not a definitive endorsement of TSA1 as the preferred analog. With more aggressive screening, TSA2 or TSA3 may ultimately yield a more active antibody not recognized by TSA1. The failure of a TSA (e.g., TSA2) to bind to a catalytic antibody (e.g., 15A10) derived from an alternate immunogenic conjugate confirmed that the tether site limits the antibodies produced and supports the general strategy of varying the site of attachment to carrier protein.
5. Characteristics of Mab 15A10 Mab 15A10 was evaluated for susceptibility to inhibition or inactivation that might limit its effectiveness in vivo. The fact that Mab 15A10 showed no affinity to TSA2 suggested that the phenyl group of cocaine might occupy a deep binding pocket that was
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inaccessible because of the TSA2 tether on the benzene ring. The alcohol hydrolysis product ecgonine methyl ester showed no inhibition up to 1 mM. Benzoic acid did inhibit with a Ki of f 250 AM. However, in human benzoic acid levels are markedly suppressed by reaction with glycine to form hippuric acid (Ambrew, 1985). Perhaps due to a difference in charge, the acid metabolite of cocaine, benzoyl ecgonine, showed no inhibition of catalysis. Deactivating side reactions in the course of repetitive turnover was excluded by demonstrating that after 6 h and >200 turnovers, the Kcat of Mab 15A10 remained >95% of its baseline value. Thus, Mab 15A10 possesses characteristics useful for an in vivo catalyst.
6. The effect of Mab 15A10 in vivo Mab 15A10 was tested in several models of cocaine intoxication, overdose and addiction. The in vivo effectiveness of Mab 15A10 was explored in its capacity to block the cardiovascular effects of acute cocaine administration in mouse (Briscoe et al., 2001). Balb/c mice, implanted with a femoral artery catheter for mean arterial pressure (MAP) monitoring, were pretreated with Mab 15A10 (10, 32, 100 and 300 mg/ kg i.v.) or vehicle prior to cocaine injection (100 mg/ kg i.p.). Increases in MAP ( f 25 mm Hg) following cocaine injection were dose-dependently attenuated by Mab 15A10 (Fig. 3). A time course analysis for Mab 15A10’s effect was also conducted in which either vehicle or 100 mg/kg Mab 15A10 was infused 1, 3, 10 and 30 days prior to cocaine treatment. The antibody was effective in attenuating cocaine-induced increases in MAP at all pretreatment times except the 30-day time point, when compared to vehicle-pretreated mice. Mab 15A10 reduced mortality at some of the time points studied. In addition, a pharmacokenetic experiment was conducted in order to determine whether any observed alterations in cocaineinduced MAP responses were related to changes in plasma cocaine or ecgonine-methyl ester levels following antibody administration. Plasma cocaine levels were significantly decreased early in the recording session, whereas the levels of ecgonine methyl ester significantly increased. These results demonstrated a significant attenuation of the cocaine-induced increase in blood pressure with Mab 15A10 pretreatment and
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Fig. 3. Mab 15A10 pretreatment dose response. Group averages ( F S.E.M.) of mean arterial pressure change from baseline for mAb 15A10 pretreatment, following 100 mg/kg i.p. cocaine, are plotted as a function of minutes following the cocaine injection. Each point represents the mean of eight mice.
suggest that the antibody has a relatively long duration of action in antagonizing the cardiovascular effects of cocaine that is related, at least in part, to its capacity to augment cocaine metabolism. To assess the effect of Mab 15A10 in a rodent model of cocaine overdose, we utilized a method based on coinfusion of catecholamines (Mets et al., 1996). The toxicity of cocaine can vary significantly among individuals depending on endogenous catecholamine levels and, thus, we standardized catecholamine levels through intravenous infusion [norepinephrine (0.725 Ag/kg/min), epinephrine (0.44 Ag/kg/min), and dopamine (0.8 Ag/kg/min)] in conscious, unrestrained animals. For continuously coinfused cocaine (1 mg/kg/ min), the LD50 was 10 mg/kg and the LD90 was 16 mg/kg. Two experiments were derived to determine the protective effects of Mab 15A10 against cocaine toxicity (Mets et al, 1998). In the first, Sprague – Dawley rats (350 –400 g) were fitted with femoral arterial and venous catheters under pentobarbital anesthesia and after 24 h, arterial pressure was transduced, and saline
or antibody (5, 15 or 50 mg/kg) was administered. This administration was followed after 15 min by coinfusion of cocaine (1 mg/kg/min) and catecholamines. The infusion was continued for 16 min to deliver the LD90 of cocaine, unless the animals expired earlier. Rats pretreated with Mab 15A10 showed a significant ( P < 0.001) dose-dependent increase in survival after the LD90 infusion (Fig. 4). Four of five animals receiving 15 mg/kg antibody, and all of five receiving 50 mg/kg antibody, survived. In contrast, all eight untreated rats expired before the cocaine infusion was complete. In the second experiment, Sprague –Dawley rats were given Mab 15A10 100 mg/kg (n = 4), Mab 1C1 100 mg/kg (n = 4) (a noncatalytic anticocaine antibody), or saline (n = 17) in equal volume. After 15 min, cocaine was infused intravenously at a rate of 1 mg/kg/min with catecholamines as above until cardiopulmonary arrest. The dose of cocaine at seizure averaged 9.48 mg/kg for saline controls and 32.5 mg/ kg for animals treated with Mab 15A10 ( P < 0.01). The mean lethal dose of cocaine was also increased less than threefolds, from 11.5 mg/kg of cocaine for controls to 37.0 mg/kg for the Mab 15A10 group ( P < 0.01) (Fig. 5A). Simple binding was an unlikely explanation for the effectiveness of Mab 15A10
Fig. 4. Log dose – response relationship for Mab 15A10 on rats’ survival after intravenous infusion of an LD90 (16 mg/kg) cocaine.
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Fig. 6. Pattern of intravenous cocaine (A), saline (B) or cocaine 1 Mab 15A10 (C) self-administration in a single rat. Each vertical line within the panels indicates a single injection, obtained on a fixed ratio, 5 time-out 10-s schedule of cocaine delivery. The three panels show infusion patterns from three consecutive sessions.
Fig. 5. Saturation of Mab 15A10 with cocaine. Mean cocaine dose at seizure and at death (A). Plasma concentration of ecgonine methyl ester (EME) and cocaine at death (B). A significant difference between the saline control group and the 15A10 group was determined using the Mann – Whitney U-test for unpaired samples.
because an antibody, Mab 1C1, that binds cocaine with comparable affinity, but is noncatalytic, was not effective. To further demonstrate catalysis in vivo, plasma concentrations of cocaine metabolites were measured and the concentration of ecgonine-methyl ester was found to be more than 10-times higher in rats treated with Mab 15A10 than in those treated with saline or the cocaine-binding antibody 1C1 (Fig. 5B). To evaluate the reinforcing effects of cocaine, Sprague – Dawley rats were trained in operant conditioning chambers (Mets et al, 1998). The rats were prepared with chronic indwelling intravenous catheters and trained to press on levers and receive intravenous injections of 0.3 mg/kg/injection cocaine during 1-h daily sessions. Cocaine maintained regular patterns of lever pressing and when saline was substituted for cocaine, lever-pressing decreased rapidly during the session (Fig. 6A and B). Mab 15A10 blocked completely the reinforcing effects of intra-
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venous cocaine in the rat; both the number of cocaine injections and pattern of responding were similar to those after saline substitution. The saline-like pattern of behavior was not due to Mab 15A10 simply disrupting behavior in general, because it did not alter milk reinforcement. The effect was also not a nonspecific effect on the dopaminergic reward pathway because Mab 15A10 did not alter the pattern or amount of bupropion self-administered by the rats. A within-session multiple-dose protocol was also used wherein rats were allowed access to saline or one of six doses of cocaine [0 (saline), 0.015, 0.03, 0.06, 0 (saline), 0.125, 0.25, or 0.5 mg/kg/injection] each hour in the order stated (Baird et al., 2000). After demonstrating stable dose – response curves over 3 consecutive days, rats were given 30-min pretreatments of saline or Mab15A10, (10, 30, or 100 mg/kg i.v.). Whereas the acute 10 mg/kg Mab 15A10 administration did not alter the cocaine dose – response curve relative to saline, both 30 and 100 mg/kg antibody pretreatments produced statistically significant decreases in the number of injections self-administered within the 0.03, 0.06 and 0.125 mg/kg dose components of the multiple-dose schedule (all P < 0.01). The 100 mg/kg Mab 15A10 pretreatment resulted in an increase in the number of self-administered cocaine infusions at the highest dose of
Fig. 7. Cocaine dose – response curves after pretreatments of mAb 15A10. The effects of 10, 30 and 100 mg/kg i.v. pretreatments of Mab 15A10 on operant responding for intravenous cocaine injections of a multiple-dose schedule are illustrated. Each point is the mean number of injections self-administered at a given dose of cocaine available within a single, 50-min component of the selfadministration session.
cocaine available on the schedule (0.5 mg/kg/injection) relative to both the 10 and 30 mg/kg Mab 15A10 pretreatment groups ( P < 0.004) (Fig. 7) consistent with peripheral blockade of cocaine that was surmounted at high cocaine dose.
7. Discussion The animal data demonstrate that a catalytic antibody raised to a transition-state analog of cocaine hydrolysis is able to increase the rate of cocaine degradation in vivo, protect against cocaine’s toxic effects, and its reinforcing effects. The potential usefulness of an effective anticocaine medication can be inferred from decades of experience with the pharmacologic treatment of heroin addiction. Heroin treatment programs that employ both counseling and methadone administration report abstinence rates at 60% and 80%, in contrast to 10% and 30% for programs that rely only on nonpharmacological approaches. In contrast to methadone, a catalytic antibody would not be expected to enter the brain and therefore would not be psychoactive. Whereas simple blockers of heroin such as naltrexone are useful for treating opiate overdose, they are not generally useful in treating addiction because the blocker requires daily administration, is easily discontinued, and rapidly clears. Thus, were an analogous blocker of cocaine developed, as a small molecule it would also likely be short-lived in vivo and, in the absence of a depot formulation, of limited value for addiction. In contrast, natural antibodies have plasma half-lives of 3 weeks and a humanized Mab with a half-life sufficient for a dosing interval of several weeks would provide an appropriate treatment for a population prone to recidivism and relapse. Since our original report on anticocaine catalytic antibodies (Landry et al., 1993), others have described variations on the concept of intercepting cocaine before the drug reaches its receptors. For example, intraperitoneal administration of the enzyme cholinesterase was shown to inhibit toxicity due to intraperitoneal cocaine in mice (Hoffman et al., 1996), and this enzyme has been proposed as treatment for cocaine overdose. Also, immunization with cocaine analogs designed to elicit noncatalytic anticocaine antibodies were shown to diminish cocaine-induced psychomotor
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effects (Carrera et al., 1995, 2000) and reinforcement (Fox et al., 1996) in rats and has been proposed as a vaccine for cocaine addiction (Fox, 1997). However, catalytic antibodies are likely to be longer-lived in plasma than natural enzymes and, in contrast to typical antibodies that can form long-lived complexes with antigen, catalytic antibodies are not susceptible to depletion by the act of complex formation with cocaine. Thus, catalytic antibodies have the unique potential to treat both the acute and chronic aspects of cocaine abuse.
8. Conclusion Using rationally designed transition-state analogs (TSA1, TSA2 and TSA3) for the hydrolysis of cocaine’s benzoyl ester, we elicited several distinct structural and functional families of anticocaine catalytic antibodies. TSA1 and TSA3 were found to be particularly effective analogs. Mab 15A10, the most potent of the catalytic antibodies elicited, was found to be sufficiently active to prevent cocaine’s reinforcing and toxic effects in rodents. MAb 15A10 is a suitable candidate for humanization and optimization by mutagenesis in preparation for clinical trials.
References Ambrew, J., 1985. J. Anal. Toxicol. 9, 241. Baird, T.J., Deng, S.X., Landry, D.W., Winger, G., Woods, J.H., 2000. Natural and artificial enzymes against cocaine: I. Monoclonal antibody 15A10 and the reinforcing effects of cocaine in rats. J. Pharmacol. Exp. Ther. 295 (3), 1127. Bonese, K.F., Wainer, B.H., Fitch, F.W., Rothberg, R.M., Schuster, C.R., 1974. Changes in heroin self-administration by a rhesus monkey after morphine immunization. Nature (Lond.) 252 (5485), 708. Briscoe, R.J., Jeanville, P.M., Cabrera, C., Baird, T.J., Woods, J.H., Landry, D.W., 2001. A catalytic antibody against cocaine attenuates cocaine’s cardiovascular effects in mice: a dose and time course analysis. Int. Immunopharmacol. 1 (6), 1189. Carrera, M.R., Ashley, J.A., Parson, L.H., Wirsching, P., Koob, G.F., Janda, K.D., 1995. Suppression of psychoactive effects of cocaine by active immunization. Nature (Lond.) 378, 727. Carrera, M.R., Ashley, J.A., Zhou, B., Wirsching, P., Koob, G.F., Janda, K.D., 2000. Cocaine vaccines: antibody protection against relapse in a rat model. Proc. Natl. Acad. Sci. U. S. A. 97, 6202.
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Denison, M.E., Paredes, A., Bacal, S., Gawin, F.H., 1998. Psychological and psychiatric consequences of cocaine. In: Tarter, R.E., Ammerman, R.T., Ott, P.J. (Eds.), Handbook of Substance Abuse: Neurobehavioral Pharmacology. Plenum, New York, pp. 201 – 213. Fox, B.S., 1997. Development of a therapeutic vaccine for the treatment of cocaine addiction. Drug Alcohol Depend. 48, 153 – 158. Fox, B.S., Kantak, K.M., Edwards, M.A., Black, K.M., Bollinger, B.K., Botka, A.J., French, T.L., Thompson, T.L., Schad, V.C., Greenstein, J.L., 1996. Nat. Med. 2, 1129. Gantenberg, N.S., Hageman, G.R., 1992. Cocaine-enhanced arrhythmogenesis: neural and nonneural mechanisms. Can. J. Physiol. Pharmacol. 70, 240. Gatley, S.J., MacGregor, R.R., Fowler, J.S., Wolf, A.P., Dewey, S.L., Schlyer, D.J., 1990. Rapid stereoselective hydrolysis of (+)-cocaine in baboon plasma prevents its uptake in the brain: implications for behavioral studies. J. Neurochem. 54 (2), 720. Gatley, S.J., Yu, D.W., Fowler, J.S., MacGregor, R.R., Schlyer, D.J., Dewey, S.L., Volkow, N.D., 1994. Studies with differentially labeled [11C]cocaine, [11C]norcocaine, [11C]benzoylecgonine, and [11C]-and 4V-[18F]fluorococaine to probe the extent to which [11C]cocaine metabolites contribute to PET images of the baboon brain. J. Neurochem. 62 (3), 1154. Gatley, S.J., Gifford, A.N., Volkow, N.D., Fowler, J.S., 1998. Pharmacology of cocaine. In: Tarter, R.E., Ammerman, R.T., Ott, P.J. (Eds.), Handbook of Substance Abuse: Neurobehavioral Pharmacology. Plenum, New York, pp. 161 – 185. Hoffman, R.S., Morasco, R., Goldfrank, L.R., 1996. J. Toxicol. Clin. Toxicol. 34, 259. Jencks, W.P., 1969. Catalysis in Chemistry and Enzymology. McGraw-Hill, New York. Landry, D.W., Zhao, K., Yang, G.X.Q., Glickman, M., Georgiadis, M., 1993. Antibody catalyzed degradation of cocaine. Science (Wash. D.C.) 259, 1899. Leshner, A.I., Koob, G.F., 1999. Drugs of abuse and the brain. Proc. Assoc. Am. Physicians 111, 99. Mets, B., Jamdar, S., Landry, D.W., 1996. Life Sci. 59, 2021. Mets, B., Winger, G., Cabrera, C., Seo, S., Jamdar, S., Yang, G., Zhao, K., Briscoe, R.J., Almonte, R., Woods, J.H., Landry, D.W., 1998. A catalytic antibody against cocaine prevents cocaine’s reinforcing and toxic effects in rats. Proc. Natl. Acad. Sci. U. S. A. 95, 10176. Office of the National Drug Control Policy, 1996. The National Drug Control Strategy Executive Office of the President of the United States, Ed.: 41 – 51, Washington, DC. Pollack, S.J., Jacobs, J.W., Schultz, P.G., 1986. Selective chemical catalysis by an antibody. Science (Wash. D.C., 1883) 234 (4783), 1570. Stewart, D.J., Inaba, T., Tang, B.K., Kalow, W., 1977. Hydrolysis of cocaine in human plasma by cholinesterase. Life Sci. 20, 1557. Stewart, D.J., Inaba, T., Lucassen, M., Kalow, W., 1979. Cocaine metabolism: cocaine and norcocaine hydrolysis by liver and serum esterases. Clin. Pharmacol. Ther. 25, 464. Tramontano, A., Janda, K.D., Lerner, R.A., 1986. Catalytic antibodies. Science 234 (4783), 1566. Verebey, K., Gold, M.S., 1988. From coca leaves to crack: the
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effects of dose and routes of administration in abuse liability. Psychiatr. Ann. 18, 513. Xie, W., Altamirano, C.V., Bartels, C.F., Speirs, R.J., Cashman, J.R., Lockridge, O., 1999. An improved cocaine hydrolase: the A328Y mutant of human butyrylcholinesterase is 4-fold more efficient. Mol. Pharmacol. 55, 83.
Yang, G., Chun, J., Arakawa, U.H., Wang, X., Gawinowicz, M.A., Zhao, K., Landry, D.W., 1996. Anti-cocaine catalytic antibodies: a synthetic approach to improved antibody diversity. J. Am. Chem. Soc. 118, 5881. Zhao, K., Landry, D.W., 1992. Tetrazole catalyzed synthesis of phosphonate esters. Tetrahedron 49 (2), 363.
Anticocaine catalytic antibodies Shi Xian Deng, Paloma de Prada, Donald W. Landry * Division of Clinical Pharmacology and Experimental Therapeutics, Department of Medicine, Columbia University, Box 84, 630 W 168th Street, New York, NY 10032, USA Received 27 March 2002; accepted 10 April 2002
Abstract Cocaine mediates its reinforcing and toxic actions through a ‘‘loss of function’’ effect at multiple receptors. The difficulties inherent in blocking a pleiotropic blocker pose a great obstacle for the classical receptor – antagonist approach and have contributed to the failure (to date) to devise specific treatments for cocaine overdose and addiction. As an alternative, we have embarked on an investigation of catalytic antibodies, a programmable class of artificial enzyme, as ‘‘peripheral blockers’’— agents designed to bind and degrade cocaine in the circulation before it partitions into the central nervous system to exert reinforcing or toxic effects. We synthesized transition-state analogs of cocaine’s hydrolysis at its benzoyl ester, immunized mice, prepared hybridomas and developed the first anticocaine catalytic antibodies with the capacity to degrade cocaine to nonreinforcing, nontoxic products. We subsequently identified several families of anticocaine catalytic antibodies and found that the most potent antibody possessed sufficient activity to block cocaine-induced reinforcement, organ dysfunction and sudden death in rodent models of addiction, toxicity and overdose, respectively. With the potential to promote cessation of use, prolong abstinence and provide a treatment for acute overdose, the artificial enzyme approach comprehensively responds to the problem of cocaine. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cocaine; Addiction; Toxicity
1. Introduction The problem of cocaine has been an intractable one for the organic chemist. The crux of the problem stems from the fact that cocaine itself is a blocker, and analogs of cocaine that can displace it from its binding site, still block function just as cocaine would. The difficulties inherent in blocking
* Corresponding author. Tel.: +1-212-305-1890; fax: +1-212305-3475. E-mail address: [email protected] (D.W. Landry).
a blocker led us to an alternative approach based on peripheral blockade rather than central blockade. Regardless of the route by which an addictive drug enters the body, the drug must pass through the blood to reach the brain. An agent that intercepted a drug in the peripheral circulation before it partitions into the central nervous system could circumvent limitations on central blocks that act directly within the central nervous system. For peripheral blocking agents, we chose catalytic antibodies, biocompatible artificial enzymes that are programmable to particular molecular targets and have the potential for an extended plasma half-life.
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 2 3 7 - 5
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2. Background For over a thousand years, Peruvian Indians chewed Coca leaves, a practice that increased their energy and enabled them to work with increased vigor at high altitude. However, it was not until the 1850s when German chemists purified the active constituent of coca leaves that cocaine arose as a potent drug of abuse. Cocaine was initially popularized by Sigmund Freud as a treatment for opium addiction and he did succeed in curing opiate addicts but in their place appeared a legion of cocaine addicts. Enthusiasm for cocaine culminated in the cocaine pandemic of the 1890s. Cocaine fell acutely from favor but appreciation for its potential faded and widespread addiction recrudesced again in the 1920s. Stimulant abuse emerged once more in the 1950s with amphetamine as the stimulant of choice, and again in the 1960s with the rise of metamphetamine. The fifth and current stimulant epidemic began in the 1980s with the reemergence of cocaine. Renewed use of cocaine was driven in part by an enormous increase in supply and in part by a new method of administration, the smoking of crack cocaine. Over the last 20 years, 60 million Americans have been exposed to cocaine, with approximately 6 million regular users and 2 –3 million hard-core addicts (Office of the National Drug Control Policy, 1996). The clinical characteristics of cocaine underscore its potential for abuse. First, there is a magnification of pleasure. The euphoria induced by cocaine is dosedependent. A consequence of heavy use is progressive social isolation and a transition to binges. Periods of abstinence also have well-defined clinical characteristics. After heavy use, there is a crash, a period of hypersomnolence and dysphoria, usually mild, lasting 12– 96 h and followed by a period of withdrawal marked by anergia (decreased energy) and anhedonia (decreased capacity to experience pleasure). The symptoms of cocaine withdrawal differ markedly from those of heroin or alcohol withdrawal, both of which can be life threatening. In the case of cocaine, withdrawal is principally experienced as craving, and is not associated with vascular collapse. In alcohol withdrawal, an addict can at any given moment indicate the progress of withdrawal, initially intense and diminishing over time. For cocaine addiction, craving is a function of perceived availability of the
drug. Thus, a cocaine addict may feel no desire for the drug, but the sudden introduction of a social cue can precipitate craving. For example, seeing a person who supplies the drug or with whom the drug was used can be a potential precipitant. One consequence of this physiology is that an agent that suddenly blocked the actions of cocaine would not have the deleterious consequences, e.g., vascular collapse anticipated for an analogous agent that suddenly blocked the actions of alcohol or heroine. At the same time, an agent capable of blocking the actions of cocaine could through a diminution in the perceived availability of the drug actually diminish the incidence or magnitude of craving. At the moment, no treatment for cocaine addiction exists and relapse is the rule. Up to 90% of cocaine addicts in treatment use cocaine while in treatment. Motivated individuals who enlist in treatment programs relapse repeatedly, but if they persist, as many as 50% can be in remission at the end of 1 year. With abstinence, cravings gradually diminish in frequency and intensity, the process of extinction. A treatment for cocaine addiction that could be applied over a period of 4– 6 months would have an excellent chance of fostering remission. The potential utility of such an agent can be appreciated by considering the experience with methadone in the treatment of heroin addiction. Without methadone, the success rate for heroin treatment programs is approximately 20– 30%, but rises to 70– 80% when methadone maintenance is included. The pattern of initial stimulant use and the transition to heavy abuse is well documented for cocaine (Denison et al., 1998). After a first exposure, a typical pattern is one of stable intermittent use. Then after some period of time, usually years and perhaps as many as 5, there is a sudden conversion to heavy use and hard-core addiction. This transition is often associated with an increase in the supply of the drug. But in this latest stimulant epidemic, the transition has been accelerated due to an improved delivery system, the smoking of crack. To appreciate the role of delivery and route of administration on cocaine addiction, we need to consider the elementary chemistry of cocaine. Cocaine hydrochloride is the form of cocaine found in coca leaves. The hydrochloride salt is soluble in water and it is the form that is snorted in the course of nasal administration and injected in intravenous
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administration. If cocaine hydrochloride is dissolved in water, treated with a base such as bicarbonate and extracted with an organic solvent, then evaporation of the separated organic phase yields a plate-like residue that cracks when tapped, i.e. ‘‘crack’’ cocaine or the free base of cocaine. The important property unique to the free base is its potential for volatilization on heating. In contrast to cocaine hydrochloride, which decomposes upon heating, the free base is efficiently volatilized by smoking. Because the surface area of the lungs is similar to that of a tennis court, the absorption of the smoked crack cocaine can be extremely rapid and of equivalent efficiency to an intravenous injection of cocaine hydrochloride. Intravenous administration is at once more potent that nasal but also an impediment to widespread use of a drug and, thus, the smoking of crack cocaine is especially pernicious and greatly expands the potential pool of addicts with access to a most addictive formulation (Verebey and Gold, 1988). The difference in these various forms of administration can be seen in the pharmacokinetic curves depicting plasma concentrations versus time. The oral administration of cocaine hydrochloride results in a slow rise to a peak level of the drug at f 60 min. The same dose of cocaine hydrochloride administered nasally results in a more rapid rise and a peak level achieved at f 30 min because cocaine hydrochloride constricts the blood vessels of the nasal mucosa, it actually retards its own absorption. In contrast, the intravenous injection of cocaine hydrochloride results in rapid rise to a peak level and a higher peak than that of oral or intranasal administration at the same dose. The smoking of crack cocaine at this dose results in a rapid rise to a peak level that is virtually identical to intravenous administration. The relationship of pharmacokinetics to physiologic effect has been studied and the concordance between a peak plasma level due to an intravenous injection and a peak effect, for example, an increase in heart rate, is well documented (Verebey and Gold, 1988). This increase in heart rate is mediated by the central nervous system and is not a direct effect on the heart. Qualitatively similar relationships have been established for cocaine peak plasma concentrations and responses to common cocaine use, such as perceived euphoria. Interestingly, the decline in physiologic response, for example the increase in heart rate,
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is more rapid than the decline in the plasma concentration, and this discordance reflects the tachyphylaxis that occurs in the course of a single administration of the drug. Parameters important for physiological and behavioral effects include the peak serum level, but also the rate of rise to the peak and the baseline from which the rise occurs. It is the rapid rise to peak serum concentration upon the smoking of crack that results in a rapid rise in the central nervous system concentration and profound reinforcing effects. Cocaine is thought to mediate reinforcement of self-administration—the hallmark of addiction—by increasing neurotransmission in the mesolimbocortical reward pathway. Cocaine’s actions have been localized to the nucleus accumbens of the striatum and, even more specifically, to a dopaminergic synapse in the nucleus acumbens (Leshner and Koob, 1999). This dopaminergic reward pathway evolved in preconscious life to be activated by behaviors conducive to survival, such as finding food or a mate. After an appropriate latency period, this pathway would provide a drive for the repetition of that beneficial behavior. Cocaine, by activating this pathway, causes that same drive for repetition but now the repetition is entrained to drug seeking and drug taking behavior. In addition to this drive to repeat, there is, for conscious life, the subjective experience of euphoria. The specific site of action of cocaine within the synapse is known to be the dopamine reuptake transporter. In dopaminergic neurotransmission, dopamine is released from a presynaptic neuron and its binding to postsynaptic dopamine receptors causes continued neurotransmission. Dopaminergic neurotransmission is turned off by the reuptake of dopamine into the presynaptic nerve terminal, a process driven by the cotransport of sodium and chloride. Cocaine’s amplification of dopaminergic neurotransmission occurs because it blocks the dopamine reuptake transporter. In some cases, this blockade appears competitive with dopamine; in others, it appears competitive with sodium binding and transport (Gantenberg and Hageman, 1992). Regardless of the molecular details, the competitive nature of this inhibition has important consequences that can be appreciated by considering the opiate receptor. Heroin binds to opiate receptors that are also present in the reward pathway thereby causing activation of neurotransmission that is the basis of heroin’s addictive effect. By modifying the
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heroin structure, organic chemists can produce a drug such as naltrexone, which binds tightly but lacks the full array of binding interactions that cause activation; the result is a heroin blocker for the treatment of overdose. Other modifications in opiate structures can provide methadone for the treatment of addiction. But consider the dopamine reuptake transporter; in this case, dopamine is the natural agonist and cocaine is the antagonist. Analogs of cocaine produced by the organic chemist capable of displacing cocaine from its binding site, will nonetheless inhibit dopamine transport. While this mapping of cocaine binding to either dopamine or sodium binding sites is not point for point, the difficulties inherent in blocking a competitive blocker have impeded the development of useful cocaine antagonists and partial agonists. As an alternative to the classic central approach to antidrug therapeutics, we considered the possibility of peripheral blockade, i.e., the use of a binder to intercept a toxic substance before it has an opportunity to reach its receptor. Antibodies—evolved for surveillance in the circulation and for binding of foreign proteins—were first investigated as peripheral blockers by Schuster et al. (Bonese et al., 1974) in rhesus monkey self-administering heroin. Rhesus monkeys addicted to heroin—and also to cocaine as a control addiction—were immunized with an opiate attached to a carrier protein. When an animal with a high titer of opiate-neutralizing antibodies was reintroduced into the self-administration protocol, cocaine self-administration was unaffected but heroin selfadministration was markedly blunted. When the serum titers of antiopiate declined, self-administration resumed. The conclusions of this trial were that, first, the binding of drug by antibody was sufficiently rapid to intercept intravenously administered drug before it partitioned into the central nervous system to exert a reinforcing effect. Second, the relevant antibody concentration appeared to be circulating antibody because the titers of antibody in cerebral spinal fluid were very low. And third, antibody is depleted by the act of binding and at lower titers self-administration resumes. A solution to the problem of stoichiometric binding would be a binder that not only bound but also destroyed what it bound to. The utility of catalytic degradation was underscored in a study of PETlabeled 11C-cocaine binding to baboon brain by
Gatley et al. (1990, 1994, 1998). Comparing the binding of ( ) cocaine, the natural form of cocaine, to its mirror image, the (+) enantiomer, Gatley expected to see specific uptake for ( ) cocaine in the striatum and only nonspecific uptake with (+) cocaine, with the difference between those two defining the specific binding sites. However, no trace of (+) cocaine was observed in the baboon brain, despite the fact that its acid –base properties, lipophilicity and CNS partition coefficient are identical to those of ( ) cocaine. Assays of ( ) and (+) cocaine in baboon blood showed a well-defined peak for ( ) cocaine but no discernable peak for (+) cocaine when the resolution was in minutes. A circulating esterase, butyrylcholinesterase (BuChE), was ultimately shown to be responsible for the degradation of (+) cocaine in seconds. Butyrylcholinesterase metabolizes ( ) cocaine, the active form of cocaine with a half-life of 10 –20 min but by chance, metabolizes (+) cocaine a 1000-fold more rapidly, with a half-life of approximately 5 s. These findings demonstrate that a circulating cocaine esterase can metabolize intravenously administered cocaine with sufficient rapidity that it never partitions into the brain (Stewart et al., 1977, 1979; Xie et al., 1999). BuChE has a plasma half-life of days, and so while a potentially useful treatment for acute cocaine overdose, its utility for treating addiction was likely to be limited, since protection lasting for weeks is essential for a population with a high propensity to recidivism. For this reason, we considered catalytic antibodies as a class of biocompatible artificial enzymes with programmable specificity and the potential for prolonged half-life. The basic principles underlying antibody catalysis can be appreciated by considering the hydrolysis of an ester. Hydrolysis of a triagonal planar ester proceeds through a tetrahedral intermediate to yield a carboxylic acid and an alcohol. According to transition state theory, the highest point on the energy diagram, the transition state, resembles the intermediate closest to it in energy, in this case the tetrahedral intermediate. Most simply, a natural enzyme is a structure that stabilizes the transition state of a chemical reaction over the ground state starting material. In the case of ester hydrolysis, an enzyme would possess a binding pocket that stabilizes the charged tetrahedral transition state over the uncharged triagonal planar ground state. This stabilization lowers the energy to the transition
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state and lowered energy results in rate acceleration. As proposed by Jencks (1969), and realized by Richard Lerner (Tramontano et al., 1986), and independently by Peter Schultz (Pollack et al., 1986), the stable analog of an evanescent transition state could elicit antibodies that favor binding of the transition state structure over the ground state structure. These antibodies placed in the presence of substrate, stabilize the transition state, accelerate the reaction and function as a catalyst. The application of catalytic antibodies to the problem of cocaine requires that the transformation of cocaine yield inactive products. The large epitope provided by the benzoyl ester, rendered this site a superior target, and the products of benzoyl hydrolysis, ecgonine methyl ester and benzoic acid, are nontoxic and nonreinforcing. A second consideration—that the desired chemical transformation fall within the purview known for antibody-based catalysts—was also satisfied. Among the over 50 transformations known for antibody catalysis, ester hydrolysis is one of the most facile. Thus, both the general chemistry and specific target supported the antibody-catalyzed degradation of cocaine.
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3. Methodology Substitution of the uncharged triagonal planar benzoyl ester carbonyl with a charged tetrahedral phosphonate monoester provided a high fidelity transition state analog (TSA) of cocaine benzoyl ester hydrolysis (Fig. 1). Because antibodies are not elicited by small molecules alone, a tether was constructed for attachment to carrier protein. Three tether sites were easily accessible, the first at the carbomethoxyl group (TSA1), the second at the 4V position of the phenyl group (TSA2) and the third at the tropane methyl group (TSA3). We favored attachment at the carbomethoxyl group because the tropane nitrogen and the phenyl group remain unencumbered for strong binding interactions. However, each tether site exposes different epitopes to the immune system, thus, we constructed all three. We envisioned the attachment to carrier protein through an activated ester and protection of the phosphonate monoester as a diester, until the penultimate step in the synthesis. Using our unique tetrazole catalysis method (Zhao and Landry, 1992; Yang et al., 1996), we prepared TSA1, TSA2
Fig. 1. Hydrolysis of cocaine at the benzoyl ester and the corresponding phosphonate monoester analogs.
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Fig. 2. Representative synthesis of one of the TSAs—TSA1.
and TSA3 (Fig. 2). The core phosphonate monoester structure was identical in each, and only the tether sites varied (Fig. 1). The nine-atom tether contained a 14 C label to permit easy assessment of the degree of incorporation into carrier protein (Fig. 2). Mice were immunized with each analog conjugated to BSA, and high-titer antisera were elicited by each. Monoclonal antibodies were prepared by standard protocols, and hybridomas secreting analog-specific antibodies were selected by an enzymelinked immunosorbent assay (ELISA). All IgG antianalog antibodies were prepared in ascites or cell culture, and purified by protein A affinity column chromatography (Landry et al., 1993; Yang et al., 1996). The phosphonate monoester was an effective immunogen and an assay was required to determine which of the antianalog antibodies was also an anticocaine esterase. We sought a sensitive assay that would not alter the chemical structure of the molecule and devised an assay based on the hydrolysis of cocaine radiolabel on the benzoyl group. After the acidification of the reaction, the benzoic acid product appears in the organic phase, and unreacted 14C-cocaine remains in the aqueous phase. Using this assay we identified 3B9 and 6A12 as the first artificial cocaine hydrolysases (Landry et al., 1993). Compared to controls, both antibodies released radiolabeled benzoic acid above background, and the release of benzoic acid was inhibited by free transition state analog (free TSA). Using analogs TSA1, TSA2 and TSA3, a total of nine
catalytic antibodies with saturation kinetics and the first order rate constant (Kcat) were obtained from the 107 antianalog antibodies elicited (Yang et al., 1996). TSA1 yielded six out of 50 antianalog antibodies; TSA2 yielded 1 out of 8 and TSA3 yielded 2 out of 49. All catalysts were inhibited by free TSA at 50 AM but not by an inhibitor of serum esterases, eserine, at 1 mM. The most potent at pH 8 was 15A10, with a Km of 220 AM and a Kcat 2.3 per min.
4. Results When we cloned and sequenced the complementarity determining regions (CDRs) of the light chains and heavy chains of the nine catalytic antibodies, we identified several structural classes. A comparison of the CDRs of the antibodies showed four discrete nonoverlapping families that were elicited specifically by TSA1 (3B9 – 6A12 – 2A10 and 9A3 – 19G8 – 15A10) and by TSA3 (8G4G and 8G4E). TSA2 yielded one antibody 12H1 highly homologous to the 3B9 family from TSA1 and without homology to the antibodies derived from TSA3. These structural families overlapped in part with two broad groups defined by competitive ELISA (Yang et al., 1996). Competitive ELISA defined a group consisting of Mabs 3B9, 6A12, 2A10, and 12H1 that displayed high affinity for TSA1, moderate affinity for TSA2, and very low affinity for TSA3. All members of this group could have been elicited by TSA1; the one
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antibody derived from TSA2, Mab 12H1, bound TSA1 with even greater affinity than TSA2. Nonetheless, most if not all members of the group could likely have been elicited by TSA2 since the range of affinities for TSA2 overlapped with the range of affinities for the TSAs that elicited each antibody. In contrast, the very low affinity of TSA3 for every member of this group suggests that TSA3 could not yield any member of the group. Thus, a strategy to obtain catalytic antibodies against cocaine based only on a TSA tethered at the tropane nitrogen would have failed to identify this group of antibodies (Yang et al., 1996). The second group defined by competitive ELISA consisted of five catalytic antibodies from three structural families: 9A3 – 19G8 – 15A10 derived from TSA1; 8G4G and 8G4E from TSA3. These five antibodies displayed equally high affinity for TSA1 and TSA3, and in principle either TSA1 or TSA3 could have elicited every catalytic antibody in this group. None of these antibodies could have been obtained with TSA2 and, thus, three of the four structural families would not have been identified with this conjugate. TSA1 elicited the most active catalytic antibody, Mab 15A10. Moreover, on the basis of the high affinity for TSA1 for all nine catalytic antibodies, TSA1 could plausibly have elicited every antibody described. This result was unexpected but not a definitive endorsement of TSA1 as the preferred analog. With more aggressive screening, TSA2 or TSA3 may ultimately yield a more active antibody not recognized by TSA1. The failure of a TSA (e.g., TSA2) to bind to a catalytic antibody (e.g., 15A10) derived from an alternate immunogenic conjugate confirmed that the tether site limits the antibodies produced and supports the general strategy of varying the site of attachment to carrier protein.
5. Characteristics of Mab 15A10 Mab 15A10 was evaluated for susceptibility to inhibition or inactivation that might limit its effectiveness in vivo. The fact that Mab 15A10 showed no affinity to TSA2 suggested that the phenyl group of cocaine might occupy a deep binding pocket that was
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inaccessible because of the TSA2 tether on the benzene ring. The alcohol hydrolysis product ecgonine methyl ester showed no inhibition up to 1 mM. Benzoic acid did inhibit with a Ki of f 250 AM. However, in human benzoic acid levels are markedly suppressed by reaction with glycine to form hippuric acid (Ambrew, 1985). Perhaps due to a difference in charge, the acid metabolite of cocaine, benzoyl ecgonine, showed no inhibition of catalysis. Deactivating side reactions in the course of repetitive turnover was excluded by demonstrating that after 6 h and >200 turnovers, the Kcat of Mab 15A10 remained >95% of its baseline value. Thus, Mab 15A10 possesses characteristics useful for an in vivo catalyst.
6. The effect of Mab 15A10 in vivo Mab 15A10 was tested in several models of cocaine intoxication, overdose and addiction. The in vivo effectiveness of Mab 15A10 was explored in its capacity to block the cardiovascular effects of acute cocaine administration in mouse (Briscoe et al., 2001). Balb/c mice, implanted with a femoral artery catheter for mean arterial pressure (MAP) monitoring, were pretreated with Mab 15A10 (10, 32, 100 and 300 mg/ kg i.v.) or vehicle prior to cocaine injection (100 mg/ kg i.p.). Increases in MAP ( f 25 mm Hg) following cocaine injection were dose-dependently attenuated by Mab 15A10 (Fig. 3). A time course analysis for Mab 15A10’s effect was also conducted in which either vehicle or 100 mg/kg Mab 15A10 was infused 1, 3, 10 and 30 days prior to cocaine treatment. The antibody was effective in attenuating cocaine-induced increases in MAP at all pretreatment times except the 30-day time point, when compared to vehicle-pretreated mice. Mab 15A10 reduced mortality at some of the time points studied. In addition, a pharmacokenetic experiment was conducted in order to determine whether any observed alterations in cocaineinduced MAP responses were related to changes in plasma cocaine or ecgonine-methyl ester levels following antibody administration. Plasma cocaine levels were significantly decreased early in the recording session, whereas the levels of ecgonine methyl ester significantly increased. These results demonstrated a significant attenuation of the cocaine-induced increase in blood pressure with Mab 15A10 pretreatment and
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Fig. 3. Mab 15A10 pretreatment dose response. Group averages ( F S.E.M.) of mean arterial pressure change from baseline for mAb 15A10 pretreatment, following 100 mg/kg i.p. cocaine, are plotted as a function of minutes following the cocaine injection. Each point represents the mean of eight mice.
suggest that the antibody has a relatively long duration of action in antagonizing the cardiovascular effects of cocaine that is related, at least in part, to its capacity to augment cocaine metabolism. To assess the effect of Mab 15A10 in a rodent model of cocaine overdose, we utilized a method based on coinfusion of catecholamines (Mets et al., 1996). The toxicity of cocaine can vary significantly among individuals depending on endogenous catecholamine levels and, thus, we standardized catecholamine levels through intravenous infusion [norepinephrine (0.725 Ag/kg/min), epinephrine (0.44 Ag/kg/min), and dopamine (0.8 Ag/kg/min)] in conscious, unrestrained animals. For continuously coinfused cocaine (1 mg/kg/ min), the LD50 was 10 mg/kg and the LD90 was 16 mg/kg. Two experiments were derived to determine the protective effects of Mab 15A10 against cocaine toxicity (Mets et al, 1998). In the first, Sprague – Dawley rats (350 –400 g) were fitted with femoral arterial and venous catheters under pentobarbital anesthesia and after 24 h, arterial pressure was transduced, and saline
or antibody (5, 15 or 50 mg/kg) was administered. This administration was followed after 15 min by coinfusion of cocaine (1 mg/kg/min) and catecholamines. The infusion was continued for 16 min to deliver the LD90 of cocaine, unless the animals expired earlier. Rats pretreated with Mab 15A10 showed a significant ( P < 0.001) dose-dependent increase in survival after the LD90 infusion (Fig. 4). Four of five animals receiving 15 mg/kg antibody, and all of five receiving 50 mg/kg antibody, survived. In contrast, all eight untreated rats expired before the cocaine infusion was complete. In the second experiment, Sprague –Dawley rats were given Mab 15A10 100 mg/kg (n = 4), Mab 1C1 100 mg/kg (n = 4) (a noncatalytic anticocaine antibody), or saline (n = 17) in equal volume. After 15 min, cocaine was infused intravenously at a rate of 1 mg/kg/min with catecholamines as above until cardiopulmonary arrest. The dose of cocaine at seizure averaged 9.48 mg/kg for saline controls and 32.5 mg/ kg for animals treated with Mab 15A10 ( P < 0.01). The mean lethal dose of cocaine was also increased less than threefolds, from 11.5 mg/kg of cocaine for controls to 37.0 mg/kg for the Mab 15A10 group ( P < 0.01) (Fig. 5A). Simple binding was an unlikely explanation for the effectiveness of Mab 15A10
Fig. 4. Log dose – response relationship for Mab 15A10 on rats’ survival after intravenous infusion of an LD90 (16 mg/kg) cocaine.
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Fig. 6. Pattern of intravenous cocaine (A), saline (B) or cocaine 1 Mab 15A10 (C) self-administration in a single rat. Each vertical line within the panels indicates a single injection, obtained on a fixed ratio, 5 time-out 10-s schedule of cocaine delivery. The three panels show infusion patterns from three consecutive sessions.
Fig. 5. Saturation of Mab 15A10 with cocaine. Mean cocaine dose at seizure and at death (A). Plasma concentration of ecgonine methyl ester (EME) and cocaine at death (B). A significant difference between the saline control group and the 15A10 group was determined using the Mann – Whitney U-test for unpaired samples.
because an antibody, Mab 1C1, that binds cocaine with comparable affinity, but is noncatalytic, was not effective. To further demonstrate catalysis in vivo, plasma concentrations of cocaine metabolites were measured and the concentration of ecgonine-methyl ester was found to be more than 10-times higher in rats treated with Mab 15A10 than in those treated with saline or the cocaine-binding antibody 1C1 (Fig. 5B). To evaluate the reinforcing effects of cocaine, Sprague – Dawley rats were trained in operant conditioning chambers (Mets et al, 1998). The rats were prepared with chronic indwelling intravenous catheters and trained to press on levers and receive intravenous injections of 0.3 mg/kg/injection cocaine during 1-h daily sessions. Cocaine maintained regular patterns of lever pressing and when saline was substituted for cocaine, lever-pressing decreased rapidly during the session (Fig. 6A and B). Mab 15A10 blocked completely the reinforcing effects of intra-
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venous cocaine in the rat; both the number of cocaine injections and pattern of responding were similar to those after saline substitution. The saline-like pattern of behavior was not due to Mab 15A10 simply disrupting behavior in general, because it did not alter milk reinforcement. The effect was also not a nonspecific effect on the dopaminergic reward pathway because Mab 15A10 did not alter the pattern or amount of bupropion self-administered by the rats. A within-session multiple-dose protocol was also used wherein rats were allowed access to saline or one of six doses of cocaine [0 (saline), 0.015, 0.03, 0.06, 0 (saline), 0.125, 0.25, or 0.5 mg/kg/injection] each hour in the order stated (Baird et al., 2000). After demonstrating stable dose – response curves over 3 consecutive days, rats were given 30-min pretreatments of saline or Mab15A10, (10, 30, or 100 mg/kg i.v.). Whereas the acute 10 mg/kg Mab 15A10 administration did not alter the cocaine dose – response curve relative to saline, both 30 and 100 mg/kg antibody pretreatments produced statistically significant decreases in the number of injections self-administered within the 0.03, 0.06 and 0.125 mg/kg dose components of the multiple-dose schedule (all P < 0.01). The 100 mg/kg Mab 15A10 pretreatment resulted in an increase in the number of self-administered cocaine infusions at the highest dose of
Fig. 7. Cocaine dose – response curves after pretreatments of mAb 15A10. The effects of 10, 30 and 100 mg/kg i.v. pretreatments of Mab 15A10 on operant responding for intravenous cocaine injections of a multiple-dose schedule are illustrated. Each point is the mean number of injections self-administered at a given dose of cocaine available within a single, 50-min component of the selfadministration session.
cocaine available on the schedule (0.5 mg/kg/injection) relative to both the 10 and 30 mg/kg Mab 15A10 pretreatment groups ( P < 0.004) (Fig. 7) consistent with peripheral blockade of cocaine that was surmounted at high cocaine dose.
7. Discussion The animal data demonstrate that a catalytic antibody raised to a transition-state analog of cocaine hydrolysis is able to increase the rate of cocaine degradation in vivo, protect against cocaine’s toxic effects, and its reinforcing effects. The potential usefulness of an effective anticocaine medication can be inferred from decades of experience with the pharmacologic treatment of heroin addiction. Heroin treatment programs that employ both counseling and methadone administration report abstinence rates at 60% and 80%, in contrast to 10% and 30% for programs that rely only on nonpharmacological approaches. In contrast to methadone, a catalytic antibody would not be expected to enter the brain and therefore would not be psychoactive. Whereas simple blockers of heroin such as naltrexone are useful for treating opiate overdose, they are not generally useful in treating addiction because the blocker requires daily administration, is easily discontinued, and rapidly clears. Thus, were an analogous blocker of cocaine developed, as a small molecule it would also likely be short-lived in vivo and, in the absence of a depot formulation, of limited value for addiction. In contrast, natural antibodies have plasma half-lives of 3 weeks and a humanized Mab with a half-life sufficient for a dosing interval of several weeks would provide an appropriate treatment for a population prone to recidivism and relapse. Since our original report on anticocaine catalytic antibodies (Landry et al., 1993), others have described variations on the concept of intercepting cocaine before the drug reaches its receptors. For example, intraperitoneal administration of the enzyme cholinesterase was shown to inhibit toxicity due to intraperitoneal cocaine in mice (Hoffman et al., 1996), and this enzyme has been proposed as treatment for cocaine overdose. Also, immunization with cocaine analogs designed to elicit noncatalytic anticocaine antibodies were shown to diminish cocaine-induced psychomotor
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effects (Carrera et al., 1995, 2000) and reinforcement (Fox et al., 1996) in rats and has been proposed as a vaccine for cocaine addiction (Fox, 1997). However, catalytic antibodies are likely to be longer-lived in plasma than natural enzymes and, in contrast to typical antibodies that can form long-lived complexes with antigen, catalytic antibodies are not susceptible to depletion by the act of complex formation with cocaine. Thus, catalytic antibodies have the unique potential to treat both the acute and chronic aspects of cocaine abuse.
8. Conclusion Using rationally designed transition-state analogs (TSA1, TSA2 and TSA3) for the hydrolysis of cocaine’s benzoyl ester, we elicited several distinct structural and functional families of anticocaine catalytic antibodies. TSA1 and TSA3 were found to be particularly effective analogs. Mab 15A10, the most potent of the catalytic antibodies elicited, was found to be sufficiently active to prevent cocaine’s reinforcing and toxic effects in rodents. MAb 15A10 is a suitable candidate for humanization and optimization by mutagenesis in preparation for clinical trials.
References Ambrew, J., 1985. J. Anal. Toxicol. 9, 241. Baird, T.J., Deng, S.X., Landry, D.W., Winger, G., Woods, J.H., 2000. Natural and artificial enzymes against cocaine: I. Monoclonal antibody 15A10 and the reinforcing effects of cocaine in rats. J. Pharmacol. Exp. Ther. 295 (3), 1127. Bonese, K.F., Wainer, B.H., Fitch, F.W., Rothberg, R.M., Schuster, C.R., 1974. Changes in heroin self-administration by a rhesus monkey after morphine immunization. Nature (Lond.) 252 (5485), 708. Briscoe, R.J., Jeanville, P.M., Cabrera, C., Baird, T.J., Woods, J.H., Landry, D.W., 2001. A catalytic antibody against cocaine attenuates cocaine’s cardiovascular effects in mice: a dose and time course analysis. Int. Immunopharmacol. 1 (6), 1189. Carrera, M.R., Ashley, J.A., Parson, L.H., Wirsching, P., Koob, G.F., Janda, K.D., 1995. Suppression of psychoactive effects of cocaine by active immunization. Nature (Lond.) 378, 727. Carrera, M.R., Ashley, J.A., Zhou, B., Wirsching, P., Koob, G.F., Janda, K.D., 2000. Cocaine vaccines: antibody protection against relapse in a rat model. Proc. Natl. Acad. Sci. U. S. A. 97, 6202.
309
Denison, M.E., Paredes, A., Bacal, S., Gawin, F.H., 1998. Psychological and psychiatric consequences of cocaine. In: Tarter, R.E., Ammerman, R.T., Ott, P.J. (Eds.), Handbook of Substance Abuse: Neurobehavioral Pharmacology. Plenum, New York, pp. 201 – 213. Fox, B.S., 1997. Development of a therapeutic vaccine for the treatment of cocaine addiction. Drug Alcohol Depend. 48, 153 – 158. Fox, B.S., Kantak, K.M., Edwards, M.A., Black, K.M., Bollinger, B.K., Botka, A.J., French, T.L., Thompson, T.L., Schad, V.C., Greenstein, J.L., 1996. Nat. Med. 2, 1129. Gantenberg, N.S., Hageman, G.R., 1992. Cocaine-enhanced arrhythmogenesis: neural and nonneural mechanisms. Can. J. Physiol. Pharmacol. 70, 240. Gatley, S.J., MacGregor, R.R., Fowler, J.S., Wolf, A.P., Dewey, S.L., Schlyer, D.J., 1990. Rapid stereoselective hydrolysis of (+)-cocaine in baboon plasma prevents its uptake in the brain: implications for behavioral studies. J. Neurochem. 54 (2), 720. Gatley, S.J., Yu, D.W., Fowler, J.S., MacGregor, R.R., Schlyer, D.J., Dewey, S.L., Volkow, N.D., 1994. Studies with differentially labeled [11C]cocaine, [11C]norcocaine, [11C]benzoylecgonine, and [11C]-and 4V-[18F]fluorococaine to probe the extent to which [11C]cocaine metabolites contribute to PET images of the baboon brain. J. Neurochem. 62 (3), 1154. Gatley, S.J., Gifford, A.N., Volkow, N.D., Fowler, J.S., 1998. Pharmacology of cocaine. In: Tarter, R.E., Ammerman, R.T., Ott, P.J. (Eds.), Handbook of Substance Abuse: Neurobehavioral Pharmacology. Plenum, New York, pp. 161 – 185. Hoffman, R.S., Morasco, R., Goldfrank, L.R., 1996. J. Toxicol. Clin. Toxicol. 34, 259. Jencks, W.P., 1969. Catalysis in Chemistry and Enzymology. McGraw-Hill, New York. Landry, D.W., Zhao, K., Yang, G.X.Q., Glickman, M., Georgiadis, M., 1993. Antibody catalyzed degradation of cocaine. Science (Wash. D.C.) 259, 1899. Leshner, A.I., Koob, G.F., 1999. Drugs of abuse and the brain. Proc. Assoc. Am. Physicians 111, 99. Mets, B., Jamdar, S., Landry, D.W., 1996. Life Sci. 59, 2021. Mets, B., Winger, G., Cabrera, C., Seo, S., Jamdar, S., Yang, G., Zhao, K., Briscoe, R.J., Almonte, R., Woods, J.H., Landry, D.W., 1998. A catalytic antibody against cocaine prevents cocaine’s reinforcing and toxic effects in rats. Proc. Natl. Acad. Sci. U. S. A. 95, 10176. Office of the National Drug Control Policy, 1996. The National Drug Control Strategy Executive Office of the President of the United States, Ed.: 41 – 51, Washington, DC. Pollack, S.J., Jacobs, J.W., Schultz, P.G., 1986. Selective chemical catalysis by an antibody. Science (Wash. D.C., 1883) 234 (4783), 1570. Stewart, D.J., Inaba, T., Tang, B.K., Kalow, W., 1977. Hydrolysis of cocaine in human plasma by cholinesterase. Life Sci. 20, 1557. Stewart, D.J., Inaba, T., Lucassen, M., Kalow, W., 1979. Cocaine metabolism: cocaine and norcocaine hydrolysis by liver and serum esterases. Clin. Pharmacol. Ther. 25, 464. Tramontano, A., Janda, K.D., Lerner, R.A., 1986. Catalytic antibodies. Science 234 (4783), 1566. Verebey, K., Gold, M.S., 1988. From coca leaves to crack: the
310
S.X. Deng et al. / Journal of Immunological Methods 269 (2002) 299–310
effects of dose and routes of administration in abuse liability. Psychiatr. Ann. 18, 513. Xie, W., Altamirano, C.V., Bartels, C.F., Speirs, R.J., Cashman, J.R., Lockridge, O., 1999. An improved cocaine hydrolase: the A328Y mutant of human butyrylcholinesterase is 4-fold more efficient. Mol. Pharmacol. 55, 83.
Yang, G., Chun, J., Arakawa, U.H., Wang, X., Gawinowicz, M.A., Zhao, K., Landry, D.W., 1996. Anti-cocaine catalytic antibodies: a synthetic approach to improved antibody diversity. J. Am. Chem. Soc. 118, 5881. Zhao, K., Landry, D.W., 1992. Tetrazole catalyzed synthesis of phosphonate esters. Tetrahedron 49 (2), 363.