Substrate Report for: Cocaine

Human carboxylesterase 1 (hCES1) is a major carboxylesterase in the human body and plays important roles in the metabolism of a wide variety of substances, including lipids and drugs, and therefore is attracting more and more attention from areas including lipid metabolism, pharmacokinetics, drug-drug interactions, and prodrug activation. In this work, we studied the catalytic hydrolysis mechanism of hCES1 by the quantum mechanics computation method, using cocaine as a model substrate. Our results support the four-step theory of the esterase catalytic hydrolysis mechanism, in which both the acylation stage and the deacylation stage include two transition states and a tetrahedral intermediate. The roles and cooperation of the catalytic triad, S221, H468, and E354, were also analyzed in this study. Moreover, orthoester intermediates were found in hCES1-catalyzed cocaine hydrolysis reaction, which significantly elevate the free energy barrier and slow down the reaction. Based on this finding, we propose that hCES1 substrates with beta-aminocarboxylester structure might form orthoester intermediates in hCES1-catalyzed hydrolysis, and therefore prolong their in vivo half-life. Thus, this study helps to clarify the catalytic mechanism of hCES1 and elucidates important details of its catalytic process, and furthermore, provides important insights into the metabolism of hCES1 substrates and drug designing.

Carboxylesterase-1 (CE-1) is a crucial enzyme responsible for metabolism/activation/inactivation of xenobiotics (therapeutic agents, prodrugs, abused drugs, and organophosphorus nerve agents etc.) and also involved in many other biological processes. In this study, we performed extensive computational modeling and simulations to understand the fundamental reaction mechanism of cocaine hydrolysis catalyzed by CE-1, revealing that CE-1-catalyzed cocaine hydrolysis follows a novel reaction pathway with only two reaction steps: a single-step acylation process and a single-step deacylation process. In the transition states of both single-step processes, the cocaine NH group joins the oxyanion hole to form an additional hydrogen bond with the negatively charged carbonyl oxygen atom of the cocaine. Thus, the transition states are stabilized by both intermolecular and intramolecular hydrogen bonds with the methyl ester of cocaine, specifically the carbonyl oxygen atom. The rate-limiting transition state is associated with the acylation process, and the activation free energy barrier was predicted to be 20.1 kcal/mol. Further, in vitro experimental kinetic analysis was performed for human CE-1-catalyzed cocaine hydrolysis. For CE-1-catalyzed cocaine hydrolysis, the computationally predicted free energy barrier (20.1 kcal/mol) is reasonably close to the experimentally derived turnover number ( kcat = 0.058 min(-1)), indicating the reasonability of the computational results. The obtained novel mechanistic insights are expected to benefit not only CE-1 related rational drug discovery but also future research on the catalytic mechanism of other esterases.

Cocaine is a widely abused and addictive drug without an FDA-approved medication. Our recently designed and discovered cocaine hydrolase, particularly E12-7 engineered from human butyrylcholinesterase (BChE), has the promise of becoming a valuable cocaine abuse treatment. An ideal anti-cocaine therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long biological half-life. However, recombinant human BChE and the known BChE mutants have a much shorter biological half-life compared to the native human BChE. The present study aimed to extend the biological half-life of the cocaine hydrolase without changing its high catalytic activity against cocaine. Our strategy was to design possible amino-acid mutations that can introduce cross-subunit disulfide bond(s) and, thus, change the distribution of the oligomeric forms and extend the biological half-life. Three new BChE mutants (E364-532, E377-516, and E535) were predicted to have a more stable dimer structure with the desirable cross-subunit disulfide bond(s) and, therefore, a different distribution of the oligomeric forms and a prolonged biological half-life. The rational design was followed by experimental tests in vitro and in vivo, confirming that the rationally designed new BChE mutants, i.e. E364-532, E377-516, and E535, indeed had a remarkably different distribution of the oligomeric forms and prolonged biological half-life in rats from approximately 7 to approximately 13h without significantly changing the catalytic activity against (-)-cocaine. This is the first demonstration that rationally designed amino-acid mutations can significantly prolong the biological half-life of a high-activity enzyme without significantly changing the catalytic activity.

Human carboxylesterase 1 (hCES1) is a major carboxylesterase in the human body and plays important roles in the metabolism of a wide variety of substances, including lipids and drugs, and therefore is attracting more and more attention from areas including lipid metabolism, pharmacokinetics, drug-drug interactions, and prodrug activation. In this work, we studied the catalytic hydrolysis mechanism of hCES1 by the quantum mechanics computation method, using cocaine as a model substrate. Our results support the four-step theory of the esterase catalytic hydrolysis mechanism, in which both the acylation stage and the deacylation stage include two transition states and a tetrahedral intermediate. The roles and cooperation of the catalytic triad, S221, H468, and E354, were also analyzed in this study. Moreover, orthoester intermediates were found in hCES1-catalyzed cocaine hydrolysis reaction, which significantly elevate the free energy barrier and slow down the reaction. Based on this finding, we propose that hCES1 substrates with beta-aminocarboxylester structure might form orthoester intermediates in hCES1-catalyzed hydrolysis, and therefore prolong their in vivo half-life. Thus, this study helps to clarify the catalytic mechanism of hCES1 and elucidates important details of its catalytic process, and furthermore, provides important insights into the metabolism of hCES1 substrates and drug designing.

Carboxylesterase-1 (CE-1) is a crucial enzyme responsible for metabolism/activation/inactivation of xenobiotics (therapeutic agents, prodrugs, abused drugs, and organophosphorus nerve agents etc.) and also involved in many other biological processes. In this study, we performed extensive computational modeling and simulations to understand the fundamental reaction mechanism of cocaine hydrolysis catalyzed by CE-1, revealing that CE-1-catalyzed cocaine hydrolysis follows a novel reaction pathway with only two reaction steps: a single-step acylation process and a single-step deacylation process. In the transition states of both single-step processes, the cocaine NH group joins the oxyanion hole to form an additional hydrogen bond with the negatively charged carbonyl oxygen atom of the cocaine. Thus, the transition states are stabilized by both intermolecular and intramolecular hydrogen bonds with the methyl ester of cocaine, specifically the carbonyl oxygen atom. The rate-limiting transition state is associated with the acylation process, and the activation free energy barrier was predicted to be 20.1 kcal/mol. Further, in vitro experimental kinetic analysis was performed for human CE-1-catalyzed cocaine hydrolysis. For CE-1-catalyzed cocaine hydrolysis, the computationally predicted free energy barrier (20.1 kcal/mol) is reasonably close to the experimentally derived turnover number ( kcat = 0.058 min(-1)), indicating the reasonability of the computational results. The obtained novel mechanistic insights are expected to benefit not only CE-1 related rational drug discovery but also future research on the catalytic mechanism of other esterases.

Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A199S/S227A/S287G/A328W/Y332G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (kcat/KM) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.

Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.

Cocaine is a widely abused and addictive drug without an FDA-approved medication. Our recently designed and discovered cocaine hydrolase, particularly E12-7 engineered from human butyrylcholinesterase (BChE), has the promise of becoming a valuable cocaine abuse treatment. An ideal anti-cocaine therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long biological half-life. However, recombinant human BChE and the known BChE mutants have a much shorter biological half-life compared to the native human BChE. The present study aimed to extend the biological half-life of the cocaine hydrolase without changing its high catalytic activity against cocaine. Our strategy was to design possible amino-acid mutations that can introduce cross-subunit disulfide bond(s) and, thus, change the distribution of the oligomeric forms and extend the biological half-life. Three new BChE mutants (E364-532, E377-516, and E535) were predicted to have a more stable dimer structure with the desirable cross-subunit disulfide bond(s) and, therefore, a different distribution of the oligomeric forms and a prolonged biological half-life. The rational design was followed by experimental tests in vitro and in vivo, confirming that the rationally designed new BChE mutants, i.e. E364-532, E377-516, and E535, indeed had a remarkably different distribution of the oligomeric forms and prolonged biological half-life in rats from approximately 7 to approximately 13h without significantly changing the catalytic activity against (-)-cocaine. This is the first demonstration that rationally designed amino-acid mutations can significantly prolong the biological half-life of a high-activity enzyme without significantly changing the catalytic activity.

Cocaine esterase (CocE) is known as the most efficient natural enzyme for cocaine hydrolysis. The major obstacle to the clinical application of wild-type CocE is the thermoinstability with a half-life of only approximately 12 min at 37 degrees C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of approximately 6 h at 37 degrees C is currently in clinical trial Phase II for cocaine overdose treatment. Through molecular modeling and dynamics simulation, we designed and characterized a promising new mutant of E172-173 with extra L196C/I301C mutations (denoted as enzyme E196-301) to produce cross-subunit disulfide bonds that stabilize the dimer structure. The cross-subunit disulfide bonds were confirmed by X-ray diffraction. The designed L196C/I301C mutations have not only considerably extended the in vitro half-life at 37 degrees C to >100 days, but also significantly improved the catalytic efficiency against cocaine by approximately 150%. In addition, the thermostable E196-301 can be PEGylated to significantly prolong the residence time in mice. The PEGylated E196-301 can fully protect mice from a lethal dose of cocaine (180 mg/kg, LD100) for at least 3 days, with an average protection time of approximately 94h. This is the longest in vivo protection of mice from the lethal dose of cocaine demonstrated within all studies using an exogenous enzyme reported so far. Hence, E196-301 may be developed to become a more valuable therapeutic enzyme for cocaine abuse treatment, and it demonstrates that a general design strategy and protocol to simultaneously improve both the stability and function are feasible for rational protein drug design.

Cocaine hydrolase gene transfer of mutated human butyrylcholinesterase (BChE) is evolving as a promising therapy for cocaine addiction. BChE levels after gene transfer can be 1,500-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. Because mutated BChE is approximately 70 % as efficient in hydrolyzing acetylcholine as wild-type enzyme, it is important to examine the impact on cholinergic function. Here, we focused on memory and cognition (Stone T-maze), basic neuromuscular function (treadmill endurance and grip strength), and coordination (Rotarod). BALB/c mice were given adeno-associated virus vector or helper-dependent adenoviral vector encoding mouse or human BChE optimized for cocaine. Age-matched controls received saline or luciferase vector. Despite high doses (up to 10(13) particles per mouse) and high transgene expression (1,000-fold above baseline), no deleterious effects of vector treatment were seen in neurobehavioral functions. The vector-treated mice performed as saline-treated and luciferase controls in maze studies and strength tests, and their Rotarod and treadmill performance decreased less with age. Thus, neither the viral vectors nor the large excess of BChE caused observable toxic effects on the motor and cognitive systems investigated. This outcome justifies further steps toward an eventual clinical trial of vector-based gene transfer for cocaine abuse.

INTRODUCTION: Addiction to cocaine is a major problem around the world, but especially in developed countries where the combination of wealth and user demand has created terrible social problems. Although only some users become truly addicted, those who are often succumb to a downward spiral in their lives from which it is very difficult to escape. From the medical perspective, the lack of effective and safe, non-addictive therapeutics has instigated efforts to develop alternative approaches for treatment, including anticocaine vaccines designed to block cocaine's pharmacodynamic effects. AREAS COVERED: This paper discusses the implications of cocaine pharmacokinetics for robust vaccine antibody responses, the results of human vaccine clinical trials, new developments in animal models for vaccine evaluation, alternative vaccine formulations and complementary therapy to enhance anticocaine effectiveness. EXPERT OPINION: Robust anti-cocaine antibody responses are required for benefit to cocaine abusers, but since any reasonably achievable antibody level can be overcome with higher drug doses, sufficient motivation to discontinue use is also essential so that the relative barrier to cocaine effects will be appropriate for each individual. Combining a vaccine with achievable levels of an enzyme to hydrolyze cocaine to inactive metabolites, however, may substantially increase the blockade and improve treatment outcomes.

Compared with naturally occurring enzymes, computationally designed enzymes are usually much less efficient, with their catalytic activities being more than six orders of magnitude below the diffusion limit. Here we use a two-step computational design approach, combined with experimental work, to design a highly efficient cocaine hydrolysing enzyme. We engineer E30-6 from human butyrylcholinesterase (BChE), which is specific for cocaine hydrolysis, and obtain a much higher catalytic efficiency for cocaine conversion than for conversion of the natural BChE substrate, acetylcholine (ACh). The catalytic efficiency of E30-6 for cocaine hydrolysis is comparable to that of the most efficient known naturally occurring hydrolytic enzyme, acetylcholinesterase, the catalytic activity of which approaches the diffusion limit. We further show that E30-6 can protect mice from a subsequently administered lethal dose of cocaine, suggesting the enzyme may have therapeutic potential in the setting of cocaine detoxification or cocaine abuse.

Cocaine addiction affects millions of people with disastrous personal and social consequences. Cocaine is one of the most reinforcing of all drugs of abuse, and even those who undergo rehabilitation and experience long periods of abstinence have more than 80% chance of relapse. Yet there is no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts. Recent studies, however, have demonstrated a promising potential treatment option with the help of the serum enzyme butyrylcholinesterase (BChE), which is capable of breaking down naturally occurring (-)-cocaine before the drug can influence the reward centers of the brain or affect other areas of the body. This activity of wild-type (WT) BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against (-)-cocaine. Plants are a promising means to produce large amounts of these cocaine hydrolase variants of BChE, cheaply, safely with no concerns regarding human pathogens and functionally equivalent to enzymes derived from other sources. Here, in expressing cocaine-hydrolyzing mutants of BChE in Nicotiana benthamiana using the MagnICON virus-assisted transient expression system, and in reporting their initial biochemical analysis, we provide proof-of-principle that plants can express engineered BChE proteins with desired properties.

It can be argued that an ideal anti-cocaine medication would be one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, wild-type BChE has a low catalytic efficiency against naturally occurring (-)-cocaine. Interestingly, wild-type BChE has a much higher catalytic activity against unnatural (+)-cocaine. According to available positron emission tomography (PET) imaging analysis using [(11)C](-)-cocaine and [(11)C](+)-cocaine tracers in human subjects, only [(11)C](-)-cocaine was observed in the brain, whereas no significant [(11)C](+)-cocaine signal was observed in the brain. The available PET data imply that an effective therapeutic enzyme for treatment of cocaine abuse could be an exogenous cocaine-metabolizing enzyme with a catalytic activity against (-)-cocaine comparable to that of wild-type BChE against (+)-cocaine. Our recently designed A199S/F227A/S287G/A328 W/Y332G mutant of human BChE has a considerably improved catalytic efficiency against (-)-cocaine and has been proven active in vivo. In the present study, we have characterized the catalytic activities of wild-type BChE and the A199S/F227A/S287G/A328 W/Y332G mutant against both (+)- and (-)-cocaine at the same time under the same experimental conditions. Based on the obtained kinetic data, the A199S/F227A/S287G/A328 W/Y332G mutant has a similarly high catalytic efficiency (kcat/KM) against (+)- and (-)-cocaine, and indeed has a catalytic efficiency (kcat/KM=1.84x10(9)M(-1)min(-1)) against (-)-cocaine comparable to that (kcat/KM=1.37x10(9)M(-1)min(-1)) of wild-type BChE against (+)-cocaine. Thus, the mutant may be used to effectively prevent (-)-cocaine from entering brain and producing physiological effects in the enzyme-based treatment of cocaine abuse.

BACKGROUND AND PURPOSE Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT-11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT-11. EXPERIMENTAL APPROACH: The hydrolysis of heroin and cocaine by recombinant human CEs was evaluated and the kinetic parameters determined. In addition, microsomal samples prepared from these tissues were subjected to chromatographic separation, and substrate hydrolysis and amounts of different CEs were determined. KEY RESULTS: In contrast to previous reports, cocaine was not hydrolysed by the human liver CE, hCE1 (CES1), either as highly active recombinant protein or as CEs isolated from human liver or intestinal extracts. These results correlated well with computer-assisted molecular modelling studies that suggested that hydrolysis of cocaine by hCE1 (CES1), would be unlikely to occur. However, cocaine, heroin and CPT-11 were all substrates for the intestinal CE, hiCE (CES2), as determined using both the recombinant protein and the tissue fractions. Again, these data were in agreement with the modelling results. CONCLUSIONS AND IMPLICATIONS: These results indicate that the human liver CE is unlikely to play a role in the metabolism of cocaine and that hydrolysis of this substrate by this class of enzymes is via the human intestinal protein hiCE (CES2). In addition, because no enzyme inhibition is observed at high cocaine concentrations, potentially this route of hydrolysis is important in individuals who overdose on this agent.

To address the problem of acute cocaine overdose, we undertook molecular engineering of butyrylcholinesterase (BChE) as a cocaine hydrolase so that modest doses could be used to accelerate metabolic clearance of this drug. Molecular modeling of BChE complexed with cocaine suggested that the inefficient hydrolysis (k(cat) = 4 min(-1)) involves a rotation toward the catalytic triad, hindered by Tyr332. To eliminate rotational hindrance and retain substrate affinity, we introduced two amino acid substitutions (Ala328Trp/Tyr332Ala). The resulting mutant BChE reduced cocaine burden in tissues, accelerated plasma clearance by 20-fold, and prevented cocaine-induced hyperactivity in mice. The enzyme's kinetic properties (k(cat) = 154 min(-1), K(M) = 18 microM) satisfy criteria suggested previously for treating cocaine overdose (k(cat) >120 min(-1), K(M) < 30 microM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.

Plasma butyrylcholinesterase (BChE) is important in the metabolism of cocaine, but natural human BChE has limited therapeutic potential for detoxication because of low catalytic efficiency with cocaine. Here we report pharmacokinetics of cocaine in rats treated with A328W/Y332A BChE, an excellent cocaine hydrolase designed with the aid of molecular modeling. Compared with wild-type BChE, this enzyme hydrolyzes cocaine with 40-fold improved k(cat) (154 min(-1) versus 4.1 min(-1)) and only slightly increased K(M) (18 microM versus 4.5 microM). In rats given this hydrolase (3 mg/kg i.v.) 10 min before cocaine challenge (6.8 mg/kg i.v.), cocaine half-life was reduced from 52 min to 18 min. Mirroring the reductions of plasma cocaine were large increases in benzoic acid, a product of BChE-mediated cocaine hydrolysis. All other pharmacokinetic parameters confirmed a large, dose-dependent acceleration of cocaine removal by the injected cocaine hydrolase. These results show that A328W/Y332A, an efficient cocaine hydrolase in vivo as well as in vitro, might promote cocaine detoxication in a clinical setting.

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.

It has been suggested that toxicity to cocaine is related to the relative rate of cocaine metabolism by cholinesterases and to activation of cholinergic receptors either directly or by reflex mechanisms. We examined these possibilities by altering cholinesterase activity and blocking cholinergic receptors in rats prone or resistant to cocaine-induced cardiovascular toxicity. Rats were instrumented with a pulsed Doppler flow probe on the ascending aorta for measurement of cardiac output and cannulated for arterial pressure and heart rate determination. In conscious rats, cocaine (5 mg/kg iv) elicited pressor responses and a delayed bradycardia but cardiac output and systemic vascular resistance responses varied greatly between rats. Pretreatment with the nonspecific cholinesterase inhibitors physostigmine (0.1-0.2 mg/kg) or neostigmine (0.1 mg/kg) reduced the pressor response by diminishing the increase in systemic vascular resistance. In contrast, inhibition of cocaine metabolism with the selective plasma cholinesterase inhibitor tetraisopropyl pyrophosphoramide (0.5 mg/kg) or increasing cholinesterase activity with human butyryl cholinesterase (9.9 mg/kg iv) did not alter hemodynamic responses to cocaine. Administration of atropine methyl bromide (0.5-1 mg/kg iv) alone or with physostigmine to prevent the cholinomimetic effects of physostigmine reduced the cocaine-induced decrease in cardiac output noted in some animals. These data suggest that the cocaine-induced decrease in cardiac output observed in some rats is, at least in part, dependent on activation of muscarinic receptors. In addition, the rate of cocaine metabolism is not critical for the initial hemodynamic responses to cocaine in conscious rats.

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 microM. 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 microM for a 4-fold 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 with human BChE, horse BChE had a 2-fold higher kcat but a lower binding affinity, cat BChE was similar to human, and 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 k(cat) and Km values similar to wild-type, but because these variants are reduced to 66 and 33% of normal levels in human blood, respectively, people with these variants may be at risk for cocaine toxicity. The atypical variant (D70G) had a 10-fold 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.

In humans, the plasma enzyme butyrylcholinesterase, BChE (EC 3.1.1.8), mediates the in vivo plasma hydrolysis of cocaine to the pharmacologically inactive metabolite ecgonine methyl ester, EME. This enzyme has been purified from human plasma to investigate the potential as a treatment for cocaine intoxication. Cocaine (2.1 micrograms mL-1) was incubated in plasma with a BChE concentration in the normal range (3.02 micrograms mL-1) and in plasma with enhanced BChE concentrations of 9.14, 20.8 and 37.8 micrograms mL-1, respectively for time periods up to 120 min. Cocaine and the hydrolytic products, ecgonine methyl ester and ecgonine, were quantified simultaneously by gas chromatography-mass spectrometry (GC-MS). The enhancement of plasma BChE concentration resulted in a dramatic increase in the rate of hydrolysis of cocaine. There was a stoichimetric conversion of cocaine to the inactive hydrolysis product, ecgonine methyl ester. Accordingly, the half-life of cocaine in plasma decreased significantly with enhanced BChE concentration. At plasma BChE concentrations of 3.02, 9.14, 20.8 and 37.8 micrograms mL-1, half-life values of 116, 35.8, 21.4 and 9.0 min, respectively were observed. The marked reduction in cocaine half-life provides evidence supporting the potential therapeutic use of BChE for the treatment of cocaine intoxication.

The synthesis and characterization of diastereomers of cocaine benzoyl thioester is described. Allococaine benzoyl thioester and allopseudococaine benzoyl thioester were synthesized by the conjugate addition of p-methoxytolyl thiol to ecgonine methyl ester followed by debenzylation and benzoylation. The absolute structure of the hydrochloride salt of the major ecgonine p-methoxytolyl sulfide formed was determined by single-crystal diffraction analysis and used to establish the addition geometry. When placed in aqueous solution, the cocaine benzoyl thioester diastereomers hydrolyzed to give thioecgonine methyl ester. The rate of cocaine benzoyl thioester hydrolysis was carefully investigated spectrophotometrically by using the Ellman reagent. At neutral pH, the hydrolysis of the diastereomers was found to proceed at detectable rates. Upon increasing pH, the rate of hydrolysis of cocaine benzoyl thioester diastereomers was increased and the reaction was catalyzed by basic buffer species. In addition to defining the kinetics of hydrolysis in aqueous solution, cocaine benzoyl thioester was utilized as a highly efficient method to monitor the activity of cholinesterases and pig liver esterase. Use of cocaine benzoyl thioester represents a rapid and sensitive way to screen for cocaine esterase activity.

Existing pharmacodynamic approaches to cocaine abuse treatment have not been widely successful. An alternative, pharmacokinetic, approach is to enhance cocaine metabolism by administration of butyrylcholinesterase (BChE), a major cocaine-metabolizing enzyme in primates. Initial studies in rodents suggest that BChE pretreatment can substantially reduce the acute physiological and behavioral effects of cocaine, at enzyme doses that themselves have no behavioral or toxic effects. A single enzyme injection may increase plasma BChE activity for several days, suggesting that exogenous administration may be practical. BChE treatment may also produce a favorable pattern of cocaine metabolites. Further research is needed to evaluate the long-term effects of BChE administration.

The ability of human plasma butyrylcholinesterase (BChE) to detoxify cocaine in vivo was evaluated. Intravenous administration of BChE, at doses sufficient to increase the plasma levels of the enzyme as much as 800-fold, produced no adverse effects on the cardiovascular, autonomic, or central nervous systems of rats. Most of the enzyme could be recovered in the plasma immediately after administration and remained active with a beta-t(1/2) of 21.6 +/- 2.4 hr. Pretreatment of chloralose-urethane anesthetized rats with BChE, 0.1-7.8 mg/kg, decreased the hypertensive and arrhythmogenic effects produced by cocaine and increased the lethal dose of cocaine by three- to fourfold. Treatment of conscious rats with 1 and 10 mg/kg BChE decreased the incidence of seizures and deaths produced by a prior dose of cocaine (80 mg/kg, i.p.). These results suggest that BChE would provide a safe and highly efficacious treatment for cocaine intoxication.

The most common complications of cocaine ingestion are on the cardiovascular and central nervous systems and produce chest pain and generalized seizures. In humans, decreased levels of butyrylcholinesterase (BChE) (EC 3.1.1.8) have been associated with sustained effects of cocaine and life-threatening complications. Administration of purified human BChE has previously been demonstrated to protect against cocaine-associated cardiovascular toxicity in rats. A shift in the metabolism of cocaine as well as enhanced metabolism may be the underlying mechanism of the enzyme. Therefore, levels of the parent drug and four metabolites were determined in rat plasma after i.p. administration of a lethal cocaine dose, followed by i.v. administration of BChE. Plasma and brain concentrations of cocaine were lowered by 80% after BChE administration. Furthermore, the metabolic profile of cocaine in the plasma was altered. The concentration of ecgonine methylester was doubled although the concentration of ecgonine, a secondary metabolite of cocaine, was reduced. The level of benzoylecgonine was reduced by one-half while norcocaine was absent. Cocaine-associated effects upon the central nervous system were also shown to be reduced by administration of BChE to conscious rats. Furthermore, our studies in the cat have also shown that purified BChE shifts the metabolic profile of cocaine (1 mg/kg) to the pharmacologically inactive products ecgonine methylester and ecgonine. Pretreatment with BChE (0.27, 1.0, and 10.0 mg/kg) ameliorated the hypertensive effects of cocaine (1 mg/kg) by reducing the duration and the extent of BP elevation by 66%. Administration of the enzyme, 1 min after cessation of cocaine infusion, resulted in an immediate attenuation in the cocaine-induced broadening of the QRS complex. These results suggest that BChE could be an effective and rapid therapy for the treatment of life-threatening cocaine-induced cardiovascular effects in human while clearing the total body burden of cocaine.

1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous absorption and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Therefore, we studied BuChE expression during placental development. 2. RT-PCR revealed both BuChEmRNA and acetylcholinesterase (AChE) mRNA throughout gestation. However, cytochemical staining detected primarily BuChE activity in first-trimester placenta but AChE activity in term placenta. 3. As the atypical variant of BuChE has a narrower specificity for substrates and inhibitors than the normal enzyme, we investigated its interactions with alpha-solanine and cocaine, and sought a correlation between the occurrence of this variant and placental malfunction. 4. Atypical BuChE of serum or recombinant origin presented > 10-fold weaker affinities than normal BuChE for cocaine and alpha-solanine. However, BuChE in the serum of the heterozygote and a homozygous normal were similar in their drug affinities. Therefore, heterozygous serum or placenta can protect the fetus from drug or poison exposure, unlike homozygous atypical serum or placenta. 5. Genotype analyses revealed that heterozygous carriers of atypical BuChE were threefold less frequent among 49 patients with placental malfunction than among 76 controls of the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.

Butyrylcholinesterase [BCHE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BCHE variant, in which Asp70 at the rim of the active site gorge is substituted by glycine, displayed a more drastically weakened interaction with tacrine than with cocaine, dibucaine, succinylcholine, BW284c51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide], or alpha-solanine. To delineate the protein domains that are responsible for this phenomenon, we mutated residues within the rim of the active site gorge, the region parallel to the peripheral site in the homologous enzyme acetylcholinesterase [AChE (acetylcholine acetyl hydrolase); EC 3.1.1.7], the oxyanion hole, and the choline-binding site. When expressed in microinjected Xenopus laevis oocytes, all mutant DNAs yielded comparable amounts of immunoreactive protein products. Most mutants retained catalytic activity close to that of wild-type BCHE and were capable of binding ligands. However, certain modifications in and around the oxyanion hole caused a dramatic loss in activity. The affinities for tacrine were reduced more dramatically than for all other ligands, including cocaine, in both oxyanion hole and choline-binding site mutants. Modified ligand affinities further demonstrated a peripheral site in residues homologous with those of AChE. BCHE mutations that prevented tacrine interactions also hampered its ability to bind other drugs and inhibitors, which suggests a partial overlap of the binding sites. This predicts that in addition to their genetic predisposition to adverse responses to tacrine, homozygous carriers of "atypical" BCHE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.

The characterization of the enzymes responsible for drug metabolism in the human placenta is of great importance in determining the possible role the placenta plays in protecting the fetus from potentially fetotoxic drugs. We speculate that the placenta metabolizes cocaine, serving to protect the fetus from the drug's ill effects. Cholinesterase, the principle enzyme that metabolizes cocaine, has been hypothesized to be present yet is not well characterized in the human placenta. The purpose of this study was to quantify human placental acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity. Human placentas were obtained from elective cesarean sections, and several lobules were thoroughly perfused with cold buffer to ensure minimal contamination from erythrocyte AChE. Subcellular fractions were then prepared from these lobules by using standard differential centrifugation techniques. Microsomes and cytosol were assayed for AChE and BChE activity by using a spectrophotometric assay. BChE activity was found in the cytosolic fraction of the placental villous tissue, whereas AChE activity was measured in the microsomal fraction. By demonstrating that BChE activity is present in human term placenta we have shown that this organ has the capacity to metabolize cocaine and may therefore serve as a metabolic barrier to fetal exposure to cocaine.

Cocaine induces acute lethal cell injury in rat hepatocytes following N-oxidative metabolic activation by cytochrome P450-dependent and flavin-dependent monooxygenases. Beside this oxidative bioactivation pathway, hepatic carboxylesterases may cleave the carboxymethylester or the benzoylester linkage which leads to molecules found to be non-toxic in vivo. To elucidate the structural requirements of the cocaine molecule for its bioactivation and inactivation, the cytotoxic potential of the natural (-)-cocaine relative to two isomeric forms, (+)-cocaine* (the unnatural enantiomer) and (-)-psi-cocaine (the C2 epimer of the unnatural cocaine) were investigated. Primary short-term cultures of rat hepatocytes obtained from phenobarbital (PB)-pretreated rats were exposed to the drugs for up to 24 h. (-)-Cocaine produced marked time- and concentration-dependent release of lactate dehydrogenase (LDH) into the extracellular medium, whereas the other forms were not cytotoxic (0-1 mM). Furthermore, depletion of cellular glutathione (GSH) with diethylmaleate enhanced LDH release in (-)-cocaine-treated cells and caused marginal cytotoxicity in hepatocytes exposed to the other isomers. To investigate the mechanisms that could be responsible for these isomer-specific effects, the time-dependent metabolic degradation was determined both in cultured hepatocytes and in hepatic microsomes in the presence or absence of the serine carboxylesterase inhibitors, phenylmethylsulfonylfluoride (PMSF) or NaF. All three cocaine analogs were enzymatically degraded, but the rates of ester cleavage greatly varied among the stereoisomers. (-)-Cocaine was primarily N-oxidized via SKF-525A-sensitive pathways, whereas (+)-cocaine was predominantly hydrolyzed by PMSF-sensitive carboxylesterases. In contrast, (-)-psi-cocaine, which is very stable in the absence of cells at 37 degrees C and pH 7.4, was subject to extremely fast enzymatic ester cleavage. In conclusion, these results indicate that the isomer-specific differential cytotoxicity of (-)-cocaine, (+)-cocaine and (-)-psi-cocaine in hepatocytes may be related to stereoselective differences in the rates of hydrolytic inactivation by hepatic carboxylesterases and that the N-oxidative pathway, resulting in hepatocyte injury, may thus be relevant only for (-)-cocaine.

The behaviors of the enantiomers of cocaine (benzoylecgonine methyl ester) and related compounds with butyrylcholinesterase (BChE; EC 3.1.1.8) were investigated spectrophotometrically at 235 nm. The unnatural enantiomer, (+)-cocaine, was hydrolyzed by BChE (extinction coefficient 6.7 L.mmol-1.cm-1) at about half the rate of benzoylcholine, but over 2000 times faster than naturally occurring (-)-cocaine. This rapid hydrolysis of (+)-cocaine may account, in part, for its pharmacological inactivity. (+)-Norcocaine, (+)-benzoylecgonine, (-)-psi-cocaine and tropacocaine were also substrates for BChE. Hydrolysis of (+)-cocaine was sensitive to several standard inhibitors of BChE, including those of competitive, carbamate and organophosphorus classes. Although (-)-cocaine was a poor substrate for debenzoylation, it was a fairly good competitive inhibitor (Ki approximately 10 microM) of the hydrolysis of other substrates. The cocaine metabolites (-)-norcocaine, (-)-benzoylecgonine and (-)-ecgonine methyl ester inhibited BChE with Ki values of 15, 76 and 1300 microM, respectively. (+)-psi-Cocaine had Ki = 3 microM, p-Nitro and p-fluoro derivatives of cocaine and analogs with phenyl and p-fluorophenyl groups in place of the benzoyl ester linkage (WIN 35,065-2 and WIN 35,428) inhibited BChE comparably to (-)-cocaine itself. Both cocaine enantiomers were weak inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) from human erythrocytes with similar Ki values (160-170 microM). Although it is unlikely that the inhibition of BChE is an important factor in the subjective effects of cocaine, it may have implications for the toxicity of cocaine to the fetus, since BChE appears in the development of the central nervous system before AChE, and has been suggested to function as an embryonic acetylcholinesterase.

People with genetic variants of cholinesterase respond abnormally to succinylcholine, experiencing substantial prolongation of muscle paralysis with apnea rather than the usual 2-6 min. The structure of usual cholinesterase has been determined including the complete amino acid and nucleotide sequence. This has allowed identification of altered amino acids and nucleotides. The variant most frequently found in patients who respond abnormally to succinylcholine is atypical cholinesterase, which occurs in homozygous form in 1 out of 3500 Caucasians. Atypical cholinesterase has a single substitution at nucleotide 209 which changes aspartic acid 70 to glycine. This suggests that Asp 70 is part of the anionic site, and that the absence of this negatively charged amino acid explains the reduced affinity of atypical cholinesterase for positively charged substrates and inhibitors. The clinical consequence of reduced affinity for succinylcholine is that none of the succinylcholine is hydrolyzed in blood and a large overdose reaches the nerve-muscle junction where it causes prolonged muscle paralysis. Silent cholinesterase has a frame shift mutation at glycine 117 which prematurely terminates protein synthesis and yields no active enzyme. The K variant, named in honor of W. Kalow, has threonine in place of alanine 539. The K variant is associated with 33% lower activity. All variants arise from a single locus as there is only one gene for human cholinesterase (EC 3.1.1.8). Comparison of amino acid sequences of esterases and proteases shows that cholinesterase belongs to a new family of serine esterases which is different from the serine proteases.

Following ingestion of [N-14CH3]cocaine (10 mg, 2.3 muCi) by 2 healthy subjects, breath, saliva, serum, and urine samples were collected serially. Labeled CO2 production was monitored as a measure of N-demethylation of cocaine. The cumulative excretion of 14CO2 in 5 hr was 2.4% and 6.2% of the administered dose with half-lives of 2.3 and 1.4 hr, respectively. The greater N-demethylation was found in a subject with lower plasma cholinesterase activity. Radioactivity excreted in 0 to 28 hr urine reached 65% to 75% of the dose. Ecgonine methyl ester, a product of cocaine hydrolysis by plasma cholinesterase, was identified as a major metabolite in the urine of both subjects and accounted for 32% to 49% of the urinary metabolites.