Accelerate Cocaine Metabolism Research Articles

Catalytic activities of a highly efficient cocaine hydrolase for hydrolysis of biologically active cocaine metabolites norcocaine and benzoylecgonine

Cocaine is a widely abused, hepatotoxic drug without an FDA-approved pharmacotherapy specific for cocaine addiction or overdose. It is recognized as a promising therapeutic strategy to accelerate cocaine metabolism which can convert cocaine to pharmacologically inactive metabolite(s) using an efficient cocaine-metabolizing enzyme. Our previous studies have successfully designed and discovered a highly efficient cocaine hydrolase, denoted as CocH5-Fc(M6), capable of rapidly hydrolyzing cocaine at the benzoyl ester moiety. In the present study, we determined the kinetic parameters of CocH5-Fc(M6) against norcocaine (kcat = 9,210 min−1, KM = 20.9 μM, and kcat/KM = 1.87 × 105 min−1 M−1) and benzoylecgonine (kcat = 158 min−1, KM = 286 μM, and kcat/KM = 5.5 × 105 min−1 M−1) for the first time. Further in vivo studies have demonstrated that CocH5-Fc(M6) can effectively accelerate clearance of not only cocaine, but also norcocaine (known as a cocaine metabolite which is more toxic than cocaine itself) and benzoylecgonine (known as an unfavorable long-lasting metabolite with some long-term toxicity concerns) in rats. Due to the desired high catalytic activity against norcocaine, CocH5-Fc(M6) is capable of quickly detoxifying both cocaine and its more toxic metabolite norcocaine after intraperitoneally administering lethal dose of 60 or 180 mg/kg cocaine. In addition, the ability of CocH5-Fc(M6) to accelerate clearance of benzoylecgonine should also be valuable for the use of CocH5-Fc(M6) in treatment of cocaine use disorder.

Development of a Highly Efficient Long-Acting Cocaine Hydrolase Entity to Accelerate Cocaine Metabolism.

It is particularly challenging to develop a truly effective pharmacotherapy for cocaine use disorder (CUD) treatment. Accelerating cocaine metabolism via hydrolysis at cocaine benzoyl ester using an efficient cocaine hydrolase (CocH) is known as a promising pharmacotherapeutic approach to CUD treatment. Preclinical and clinical studies on our first CocH (CocH1), in its human serum albumin-fused form known as TV-1380, have demonstrated the promise of a general concept of CocH-based pharmacotherapy for CUD treatment. However, the biological half-life of TV-1380 (t1/2 = 8 h in rats, associated with t1/2 = 43-77 h in humans) is not long enough for practical treatment of cocaine dependence, which requires enzyme injection for no more than once weekly. Through protein fusion of a human butyrylcholinesterase mutant (denoted as CocH5) with a mutant (denoted as Fc(M6)) of Fc from human IgG1, we have designed, prepared, and tested a new fusion protein (denoted as CocH5-Fc(M6)) for its pharmacokinetic profile and in vivo catalytic activity against (-)-cocaine. CocH5-Fc(M6) represents the currently most efficient long-acting cocaine hydrolase with both the highest catalytic activity against (-)-cocaine and the longest elimination half-life (t1/2 = 229 ± 5 h) in rats. As a result, even at a single modest dose of 3 mg/kg, CocH5-Fc(M6) can significantly and effectively accelerate the metabolism of cocaine in rats for at least 60 days. In addition, ∼70 nM CocH5-Fc(M6) in plasma was able to completely block the toxicity and physiological effects induced by intraperitoneal injection of a lethal dose of cocaine (60 mg/kg).

Reengineering of Albumin-Fused Cocaine Hydrolase CocH1 (TV-1380) to Prolong Its Biological Half-Life.

Therapeutic treatment of cocaine toxicity or addiction is a grand medical challenge. As a promising therapeutic strategy for treatment of cocaine toxicity and addiction to develop a highly efficient cocaine hydrolase (CocH) capable of accelerating cocaine metabolism to produce physiologically/biologically inactive metabolites, our previously designed A199S/S287G/A328W/Y332G mutant of human butyrylcholinesterase (BChE), known as cocaine hydrolase-1 (CocH1), possesses the desirably high catalytic activity against cocaine. The C-terminus of CocH1, truncated after amino acid #529, was fused to human serum albumin (HSA) to extend the biological half-life. The C-terminal HSA-fused CocH1 (CocH1-HSA), known as Albu-CocH1, Albu-CocH, AlbuBChE, Albu-BChE, or TV-1380 in literature, has shown favorable preclinical and clinical profiles. However, the actual therapeutic value of TV-1380 for cocaine addiction treatment is still limited by the short half-life. In this study, we designed and tested a new type of HSA-fused CocH1 proteins, i.e., N-terminal HSA-fused CocH1, with or without a linker between the HSA and CocH1 domains. It has been demonstrated that the catalytic activity of these new fusion proteins against cocaine is similar to that of TV-1380. However, HSA-CocH1 (without a linker) has a significantly longer biological half-life (t1/2 = 14 ± 2h) compared to the corresponding C-terminal HSA-fused CocH1, i.e., CocH1-HSA (TV-1380 with t1/2 = 5-8h), in rats. Further, the N-terminal HSA-fused CocH1 proteins with a linker have further prolonged biological half-lives: t1/2 = 17 ± 2h for both HSA-EAAAK-CocH1 and HSA-PAPAP-CocH1, and t1/2 = 18 ± 3h for HSA-(PAPAP)2-CocH1. These N-terminal HSA-fused CocH1 proteins may serve as more promising protein drug candidates for cocaine addiction treatment.

Evaluation of the cholinesterase activity of a potential therapeutic cocaine esterase for cocaine overdose

BackgroundCocaine is a commonly abused drug and there is no approved medication specifically to treat its addiction or overdose. Bacterial cocaine esterase (CocE)-derived RBP-8000 is currently under clinical development for cocaine overdose treatment. It is proven to be effective for human use to accelerate cocaine metabolism into physiologically inactive products. Besides cocaine, RBP-8000 may hydrolyze the neurotransmitter acetylcholine (ACh), however, no study has reported its cholinesterase activity. The present study aims to examine RBP-8000′s cholinesterase activity and substrate selectivity to address the potential concern that this enzyme therapy might produce cholinergic side-effects. MethodsBoth computational modeling and experimental kinetic analysis were carried out to characterize the potential cholinesterase activity of RBP-8000. Substrates interacting with RBP-8000 were modeled for their enzyme-substrate binding complexes. In vitro enzymatic kinetic parameters were measured using Ellman’s colorimetric assay and analyzed by Michaelis–Menten kinetics. ResultsIt is the first demonstration that RBP-8000 catalyzes the hydrolysis of acetylthiocholine (ATC). However, its catalytic efficiency (kcat/KM) against ATC is 1000-fold and 5000-fold lower than it against cocaine at 25 °C and 37 °C, respectively, suggesting RBP-8000 has the desired substrate selectivity for cocaine over ACh. ConclusionGiven the fact that clinically relevant dose of RBP-8000 displays insignificant cholinesterase activity relative to endogenous cholinesterases in human, administration of RBP-8000 is unlikely to produce any significant cholinergic side-effects. This study provides supplemental evidences in support of further development of RBP-8000 towards a clinically used pharmacotherapy for cocaine overdose.

Highly efficient long‐acting cocaine hydrolases as a treatment for cocaine addiction

AimsEnzyme therapy using a highly efficient exogenous cocaine‐metabolizing enzyme is recognized as a promising new therapeutic strategy for cocaine abuse. Our recently designed highly efficient, long‐acting cocaine hydrolases (LA‐CocHs), engineered from human butyrylcholinesterase, have a considerably improved catalytic efficiency against cocaine and a significantly improved biological half‐life. Further, we want to know whether LA‐CocHs are effective in blocking the physiological and psychological effects of cocaine. Based on the animal data, we may select the LA‐CocH with the best overall in vivo profile for further development.MethodsStructure and mechanism‐based computational design was followed by laboratory experimental studies. LA‐CocHs were produced in a bioreactor using stable cell lines that we developed previously, and were purified. The purified LA‐CocHs were used to test their actual in vivo activities in animals (rats and monkeys), including pharmacokinetics (PK), pharmacodynamics (PD), hyperactivity, drug discrimination, and drug self‐administration studies.ResultsWe have successfully designed, prepared, and characterized a set of LA‐CocHs, including CocH5‐Fc(6M). According to the animal data (including those from extensive PK/PD, locomotor activity, drug discrimination, and drug self‐administration tests), a single dose of LA‐CocH was able to effectively accelerate cocaine metabolism in animals even after 30 days and, thus, block cocaine‐induced physiological effects and toxicity for a long period of time. In consideration of the general observation that the biological half‐life of a protein drug in humans is generally longer than that in animals, CocH5‐Fc(6M) examined in this study could allow dosing once every month for cocaine addiction treatment in humans.ConclusionThe animal data suggest that CocH5‐Fc(6M) has the desirable potency of a therapeutic enzyme. Hence, CocH5‐Fc(6M) could be developed further to become the desirable enzyme therapy for cocaine addiction treatment.Support or Funding InformationThis work was supported by an NIDA Translational Avant‐Garde Award (grant UH2/UH3 DA041115) and NIDA R01 grants (DA035552, DA032910, DA013930, and DA025100).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of Fc-Fused Cocaine Hydrolase for Cocaine Addiction Treatment: Catalytic and Pharmacokinetic Properties.

Cocaine abuse is a worldwide public health and social problem without a US Food and Drug Administration (FDA)-approved medication. Accelerating cocaine metabolism that produces biologically inactive metabolites by administration of an efficient cocaine hydrolase (CocH) has been recognized as a promising strategy for cocaine abuse treatment. However, the therapeutic effects of CocH are limited by its short biological half-life (e.g., 8h or shorter in rats). In this study, we designed and prepared a set of Fc-fusion proteins constructed by fusing Fc(M3) with CocH3 at the N-terminus of CocH3. A linker between the two protein domains was optimized to improve both the biological half-life and catalytic activity against cocaine. It has been concluded that Fc(M3)-G6S-CocH3 not only has fully retained the catalytic efficiency of CocH3 against cocaine but also has the longest biological half-life (e.g., ∼ 136h in rats) among all of the long-acting CocHs identified so far. A single dose (0.2mg/kg, IV) of Fc(M3)-G6S-CocH3 was able to significantly attenuate 15mg/kg cocaine-induced hyperactivity for at least 11days (268h) after the Fc(M3)-G6S-CocH3 administration.

Effects of a cocaine hydrolase engineered from human butyrylcholinesterase on metabolic profile of cocaine in rats

Accelerating cocaine metabolism through enzymatic hydrolysis at cocaine benzoyl ester is recognized as a promising therapeutic approach for cocaine abuse treatment. Our more recently designed A199S/F227A/S287G/A328W/Y332G mutant of human BChE, denoted as cocaine hydrolase-3 (CocH3), has a considerably improved catalytic efficiency against cocaine and has been proven active in blocking cocaine-induced toxicity and physiological effects. In the present study, we have further characterized the effects of CocH3 on the detailed metabolic profile of cocaine in rats administrated intravenously (IV) with 5 mg/kg cocaine, demonstrating that IV administration of 0.15 mg/kg CocH3 dramatically changed the metabolic profile of cocaine. Without CocH3 administration, the dominant cocaine-metabolizing pathway in rats was cocaine methyl ester hydrolysis to benzoylecgonine (BZE). With the CocH3 administration, the dominant cocaine-metabolizing pathway in rats became cocaine benzoyl ester hydrolysis to ecgonine methyl ester (EME), and the other two metabolic pathways (i.e. cocaine methyl ester hydrolysis to BZE and cocaine oxidation to norcocaine) became insignificant. The CocH3-catalyzed cocaine benzoyl ester hydrolysis to EME was so efficient such that the measured maximum blood cocaine concentration (∼38 ng/ml) was significantly lower than the threshold blood cocaine concentration (∼72 ng/ml) required to produce any measurable physiological effects.

Long-acting cocaine hydrolase for addiction therapy

Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2-4 wk, or longer, for treatment of cocaine addiction in humans.

Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine

Cocaine is a widely abused drug without an FDA (Food and Drug Administration)-approved medication. It has been recognized that an ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. human BChE (butyrylcholinesterase)-catalysed hydrolysis. However, the native human BChE has a low catalytic activity against cocaine. We recently designed and discovered a BChE mutant (A199S/F227A/S287G/A328W/Y332G) with a high catalytic activity (kcat=5700 min-1, Km=3.1μM) specifically for cocaine, and the mutant was proven effective in protecting mice from acute cocaine toxicity of a lethal dose of cocaine (180mg/kg of body weight, LD100). Further characterization in animal models requires establishment of a high-efficiency stable cell line for the BChE mutant production at a relatively larger scale. It has been extremely challenging to develop a high-efficiency stable cell line expressing BChE or its mutant. In the present study, we successfully developed a stable cell line efficiently expressing the BChE mutant by using a lentivirus-based repeated-transduction method. The scaled-up protein production enabled us to determine for the first time the invivo catalytic activity and the biological half-life of this high-activity mutant of human BChE in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant (administered to mice 1min prior to cocaine) can quickly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Substrate selectivity of high-activity mutants of human butyrylcholinesterase.

Cocaine is one of the most addictive drugs, and there is still no FDA (Food and Drug Administration)-approved medication specific for cocaine abuse. A promising therapeutic strategy is to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, the native BChE has a low catalytic efficiency against the abused cocaine, i.e. (-)-cocaine. Our recently designed and discovered A199S/F227A/S287G/A328W/Y332G mutant and other mutants of human BChE have a considerably improved catalytic efficiency against (-)-cocaine. In the present study, we carried out both computational modeling and experimental kinetic analysis on the catalytic activities of these promising new BChE mutants against other known substrates, including neurotransmitter acetylcholine (ACh), acetylthiocholine (ATC), butyrylthiocholine (BTC), and (+)-cocaine, in comparison with the corresponding catalytic activity against (-)-cocaine. Both the computational modeling and kinetic analysis have consistently revealed that all the examined amino acid mutations only considerably improve the catalytic efficiency of human BChE against (-)-cocaine, without significantly improving the catalytic efficiency of the enzyme against any of the other substrates examined. In particular, all the examined BChE mutants have a slightly lower catalytic efficiency against neurotransmitter ACh compared to the wild-type BChE. This observation gives us confidence in developing an anti-cocaine enzyme therapy by using one of these BChE mutants, particularly the A199S/F227A/S287G/A328W/Y332G mutant.

Accelerating Cocaine Metabolism as an Approach to the Treatment of Cocaine Abuse and Toxicity

One pharmacokinetic approach to the treatment of cocaine abuse and toxicity involves the development of compounds that can be safely administered to humans and that accelerate the metabolism of cocaine to inactive components. Catalytic antibodies have been developed and shown to accelerate cocaine metabolism, but their catalytic efficiency for cocaine is relatively low. Mutations of human butyrylcholinesterase and a bacterial cocaine esterase found in the soil of coca plants have also been developed. These compounds accelerate cocaine metabolism and antagonize the behavioral and toxic effects of cocaine in animal models. Of these two approaches, the human butyrylcholinesterase mutants show the most immediate promise as they would not be expected to evoke an immune response in humans.

Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine.

Cocaine is a widely abused drug without a U.S. Food and Drug Administration-approved medication. There is a recognized, promising anticocaine medication to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway [i.e., cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma]. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against (-)-cocaine but also certain selectivity for (-)-cocaine over neurotransmitter acetylcholine (ACh), such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. The present study accounting for the mutation-caused changes of the catalytic activities of BChE against both (-)-cocaine and ACh by means of molecular modeling and site-directed mutagenesis has led to identification of three BChE mutants that have not only a considerably improved catalytic efficiency against (-)-cocaine but also the desirable selectivity for (-)-cocaine over ACh. Two representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg). Pretreatment with the BChE mutant (i.e., 1 min before cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. In particular, all mice pretreated with the mutant (e.g., 0.02 mg or more of A199S/F227A/S287G/A328W/E441D BChE) survived. The in vivo data reveal the primary factor (i.e., the relative catalytic efficiency), determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment.

Recent Progress in Protein Drug Design and Discovery with a Focus on Novel Approaches to the Development of Anticocaine Medications

Cocaine is highly addictive and no anti-cocaine medication is currently available. Accelerating cocaine metabolism, producing biologically inactive metabolites, is recognized as an ideal anti-cocaine medication strategy, especially for the treatment of acute cocaine toxicity. However, currently known wild-type enzymes have either too low a catalytic efficiency against the abused cocaine, in other words (-)-cocaine, or the in vivo half-life is too short. Novel computational strategies and design approaches have been developed recently to design and discover thermostable or high-activity mutants of enzymes based on detailed structures and catalytic/inactivation mechanisms. The structure- and mechanism-based computational design efforts have led to the discovery of high-activity mutants of butyrylcholinesterase and thermostable mutants of cocaine esterase as promising anti-cocaine therapeutics. The structure- and mechanism-based computational strategies and design approaches may be used to design high-activity and/or thermostable mutants of many other proteins that have clear therapeutic potentials and to design completely new therapeutic enzymes.

Free-Energy Perturbation Simulation on Transition States and Redesign of Butyrylcholinesterase

It is recognized that an ideal anti-cocaine treatment is to accelerate cocaine metabolism by producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., butyrylcholinesterase (BChE)-catalyzed hydrolysis of cocaine. BChE mutants with a higher catalytic activity against (-)-cocaine are highly desired for use as an exogenous enzyme in humans. To develop a rational design for high-activity mutants, we carried out free-energy perturbation (FEP) simulations on various mutations of the transition-state structures in addition to the corresponding free-enzyme structures by using an extended FEP procedure. The FEP simulations on the mutations of both the free-enzyme and transition-state structures allowed us to calculate the mutation-caused shift of the free-energy change from the free enzyme (BChE) to the transition state, and thus to theoretically predict the mutation-caused shift of the catalytic efficiency (kcat/KM). The computational predictions are supported by the kinetic data obtained from the wet experiments, demonstrating that the FEP-based computational design approach is promising for rational design of high-activity mutants of an enzyme. One of the BChE mutants designed and discovered in this study has an ∼1800-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. The high-activity mutant may be therapeutically valuable.

Gene Transfer of Cocaine Hydrolase Suppresses Cardiovascular Responses to Cocaine in Rats

We previously found that injection of a cocaine hydrolase (CocE) engineered from human butyrylcholinesterase will transiently accelerate cocaine metabolism in rats while reducing physiological and behavioral responses. To investigate more extended therapeutic effects, CocE cDNA was incorporated into a replication-incompetent type-5 adenoviral vector with a cytomegalovirus promoter. In rats dosed with this agent (2.2 x 10(9) plaque-forming units), the time course of expression was characterized by reverse transcription polymerase chain reaction for CocE mRNA and by radiometric assay for enzyme activity. Liver and plasma showed comparable expression, beginning 2 days after vector administration and peaking between 5 and 7 days. Plasma CocE content was up to 100 mU/ml, with total cocaine hydrolyzing activity 3000-fold greater than in "empty vector" or untreated controls. This level of expression approximated that found immediately after i.v. injection of purified hydrolase, 3 mg/kg, a dose that shortened cocaine halflife and blunted cardiovascular effects. Sucrose density gradient analysis showed that 96% of the circulating CocE activity was associated with tetrameric enzyme forms, expected to be stable in vivo. Consistent with this expectation, CocE from vector-treated rats showed a plasma t(1/2) of 33 h when reinjected into naive rats. Transduction of another mutant butyrylcholinesterase, Applied Molecular Evolution mutant 359 (AME(359)), caused plasma cocaine hydrolase activity to rise 50,000-fold. At the point of peak AME(359) expression, cocaine was cleared from the blood too rapidly for accurate measurement, and pressor responses to the injection of drug were greatly impaired.