High-Dose Methylphenidate and Carboxylesterase 1 Genetic Variability in Patients With Attention-Deficit/Hyperactivity Disorder: A Case Series.

PURPOSE/BACKGROUND: Methylphenidate (MPH) is widely used to reduce symptoms of attention-deficit/hyperactivity disorder. Methylphenidate is metabolized by the carboxylesterase 1 (CES1) enzyme. Some patients need a very high dose of MPH to reach desired clinical effects, without having adverse effects. This may be due to differences in MPH pharmacokinetics (PK), potentially caused by DNA variants in CES1 , the gene encoding the enzyme that metabolizes MPH. Here we describe 3 patients requiring high-dose MPH and investigated the CES1 gene. METHODS/PROCEDURES: The 3 patients were using short-acting MPH in a dose of 180 to 640 mg instead of the maximum advised dose of around 100 mg MPH in the Netherlands. Plasma concentrations of MPH were determined at scheduled time points (day-curve). Methylphenidate plasma concentrations were used for PK analysis using an earlier published 2-compartment PK population model of MPH. Individual data of the 3 patients were compared with simulated population data, when equivalent doses were used. In addition, CES1 was genotyped (number of gene copies and single nucleotide polymorphisms) using real-time polymerase chain reaction. FINDINGS/RESULTS: Pharmacokinetic analysis in all 3 patients showed lower plasma concentrations of MPH in comparison with the population data. The mean absorption time and volume of distribution of the central compartment were equal, but the elimination clearance was higher. However, CES1 genotyping revealed no variations that could explain a higher metabolism of MPH. IMPLICATIONS/CONCLUSIONS: In these 3 cases, we could not demonstrate a correlation between MPH clearance and known genetic variants of the CES1 gene.

Interfering with lipid metabolism through targeting CES1 sensitizes hepatocellular carcinoma for chemotherapy.

Hepatocellular carcinoma (HCC) is the most common lethal form of liver cancer. Apart from surgical removal and transplantation, other treatments have not yet been well established for patients with HCC. In this study, we found that carboxylesterase 1 (CES1) is expressed at various levels in HCC. We further revealed that blockage of CES1 by pharmacological and genetical approaches leads to altered lipid profiles that are directly linked to impaired mitochondrial function. Mechanistically, lipidomic analyses indicated that lipid signaling molecules, including polyunsaturated fatty acids (PUFAs), which activate PPARα/γ, were dramatically reduced upon CES1 inhibition. As a result, the expression of SCD, a PPARα/γ target gene involved in tumor progression and chemoresistance, was significantly downregulated. Clinical analysis demonstrated a strong correlation between the protein levels of CES1 and SCD in HCC. Interference with lipid signaling by targeting the CES1-PPARα/γ-SCD axis sensitized HCC cells to cisplatin treatment. As a result, the growth of HCC xenograft tumors in NU/J mice was potently slowed by coadministration of cisplatin and CES1 inhibition. Our results, thus, suggest that CES1 is a promising therapeutic target for HCC treatment.

The impact of ABCB1 and CES1 polymorphisms on the safety of dabigatran in patients with non-valvular atrial fibrillation.

BACKGROUND: This study aimed to analyze associations between genetic variants and plasma concentrations along with clinical outcomes in dabigatran in patients with non-valvular atrial fibrillation (NVAF). METHODS: We conducted a prospective study and enrolled NVAF patients treated with dabigatran in the real world. A total of 86 patients treated with 110 mg DE twice daily were recruited for this study. Blood samples were obtained from each patient and used for genotyping and determination of plasma dabigatran concentration. All bleeding and thromboembolic complications were recorded during the 1.5 years of follow-up. RESULTS: Eighty-three patients provided samples at the trough plasma level of dabigatran, and 58 patients provided samples at the peak plasma level of dabigatran. There was a significant association between the CES1 SNP rs8192935 and trough plasma concentrations of dabigatran (P = 0.013). Our results showed that the CES1 SNP rs8192935 significantly influenced dabigatran trough concentrations in the Chinese population, and carriers of the G allele had increased trough plasma concentrations of dabigatran compared to noncarriers. The ABCB1 SNP c.2482-2236G > A (rs4148738) was associated with major bleeding events in the addictive model (P = 0.046, OR = 3.29) and dominant model (P = 0.040, OR = 8.17). Additionally, the ABCB1 SNP c.3435 C > T (rs1045642) was associated with the incidence of major bleeding events in the addictive model (P = 0.043, OR = 3.34) and dominant model (P = 0.046, OR = 7.77). However, no significant associations were found between all the SNPs and the incidence of minor bleeding events. CONCLUSION: Our results indicated that the CES1 polymorphism rs8192935 was associated with trough plasma concentrations of dabigatran. Carriers of the G allele had increased trough plasma concentrations of dabigatran compared to noncarriers. The ABCB1 polymorphisms rs4148738 and rs1045642 were associated with an increased risk for major bleeding events for the first time in a Chinese population.

AADAC promotes therapeutic activity of cisplatin and imatinib against ovarian cancer cells.

OBJECTIVE: To explore how AADAC functions in the malignant progression of ovarian cancer, and the effect of AADAC on drug therapeutic activity against ovarian cancer cells. METHODS: AADAC level in tumor and normal samples from TCGA-OV dataset and its survival significance were analyzed by bioinformatics methods. Signaling pathway enrichment analysis for the high- and low-AADAC patients was achieved by using GSEA software. AADAC expression in the cell lines with different treatments was evaluated via qRT-PCR. Cell proliferative ability was assessed via MTT assay Cell migratory and invasive abilities were evaluated via transwell assay. Angiogenesis assay was performed to examine the angiogenetic ability. RESULTS: AADAC was upregulated in ovarian cancer tissues, and patients with high expression of AADAC had favorable survival conditions compared to the low AADAC expression ones. Overexpression of AADAC inhibited the malignant progression of ovarian cancer cells. Both cisplatin and imatinib suppressed cancer cell malignant progression, while overexpressed AADAC synergistically enhanced such inhibition. CONCLUSIONS: The study demonstrated that AADAC could somehow suppress the malignant progression of ovarian cancer, especially at the cellular level. In addition, synergic tumor-inhibitory effects between AADAC and the anti-cancer drugs were identified. All the above results proposed a novel idea and candidate biomarker for ovarian cancer therapy.

A plant-derived cocaine hydrolase prevents cocaine overdose lethality and attenuates cocaine-induced drug seeking behavior.

Cocaine use disorders include short-term and acute pathologies (e.g. overdose) and long-term and chronic disorders (e.g. intractable addiction and post-abstinence relapse). There is currently no available treatment that can effectively reduce morbidity and mortality associated with cocaine overdose or that can effectively prevent relapse in recovering addicts. One recently developed approach to treat these problems is the use of enzymes that rapidly break down the active cocaine molecule into inactive metabolites. In particular, rational design and site-directed mutagenesis transformed human serum recombinant butyrylcholinesterase (BChE) into a highly efficient cocaine hydrolase with drastically improved catalytic efficiency toward (-)-cocaine. A current drawback preventing the clinical application of this promising enzyme-based therapy is the lack of a cost-effective production strategy that is also flexible enough to rapidly scale-up in response to continuous improvements in enzyme design. Plant-based expression systems provide a unique solution as this platform is designed for fast scalability, low cost and the advantage of performing eukaryotic protein modifications such as glycosylation. A Plant-derived form of the Cocaine Super Hydrolase (A199S/F227A/S287G/A328W/Y332G) we designate PCocSH protects mice from cocaine overdose, counters the lethal effects of acute cocaine overdose, and prevents reinstatement of extinguished drug-seeking behavior in mice that underwent place conditioning with cocaine. These results demonstrate that the novel PCocSH enzyme may well serve as an effective therapeutic for cocaine use disorders in a clinical setting.

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

BACKGROUND: Cocaine 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. METHODS: Both 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. RESULTS: It 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. CONCLUSION: Given 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.

Plant expression of cocaine hydrolase-Fc fusion protein for treatment of cocaine abuse.

BACKGROUND: A recently reported cocaine hydrolase (CocH3) fused with fragment crystallizable (Fc) region of human immunoglobulin G1, denoted as CocH3-Fc, is known as a promising therapeutic candidate for the treatment of cocaine overdose and addiction. A challenge for practical therapeutic use of this enzyme exists in the large-scale protein production and, therefore, it is interesting to identify a low-cost and feasible, sustainable source of CocH3-Fc production. RESULTS: CocH3-Fc was transiently expressed in plant Nicotiana benthamiana leaves. The plant-expressed protein, denoted as pCocH3-Fc, was as active as that expressed in mammalian cells both in vitro and in vivo. However, compared to the mammalian-cell expressed CocH3-Fc protein, pCocH3-Fc had a shorter biological half-life, probably due to the lack of protein sialylation in plant. Nevertheless, the in vivo half-life was significantly extended upon the PEGylation of pCocH3-Fc. The Fc fusion did not prolong the biological half-life of the plant-expressed enzyme pCocH3-Fc, but increased the yield of the enzyme expression in the plant under the same experimental conditions. CONCLUSIONS: It is feasible to express pCocH3-Fc in plants. Further studies on the pCocH3-Fc production in plants should focus on the development of vectors with additional genes/promoters for the complete protein sialylation and for a better yield.

Roles of Triolein and Lipolytic Protein in the Pathogenesis and Survival of Mycobacterium tuberculosis: a Novel Therapeutic Approach.

Discovery of novel secreted enzymes and proteins in Mycobacterium tuberculosis (M. tuberculosis) are imperative to understanding the pathogenic system for pathogenesis requires attention. Till date, the groups of these secreted enzymes are not meaningfully characterized in terms of M. tuberculosis. In this way, cutinase, a small lipolytic protein, exists in both bacteria and fungi as well which have a potential catalytic activity. During our search, we have found a few genes of M. tuberculosis demonstrating a same significant lipase action as fungi Fusarium solani cutinase contain. Genome sequencing of M. tuberculosis uncover a lot of proteins, wherein (Rv1758, Rv1984c, Rv2301, Rv3451, Rv3452, Rv3724A, Rv3724B, and Rv3802c) genes have been noted which are exhibiting a cutinase-like activity and closely homologous to that of F. solani cutinase and having the ability to hydrolyze model substrates including p-nitrophenyl butyrate (p-PNB), cutin, triacylglycerols (TAGs), and triolein (TO), yet their biological significance in pathogenesis stays subtle and uncharacterized. In a basic perspective, the measure of cutinase expressed by M. tuberculosis and part of these small lipolytic enzymes in the pathologic discipline require thorough characterization. So, through focusing on cutinase-encoding genes in M. tuberculosis and their active catalytic motif could help to build up a novel therapeutic approach.

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.

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target.

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.

Efficacy of AiiM, an N-acylhomoserine lactonase, against Pseudomonas aeruginosa in a mouse model of acute pneumonia.

Quorum sensing (QS) in Pseudomonas aeruginosa regulates the production of many virulence factors and plays an important role in the pathogenesis of P. aeruginosa infection. N-acyl homoserine lactones (AHL) are major QS signal molecules. Recently, a novel AHL-lactonase enzyme, AiiM, has been identified. The aim of this study was to evaluate the effect of AiiM on the virulence of P. aeruginosa in a mouse model of acute pneumonia. We developed a P. aeruginosa PAO1 strain harboring an AiiM-expressing plasmid. The production of several virulence factors by the AiiM-expressing strain was examined. Mice were intratracheally infected with an AiiM-expressing PAO1 strain. Lung histopathology, bacterial burden, and bronchoalveolar lavage (BAL) fluid were assessed at 24 h postinfection. AiiM expression in PAO1 reduced production of AHL-mediated virulence factors and attenuated cytotoxicity against human lung epithelial cells. In a mouse model of acute pneumonia, AiiM expression reduced lung injury and greatly improved the survival rates. The levels of proinflammatory cytokines and myeloperoxidase activity in BAL fluid were significantly lower in mice infected with AiiM-expressing PAO1. Thus, AiiM can strongly attenuate P. aeruginosa virulence in a mammalian model and is a potential candidate for use as a therapeutic agent against P. aeruginosa infection.

Modeling of pharmacokinetics of cocaine in human reveals the feasibility for development of enzyme therapies for drugs of abuse.

A promising strategy for drug abuse treatment is to accelerate the drug metabolism by administration of a drug-metabolizing enzyme. The question is how effectively an enzyme can actually prevent the drug from entering brain and producing physiological effects. In the present study, we have developed a pharmacokinetic model through a combined use of in vitro kinetic parameters and positron emission tomography data in human to examine the effects of a cocaine-metabolizing enzyme in plasma on the time course of cocaine in plasma and brain of human. Without an exogenous enzyme, cocaine half-lives in both brain and plasma are almost linearly dependent on the initial cocaine concentration in plasma. The threshold concentration of cocaine in brain required to produce physiological effects has been estimated to be 0.22±0.07 µM, and the threshold area under the cocaine concentration versus time curve (AUC) value in brain (denoted by AUC2(∞)) required to produce physiological effects has been estimated to be 7.9±2.7 µM·min. It has been demonstrated that administration of a cocaine hydrolase/esterase (CocH/CocE) can considerably decrease the cocaine half-lives in both brain and plasma, the peak cocaine concentration in brain, and the AUC2(∞). The estimated maximum cocaine plasma concentration which a given concentration of drug-metabolizing enzyme can effectively prevent from entering brain and producing physiological effects can be used to guide future preclinical/clinical studies on cocaine-metabolizing enzymes. Understanding of drug-metabolizing enzymes is key to the science of pharmacokinetics. The general insights into the effects of a drug-metabolizing enzyme on drug kinetics in human should be valuable also in future development of enzyme therapies for other drugs of abuse.

Effects of a long-acting mutant bacterial cocaine esterase on acute cocaine toxicity in rats.

BACKGROUND: A longer acting, double mutant bacterial cocaine esterase (CocE T172R/G173Q; DM CocE) has been shown to protect mice from cocaine-induced lethality, inhibit the reinforcing effects of cocaine in rats, and reverse cocaine's cardiovascular effects in rhesus monkeys. The current studies evaluated the effectiveness of DM CocE to protect against, and reverse cocaine's cardiovascular, convulsant, and lethal effects in male and female rats. METHODS: Pretreatment studies were used to determine the effectiveness and in vivo duration of action for DM CocE to protect rats against the occurrence of cardiovascular changes, convulsion and lethality associated with acute cocaine toxicity. Posttreatment studies were used to evaluate the capacity of DM CocE to rescue rats from the cardiovascular and lethal effects of large doses of cocaine. In addition, male and female rats were studied to determine if there were any potential effects of sex on the capacity of DM CocE to protect against, or reverse acute cocaine toxicity in rats. RESULTS: Pretreatment with DM CocE dose-dependently protected rats against cocaine-induced cardiovascular changes, convulsion and lethality, with higher doses active for up to 4h, and shifting cocaine-induced lethality at least 10-fold to the right. In addition to dose-dependently recovering rats from an otherwise lethal dose of cocaine, post-treatment with DM CocE also reversed the cardiovascular effects of cocaine. There were no sex-related differences in the effectiveness of DM CocE to protect against, or reverse acute cocaine toxicity. CONCLUSIONS: Together, these results support the development of DM CocE for the treatment of acute cocaine toxicity.

A thermally stable form of bacterial cocaine esterase: a potential therapeutic agent for treatment of cocaine abuse.

Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 degrees C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 degrees C, which represents a 340-fold improvement over wt and is 15-fold greater than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-A resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.

Prevention and reversal by cocaine esterase of cocaine-induced cardiovascular effects in rats.

The present study is the first to utilize bacterial cocaine esterase (CocE) to increase elimination of a lethal dose of cocaine and evaluate its cardioprotective effects. Rats received one of 5 treatments: CocE 1 min after saline; CocE 1 min after a lethal i.p. dose of cocaine; saline 1 min after a lethal i.p. dose of cocaine; CocE immediately after observing a cocaine-induced convulsion; and CocE 1 min after observing a cocaine-induced convulsion. Measures were taken of ECG, blood pressure, and cardiac troponin I (cTnI). The specificity of CocE against cocaine was determined by evaluating its actions against the cocaine analogue, WIN-35,065-2, which lacks an ester attack point for CocE. In addition, CocE's effects were compared with those of midazolam, a benzodiazepine often used to manage cocaine overdose. Whereas CocE alone had negligible cardiovascular effects, it blocked or reversed cocaine-induced QRS complex widening, increased QTc interval, ST elevation, bradycardia, and hypertension. When administered 1 min after cocaine, CocE inhibited myocardial damage; however, administered 1 min after a cocaine-induced convulsion (approximately 40s before cocaine-induced death), CocE did not block cTnI release, but did restore cardiac function. Midazolam blocked convulsions, but exhibited inadequate protection against cocaine-induced cardiotoxicity. The majority of rats given cocaine plus midazolam died. CocE did not prevent the lethal cardiovascular effects of WIN-35,065-2. In all likelihood, CocE rapidly and specifically reduced the body burden of cocaine and inhibited or reversed the cardiovascular consequences of high-dose cocaine. These results support CocE as a potential therapeutic avenue in cocaine overdose.

PEGylation of bacterial cocaine esterase for protection against protease digestion and immunogenicity.

Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been considered as a promising treatment strategy for cocaine overdose and addiction, as CocE is the most efficient native enzyme yet identified for metabolizing the naturally occurring cocaine. A major obstacle to the clinical application of CocE, however, lies in its thermo-instability, rapid degradation by circulating proteases, and potential immunogenicity. PEGylation, namely by modifying a protein or peptide compound via attachment of polyethylene glycol (PEG) chains, has been proven to overcome such problems and was therefore exploited in this CocE investigation. The PEG-CocE conjugates prepared in this study showed a purity of greater than 93.5%. Attachment of PEG to CocE apparently inhibited the binding of anti-CocE antibodies to the conjugate, as demonstrated by the enzyme-linked immunosorbent assay (ELISA) assay. In addition, PEGylation yielded protection to CocE against thermal degradation and protease digestion. Furthermore, preliminary in vivo results suggested that, similarly to native CocE, the PEG-CocE conjugates were able to protect animals from cocaine-induced toxic effects. Overall, this study provides evidence that the PEGylation may serve as a tool to prolong CocE functionality in the circulation and reduce its potential immunogenicity.

Effects of cocaine esterase following its repeated administration with cocaine in mice.

BACKGROUND: A bacterial cocaine esterase (CocE) produces robust protection and reversal of cocaine toxicity. The aim of this study was to investigate how effectiveness of CocE was changed following its repeated administration together with cocaine. METHODS: Cocaine toxicity was quantified by measuring the occurrence of convulsions and lethality in mice. Immunologic responses of CocE were determined using ELISA. In the protection experiment, i.v. CocE 0.3mg was given 1min before a lethal dose of i.p. cocaine 180mg/kg. In the rescue experiment, i.v. CocE 0.3mg was given 1min after the occurrence of convulsions elicited by i.p. cocaine 100mg/kg. In both treatment paradigms, four trials were conducted in the same animals with a 2-week interval. RESULTS: CocE retained its effectiveness to protect or rescue mice during the first two trials and these mice did not show an immune response. In contrast, CocE's effectiveness was gradually reduced in the last two trials, accompanied by 10- and 100-fold increases in anti-CocE antibody titers. Nevertheless, effectiveness of CocE could be partially recovered by increasing the dose of CocE. In addition, escalating the dose of CocE from the minimum effective dose for repeated administration could also retain CocE's effectiveness longer and slow the production of anti-CocE antibodies. CONCLUSIONS: These results indicate that CocE is a weak antigen and it can maintain its protective and rescuing ability initially against cocaine-induced toxicity. Decreased effectiveness of CocE following repeated use can be partially improved by adjusting the dose and frequency of CocE treatment.

Thermostable variants of cocaine esterase for long-time protection against cocaine toxicity.

Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been recognized as a promising treatment strategy for cocaine overdose and addiction, because CocE is the most efficient native enzyme for metabolizing the naturally occurring cocaine yet identified. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only a few minutes at physiological temperature (37 degrees C). Here we report thermostable variants of CocE developed through rational design using a novel computational approach followed by in vitro and in vivo studies. This integrated computational-experimental effort has yielded a CocE variant with a approximately 30-fold increase in plasma half-life both in vitro and in vivo. The novel design strategy can be used to develop thermostable mutants of any protein.

A bacterial cocaine esterase protects against cocaine-induced epileptogenic activity and lethality.

STUDY OBJECTIVE: Cocaine toxicity results in cardiovascular complications, seizures, and death and accounts for approximately 20% of drug-related emergency department visits every year. Presently, there are no treatments to eliminate the toxic effects of cocaine. The present study hypothesizes that a bacterial cocaine esterase with high catalytic efficiency would provide rapid and robust protection from cocaine-induced convulsions, epileptogenic activity, and lethality. METHODS: Cocaine-induced paroxysmal activity and convulsions were evaluated in rats surgically implanted with radiotelemetry devices (N=6 per treatment group). Cocaine esterase was administered 1 minute after a lethal dose of cocaine or after cocaine-induced convulsions to determine the ability of the enzyme to prevent or reverse, respectively, the effects of cocaine. RESULTS: The cocaine esterase prevented all cocaine-induced electroencephalographic changes and lethality. This effect was specific for cocaine because the esterase did not prevent convulsions and death induced by a cocaine analog, (-)-2beta-carbomethoxy-3beta-phenyltropane. The esterase prevented lethality even after cocaine-induced convulsions occurred. In contrast, the short-acting benzodiazepine, midazolam, prevented cocaine-induced convulsions but not the lethal effects of cocaine. CONCLUSION: The data showed that cocaine esterase successfully degraded circulating cocaine to prevent lethality and that cocaine-induced convulsions alone are not responsible for the lethal effects of cocaine in this model. Therefore, further investigation into the use of cocaine esterase for treating cocaine overdose and its toxic effects is warranted.

Mammalian carboxylesterases: from drug targets to protein therapeutics.

Our understanding of the detailed recognition and processing of clinically useful therapeutic agents has grown rapidly in recent years, and we are now able to begin to apply this knowledge to the rational treatment of disease. Mammalian carboxylesterases (CEs) are enzymes with broad substrate specificities that have key roles in the metabolism of a wide variety of clinical drugs, illicit narcotics and chemical nerve agents. Here, the functions, mechanism of action and structures of human CEs are reviewed, with the goal of understanding how these proteins are able to act in such a non-specific fashion, yet catalyze a remarkably specific chemical reaction. Current approaches to harness these enzymes as protein-based therapeutics for drug and chemical toxin clearance are described, as well as their uses for targeted chemotherapeutic prodrug activation. Also included is an outline of how selective CE inhibitors could be used as co-drugs to improve the efficacy of clinically approved agents.