Xiabin Chen

Gene transfer of mutant mouse cholinesterase provides high lifetime expression and reduced cocaine responses with no evident toxicity

Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with 3H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3×1011 particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (1010 particles) to 20,000 fold (1013 particles), while the hdAD vector (1.7×1012 particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7×1012 particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose.

Long-acting cocaine hydrolase for addiction therapy.

Significance It is essential for a truly effective addiction medication to block the drugs physiological effects effectively without affecting normal functions of the brain and other critical organs such as the heart and while still preventing relapse during abstinence. Most popularly used pharmacological approaches to addiction treatment, including all currently available addiction therapies, either affect normal functions of brain receptors/transporters or are unable to prevent relapse. The long-acting enzyme approach may provide a novel, truly promising therapy capable of effectively blocking the physiological and toxic effects of cocaine without affecting normal functions of the brain and other critical organs and prevent relapse during abstinence. New insights obtained in this study also may be valuable in guiding development of other therapeutic proteins. 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.

Rational Design, Preparation, and Characterization of a Therapeutic Enzyme Mutant with Improved Stability and Function for Cocaine Detoxification

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 ∼12 min at 37 °C. The previously designed T172R/G173Q mutant (denoted as enzyme E172–173) with an improved in vitro half-life of ∼6 h at 37 °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 °C to >100 days, but also significantly improved the catalytic efficiency against cocaine by ∼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 ∼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.

Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase.

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 (k(cat)/K(M)) 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.

Mccrearamycins A–D, Geldanamycin-Derived Cyclopentenone Macrolactams from an Eastern Kentucky Abandoned Coal Mine Microbe

Four cyclopentenone-containing ansamycin polyketides (mccrearamycins A-D), and six new geldanamycins (Gdms B-G, including new linear and mycothiol conjugates), were characterized as metabolites of Streptomyces sp. AD-23-14 isolated from the Rock Creek underground coal mine acid drainage site. Biomimetic chemical conversion studies using both simple synthetic models and Gdm D confirmed that the mccrearamycin cyclopentenone derives from benzilic acid rearrangement of 19-hydroxy Gdm, and thereby provides a new synthetic derivatization strategy and implicates a potential unique biocatalyst in mccrearamycin cyclopentenone formation. In addition to standard Hsp90α binding and cell line cytotoxicity assays, this study also highlights the first assessment of Hsp90α modulators in a new axolotl embryo tail regeneration (ETR) assay as a potential new whole animal assay for Hsp90 modulator discovery.

A quantitative LC–MS/MS method for simultaneous determination of cocaine and its metabolites in whole blood

HIGHLIGHTSA LC–MS/MS method to simultaneously detect cocaine and 9 metabolites is developed.The method is highly sensitive and reliable for all of the 10 compounds detected.Metabolic profile of cocaine in rats with co‐administration of alcohol is determined. ABSTRACT As new metabolic pathways of cocaine were recently identified, a high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously determine cocaine and nine cocaine‐related metabolites in whole blood samples. One‐step solid phase extraction was used to extract all of the ten compounds and corresponding internal standards from blood samples. All compounds and internal standards extracted were separated on an Atlantis T3 (100 Å, 3 &mgr;m, 2.1 mm × 150 mm I.D) column and detected in positive ion and high sensitivity mode with multiple reaction monitoring. This method was validated for its sensitivity, linearity, specificity, accuracy, precision, recovery, and stability. All of the ten compounds were quantifiable ranging from the lower limit of quantification (LLOQs) of ˜10 nM (1.9–3.2 ng/ml) to ˜1000 nM (190–320 ng/ml) without any interfering substance. Accuracy and precision were determined, and both of them were within the acceptance criteria of the United States (US) Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. The recovery was above 66.7% for all compounds. Stability tests demonstrated the stability of compounds under different storage conditions in whole blood samples. The method was successfully applied to a pharmacokinetic study with co‐administration of cocaine and alcohol in rats.

Clinical Potential of an Enzyme-Based Novel Therapy for Cocaine Overdose

It is a grand challenge to develop a truly effective medication for treatment of cocaine overdose. The current available, practical emergence treatment for cocaine overdose includes administration of a benzodiazepine anticonvulsant agent (e.g. diazepam) and/or physical cooling with an aim to relieve the symptoms. The inherent difficulties of antagonizing physiological effects of drugs in the central nervous system have led to exploring protein-based pharmacokinetic approaches using biologics like vaccines, monoclonal antibodies, and enzymes. However, none of the pharmacokinetic agents has demonstrated convincing preclinical evidence of clinical potential for drug overdose treatment without a question mark on the timing used in the animal models. Here we report the use of animal models, including locomotor activity, protection, and rescue experiments in rats, of drug toxicity treatment with clinically relevant timing for the first time. It has been demonstrated that an efficient cocaine-metabolizing enzyme developed in our previous studies can rapidly reverse the cocaine toxicity whenever the enzyme is given to a living rat, demonstrating promising clinical potential of an enzyme-based novel therapy for cocaine overdose as a successful example in comparison with the commonly used diazepam.

Metabolic Enzymes of Cocaine Metabolite Benzoylecgonine

Cocaine is one of the most addictive drugs without a U.S. Food and Drug Administration (FDA)-approved medication. Enzyme therapy using an efficient cocaine-metabolizing enzyme is recognized as the most promising approach to cocaine overdose treatment. The actual enzyme, known as RBP-8000, under current clinical development for cocaine overdose treatment is our previously designed T172R/G173Q mutant of bacterial cocaine esterase (CocE). The T172R/G173Q mutant is effective in hydrolyzing cocaine but inactive against benzoylecgonine (a major, biologically active metabolite of cocaine). Unlike cocaine itself, benzoylecgonine has an unusually stable zwitterion structure resistant to further hydrolysis in the body and environment. In fact, benzoylecgonine can last in the body for a very long time (a few days) and, thus, is responsible for the long-term toxicity of cocaine and a commonly used marker for drug addiction diagnosis in pre-employment drug tests. Because CocE and its mutants are all active against cocaine and inactive against benzoylecgonine, one might simply assume that other enzymes that are active against cocaine are also inactive against benzoylecgonine. Here, through combined computational modeling and experimental studies, we demonstrate for the first time that human butyrylcholinesterase (BChE) is actually active against benzoylecgonine, and that a rationally designed BChE mutant can not only more efficiently accelerate cocaine hydrolysis but also significantly hydrolyze benzoylecgonine in vitro and in vivo. This sets the stage for advanced studies to design more efficient mutant enzymes valuable for the development of an ideal cocaine overdose enzyme therapy and for benzoylecgonine detoxification in the environment.

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

BackgroundA 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.ResultsCocH3-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.ConclusionsIt 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.