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

HUMAN BUTYRYLCHOLINESTERASE MUTANTS FOR COCAINE DETOXIFICATION

Cocaine is one of the most reinforcing drugs of abuse and has caused serious medical and social problems. There is no FDA-approved medication specific for cocaine. It is of a high priority to develop an effective therapeutic treatment for cocaine abuse. Human butyrylcholinesterase (BChE) has been recognized as a promising candidate of enzyme therapy to metabolize cocaine into biologically inactive metabolites and prevent it from reaching central nervous system (CNS). However, the catalytic activity of wide-type human BChE against cocaine is not sufficiently high for treatment of cocaine abuse. Dr. Zhan’s lab has successfully designed and discovered a series of high-activity mutants of human BChE specific for cocaine metabolism. This dissertation is mainly focused to address the possible concerns in further development of promising human BChE mutants for cocaine detoxification, including whether the administration of this exogenous enzyme will affect the cholinergic system, whether it can efficiently hydrolyze cocaine’s toxic metabolites, and whether the commonly used therapeutic agents will significantly affect the catalytic activity of the BChE mutants against cocaine when they are co-administered. According to the results obtained, all of the examined BChE mutants have a considerably improved catalytic efficiency against (-)-cocaine, without significantly improving the catalytic efficiency against any of the other examined substrates, including neurotransmitter acetylcholine. Two representative mutants (including E12-7) also have a considerably improved catalytic activity against cocaethylene (formed from combined use of cocaine and alcohol) compared to wild-type BChE, and E12-7 can rapidly metabolize cocaethylene, in addition to cocaine, in rats. Further evaluation of possible drug-drug interactions between E12-7 and some other commonly used therapeutic agents revealed that all of the examined agents, except some tricyclic antidepressants, do not significantly inhibit E12-7. In addition, an effort to discover new mutants with further improved activity against cocaine led to the discovery of a new BChE mutant, denoted as E20-7, according to both the in vitro and in vivo assays. The encouraging outcomes of the present investigation suggest that it is possible to develop a more effective enzyme therapy for cocaine abuse treatment using one of the most promising BChE mutants, such as E12-7 or E20-7

HIGH-ACTIVITY MUTANTS OF HUMAN BUTYRYLCHOLINESTERASE FOR COCAINE ABUSE TREATMENT

Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native BChE has a low catalytic activity against cocaine. We recently designed and discovered a set of BChE mutants with a high catalytic activity specifically for cocaine. 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. Through integrated computational-experimental studies, several BChE mutants were identified to have not only a considerably improved catalytic efficiency against cocaine, but also the desirable selectivity for cocaine over ACh. 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, LD100). Pretreatment with the BChE mutant (i.e. 1 min prior to cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. 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. For further characterization in animal models, we successfully developed high-efficiency stable cell lines efficiently expressing the BChE mutants by using a lentivirus-based repeated-transduction method. The large-scale protein production enabled us to further characterize the in vivo profiles of the BChE mutant concerning the biological half-life and potency in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant can rapidly 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

CHARACTERIZATION AND ENGINEERING OF HUMAN PROTEINS AS THERAPEUTIC CANDIDATES

Protein engineering has been a useful tool in the fight against human diseases. Human insulin was the first recombinant DNA-derived therapeutic protein (Humulin®) approved by the US FDA in 1982. However, many of the early protein drugs were only recombinant versions of natural proteins with no modification of their primary amino acid sequence and most of them did not make optimal drug products mainly due to their short half-life or suboptimal affinity, leading to poor therapeutic efficacy. The difficulty in the large-scale production of some therapeutic proteins was another important issue. In the past three decades, different protein engineering platforms have been developed to overcome the obstacles seen in the first generations of these treatments. With the help of these new techniques, proteins have been purposefully modified to improve their clinical potential. The focus of my dissertation is the engineering of potential protein drugs to make them therapeutically useful and more valuable. Previously, our research group has developed cocaine hydrolases (CocHs) from human butyrylcholinesterase (BChE) for treatment of cocaine addiction and prevention of acute cocaine intoxication. In the first project, CocHs were further engineered to improve their performance, e.g., Fc-fused CocHs with an extended serum half-life. Then, I investigated the potential application of a long-lasting CocH for protection against the acute toxic and stimulant effects of cocaine. In the second project, I investigated the potential inhibition of CocH-mediated cocaine hydrolysis by heroin (3,6-diacetylmorphine) or its initial host metabolite, 6-monoacetylmorphine (6-MAM). The investigation of this possible inhibition was important to determine the in vivo efficacy of CocHs, as heroin is one of the most commonly co-abused drugs by cocaine-dependent individuals, as well as a possible metabolite of CocHs. In the third project, I expressed and characterized the recombinant human UDP-glucuronosyltransferase 1A10 (UGT1A10) enzyme, which can inactivate many therapeutically valuable substances. In the fourth and final project, prostate apoptosis response-4 (Par-4), a tumor suppressor protein, was engineered to have a prolonged duration of action so that it may be more therapeutically valuable for cancer treatment

DEVELOPMENT OF COCAINE HYDROLASE FOR THERAPEUTIC TREATMENT OF COCAINE ABUSE

Cocaine abuse is a world-wide public health and social problem without a U.S. Food and Drug Administration (FDA)-approved medication. An ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Recent studies in our lab have led to discovery of the desirable, highly efficient human cocaine hydrolases (hCocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of central nervous system (CNS). Preclinical and clinical data have demonstrated that these hCocHs are safe for use in humans and effective for accelerating cocaine metabolism. However, the actual therapeutic use of a hCocH in cocaine addiction treatment is limited by the short biological half-life (e.g. 8 hours or shorter in rats) of the hCocH. In the investigation described in this thesis, we have 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. In addition, we have identified the first benzoylecgonine-metabolizing enzymes that can hydrolyze benzoylecgonine and accelerate its clearance in rats. The developed LC-MS/MS method has enabled us to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. In development of the long-acting hCocHs, we have designed and discovered a novel hCocH form, catalytic antibody analog, which is an Fc-fused hCocH dimer (hCocH-Fc). The hCocH-Fc has not only a high catalytic efficiency against cocaine, but also a considerably longer biological half-life. A single dose of hCocH-Fc was able to accelerate cocaine metabolism in rats even after 20 days and, thus, block cocaine-induced hyperactivity for a long period of time. In consideration of the general observation that the biological half-life of a protein drug in humans is significantly longer than that in rodents, the hCocH-Fc could allow dosing once every 2-4 weeks, or longer for cocaine addiction treatment in humans

DEVELOPMENT AND PRECLINICAL EVALUATION OF LONG-LASTING COCAINE HYDROLASES FOR COCAINE OVERDOSE AND COCAINE USE DISORDER TREATMENT

Cocaine is a plant-based illicit drug commonly involved in substance use disorder. Although cocaine overdose and cocaine use disorders cause adverse health consequences to individuals and the economic burden on their family and society, there are no FDA (Food and Drug Administration) approved medications for treatment. Recently, it has been recognized that delivery of cocaine hydrolase (CocH) is a promising therapeutic strategy. Human butyrylcholinesterase (hBChE), the primary enzyme involved in cocaine metabolism in human, have advantages over other candidates for the development of CocH. Previous studies in our laboratory have designed and characterized hBChE mutants that have ~4,000-fold improved catalytic efficiency against naturally occurring (-)-cocaine as compared to the wild-type hBChE. Besides the catalytic efficiency, the biological half-life is another essential factor that influences the desired therapeutic value in the long-term treatment of cocaine use disorder. In order to provide prolonged effects to reduce administration frequency in clinical use, efforts have been made to increase the retention time of CocHs in blood circulation by fusing CocHs with other thermostable proteins or their mutants, including human serum albumin (Albu) or the Fc region of the human IgG (Fc). In this dissertation, we demonstrated the clinical potential and the benefits of long-lasting CocHs for cocaine overdose treatment. We used rodent models to show the ability of AlbuCocH1 to block or reverse manifestations of toxic effects of cocaine. In addition, a concomitant LC-MS/MS-based analysis was conducted to investigate the pharmacokinetic profile of a lethal dose of cocaine with the presence of AlbuCocH1. These experimental data demonstrated AlbuCocH1 as an effective cocaine detoxification agent by accelerating the metabolism of cocaine. In order to examine the potential therapeutic value of Fc-fused CocHs in the treatment of cocaine use disorder, we conducted a series of behavioral experiments in rats to evaluate the effectiveness and duration of Fc-fused CocHs in blocking or attenuating cocaine-induced psychostimulant and discriminative stimulus effects. In addition, the intravenous self-administration model was used to investigate the long-term effectiveness of Fc-fused CocHs in blocking or attenuating the reinforcing effects of cocaine. It has been shown that a single dose of E30-6-Fc (3 mg/kg) was able to effectively alter the cocaine dose-response curve and attenuate the reinforcing efficacy of cocaine for at least a month in both male and female rats. In summary, AlbuCocH1 (TV-1380), which failed to meet the primary efficacy endpoint in clinical trials for facilitating abstinence in cocaine-dependent subjects with a weekly dosing schedule (due to the short biological half-life), is more suitable to be developed as a cocaine detoxification agent. On the contrary, the newly designed Fc-fused CocH (e.g. CocH3-Fc, E30-6-Fc) with higher catalytic efficiency and longer biological half-life will be beneficial for long-term abstinence management in cocaine-dependent individuals

Mutants of Cocaine Esterase

Provided are mutant cocaine esterase polypeptides and PEGylated formulations thereof

Cocaine Hydrolase-Fc Fusion Proteins for Cocaine and Methods for Utilizing the Same

The presently-disclosed subject matter includes isolated polypeptides that comprise a butyrylcholinestrase (BChE) polypeptide and a second polypeptide. The BChE polypeptide as well as the second polypeptide can be variants and/or fragments thereof. The presently-disclosed subject matter also includes a pharmaceutical composition that comprises the present isolated polypeptide and a suitable pharmaceutical carrier. Further still, methods are provided for treating cocaine-induced conditions, and comprise administering the isolated polypeptide and/or pharmaceutical compositions thereof to an individual

The Effect Of Abstinence From Smoking On Stress Reactivity

Subjective stress is a well-documented predictor of early smoking relapse, yet our understanding of stress and tobacco use is limited by the reliability of current available measures of stress. Functional magnetic reasoning imaging (fMRI) could provide a much-needed objective measure of stress reactivity. The goal of this dissertation is to contribute to the understanding of abstinence-induced changes in stress reactivity by examining neural, neuroendocrine (cortisol), and subjective measures of stress response during abstinence. In addition, this study investigated the influence of individual variation in nicotine metabolism rates on these measures of stress reactivity. Seventy-five treatment-seeking smokers underwent blood oxygen level dependent (BOLD) fMRI during the Montreal Imaging Stress Task (MIST) on two occasions: once during smoking satiety and once following biochemically confirmed 24-hour abstinence (order counter-balanced). The primary outcome measure was brain response during stress (vs. control) blocks of the MIST. Neural stress reactivity during abstinence (vs. satiety) was associated with significantly increased activation in the left inferior frontal gyrus (IFG), a brain region previously associated with inhibitory control. Greater abstinence-induced change in brain response to stress was associated with greater abstinence-induced change in subjective stress. However, there was no association with abstinence-induced change in cortisol response. In addition, higher rates of nicotine metabolism were associated with increased abstinence-induced change in self-reported stress, but not with brain or cortisol response. This study provides novel evidence that the brain response to stress is altered during the first 24 hours of a quit attempt compared to smoking satiety. These results underscore the importance of stress response during abstinence, and suggest that neuroimaging may provide a useful biomarker of stress response during the early smoking cessation, a period when smokers are most vulnerable to relapse

Deskripsi model farmakokinetik kokain

INDONESIA: Penelitian ini membahas tentang bagaimana pengaruh terapi antibodi pada model farmakokinetik dalam menetralkan konsentrasi kokain pada tubuh. Farmakokinetika adalah cabang ilmu dari farmakologi yang mempelajari tentang perjalanan obat mulai sejak diminum hingga keluar melalui organ ekskresi di tubuh manusia Tujuan dari penelitian ini untuk mengetahui proses terbentuknya model farmakokinetik kokain dalam tubuh dengan memasukkan terapi antibodi monoklonal serta mendapatkan hasil simulasi model farmakokinetik kokain dalam tubuh. Pada penelitian ini model farmakokinetik merupakan persamaan diferensial biasa orde satu bergantung pada waktu t. Model ini merupakan persamaan diferensial yang terdiri dari tujuh persamaan dan membentuk sebuah sistem. Sistem persamaan diferensial yang digunakan dalam model tersebut terdiri dari tujuh variabel bergantung, yaitu x_1 merupakan banyaknya konsentrasi antibodi di sentral, x_2 merupakan banyaknya konsentrasi kokain di sentral, x_3 merupakan banyaknya konsentrasi kokain antibodi kompleks di sentral, x_4 merupakan banyaknya konsentrasi kokain di otak, x_5 merupakan banyaknya konsentrasi kokain di perifer, x_6 merupakan banyaknya konsentrasi antibodi di perifer, x_7 merupakan banyaknya konsentrasi kokain antibodi kompleks di perifer dengan parameter konsentrasi kokain (β), konsentrasi antibodi (α) dan konstanta asosiasi/disasosiasi (k). Kemudian disimulasikan menggunakan software MATLAB dengan metode ode45. Disimpulkan bahwa hasil simulasi menunjukkan bahwa efek terapi antibodi berhasil menetralkan konsentrasi kokain pada organ pusat dan perifer, namun tidak di dalam otak. ENGLISH: This study discusses how the effect of antibody therapy on pharmacokinetic models in neutralizing cocaine concentrations in the body. Pharmacokinetics is a branch of pharmacology that studies drug travel from ingestion to excretion through the excretory organs in the human body. The purpose of this study was to determine the process of establishing a cocaine pharmacokinetic model in the body by incorporating monoclonal antibody therapy and obtaining simulation results of cocaine pharmacokinetic models in the body. In this study, the pharmacokinetic model is a first-order ordinary differential equation that depends on time t. This model is a differential equation which consists of seven equations and forms a system. The system of differential equations used in the model consists of seven dependent variables, namely x_1 is the amount of antibody complex in the center, x_2 is the amount of cocaine concentration in the center, x_3 is the amount of cocaine antibody complex in the center, x_4 is the amount of cocaine concentration in the brain, x_5 is the amount of cocaine concentration in the periphery, x_6 is the amount of antibody complex in the periphery, x_7 is the amount of cocaine antibody complex concentration in the periphery with parameters of cocaine concentration (β), antibody concentration (α) and association/disassociation constant (k). Then simulated using MATLAB software with the ode45 method. It was concluded that the simulation results showed that the effect of antibody therapy managed to neutralize cocaine concentrations in central and peripheral organs, but not in the brain. ARABIC: تناقش هذه الدراسة كيفية تأثير العلاج بالأجسام المضادة على نماذج الحرائك الدوائية في تحييد تركيزات الكوكايين في الجسم. الحرائك الدوائية هي فرع من فروع علم الأدوية يدرس انتقال الدواء من الابتلاع إلى الإخراج عبر أعضاء الإخراج في جسم الإنسان. وكان الغرض من هذه الدراسة هو تحديد عملية إنشاء نموذج حركية دوائية للكوكايين في الجسم من خلال دمج العلاج بالأجسام المضادة أحادية النسيلة والحصول على المحاكاة نتائج نماذج الحرائك الدوائية للكوكايين في الجسم. في هذه الدراسة ، يعد نموذج الحرائك الدوائية معادلة تفاضلية عادية من الدرجة الأولى تعتمد على الوقت t. هذا النموذج هو معادلة تفاضلية تتكون من سبع معادلات وتشكل نظامًا. يتكون نظام المعادلات التفاضلية المستخدمة في النموذج من سبعة متغيرات تابعة ، وهي x_1 هي كمية معقد الجسم المضاد في المركز ، و x_2 هي كمية تركيز الكوكايين في المركز ، و x_3 هي كمية مركب الجسم المضاد للكوكايين في المركز ، x_4 هي كمية تركيز الكوكايين في الدماغ ، x_5 هي كمية تركيز الكوكايين في المحيط ، x_6 هي كمية معقد الأجسام المضادة في المحيط ، x_7 هي كمية تركيز معقد الأجسام المضادة للكوكايين في المحيط مع معلمات تركيز الكوكايين (β) وتركيز الجسم المضاد (α) وثابت الارتباط / التفكك (k). م تمت محاكاته باستخدام برنامج MATLAB. وخلص إلى أن نتائج المحاكاة أظهرت أن تأثير العلاج بالأجسام المضادة نجح في معادلة تركيزات الكوكايين في الأعضاء المركزية والمحيطية ، ولكن ليس في الدماغ