The aim of this thesis was to optimize the proenzyme and enzyme therapy of the healthy and mice suffering of cancer and to clarify the mechanism of this therapy. We found optimal mode of application of therapeutic preparations. We studied changes of serum {$\alpha$}2-macroglobulin, total level of serum protease inhibitors, and free or latent TGF-{$\beta$} in various modes of experimental tumor therapies.

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.

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.

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.