Cocaine Hydrolase Research Articles

In vitro and in vivo stability of a highly efficient long-acting cocaine hydrolase

It is recognized as a promising therapeutic strategy for cocaine use disorder to develop an efficient enzyme which can rapidly convert cocaine to physiologically inactive metabolites. We have designed and discovered a series of highly efficient cocaine hydrolases, including CocH5-Fc(M6) which is the currently known as the most efficient cocaine hydrolase with both the highest catalytic activity against (−)-cocaine and the longest biological half-life in rats. In the present study, we characterized the time courses of protein appearance, pH, structural integrity, and catalytic activity against cocaine in vitro and in vivo of a CocH5-Fc(M6) bulk drug substance produced in a bioreactor for its in vitro and in vivo stability after long-time storage under various temperatures (− 80, − 20, 4, 25, or 37 °C). Specifically, all the tested properties of the CocH5-Fc(M6) protein did not significantly change after the protein was stored at any of four temperatures including − 80, − 20, 4, and 25 °C for ~ 18 months. In comparison, at 37 °C, the protein was less stable, with a half-life of ~ 82 days for cocaine hydrolysis activity. Additionally, the in vivo studies further confirmed the linear elimination PK profile of CocH5-Fc(M6) with an elimination half-life of ~ 9 days. All the in vitro and in vivo data on the efficacy and stability of CocH5-Fc(M6) have consistently demonstrated that CocH5-Fc(M6) has the desired in vitro and in vivo stability as a promising therapeutic candidate for treatment of cocaine use disorder.

Long-lasting blocking of interoceptive effects of cocaine by a highly efficient cocaine hydrolase in rats

Cocaine dependence is a serious world-wide public health problem without an FDA-approved pharmacotherapy. We recently designed and discovered a highly efficient long-acting cocaine hydrolase CocH5-Fc(M6). The present study examined the effectiveness and duration of CocH5-Fc(M6) in blocking interoceptive effects of cocaine by performing cocaine discrimination tests in rats, demonstrating that the duration of CocH5-Fc(M6) in blocking cocaine discrimination was dependent on cocaine dose and CocH5-Fc(M6) plasma concentration. Particularly, a dose of 3 mg/kg CocH5-Fc(M6) effectively attenuated discriminative stimulus effects of 10 mg/kg cocaine, cumulative doses of 10 and 32 mg/kg cocaine, and cumulative doses of 10, 32 and 56 mg/kg cocaine by ≥ 20% for 41, 19, and 10 days, and completely blocked the discriminative stimulus effects for 30, 13, and 5 days with corresponding threshold plasma CocH5-Fc(M6) concentrations of 15.9, 72.2, and 221 nM, respectively, under which blood cocaine concentration was negligible. Additionally, based on the data obtained, cocaine discrimination model is more sensitive than the locomotor activity to reveal cocaine effects and that CocH5-Fc(M6) itself has no long-term toxicity regarding behavioral activities such as lever pressing and food consumption in rats, further demonstrating that CocH5-Fc(M6) has the desired properties as a promising therapeutic candidate for prevenance of cocaine dependence.

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.

Kinetic characterization of an efficient cocaine hydrolase against toxic metabolites of cocaine.

In the presence of alcohol, cocaine metabolism produces a number of metabolites, including three toxic ones (cocaethylene, norcocaine, and norcocaethylene) which are all more toxic than cocaine itself, with the toxicity in the order of cocaine < cocaethylene < norcocaine < norcocaethylene. In this study, we performed kinetic analysis on our previously reported cocaine hydrolase (E30-6) for its catalytic activities accelerating the hydrolysis of the three toxic metabolites in comparison with cocaine. Based on the obtained kinetic data, the in vitro catalytic efficiencies of the enzyme against these substrates are in the order of cocaine > cocaethylene > norcocaine > norcocaethylene. It has been demonstrated that E30-6 can efficiently accelerate the hydrolysis of not only cocaine itself, but also all three toxic metabolites in vitro and in vivo. E30-6 is the most efficient enzyme for each of these toxic substrates (cocaine, cocaethylene, norcocaine, and norcocaethylene) among all the reported enzymes as far as we know at this point. These findings suggest that E30-6 is capable of efficiently treating cocaine toxicity even when alcohol and cocaine are used concurrently.

Gender differences in cocaine-induced hyperactivity and dopamine transporter trafficking to the plasma membrane.

As well known, cocaine induces stimulant effects and dopamine transporter (DAT) trafficking to the plasma membrane of dopaminergic neurons. In the present study, we examined cocaine-induced hyperactivity along with cocaine-induced DAT trafficking and the recovery rate of the dopaminergic system in female rats in comparison with male rats, demonstrating interesting gender differences. Female rats are initially more sensitive to cocaine than male rats in terms of both the DAT trafficking and hyperactivity induced by cocaine. Particularly, intraperitoneal (i.p.) administration of 5 mg/kg cocaine induced significant hyperactivity and DAT trafficking in female rats but not in male rats. After repeated cocaine exposures (i.e., i.p. administration of 20 mg/kg cocaine every other day from Day 0 to Day 32), cocaine-induced hyperactivity in female rats gradually became a clear pattern of two phases, with the first phase of the hyperactivity lasting for only a few minutes and the second phase lasting for over an hour beginning at ~30 min, which is clearly different from that of male rats. It has also been demonstrated that the striatal DAT distribution of female rats may recover faster than that of male rats after multiple cocaine exposures. Nevertheless, despite the remarkable gender differences, our recently developed long-acting cocaine hydrolase, known as CocH5-Fc(M6), can similarly and effectively block cocaine-induced DAT trafficking and hyperactivity in both male and female rats.

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).

Recovery of dopaminergic system after cocaine exposure and impact of a long-acting cocaine hydrolase.

Dysregulation of dopamine transporters (DAT) within the dopaminergic system is an important biomarker of cocaine exposure. Depending on cocaine amount in-taken, one-time exposure in rats could lead to most (>95% of total) of DAT translocating to plasma membrane of the dopaminergic neurons compared to normal DAT distribution (~5.7% on the plasma membrane). Without further cocaine exposure, the time course of striatal DAT distribution, in terms of intracellular and plasma membrane fractions of DAT, represents a recovery process of the dopaminergic system. In this study, we demonstrated that after an acute cocaine exposure of 20 mg/kg (i.p.), the initial recovery process from days 1 to 15 in rats was relatively faster (from >95% on day 1 to ~35.4% on day 15). However, complete recovery of the striatal DAT distribution may take about 60 days. In another situation, with repeated cocaine exposures for once every other day for a total of 17 doses of 20 mg/kg cocaine (i.p.) from days 0 to 32, the complete recovery of striatal DAT distribution may take an even longer time (about 90 days), which represents a consequence of chronic cocaine use. Further, we demonstrated that a highly efficient Fc-fused cocaine hydrolase, CocH5-Fc(M6), effectively blocked cocaine-induced hyperactivity and DAT trafficking with repeated cocaine exposures by maintaining a plasma CocH5-Fc(M6) concentration ≥58.7±2.9nM in rats. The cocaine hydrolase protected dopaminergic system and helped the cocaine-altered DAT distribution to recover by preventing the dopaminergic system from further damage by cocaine.

Cocaine hydrolase blocks cocaine-induced dopamine transporter trafficking to the plasma membrane.

Cocaine blocks dopamine uptake via dopamine transporter (DAT) on plasma membrane of neuron cells and, as a result, produces the high and induces DAT trafficking to plasma membrane which contributes to the drug seeking or craving. In this study, we first examined the dose dependence of cocaine-induced DAT trafficking and hyperactivity in rats, demonstrating that cocaine at an intraperitoneal dose of 10mg/kg or higher led to redistribution of most DAT to the plasma membrane while inducing significant hyperactivity in rats. However, administration of 5-mg/kg cocaine (ip) did not significantly induce DAT trafficking or hyperactivity in rats. So the threshold (intraperitoneal) dose of cocaine that can significantly induce DAT trafficking or hyperactivity should be between 5 and 10mg/kg. These data suggest that when a cocaine dose is high enough to induce significant hyperactivity, it can also significantly induce DAT trafficking to the plasma membrane. Further, the threshold brain cocaine concentration required to induce significant hyperactivity and DAT trafficking was estimated to be ~2.0 ± 0.8μg/g. Particularly, for treatment of cocaine abuse, previous studies demonstrated that an exogenous cocaine-metabolizing enzyme, for example, CocH3-Fc(M3), can effectively block cocaine-induced hyperactivity. However, it was unknown whether an enzyme could also effectively block cocaine-induced DAT trafficking to the plasma membrane. This study demonstrates, for the first time, that the enzyme is also capable of effectively blocking cocaine from reaching the brain even with a lethal dose of 60-mg/kg cocaine (ip) and, thus, powerfully preventing cocaine-induced physiological effects such as the hyperactivity and DAT trafficking.

Clinical potential of a rationally engineered enzyme for treatment of cocaine dependence: Long-lasting blocking of the psychostimulant, discriminative stimulus, and reinforcing effects of cocaine

It is a grand challenge to develop a truly effective treatment of substance use disorder (SUD), particularly for cocaine and other drugs without an FDA-approved treatment available, because a truly effective therapy must effectively block the drug's physiological and reinforcing effects during the entire period of treatment in order to achieve the long-time abstinence required by the FDA. Whether a biologic, such as monoclonal antibody, vaccine, or therapeutic enzyme, can be truly effective for SUD treatment or not has been the subject of extensive debate. The main debate question is whether a biologic, particularly an exogenous enzyme, can effectively block the drug's reinforcing effect. In this report, we demonstrate that a modest dose of a recently redesigned long-acting cocaine hydrolase, CocH3-Fc(M6), can be used to effectively block the psychostimulant, discriminative stimulus, and reinforcing effects of cocaine for a sufficiently long period of time. For example, a dose of 3 mg/kg CocH3-Fc(M6) completely blocked the discriminative stimulus and reinforcing effects for 24/25 days and continued to significantly attenuate/decrease the cocaine effects for at least 29 days in rats. All the animal data consistently suggest that the long-acting cocaine hydrolase is a truly promising candidate of enzyme therapy for treatment of cocaine use disorder.

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.

Structure-Based Design and Discovery of a Long-Acting Cocaine Hydrolase Mutant with Improved Binding Affinity to Neonatal Fc Receptor for Treatment of Cocaine Abuse.

Despite decades of efforts to develop a pharmacotherapy for cocaine abuse treatment, there is still no FDA-approved treatment of diseases associated with this commonly abused drug. Our previously designed highly efficient cocaine hydrolases (CocHs) and the corresponding Fc-fusion proteins (e.g., CocH3-Fc) are recognized as potentially promising therapeutic enzyme candidates for cocaine abuse treatment, but all with limited biological half-lives. In order to prolong the biological half-life and, thus, decrease the required frequency of the enzyme administration for cocaine abuse treatment, we have modeled the Fc-fusion CocH binding with neonatal Fc receptor (FcRn) in the present study. This approach led to the design and testing of CocH3-Fc(M6), a CocH3-Fc mutant with nearly 100-fold increased binding affinity: from Kd = ~ 4μM to Kd = 43nM. As a result, CocH3-Fc(M6) indeed revealed a markedly prolonged biological half-life (t1/2 = 206 ± 7h or ~ 9days) in rats, longer than other known Fc-fusion protein drugs such as abatacept and alefacept (for other therapeutic purposes) in the same species (rats). It has been demonstrated that a single dose of 3mg/kg CocH3-Fc(M6) effectively blocked 20mg/kg cocaine-induced hyperactivity on day 18 after CocH3-Fc(M6) administration. This is the first attempt to rationally design long-acting Fc-fusion enzyme mutant based on combined computational modeling and experimental measurement of the Fc-fusion CocH binding with FcRn. The similar structure-based design strategy may be used to prolong the biological half-lives of other Fc-fusion protein drugs.

Catalytic activities of cocaine hydrolases against the most toxic cocaine metabolite norcocaethylene.

A majority of cocaine users also consume alcohol. The concurrent use of cocaine and alcohol produces the pharmacologically active metabolites cocaethylene and norcocaethylene, in addition to norcocaine. Both cocaethylene and norcocaethylene are more toxic than cocaine itself. Hence, a truly valuable cocaine-metabolizing enzyme for cocaine abuse/overdose treatment should be effective for the hydrolysis of not only cocaine, but also its metabolites norcocaine, cocaethylene, and norcocaethylene. However, there has been no report on enzymes capable of hydrolyzing norcocaethylene (the most toxic metabolite of cocaine). The catalytic efficiency parameters (kcat and KM) of human butyrylcholinesterase (BChE) and two mutants (known as cocaine hydrolases E14-3 and E12-7) against norcocaethylene have been characterized in the present study for the first time, and they are compared with those against cocaine. According to the obtained kinetic data, wild-type human BChE showed a similar catalytic efficiency against norcocaethylene (kcat = 9.5 min-1, KM = 11.7 μM, and kcat/KM = 8.12 × 105 M-1 min-1) to that against (-)-cocaine (kcat = 4.1 min-1, KM = 4.5 μM, and kcat/KM = 9.1 × 105 M-1 min-1). E14-3 and E12-7 showed an improved catalytic activity against norcocaethylene compared to wild-type BChE. E12-7 showed a 39-fold improved catalytic efficiency against norcocaethylene (kcat = 210 min-1, KM = 6.6 μM, and kcat/KM = 3.18 × 107 M-1 min-1). It has been demonstrated that E12-7 as an exogenous enzyme can efficiently metabolize norcocaethylene in rats.

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.

Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression

Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP)2-CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP)2-CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ± 7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)2-CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP)2-CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.

Kinetic characterization of cholinesterases and a therapeutically valuable cocaine hydrolase for their catalytic activities against heroin and its metabolite 6-monoacetylmorphine

As the most popularly abused one of opioids, heroin is actually a prodrug. In the body, heroin is hydrolyzed/activated to 6-monoacetylmorphine (6-MAM) first and then to morphine to produce its toxic and physiological effects. It has been known that heroin hydrolysis to 6-MAM and morphine is accelerated by cholinesterases, including acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). However, there has been controversy over the specific catalytic activities and functional significance of the cholinesterases, which requires for the more careful kinetic characterization under the same experimental conditions. Here we report the kinetic characterization of AChE, BChE, and a therapeutically promising cocaine hydrolase (CocH1) for heroin and 6-MAM hydrolyses under the same experimental conditions. It has been demonstrated that AChE and BChE have similar kcat values (2100 and 1840 min−1, respectively) against heroin, but with a large difference in KM (2170 and 120 μM, respectively). Both AChE and BChE can catalyze 6-MAM hydrolysis to morphine, with relatively lower catalytic efficiency compared to the heroin hydrolysis. CocH1 can also catalyze hydrolysis of heroin (kcat = 2150 min−1 and KM = 245 μM) and 6-MAM (kcat = 0.223 min−1 and KM = 292 μM), with relatively larger KM values and lower catalytic efficiency compared to BChE. Notably, the KM values of CocH1 against both heroin and 6-MAM are all much larger than previously reported maximum serum heroin and 6-MAM concentrations observed in heroin users, implying that the heroin use along with cocaine will not drastically affect the catalytic activity of CocH1 against cocaine in the CocH1-based enzyme therapy for cocaine abuse.

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.

Treating Cocaine Addiction, Obesity, and Emotional Disorders by Viral Gene Transfer of Butyrylcholinesterase.

Butyrylcholinesterase (BChE), a plasma enzyme that hydrolyses the neurotransmitter, acetylcholine relatively well, with far lower efficiency than acetylcholinesterase (AChE) but with the capability to degrade a broad range of bioactive esters. AChE is universally understood as essential to cholinergic neurotransmission, voluntary muscle performance, and cognition, among other roles, and its catalytic impact is essential for life. A total absence of BChE activity, whether by enzyme inhibition or simple lack of enzyme protein is not only compatible with life, but does not lead to obvious physiologic disturbance. However, very recent studies at Mayo Clinic have amassed support for the concept that BChE does have a true physiological role as a “ghrelin hydrolase” and, pharmacologically, as a cocaine hydrolase. Human subjects and animal mutations that lack functional BChE show higher than normal levels of ghrelin, an acylated peptide that drives hunger and feeding, along with certain emotional behaviors. Mice treated by viral gene transfer of BChE show higher plasma levels of enzyme and lower levels of ghrelin. Ghrelin is acknowledged as a driver of food-seeking and stress. This brief review examines some key phenomena and considers means of modulating BChE as treatments for cocaine addiction, anxiety, aggression, and obesity.

Effectiveness of a Cocaine Hydrolase for Cocaine Toxicity Treatment in Male and Female Rats.

Development of a truly effective medication for treatment of cocaine abuse has been a grand challenge. There is no FDA-approved therapeutic agent specific for cocaine addiction or overdose. An enzyme therapy using an efficient cocaine-metabolizing enzyme could be a promising treatment strategy for cocaine overdose and addiction. One of our previously designed cocaine hydrolases (CocHs), known as CocH1, was fused with human serum albumin (HSA) to prolong the biological half-life. The fusion protein CocH1-HSA is an investigational new drug (IND) approved by the FDA for clinical trials in cocaine addiction treatment, but not in cocaine overdose/toxicity treatment. In the present study, we aimed to evaluate the pharmacokinetic profile of CocH1-HSA and its effectiveness for cocaine toxicity treatment in male and female rats and demonstrate the clinical potential. The data demonstrate that enzyme CocH1-HSA has very similar pharmacokinetic profile in male and female rats. For both male and female rats, the enzyme can rapidly eliminate cocaine even if the cocaine dose is as high as 180mg/kg (LD100). Based on the animal data, whenever the enzyme is given to a living subject, the remaining cocaine in the body will be converted rapidly to physiologically inactive metabolites and, thus, reverse the cocaine toxicity and help the subject to recover. So, an enzyme therapy using CocH1-HSA can effectively treat cocaine toxicity and prevent the subject from further damage by cocaine. The data obtained clearly demonstrate the promising clinical potential of CocH1-HSA in cocaine overdose treatment for both genders.

Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme’s ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic.