Novel pharmacological approaches to treatment of drug overdose and addiction

Drug abuse is a major public health problem. It is still a challenge to develop a truly effective therapeutic agent for the treatment of drug overdose and addiction. Let us first take cocaine as an example. Cocaine is highly addictive and may be the most reinforcing of all drugs of abuse [1]. Despite huge advances in the neuroscience of drug abuse and dependence in the past decades, no approved pharmacological treatment exists for cocaine abuse. Cocaine reinforces self-administration in relation to the peak serum concentration of the drug, the rate of rise to the peak and the degree of change of the serum level. Potent CNS stimulation is followed by depression. With drug overdose, respiratory depression, cardiac arrhythmia and acute hypertension are common effects. The disastrous medical and social consequences of cocaine abuse have made the development of an effective pharmacological treatment a high priority [2,3]. Pharmacological treatment for cocaine overdose and addiction can be either pharmacodynamic or pharmacokinetic. Most of currently employed anti-addiction strategies use the classical pharmacodynamic approach, that is, developing small molecules that interact with one or more neuronal binding sites, with the goal of blocking or counteracting a drug’s neuropharmacological actions. However, no pharmacodynamic agent has been proven successful for cocaine abuse treatment [2,4]. Novel pharmacological approaches to the treatment of cocaine overdose and addiction are highly desirable.

The inherent difficulties in antagonizing a blocker such as cocaine have led to the development of the pharmacokinetic approach, which aims to act directly on the drug itself to alter its distribution or accelerate its clearance [2,5,6]. Pharmacokinetic antagonism of cocaine could be implemented by administration of a molecule, such as a cocaine antibody, which binds tightly to cocaine so as to prevent cocaine from crossing the blood–brain barrier [7]. The blocking action could also be implemented by administration of an enzyme or a catalytic antibody (regarded as an artificial enzyme) that not only binds but also accelerates cocaine metabolism, thereby freeing itself for further binding [8,9]. Usually, a pharmacokinetic agent would not be expected to cross the blood–brain barrier and thus would itself have no direct pharmacodynamic action, such as abuse liability [2,4].

The primary pathway for cocaine metabolism in primates is hydrolysis at the benzoyl ester or methyl ester group [2]. Benzoyl ester hydrolysis generates ecgonine methyl ester, whereas the methyl ester hydrolysis yields benzoylecgonine. The major cocaine-metabolizing enzymes in humans are butyrylcholinesterase (BChE) that catalyzes benzoyl ester hydrolysis and two liver carboxylesterases (denoted by hCE-1 and hCE-2) that catalyze hydrolysis at the methyl ester and the benzoyl ester, respectively. Among the three, BChE is the principal cocaine hydrolase in human serum. Hydrolysis accounts for approximately 95% of cocaine metabolism in humans. The remaining 5% is deactivated through oxidation by the liver microsomal cytochrome P450 system, producing norcocaine [2,10]. Ecgonine methyl ester appears the least pharmacologically active of the cocaine metabolites and may even cause vasodilation, whereas both benzoylecgonine and norcocaine appear to cause vasoconstriction and lower the seizure threshold, similar to cocaine itself. Norcocaine is hepatotoxic and a local anesthetic [11]. Thus, hydrolysis of cocaine at the benzoyl ester by an enzyme is the pathway most suitable for amplification.

Besides hCE-2, two types of known native enzymes may be used to catalyze hydrolysis of cocaine at the benzoyl ester: BChE and cocaine esterase (CocE). The use of an exogenous enzyme has some potential advantages over active immunization, as the enzyme administration would immediately enhance cocaine metabolism and would not require an immune response to be effective.

In the older literature, BChE was known as pseudocholinesterase or plasma cholinesterase, to distinguish it from its close cousin acetylcholinesterase. It is synthesized in the liver and widely distributed in the body, including in the plasma, brain and lung [2,12]. Studies in animals and humans demonstrate that enhancement of BChE activity by administration of exogenous enzyme substantially decreases cocaine half-life [2]. Clinical studies suggest that BChE has unique advantages. First, human BChE has a long history of clinic use, and no adverse effects have been noted with increased BChE plasma activity. Second, more than 20 different naturally occurring mutants of human BChE have been identified [13], and there is no evidence that these mutants are antigenic. BChE also has potential advantages over active immunization, as BChE administration would immediately enhance cocaine metabolism and would not require an immune response to be effective. For these reasons, enhancement of cocaine metabolism by administration of BChE is considered a promising pharmacokinetic approach for the treatment of cocaine abuse and dependence [2,4]. However, the catalytic activity of this plasma enzyme is three orders of magnitude lower against the naturally occurring (-)-cocaine than that against the biologically inactive (+)-cocaine enantiomer [2]. (+)-cocaine can be cleared from plasma in seconds and prior to partitioning into the CNS, whereas (-)-cocaine has a plasma half-life of approximately 47 min or longer (for an intravenous dose of 0.2 mg/kg cocaine), long enough for manifestation of the CNS effects, which peak in minutes [9]. Thus, PET, applied to mapping of the binding of (-)-cocaine and (+)-cocaine in baboon CNS, indicated marked uptake corresponding to (-)-cocaine at the striatum along with other areas of low uptake, whereas no CNS uptake corresponding to (+)-cocaine was observed [2]. (+)-cocaine was hydrolyzed by BChE so rapidly that it never reached the CNS for PET visualization. Furthermore, we note that the actual half-life of (-)-cocaine in plasma is dependent on the dose of cocaine received. This is because the enzyme BChE should be saturated even with a low intravenous dose of 0.2 mg/kg cocaine, as the concentration of (-)-cocaine in plasma is much larger than the Michaelis–Menten constant (K_M = 4.1 µM) of BChE against (-)-cocaine. When the enzyme is saturated, the (-)-cocaine hydrolysis speed has already reached the maximum and will not change with increasing the dose of (-)-cocaine. Thus, the actual half-life of (-)-cocaine in plasma should be proportional to the actual dose of (-)-cocaine in the case of overdose.

Cocaine esterase, expressed by Rhodococcus spp. strain MB1, a bacterium isolated from the rhizosphere soil of coca plants, hydrolyzes the benzoyl ester of cocaine [14]. This bacterial enzyme hydrolyzes (-)-cocaine with a catalytic constant (k_cat) value of 468 min^-1 (7.8 s^-1) and a K_Mvalue of 640 nM. Therefore, the catalytic efficiency (k_cat/K_M) of native CocE is approximately 800-fold greater than that of native BChE against (-)-cocaine. The potential of CocE as an enzyme-based therapy for cocaine abuse was demonstrated originally in rodent models. First, in a rat model, 1 mg intravenous CocE, when administered 1 min prior to cocaine injection (intraperitoneal 180 mg/kg), protected 100% of rats as opposed to BChE 13 mg intravenously, which offered no protection from the lethal dose of cocaine [15]. Second, in a mouse model, pretreatment of intravenous CocE at 0.32 mg and 1 mg doses resulted in ten- and 18-fold shifts in the dose–response curve for cocaine-induced convulsions, and 8- and 14-fold shifts in the dose–response curve for cocaine-induced lethality, respectively [16]. CocE has a relatively short half-life in vivo and the main reason for the very short half-life is thought to be its low thermostability. CocE evolved to function at a much lower temperature than a physiological temperature of 37°C. In rat plasma, CocE has an approximately 10 min half-life, and the effectiveness of CocE decreases with time in vivo. Prior treatment with CocE for 10 and 30 min before cocaine administration saved only 66.7 and 33.3% of rats, respectively, in contrast to a 100% survival rate with a 1 min prior treatment. CocE, when given 100 min before cocaine injection, failed to protect any rats [15]. In mice, CocE given 10 min before cocaine only protected 50% of the animals [16]. In addition, being a bacterial protein, CocE could elicit a robust immune response when used as a therapy.

For the use of these enzymes in anticocaine therapeutics, both BChE and CocE have their own advantages and disadvantages. Concerning the advantages of using human BChE, first of all, BChE from a human source can be tolerated perfectly in the human body. In addition, BChE is very stable in a physiological condition, thus, BChE has a relatively longer half-life in the human body. The disadvantages of using BChE are associated with the low catalytic efficiency of native BChE against the naturally occurring (-)-cocaine. Compared with native BChE, the catalytic efficiency of native CocE against (-)-cocaine is approximately 800-fold higher. A disadvantage for the use of native CocE is its bacterial origin and low thermostability. Hence, the two types of potential treatment agents, BChE and CocE, can complement each other. Both BChE and CocE could be engineered to become valuable anticocaine therapeutic agents.

The use of CocE as anticocaine therapeutic requires a decrease in the immunogenicity and an increase in the thermostability of the enzyme. Encouraging progress has been made in the development of both BChE and CocE for the treatment of cocaine overdose. For CocE development, promising thermostable mutants of CocE have been designed and discovered through computational modeling and integrated computational–experimental studies [17]. A double-mutant of CocE demonstrated a half-life of approximately 5 h at a physiological temperature (37^oC) [17].

The use of an engineered BChE as anticocaine therapeutic requires an improved catalytic efficiency against (-)-cocaine [18–20]. Design of a high-activity enzyme mutant is extremely challenging, particularly when the chemical reaction process becomes rate determining for the enzymatic reaction [21]. Generally speaking, for rational design of a mutant enzyme with an improved catalytic activity for a given substrate, one needs to design possible mutations that can accelerate the rate-determining step of the entire catalytic reaction process while the other steps are not slowed down by the mutations. In order to improve the catalytic activity of BChE against (-)-cocaine, a number of BChE mutants have been made through site-directed mutagenesis and their catalytic activity for (-)-cocaine hydrolysis has been measured [22–24]. Notably, novel computational design strategies/approaches have been developed and used to design the high-activity mutants based on the detailed structural and mechanistic understanding and virtual screening of the transition states of the enzymatic reaction [25–27]. The structure-and-mechanism-based computational design was followed by wet experimental tests. The high-activity mutants of human BChE reported so far include A199S/S287G/A328W/Y332G [25] and A199S/F227A/S287G/A328W/Y332G [27]. Each of these high-activity mutants of human BChE may be called a cocaine hydrolase (CocH). In fact, the A199S/S287G/A328W/Y332G mutant [25] has been recognized by independent researchers as: “a true CocH with a catalytic efficiency that is 1000-fold greater than wild-type BChE” [28]. The independent researchers also demonstrated that the A199S/S287G/A328W/Y332G mutant can indeed selectively block cocaine toxicity and reinstatement of drug seeking in rats. The most efficient CocH (i.e., the A199S/F227A/S287G/A328W/Y332G mutant [k_cat = 5700 min^-1 and K_M = 3.1 µM]) has an approximately 2000-fold improved catalytic efficiency (k_cat/K_M) against (-)-cocaine compared with wild-type BChE [27]. The high activity of this mutant has been confirmed by in vivo tests on mice [27]. Pretreatment with the A199S/F227A/S287G/A328W/Y332G mutant (i.e., 1 min prior to cocaine administration) dose dependently protected mice against cocaine-induced convulsions and lethality. In particular, the A199S/F227A/S287G/A328W/Y332G mutant 0.01 mg (per mouse) was good enough to produce full protection in mice from cocaine overdose, induced by a lethal dose of cocaine 180 mg/kg (p < 0.05) [27]. Clearly, these cocaine hydrolases, that is, the high-activity mutants of human BChE, are promising for therapeutic treatment of cocaine overdose and addiction.

The general concept of pharmacokinetic treatment of cocaine abuse targeting the metabolism may be extended, to explore possible enzymes suitable for treatment of other drugs of abuse. In order to explore a therapeutically useful enzyme for a given drug of abuse, one will first need to examine all possible metabolic pathways of the drug and identify a favorable metabolic pathway producing biologically inactive metabolites. If a favorable metabolic pathway and the corresponding native enzyme (or catalytic antibody that is suitable for use in human) can be identified, then the aforementioned novel, general computational design strategy and protocol of the structure-and-mechanism-based design may be used to design high-activity mutants of the chosen drug metabolizing enzyme (or catalytic antibody) against the drug. The computational design should be followed by wet experimental tests in vitro and in vivo. It is essential to ensure that the designed mutant will only specifically amplify the desirable drug-metabolizing pathway without causing any harmful chemical reactions or binding in the body. This is certainly challenging, but may be possible, at least for some drugs of abuse.

Financial & competing interests disclosure

Our US Patent No. 7,438,904 and PCT Int. Appl. WO/2008/008358 cover the above-discussed high-activity mutants of human BChE and the thermostable mutants of CocE, respectively. Financial support from the National Institute on Drug Abuse (NIDA) of NIH grants R01 DA013930, R01 DA021416, and R01 DA025100) are gratefully acknowledged. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.