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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Expert Rev Clin Pharmacol. Author manuscript; available in PMC 2010 November 8.
Published in final edited form as:
PMCID: PMC2975360

Novel pharmacological approaches to treatment of drug overdose and addiction

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Drug abuse is a major public health problem. It is still a challenge to develop a truly effective therapeutic for 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 central nervous system (CNS) stimulation is followed by depression. With the 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, i.e. 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 treatment of cocaine overdose and addiction are highly desirable.

The inherent difficulties in antagonizing a blocker like cocaine have led to the development of the pharmacokinetic approach that aims at acting 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 and 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 (EME), whereas the methyl ester hydrolysis yields benzoylecgonine (BE). The major cocaine-metabolizing enzymes in humans are butyrylcholinesterase (BChE) which catalyzes benzoyl ester hydrolysis and two liver carboxylesterases (denoted by hCE-1 and hCE-2) which 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 about 95% of cocaine metabolism in humans. The remaining 5% is deactivated through oxidation by the liver microsomal cytochrome P450 system, producing norcocaine.2,10 EME appears the least pharmacologically active of the cocaine metabolites and may even cause vasodilation, whereas both BE 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.

Ignoring hCE-2, so far, 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 since the enzyme administration would immediately enhance cocaine metabolism and would not require an immune response to be effective.

BChE, designated in older literature as pseudo-cholinesterase or plasma cholinesterase to distinguish it from its close cousin acetylcholinesterase (AChE), is synthesized in the liver and widely distributed in the body, including 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 since BChE administration would immediately enhance cocaine metabolism and would not require an immune response to be effective. For the reasons, enhancement of cocaine metabolism by administration of BChE is considered a promising pharmacokinetic approach for 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 central nervous system (CNS), whereas (-)-cocaine has a plasma half-life of ~47 min or longer (for an i.v. dose of 0.2 mg/kg cocaine), long enough for manifestation of the CNS effects which peak in minutes.9 Thus, positron emission tomography (PET), applied to mapping of the binding of (-)-cocaine and (+)-cocaine in baboon CNS, showed 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. Further, 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 i.v. dose of 0.2 mg/kg cocaine, as the concentration of (-)-cocaine in plasma is much larger than the Michaelis-Menten constant (KM = 4.1μM) 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 (CocE), expressed by Rhodococcus sp. 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 kcat value of 468 min-1 (7.8 s-1) and a KM value of 640 nM. Therefore, the catalytic efficiency (kcat/KM) of native CocE is ~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 of CocE i.v, when given 1 min prior to cocaine injection (180 mg/kg, i.p) protected 100% of rats as opposed to 13 mg of BChE i.v., which offered no protection from the lethal dose of cocaine.15 Second in a mice model, pretreatment of CocE at 0.32 mg and 1 mg doses i.v, resulted in 10- 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 a ~10 min half-life, and the effectiveness of CocE decreases with time in vivo. Prior treatment with CocE for 10 min 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 anti-cocaine therapeutics, both BChE and CocE have their own advantages and disadvantages. Concerning the advantages of using human BChE, first of all, BChE from human source can be tolerated perfectly in human body. In addition, BChE is very stable in physiological condition and, thus, BChE has a relatively longer half-life in human body. The disadvantages of using BChE are associated with the low catalytic efficiency of native BChE against the naturally occurring (-)-cocaine. Compared to native BChE, the catalytic efficiency of native CocE against (-)-cocaine is ~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, i.e. BChE and CocE, can complement each other. Both BChE and CocE could be engineered to become valuable anti-cocaine therapeutic agents.

The use of CocE as anti-cocaine 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 ~5 hours at a physiological temperature (37°C).17

The use of an engineered BChE as anti-cocaine therapeutic requires an improved catalytic efficiency against (-)-cocaine.18,19,20 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 process22,23,24 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. Notably, novel computational design strategies/approaches25,26,27 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. 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/Y332G25 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 mutant25 has been recognized by independent researchers as “a true CocH with a catalytic efficiency that is 1,000-fold greater than wild-type BChE”.28 The independent researchers28 also demonstrated that the A199S/S287G/A328W/Y332G mutant indeed can selectively block cocaine toxicity and reinstatement of drug seeking in rats. The most efficient CocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant (kcat = 5700 min-1 and KM = 3.1 μM), has a ~2,000-fold improved catalytic efficiency (kcat/KM) against (-)-cocaine compared to 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, i.e. 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 which 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 make sure 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 might be doable at least for some drugs of abuse.

Financial disclosure and acknowledgements

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 National Institutes of Health (NIH) (grants R01 DA013930, R01 DA021416, and R01 DA025100) are gratefully acknowledged.


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