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

Abstract

Majority of cocaine users also consume alcohol. Concurrent use of cocaine and alcohol produces 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 hydrolysis of not only cocaine itself, but also norcocaine, cocaethylene, and norcocaethylene. However, there has been no report of enzymes capable of hydrolyzing norcocaethylene (the most toxic metabolite of cocaine). The catalytic parameters (k_cat and K_M) of human butyrylcholinesterase (BChE) and two mutants (known as cocaine hydrolases E14–3 and E12–7) for norcocaethylene have been characterized in the present study, for the first time, in comparison with those for cocaine itself. According to the obtained kinetic data, wild-type human BChE has a similar catalytic efficiency against norcocaethylene (k_cat = 9.5 min⁻¹, K_M = 11.7 μM, and k_cat/K_M = 8.12 × 10⁵ M⁻¹ min⁻¹) compared to that against (−)-cocaine (k_cat = 4.1 min⁻¹, K_M = 4.5 μM, and k_cat/K_M = 9.1 × 10⁵ M⁻¹ min⁻¹). E14–3 and E12–7 have an improved catalytic activity against norcocaethylene compared to the wild-type BChE. E12–7 has a 39-fold improved catalytic efficiency against norcocaethylene (k_cat = 210 min⁻¹, K_M = 6.6 μM, and k_cat/K_M = 3.18 × 10⁷ M⁻¹ min⁻¹). It has been demonstrated that E12–7 as an exogenous enzyme can efficiently metabolize norcocaethylene in rats.

Graphical abstract

Combined molecular modeling, in vitro kinetic analysis, and in vivo activity testing have consistently revealed that two cocaine hydrolases engineered from human butyrylcholinesterase can efficiently catalyze hydrolysis of the most toxic cocaine metabolite norcocaethylene.

INTRODUCTION

Cocaine is a widely abused drug which is highly addictive and hepatotoxic. Traditional pharmacodynamic approach to drug dependence treatment is to modulate neurological processes by development of small-molecule therapeutic agents targeting specific subtypes of transporters/receptors. The small-molecule agents enter the brain and interact with the neurotransmitter systems, such as dopaminergic, serotoninergic, noradrenergic, cholinergic, glutamatergic, GABAergic, or/and opioidergic pathways to offset cocaine effects¹. However, despite decades of effort, there is still no U.S. Food and Drug Administration (FDA)-approved therapeutic treatment specific for cocaine overdose or dependence^2–5. The inherent difficulties in antagonizing cocaine in central nervous system (CNS) have led to the development of biologics to alter the pharmacokinetics of cocaine. A particularly promising pharmacokinetic approach would use an efficient cocaine-metabolizing enzyme to accelerate cocaine metabolism producing biologically inactive metabolites¹ via cocaine hydrolysis at the benzoyl ester^6–13, because cocaine hydrolysis at the benzoyl ester generates ecgonine methyl ester (EME) and benzoic acid that are all biologically inactive.

As well known, the primary metabolic pathways of cocaine in human are hydrolysis catalyzed by plasma enzyme butyrylcholinesterase (BChE) and two liver carboxylesterases (denoted by CE-1 and CE-2). BChE and CE-2 catalyze cocaine hydrolysis at the benzoyl ester group and, thus, convert cocaine to EME and benzoic acid, with BChE as the principal enzyme in this metabolic pathway. CE-1 catalyzes cocaine hydrolysis at the methyl ester group to produce benzoylecgonine which was regarded as a vasoconstrictor^{14, 15}. In addition, cocaine is also metabolized via oxidation by liver microsomal cytochrome P450 (CYP) 3A4 to generate norcocaine which is more toxic than cocaine itself^{11, 16}. Clearly, BChE-catalyzed cocaine hydrolysis is the most desirable metabolic pathway suitable for amplification. On the other hand, wild-type BChE has a low catalytic efficiency against naturally occurring (−)-cocaine (k_cat = 4.1 min⁻¹ and K_M = 4.5 μM)^17–21. It is highly desired to develop a BChE mutant with significantly improved catalytic efficiency against cocaine.

In addition, it has been known that majority of cocaine users (e.g. 92% as of August 2013)²² also consume alcohol (ethanol). The alcohol can react with cocaine in the presence of CE-1 to produce a significantly more cytotoxic metabolite, known as cocaethylene, through transesterification. It was reported that, with alcohol co-administration, ~24% (intravenous), ~34% (oral), or ~18% (smoked) of cocaine was converted to cocaethylene via transesterification²³. Further, the ethylated metabolite cocaethylene can undergo N-demethylation to produce norcocaethylene, an extremely toxic metabolite²⁴. Norcocaethylene has been detected in liver and lung²⁵, which provides the explanation of the phenomenon that cocaine hepatotoxicity was potentiated by alcohol²⁶. Hence, a truly valuable mutant of human BChE as an enzyme therapy for treatment of cocaine dependence and overdose should be highly efficient for hydrolysis of not only cocaine, but also the more toxic metabolites norcocaine, cocaethylene, and norcocaethylene (Figure 1).

Figure 1.

Schematic representation of cocaine and its toxic metabolites produced in the body after concurrent use of cocaine and alcohol.

In development of effective enzyme therapy for treatment of cocaine dependence and overdose, our previous computational design has led to discovery of BChE mutants with considerably improved catalytic efficiency against cocaine (which refers to naturally occurring (−)-cocaine)^27–39, norcocaine⁴⁰, and cocaethylene⁴¹. The first one of our designed promising BChE mutants, i.e. the A199S/S287G/A328W/Y332G mutant²⁷, is recognized as a true cocaine hydrolase (CocH) suitable for clinical development^42–46. Our more recently designed new BChE mutants^{30, 32} have further improved catalytic efficiency against (−)-cocaine. However, whether any of these BChE mutants can also catalyze norcocaethylene hydrolysis as proposed in Figure 2 remains unknown. To our best knowledge, we have not seen a report of any kinetic parameters for wild-type human BChE or any BChE mutant against norcocaethylene. What has been known in literature is that norcocaethylene produces cocaine-like cytotoxicity^{4, 5, 47} and has a longer in vivo half-life than that of cocaine^{23, 48–51}, and that norcocaethylene is the most toxic chemical among cocaine and all cocaine metabolites²⁴.

Figure 2.

Proposed hydrolysis of norcocaethylene catalyzed by BChE or its mutant.

An interesting question is whether the BChE mutants with a considerably improved catalytic efficiency for (−)-cocaine will also have a considerably improved catalytic efficiency for norcocaethylene or not. Our previously reported kinetic analysis on the BChE mutants for acetylcholine (ACh), which is the only known natural substrate of BChE in the body, revealed that the same mutations did not improve the catalytic efficiency of BChE for ACh^{33, 52}, and that the catalytic efficiency of the examined BChE mutants for ACh was even slightly lower than that of the wild-type BChE. Based on this background, it is unknown whether the BChE mutants reported so far have significantly improved catalytic activity for norcocaethylene compared to the wild-type enzyme.

In this study, we have characterized the catalytic activities of wild-type BChE and our computationally designed and discovered A199S/S287G/A328W/Y332G and A199S/F227A/S287G/A328W/Y332G mutants (that are also known as E14–3 and E12–7, respectively) against norcocaethylene in comparison with (−)-cocaine. According to kinetic data obtained, both E14–3 and E12–7 have not only a considerably improved catalytic efficiency against (−)-cocaine, but also a markedly improved catalytic efficiency against norcocaethylene in vitro and in vivo compared to the wild-type BChE.

MATERIALS AND METHODS

Molecular modeling.

The structures of norcocaethylene binding with wild-type human BChE, E14–3, and E12–7 were modeled by using our previously modeled structures of the same proteins^{27–33, 40}. The molecular dynamics (MD) simulations carried out previously³³ on the enzyme-cocaine binding structures⁴⁰ used the X-ray crystal structure deposited in the Protein Data Bank (pdb code: 1P0P) as the initial structure. For each protein (wild-type BChE or E14–3 or E12–7), norcocaethylene was docked into the active site cavity of the enzyme by using the AutoDock 4.2 program⁵³, as we previously did for the enzyme binding with (−)-cocaine or its metabolites^{15, 40} such as norcocaine and cocaethylene. During the process of molecular docking, we used the Solis and Wets local search method ⁵⁴ for the conformational search and the Lamarkian genetic algorithm (LGA)⁵³ to deal with the protein-ligand (norcocaethylene) interactions, with the grid size of 120 × 120 × 120. The finally obtained enzyme-norcocaethylene binding structures were the ones associated with the lowest binding free energies.

Protein preparation and in vitro activity assays.

Wild-type human BChE, E14–3, and E12–7 proteins were prepared and their enzyme activities against norcocaethylene were assayed at the same time under the same experimental conditions. First, the proteins (wild-type human BChE, E14–3, and E12–7) were expressed in Chinese Hamster Ovary (CHO) cells, and the proteins were purified by using a two-step purification procedure (ion exchange chromatography followed by affinity chromatography), as described previously in detail⁴⁰. The purified protein was dialyzed against phosphate-buffered saline and stored at −70°C before use.

The catalytic activities of the enzymes for norcocaethylene and (−)-cocaine were measured by carrying out a UV-Vis spectrophotometric assay. Using the UV-Vis spectrophotometric assay, the catalytic activities of the enzymes for norcocaethylene and (−)-cocaine were determined at the same time under the same experimental conditions except for the concentrations of the enzymes. The final concentration of enzyme (E12–7 or E14–3) was 0.11 nM against (−)-cocaine, whereas the concentration of enzyme (E12–7 or E14–3) against norcocaethylene was 20-fold higher. The concentration of the wild-type BChE was 82 nM against both (−)-cocaine and norcocaethylene. The enzymatic reaction was initiated by adding 180 μl of the substrate (norcocaethylene or (−)-cocaine) solution to 20 μl of an enzyme solution. The final norcocaethylene/(−)-cocaine concentrations of the reaction systems at the time zero (t=0) were 100, 50, 20, 10, 5, 2, and 1 μM. The enzymatic reaction was carried out at the temperature of 25 °C under pH 7.4 (with the 0.1 M potassium phosphate). The initial reaction rates of the enzymatic hydrolysis of norcocaethylene/(−)-cocaine in various initial substrate concentrations were estimated by tracking the time-dependent intrinsic absorbance peak of norcocaethylene/(−)-cocaine at 230 nm (see below for the UV-Vis absorption spectra) using a GENios Pro Microplate Reader (TECAN, Research Triangle Park, NC) with the XFluor software. The initial reaction rate was estimated from the slop of the linear portion of the progress curve. All assays were carried out in triplicate (n=3). The Michaelis-Menten kinetic analysis was carried out by using the GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA) to determine the V_max and K_M values.

Subjects used for in vivo studies.

All in vivo experiments were conducted in the animal laboratories (in the TODD Building) of the University of Kentucky’s Division of Laboratory Animal Resources (DLAR) facility (PHS assurance number A3336–01; USDA number 61-R-0002; AAALAC, Intl. Unit # 13). Male Sprague-Darley rats (200–250 g) were ordered from Harlan (Harlan, Indianapolis, IN) and were housed initially in 2 rats per cage. All rats were allowed ad libitum access to food and water and were maintained on a 12-hour light and dark cycle with lights on at 8 AM in a room kept at a temperature of 21 to 22°C. Each rat was used only once. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky.

Characterization of E12–7 in accelerating norcocaethylene clearance in rats.

Norcocaethylene fumarate (a salt formulation of norcocaethylene) was provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program (Bethesda, MD), and the protein material (purified E12–7 protein) used for in vivo studies in rats was expressed and purified in our reported study⁵⁵ using the CHO cells. Rats were injected with saline, 0.15 or 5 mg/kg of E12–7 via tail vein 1 min before i.v. injection of 2 mg/kg norcocaethylene. Four rats were used for each set of experiments (n=4). About 50 to 75 μl of blood samples were collected from saphenous veins into a heparin-coated capillary tubes and immediately diluted in 100 μl of 250 μM paraoxon (in 0.1% formic acid) at 3, 5, 10, 30, 60, 90, 120, 150, and 180 min after the i.v. injection of norcocaethylene. The diluted blood samples were stored at −70°C and analyzed by using our established LC-MS/MS approach⁵⁶ to simultaneously determine the concentrations of norcocaethylene (NCE) and the hydrolysis product norecgonine ethyl ester (NEEE) in the whole blood. According to the Guide for the Care and Use of Laboratory Animals, anesthesia is not required for the IV and IP injections and blood collection from saphenous vein in rats and mice, although anesthesia is required for blood collection from many other types of veins^{57, 58}. So, the animal studies were performed without anesthesia. At the end of the experiments, rats were euthanized by CO₂ inhalation and a secondary physical measure to confirm the death.

RESULTS AND DISCUSSION

Insights from molecular docking.

Through molecular docking, we wanted to understand how norcocaethylene may bind with human BChE and its mutants compared to cocaine. For convenience, from now on, cocaine will always refer to the naturally occurring (−)-cocaine. As shown in Figure 1, there are some differences in molecular structure between norcocaethylene and cocaine, i.e. the methyl group on cocaine’s methyl ester changes to an ethyl group in norcocaethylene, and the methyl group on the nitrogen atom in cocaine becomes a hydrogen atom in norcocaethylene. Our molecular docking revealed that norcocaethylene is capable of binding with each enzyme (wild-type human BChE or its mutant) in a similar way like cocaine. As well-known, BChE has a catalytic triad consisting of S198, H438, and E325^{30, 59, 60} and an oxyanion hole consisting of the backbone NH groups of G116, G117, and A199. As depicted in Figure 3, the carbonyl carbon of the benzoyl ester group of the substrate (norcocaethylene or cocaine) is always close to the hydroxyl oxygen of S198 side chain, ready for the anticipated nucleophilic attack from the hydroxyl oxygen of S198. Meanwhile, carbonyl oxygen of the benzoyl ester group of the substrate (norcocaethylene or cocaine) is able to stay within the oxyanion hole. Based on these binding features, we may reasonably expect that the enzyme (wild-type human BChE or its mutant) should be able to catalyze the norcocaethylene hydrolysis in the similar way as it catalyzes the cocaine hydrolysis.

Figure 3.

Docked structures of the wild-type BChE and BChE mutant E12–7 binding with cocaine and norcocaethylene: (A) Wild-type human BChE binding with cocaine; (B) Wild-type human BChE binding with norcocaethylene; (C) E12–7 binding with cocaine; (D) E12–7 binding with norcocaethylene. Indicated in the figure are the key distances (in Å) between the carbonyl oxygen of the substrate and the hydrogen atoms within the oxyanion hole of the enzyme.

Concerning specific interactions of the substrate with the oxyanion hole of wild-type BChE, similar to the binding mode of cocaine (Figure 3A), the carbonyl oxygen of norcocaethylene also has two potential weak hydrogen bonds with the NH groups of G117 and A199 (Figure 3B). For specific interactions of the substrate with the oxyanion hole of E12–7, the hydroxyl group (OH), instead of the NH group of S199 may form a stronger hydrogen bond with the carbonyl oxygen of substrate (cocaine or norcocaethylene) (Figure 3C and D). For this reason, E12–7 is expected to have an improved catalytic activity against both cocaine and norcocaethylene, compared to the wild-type BChE, although it has been known that these BChE mutants do not have an improved catalytic efficiency against ACh⁵². Further, compared to the hydrogen bonds of cocaine with E12–7 (Figure 3C) within the oxyanion hole, the corresponding hydrogen bonds of norcocaethylene with E12–7 (Figure 3D) are relatively weaker, suggesting that the catalytic activity of E12–7 against norcocaethylene might be relatively lower than that of E12–7 against cocaine.

Kinetic parameters.

In light of the computational insights, we carried out in vitro experimental tests, including the protein expression and enzyme activity assays, on wild-type human BChE, E14–3, and E12–7. The in vitro activity assays were based on our observation (Figure 4) that norcocaethylene also had an UV-Vis absorption peak at 230 nm as cocaine, and that the absorption at 230 nm is linearly proportional to the concentration of norcocaethylene or cocaine. The in vitro assays enabled us to determine the catalytic activity of the enzymes against norcocaethylene in comparison with the corresponding activity against cocaine. To minimize the possible systematic experimental errors of the kinetic data, for each enzyme the catalytic activities against both norcocaethylene and cocaine were assayed at the same time under the same experimental conditions, except the concentration of enzymes, so as to reliably determine the catalytic activity of the enzyme against norcocaethylene relative to the known activity against cocaine. Depicted in Figure 5 are the measured kinetic data. Summarized in Table 1 are the kinetic parameters of the enzymes against norcocaethylene in comparison with those against cocaine, cocaethylene, and norcocaine.

Figure 4.

UV-visible absorption of the enzyme and substrates: (A) UV-visible absorption of cocaine, norcocaethylene, and E12–7 (denoted as CocH3); (B) UV absorption at 230 nm vs the concentration of cocaine; (C) UV absorption at 230 nm vs the concentration of norcocaethylene.

Figure 5.

Kinetic data obtained in vitro for enzymatic hydrolysis of cocaine and norcocaethylene (all in triplicate): (A) wild-type human BChE; (B) E14–3; (C) E12–7; (D) E14–3 and E12–7 against norcocaethylene only. The reaction rate (represented in μM min⁻¹ per nM enzyme) was determined by measuring by the change of the absorbance at 230 nm per minute.

Table 1.

Kinetic parameters of wild-type human BChE and its mutants (E14–3 and E-12–7) against cocaine, norcocaine, cocaethylene, and norcocaethylene.

Substrate	Enzymea	KM (μM)	kcat (min−1)	kcat/KM (M−1min−1)	RCEd
Cocaineb	WT BChE	4.5	4.1	9.11 × 105	1
	E14–3	3.1	3,060	9.87 × 108	1,080
	E12–7	3.1	5,700	1.84 × 109	2,020
Norcocaineb	WT BChE	15	2.8	1.87 × 105	1
	E14–3	12	766	6.38 × 107	343
	E12–7	13	2,610	2.01 × 108	1,080
Cocaethyleneb	WT BChE	7.5	3.3	4.40 × 105	1
	E14–3	8.0	1,820	2.28 × 108	517
	E12–7	9.5	3,600	3.79 × 108	861
Norcocaethylenec	WT BChE	11.7	9.5	8.12 × 105	1
	E14–3	5.7	71	1.25 × 107	15
	E12–7	6.6	210	3.18 × 107	39

^aThe enzyme under the study was wild-type human BChE (WT BChE), the A199S/S287G/A328W/Y332G mutant (E14–3), or the A199S/F227A/S287G/A328W/Y332G mutant (E12–7).

^bData for wild-type BChE against cocaine came from reference⁶⁷, data for E14–3 against cocaine came from reference⁶⁸, data for CocH3 against cocaine came from reference³⁰, data for all enzymes against norcocaine came from reference⁴⁰, and data for all enzymes against cocaethylene came from reference⁴¹.

^cAll of the kinetic data for norcocaethylene were determined in the present study for the first time.

^dRCE refers to the Relative Catalytic Efficiency (k_cat/K_M), i.e. the ratio of the k_cat/K_M value of the mutant to that of wild-type BChE against the same substrate.

A survey of the kinetic parameters summarized in Table 1 reveals that both E14–3 and E12–7 examined in this study have an improved catalytic efficiency (k_cat/K_M) against norcocaethylene. Wild-type BChE has a similar catalytic activity against norcocaethylene (k_cat = 9.5 min⁻¹, K_M = 11.7 μM, and k_cat/K_M = 8.12 × 10⁵ M⁻¹ min⁻¹) compared to its catalytic activity against cocaine (k_cat = 4.1 min⁻¹, K_M = 4.5 μM, and k_cat/K_M = 9.11 × 10⁵ M⁻¹ min⁻¹). According to the kinetic parameters summarized in Table 1, E14–3 and E12–7 indeed have markedly improved catalytic efficiency against norcocaethylene compared to the wild-type BChE: 15-fold for E14–3 and 39-fold for E12–7. In comparison with the catalytic activities of a same mutant for different substrates, for both E14–3 and E12–7, the catalytic efficiency of the enzyme against norcocaethylene is relatively lower than that against cocaine, but higher than that of wild-type BChE against any substrate listed in Table 1. Within the enzymes examined in this study, the most efficient BChE mutant (E12–7) against norcocaethylene is also the most efficient mutant against cocaine, norcocaine, and cocaethylene. E12–7 has a 39-fold improved catalytic efficiency against norcocaethylene, an 861-fold improved catalytic efficiency against cocaethylene, a 1080-fold improved catalytic efficiency against norcocaine, and a 2020-fold improved catalytic efficiency against cocaine. So, E12–7 is identified as the most promising enzyme (BChE mutant) for metabolizing all the four toxic substrates: norcocaethylene, cocaethylene, cocaine, and norcocaine.

Concerning why E12–7 has only a 39-fold improved catalytic efficiency against norcocaethylene whereas it has a 1080-fold improved catalytic efficiency against norcocaine compared to the wild-type enzyme, we note that the major difference exists in k_cat rather than K_M. In fact, E12–7 has an even lower K_M (6.6 μM) against norcocaethylene compared to the K_M (13 μM) against norcocaine. As well known, K_M is mainly determined by the enzyme-substrate binding affinity in the Michaelis-Menten complex, whereas k_cat is determined by the free energy barrier (which may be the free energy change from the Michaelis-Menten complex to the rate-determining transition state).^{30, 60} According to the experimentally determined kinetic parameters (K_M values) listed in Table 1, norcocaethylene can more favorably bind with E12–7 than norcocaine, but with a higher free energy barrier (associated with the lower k_cat). In other words, compared to the E12–7-catalyzed hydrolysis of norcocaine, the Michaelis-Menten complex for the E12–7-catalyzed hydrolysis of norcocaethylene is a more stable structure associated with a “lower” local minimum on the potential energy surface (PES),³⁰ but its rate-determining transition state is a less stable structure associated with a “higher” saddle point on the PES. To further understand why the rate-determining transition-state structure for E12–7-catalyzed hydrolysis of norcocaethylene is less stable than that for E12–7-catalyzed hydrolysis of norcocaine, one will have to simulate the rate-determining transition-state structure by carrying out quantum mechanical/molecular mechanism (QM/MM) reaction-coordinate calculations as we did previously for the enzymatic hydrolysis of (−)-cocaine.³⁰ With the time-consuming, but more insightful, QM/MM reaction-coordinate calculations, one may further rationally design potentially more active BChE mutants associated with a more stable rate-determining transition state against norcocaethylene. Hence, and the detailed mechanistical understanding and design of the improved mutants might be an interesting future computational study.

Norcocaethylene clearance is accelerated by E12–7.

Our recently reported in vivo studies^{40, 55} demonstrated that E12–7 was able to efficiently metabolize cocaine, cocaethylene, and norcocaine in rats. With the encouraging in vitro activity data discussed above, we would like to know whether E12–7 could also efficiently metabolize norcocaethylene in rats. We characterized the pharmacokinetic (PK) profiles of norcocaethylene clearance with and without the presence of E12–7 in rats. Since information about the toxicity of norcocaethylene was rare, concerning the dose of norcocaethylene we first tried to intravenously (i.v.) administer 3 mg norcocaethylene per kg of body weight in control animals. It turned out that 3 mg/kg norcocaethylene (i.v.) was toxic and even lethal; within the four rats injected i.v. with 3 mg/kg norcocaethylene, the first rat died immediately after injection, the second rat was immobile for around 15 min with enlarged pupil, and the other two were very weak, taking a long time to start walking. As we reported earlier, 3 mg/kg norcocaine was significantly more toxic than 5 mg/kg cocaine to rats⁴⁰. This observation suggested that 3 mg/kg norcocaethylene is even more toxic than 3 mg/kg norcocaine to rats. Therefore, we finally decided to test additional rats under a lower dose, i.e. 2 mg/kg norcocaethylene and saline (control). Even if a lower dose of norcocaethylene was used, the toxicity of norcocaethylene was still evident as collection of blood samples from the tested rats was more difficult during the first 30 minutes after the i.v. injection of norcocaethylene compared to cocaine, norcocaine, and cocaethylene. The first time-point for blood collection was originally planned at 2 min to be consistent with our previous study on other toxic cocaine metabolites^{40, 41}, but we actually got the blood collected at 3 min. This implies that norcocaethylene may contribute to coronary vasospasm related to the combined use of cocaine and alcohol.

During the in vivo experiment, four rats (n=4) were used for each group. One group of rats were injected with saline first and then 2 mg/kg norcocaethylene (i.v.). The other two groups of rats (n=4 for each group) were injected with 0.15 or 5 mg/kg E12–7 and then 2 mg/kg norcocaethylene (i.v.). The i.v. injection of 0.15 and 5 mg/kg E12–7 led to a plasma E12–7 concentration of ~0.02 μM and ~1.0 μM, respectively, at ~2 min after the i.v. injection of E12–7 in this study⁵⁵. For each rat, the blood was collected at 3, 5, 10, 30, 60, 90, 120, 150, and 180 min after i.v. injection of norcocaethylene. Depicted in Figure 6 are the in vivo data.

Figure 6.

Metabolic profiles of norcocaethylene (NCE) in the presence and absence of an exogenous enzyme E12–7 (0.15 or 5 mg/kg) in rats (n=4 for each group). (A) Time course of NCE concentration in rat blood. (B) Time course of the metabolite NEEE formed from the NCE hydrolysis in rat blood. (C) Time course of plasma E12–7 concentration in rat plasma after IV injection of 0.15 or 5 mg/kg E12–7.

According to the data in Figure 6, E12–7 can accelerate norcocaethylene hydrolysis to produce norecgonine ethyl ester (NEEE) and benzoic acid. The control curves in Figure 6 reflect the overall effects of all possible norcocaethylene elimination pathways without an exogenous enzyme⁶¹. As seen in Figure 6, in the control group of rats, the average blood concentration of norcocaethylene at the first time point (3 min) was ~1.3 μM, while the average blood concentration of NEEE (metabolite) was ~0.007 μM. In the presence of an exogenous enzyme after i.v. injection of 0.15 mg/kg E12–7, the average blood concentration of norcocaethylene at ~3 min was ~1.0 μM (with a ~23% decrease compared to the control group, as seen in Figure 6A), while the average blood concentration of NEEE at the first time point (3 min) was ~0.18 μM (Figure 6B). Most (~77%) of the norcocaethylene was not hydrolyzed by E12–7 (Figure 6), indicating that the E12–7 dose (0.15 mg/kg) was not sufficiently high for effectively accelerating the hydrolysis of norcocaethylene in the blood. Increasing the E12–7 dose from 0.15 mg/kg to 5 mg/kg so as to increase the E12–7 concentration in the plasma (Figure 6C), the average blood concentration of norcocaethylene at ~3 min was only 0.045 μM (with a ~97% decrease compared to the control group, as seen in Figure 6A), while the average blood concentration of NEEE at the first time point (3 min) was as high as ~2.0 μM. So, the i.v. injection of 5 mg/kg E12–7 effectively accelerated norcocaethylene hydrolysis in the blood.

Finally, we note that the BChE mutants E14–3 and E12–7 are not expected to be cytotoxic. In fact, all our previous (and present) in vivo studies^{30, 41, 62–66} on these mutants did not cause any toxicity signs in animals. As there is still no enzyme approved by the FDA for treatment of cocaine overdose or dependence, these BChE mutants can be considered as promising candidates of an effective enzyme therapy to treat cocaine overdose.

CONCLUSION

We have characterized wild-type human BChE and its mutants E14–3 and E12–7 for their catalytic activity against norcocaethylene in comparison with the corresponding catalytic activity against cocaine. It has been demonstrated that wild-type human BChE has a similar catalytic efficiency (k_cat/K_M) against norcocaethylene (k_cat = 9.5 min⁻¹, K_M = 11.7 μM, and k_cat/K_M = 8.12 × 10⁵ M⁻¹ min⁻¹) compared to that against (−)-cocaine, but has larger individual k_cat and K_M values against norcocaethylene. Further, according to the kinetic characterization, human BChE mutants E14–3 and E12–7, known as cocaine hydrolases, have not only considerably improved catalytic activities against cocaine, norcocaine, and cocaethylene, but also a markedly improved catalytic activity against norcocaethylene in comparison with the wild-type enzyme. In particular, the in vitro activity data revealed that human BChE mutant E12–7 is the most active enzyme against norcocaethylene, with k_cat = 210 min⁻¹, K_M = 6.6 μM, and k_cat/K_M = 3.18 × 10⁷ M⁻¹ min⁻¹. Further, E12–7 is identified as the most active enzyme (BChE mutant) in the study for metabolizing all the four toxic substrates: norcocaethylene, cocaethylene, cocaine, and norcocaine. Based on our in vivo evaluation, E12–7 was able to significantly accelerate norcocaethylene hydrolysis in rats. Thus, E12–7 may be used to effectively eliminate not only cocaine itself, but also all the three toxic metabolites (norcocaine, cocaethylene, and norcocaethylene) from the body for potential therapeutic treatment of drug overdose associated with the concurrent use of cocaine and alcohol.