Introduction

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Because classic pharmacological treatments for cocaine overdose are not fully effective (Hollander, 1995), there is reason to consider alternative therapeutic strategies. Although fatal cocaine toxicity can present with widely varying drug levels (Wetli and Wright, 1979), some studies have found a correlation between cocaine response and plasma cocaine concentration (Javaid et al., 1978; Cone et al., 1988; Lau et al., 1991). Also, it has been reported (Hoffman et al., 1992) that cocaine toxicity tends to vary inversely with the levels of plasma butyrylcholinesterase (BChE), a major factor in cocaine metabolism. Such observations led to the idea that enhancing the metabolic conversion of cocaine to less toxic derivatives could be therapeutically useful (Gorelick, 1997). Plasma BChE can hydrolyze cocaine to ecgonine methyl ester and benzoic acid (Stewart et al., 1977) (Fig. 1), which lack the pharmacological activity of cocaine (Madden and Powers, 1990). In doses that increase plasma BChE levels 400-fold, BChE is claimed to enhance cocaine metabolism in monkeys (Carmona et al., 2000). Large quantities of exogenous BChE also protect rodents and monkeys against cocaine toxicity (Hoffman et al., 1996; Lynch et al., 1997; Mattes et al., 1997; Carmona et al., 1998), and even lethal overdose (Hoffman et al., 1996).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Metabolic pathways. Structures of cocaine and principal metabolites together with their relative abundance in primates, as reported by Lockridge and coworkers (Xie et al., 1999).

In search of a cocaine hydrolase that would have a useful impact at more practical dose levels, novel mutants of human plasma BChE have been explored (Xie et al., 1999). Recently, we performed molecular modeling studies of enzyme-cocaine complexes (Sun et al., 2001) and used the results to engineer a powerful cocaine hydrolase by site-directed mutagenesis of BChE (Sun et al., 2002). The new enzyme, A328W/Y332A, showed 40-fold improvement in k_cat over wild-type BChE and only slightly increased K_M (18 µM versus 4.5 µM). This hydrolase is the first BChE mutant whose kinetic properties meet previously suggested criteria for clinical utility in treating cocaine overdose (Landry et al., 1993). Compared with a recently reported bacterial cocaine hydrolase of even higher catalytic efficiency (Larsen et al., 2002), the modified BChE is attractive in that, as a nearly natural human protein, it is less likely to provoke immunological reactions.

We previously observed that A328W/Y332A BChE dramatically accelerates cocaine clearance in isolated plasma and, when injected into mice, it abolishes cocaine-induced hyperactivity (Sun et al., 2002). Here we report effects of A328W/Y332A BChE on plasma cocaine in rats. Although physiological studies have yet to be performed, the pharmacokinetic and metabolic data suggest that treatment with A328W/Y332A (or other cocaine hydrolases with equal or better catalytic properties) can substantially hasten drug elimination in vivo.

	Experimental Procedures

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Materials. Natural ()-cocaine was purchased from Sigma-Aldrich (St. Louis, MO) under an institutional license from the U.S. Drug Enforcement Administration, while ³H-()-cocaine (50 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Other reagents were echothiophate iodide from Wyeth-Ayerst (Rouses Point, NY), and butyrylthiocholine iodide, diisopropyl fluorophosphate, and tetraisopropyl pyrophosphoramide (iso-OMPA), all from Sigma-Aldrich.

Cocaine Hydrolase. Recombinant cocaine hydrolase was prepared in a stable, predominantly tetrameric form by bulk culture of CHO K1 cells (61-CCL; American Type Culture Collection, Manassas, VA) cotransfected with cDNAs for A328W/Y332A BChE and a portion of the rat COLQ gene (Krejci et al., 1997) as described previously (Altamirano and Lockridge, 1999; Xie et al., 1999). Secreted enzyme was purified by affinity chromatography on procainamide-Sepharose eluted with 0.2 M procainamide, followed by ion exchange chromatography on DE52 and elution with a NaCl gradient in 20 mM Tris-HCl, pH 7.5 (Arpagaus et al., 1990). Purified BChE was dialyzed, concentrated to 1 mg/ml, filter sterilized, and stored at 4°C.

Cocaine Levels in Plasma and Tissue. Animal studies, conducted under a protocol approved by the Mayo Institutional Animal Care and Use Committee, employed male Sprague-Dawley rats weighing 250 to 350 g (Harlan, Madison WI). Under urethane anesthesia (1.45 mg/kg i.p.), catheters were placed in the tail vein (to deliver drugs or BChE) and carotid artery (to sample blood). Rats remained anesthetized for the duration of the experiment and were finally euthanized with sodium pentobarbital (250 mg/kg i.v.). BChE (1 or 3 mg/kg) was administered first. Cocaine injections consisted of unlabeled drug mixed with exactly 30 µCi of [³H]cocaine (50 Ci/mmol). The dose (6.8 mg/kg i.v. or 60 mg/kg i.p) corresponded to 40 or 80% of reported LD₅₀ values for respective routes of administration (Borchard et al., 1990).

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Radiometric Assay. To measure cocaine and benzoic acid, the product of cocaine hydrolysis by BChE, we used sensitive radiometric assays based on toluene extraction of [³H]cocaine labeled on its benzene ring (Sun et al., 2001, 2002). In brief, 200-µl plasma aliquots were acidified with 300 µl of 0.02 M HCl for extraction of benzoic acid while paired aliquots were alkalinized with 300 µl of 1 M Na₂CO₃ for extraction of cocaine. These samples were vigorously mixed for 10 s with 4 ml of toluene-based scintillation fluor. After phase separation by centrifugation, organic phases were collected for scintillation counting. Under the extraction conditions of the cocaine assay, authentic [³H]cocaine was quantitatively detected, whereas [³H]benzoic acid was almost undetected (1%). The reverse was true of the benzoic acid assay. After the results were confirmed by mass spectrometry (see below), the assays were deemed suitable for rapid determination of plasma drug levels.

Liquid Chromatography-Mass Spectrometry (LC-MS). Plasma samples were prepared for LC-MS as described by Singh et al. (1999). Frozen plasma was quickly thawed, mixed, and microcentrifuged for 2 min at 14,000 rpm; 300-µl supernatant aliquots were then transferred to clean tubes with 1 ml of acetonitrile, mixed for 10 s, and centrifuged for 10 min at 2,500g, 4°C. Supernatants were transferred into clean tubes with 15 µl of formic acid. After vigorous mixing for 10 s, samples were evaporated to dryness at room temperature in a vacuum centrifuge. Dried samples were reconstituted with 1 ml of water and passed through a 0.2-µm syringe filter before analysis by LC-MS. Analysis after the fact indicated no appreciable loss of cocaine or metabolites attributable to "online decomposition" of samples waiting in queue.

Statistical Analysis and Pharmacokinetics. Treatment effects were subjected to analysis of variance using StatView 4.5 (Abacus Concepts, Berkeley, CA). Post hoc testing was based on Fisher's protected least significant difference; p < 0.05 was considered statistically significant. Cocaine plasma concentration-time profiles were analyzed with WinNonlin (SCI Software, Lexington, KY). Cocaine levels after i.p. administration were described by a one-compartment model because absorption was slower than redistribution. Derived parameters included absorption half-life (0.693/k_a.), peak cocaine concentration (C_max) and time to peak concentration (t_max). Data from i.v. bolus administration were analyzed by an open two-compartment model, with elimination from the central compartment. Apparent rate constants for redistribution () and elimination () were calculated along with the associated concentration parameters (A and B) by fitting plasma cocaine level, Cp, to the following equation.
Clearance (CL) and volume of distribution at steady state (V_ss) were calculated using standard noncompartment methodology. Area under the curve (AUC_0t) and area under the first moment curve (AUMC_0t) from time 0 to final sample were determined by the trapezoidal method. Terminal areas from the last measured concentration (Ct) to infinity were calculated as Ct/ for AUC_t and as Ct × t/ + Ct/² for AUMC_t. Total clearance from plasma (CL_total) was defined as dose/AUC₀, and V_ss was defined as dose × AUMC_t /AUC²_0.

	Results

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Fate of A328W/Y332A in Vivo. First we determined the distribution and stability of recombinant A328W/Y332A BChE after an i.v. injection (Fig. 2). Two minutes after the injection (0.5 mg/kg), plasma BChE activity with butyrylthiocholine as substrate had increased 80-fold over basal levels. Over the next 12 h, BChE activity decayed with a biphasic exponential course involving a rapid phase (half-life, 22 min) and a slower phase (half-life, 9 h). These characteristics indicated that BChE activity remained reasonably stable during the first hours after the injection. Interestingly, the apparent steady-state volume of distribution was 48 ml, about 3 times the expected plasma volume of a 300-g rat.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Elimination of BChE from rat plasma. A328W/Y332A, a purified double mutant recombinant BChE (70% tetrameric form) was injected intravenously into rats at a dose of 0.5 mg/kg. Plasma BChE activity was measured using butyrylthiocholine as substrate and is shown in units per milliliter. Endogenous BChE activity at time 0 was 0.04 U/ml.

Pharmacokinetics of Intravenous Cocaine. Plasma was repeatedly sampled between 2 min and 2 h after a cocaine injection of 6.8 mg/kg, and cocaine levels were monitored by a radiometric assay. From the initial value of 5.8 µM, the plasma drug concentration decayed with a biexponential time course (Fig. 3). The data fit well to a standard pharmacokinetic model that assumed redistribution between a central and a peripheral compartment, with elimination from the central compartment. Pharmacokinetic calculations with this model (Table 1) indicated an elimination half-life of 52 min in control rats. Pretreatment with wild-type BChE, 1 mg/kg, caused a slight increase in levels of the cocaine breakdown product, benzoic acid, but no change in cocaine's pharmacokinetic parameters, including elimination half-life and AUC. This result was anticipated because of the relatively small enzyme dosage. An identical dose of A328W/Y332A, however, accelerated cocaine metabolism markedly (Fig. 3A). With this pretreatment, drug levels in early plasma samples were 40% below control and continued to drop steeply with time. Pharmacokinetic calculations (Table 1) showed a halving of AUC, a 34% reduction of elimination half-life, and a doubling of cocaine clearance. Accompanying these changes was a 10-fold rise in levels of benzoic acid (Fig. 3B). Even larger effects were generated by a 3 mg/kg dose of A328W/Y332A (Fig. 3). In fact, cocaine clearance, t_1/2, and AUC all showed a significant and near-linear dependence on BChE dosage (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Pharmacokinetics of i.v. cocaine after enzyme treatment. Rats were given BChE (1 or 3 mg/kg i.v.) or saline 10 min before i.v. cocaine challenge (6.8 mg/kg). Mean plasma concentrations (in micromolar concentration) ± S.E.M are shown. In rats pretreated with A328W/Y332A, a purified double mutant recombinant BChE (n = 6), the initial level of cocaine (A) was reduced significantly (p < 0.01) compared with controls (n = 6). Note the large increase in benzoic acid levels (B) and its dependence on dosage of mutant BChE (in the control group, levels were below scale at most time points). With wild-type BChE (1 mg/kg), there were no significant effects on cocaine peak level, AUC, or t1/2. , control; , wild type 1 mg; , mutant 1 mg; , mutant 3 mg.

View this table: [in this window] [in a new window]   TABLE 1 Pharmacokinetics of intravenous cocaine [3H]Cocaine was administered i.v. to rats at a dose of 6.8 mg/kg (30 µCi total). The time course of plasma cocaine was then monitored by multiple radiometric determinations of drug levels over a 2-h sampling period in five to six rats per group. Pharmacokinetic parameters were calculated by WinNonLin software (see Experimental Procedures): t1/2 (redistribution half-life), t1/2 (elimination half-life), AUC, Cl, MRT (mean retention time), and Vss.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Dependence of cocaine elimination on dosage of cocaine hydrolase. In rats pretreated with the indicated doses of double mutant BChE, three pharmacokinetic parameters were calculated to describe the time course of cocaine after rapid bolus i.v. injection: AUC, CL, and t1/2. Regression analysis showed that all three parameters were strongly dependent on the dosage of BChE (p < 0.0001).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Plasma cocaine and metabolites after cocaine challenge. Control rats () or rats pretreated 10 min earlier with double mutant A328W/Y332A BChE () (1 mg/kg i.v.) were challenged with cocaine (6.8 mg/kg i.v.). After 30 min (A) or 60 min (B), plasma was collected, and cocaine, BE, and EME were assayed by LC-MS. Concentrations are indicated as mean ± S.E.M. in micromolar concentration. Control values for EME were below quantitation limit.

Pharmacokinetics of Intraperitoneal Cocaine. Returning to the radiometric approach, for ease of quantitation, we attempted to determine whether the effects of A328W/Y332A might be influenced by the route of cocaine administration. Specifically, in rats challenged with cocaine in an i.p. dose of 60 mg/kg, we examined the effects of BChE pretreatment on elimination half-life, AUC, absorption half-life, peak plasma concentration, and time to peak. Absorption half-life and time to peak remained stable under all tested conditions (Table 2, Fig. 6). In contrast, elimination half-life, AUC, peak plasma concentration, and benzoic acid levels depended on the type of pretreatment. Wild-type BChE in a dose of 1 mg/kg had no effect, but the same dose of A328W/Y332A BChE enhanced all these measures of cocaine disposal. It is worth stressing that the experiments with i.p and i.v. cocaine were not only performed independently but also were analyzed by different pharmacokinetic models (see Experimental Procedures). For this reason, the close agreement between pharmacokinetic parameters in Tables 1 and 2 is remarkable. The combined data show convincingly that a modest dose of A328W/Y332A can accelerate cocaine disposal to an extent that should be clinically significant.

View this table: [in this window] [in a new window]   TABLE 2 Pharmacokinetics of intraperitoneal cocaine [3H]Cocaine was administered i.p. to rats at a dose of 60 mg/kg (30 µCi total). Assays, group sizes, and pharmacokinetic calculations were as indicated in the legend to Table 1: t1/2 (elimination half-life), t1/2absorp (absorption half-life), AUC, Tmax (time of peak plasma concentration), and Cmax (peak plasma cocaine concentration).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Pharmacokinetics of i.p. cocaine after enzyme treatment. Rats were given saline or A328W/Y332A, a purified double mutant recombinant BChE (1 mg/kg i.v.), 10 min before i.p. cocaine challenge (60 mg/kg). Concentrations of plasma cocaine (A) and benzoic acid (B) are indicated (mean ± S.E.M.). In A328W/Y332A-pretreated rats (n = 6), peak level of cocaine was reduced significantly (p < 0.01) compared with controls (n = 6). Note the large increase in benzoic acid levels in the A328W/Y332A-pretreated rats. Wild-type BChE (1 mg/kg) caused no significant effects on cocaine peak level, AUC, or cocaine t1/2. , control; , wild-type (1 mg); , mutant (1 mg).

	Discussion

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Assay and Properties of Cocaine Hydrolase. We have used a simple radiometric assay validated by LC-MS to demonstrate cocaine removal by an effective hydrolase and to characterize the pharmacokinetics. The ability to assay blood samples as small as 100 µl enabled repeated measurements without hemodynamic disturbance. A second prerequisite for these experiments was a recombinant BChE that would continue to function for 1 or 2 h after an i.v. injection. Native human plasma BChE is a tetrameric enzyme with a plasma half-life of about 24 h after injection into rats (Lynch et al., 1997; Mattes et al., 1997), but recombinant BChE is typically much less stable. When prepared by ordinary methods, recombinant BChE is mainly monomeric or dimeric and, for some reason, has a very short residence time in the blood circulation, often disappearing within minutes (Saxena et al., 1998). Since recombinant tetramers are more stable, we prepared a mostly tetrameric A328W/Y332A BChE by cotransfection with COLQ cDNA. In line with previous observations in mice (O. Lockridge, unpublished data), the resulting enzyme had a half-life in excess of 9 h in rats. This BChE exhibited an unexpectedly large volume of distribution, however, as if there had been significant transfer out of the vascular system. Native BChE, being a sizeable protein (monomer molecular mass, 85 kDa), does not readily leave the circulation (Mattes et al., 1997). One explanation for the large apparent volume of distribution, therefore, is that the injected BChE contained appreciable amounts of short-lived forms that were rapidly cleared from plasma. Nonetheless, the demonstrated average stability should be ample for cocaine detoxification in a clinical setting.

Pharmacokinetics of Cocaine. Our baseline data agree well in general with the literature on uptake, distribution, and elimination of cocaine in many species, including rats (Barber et al., 1992; Pan and Heady, 1997; Barat and Abdel-Rahman, 1998; Lau et al., 1999). A consistent finding with cocaine is rapid redistribution from plasma into a theoretical volume that exceeds total body water, probably because the drug accumulates in lipid-rich tissues such as the brain. In our hands, regardless of injection route, the observed peak levels of plasma cocaine were lower than would be expected if the drug had mixed instantaneously with total body water. We are confident that this feature does not reflect aberrant behavior of radiolabeled cocaine, such as selective retention at the site of injection. In the first place, a pilot experiment with samples taken 15 min after tail vein injection recovered less than 5% of the total injected radioactivity in digested tail tissue (not shown). In the second place, almost identical peak values were reported recently by others using HPLC methods to characterize the pharmacokinetics of cocaine after i.v. injection in the rat (Pan and Hedaya, 1999; Sun and Lau, 2001). Given the rapidity of the redistribution of cocaine, we did not expect the apparent 50% shortening of t_1/2 after treatment with A328W/Y332A (Table 1). This outcome may merely reflect the limitations of bi-exponential curve fitting when elimination is fast enough to overlap with redistribution.

A328W/Y332A for Cocaine Detoxication. Normal levels of endogenous BChE are saturated immediately by bolus administration of a stoichiometric excess of cocaine, as in the pattern of bingeing among cocaine abusers. Administered in large amounts, wild-type human BChE can reduce cocaine levels in plasma and important target organs and appears to confer some protection against cocaine toxicity (Mattes et al., 1997). Our results suggest that smaller amounts of the catalytically improved hydrolase, A328W/Y332A, will be able to confer equivalent or greater protection. In particular, the finding of reduced cocaine levels in brain and heart after treatment with this enzyme (Sun et al., 2001, 2002) suggest a potential role in combating cocaine-induced seizures and cardiac arrhythmias.