Download butyrylcholinesterase accelerates cocaine metabolism: in vitro and

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DRUG METABOLISM AND DISPOSITION
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Vol. 28, No. 3
Printed in U.S.A.
BUTYRYLCHOLINESTERASE ACCELERATES COCAINE METABOLISM: IN VITRO AND
IN VIVO EFFECTS IN NONHUMAN PRIMATES AND HUMANS
GILBERTO N. CARMONA, REBECCA A. JUFER, STEVEN R. GOLDBERG, DAVID A. GORELICK, NIGEL H. GREIG,
QIAN-SHENG YU, EDWARD J. CONE, AND CHARLES W. SCHINDLER
Preclinical Pharmacology Section (G.N.C., S.R.G., C.W.S.), Chemistry and Drug Metabolism Section (R.A.J., E.J.C.), and Clinical Pharmacology
Section (D.A.G.), Intramural Research Program, National Institute on Drug Abuse; and Laboratory of Cellular and Molecular Biology
(N.H.G., Q.-S.Y.), National Institute on Aging, National Institutes of Health, Baltimore, Maryland
(Received May 24, 1999; accepted December 7, 1999)
This paper is available online at http://www.dmd.org
ABSTRACT:
reduced peak cocaine concentrations when given to anesthetized
squirrel monkeys. Finally, incubation of cocaine with added BChE
in human plasma in vitro resulted in a decrease in cocaine half-life
similar to that observed with squirrel monkey plasma. The magnitude of the decrease in cocaine half-life was proportional to the
amount of added BChE. Together, these results indicate that exogenously administered BChE can accelerate cocaine metabolism
in such a way as to potentially lessen the behavioral and toxic
effects of cocaine. Therefore, BChE may be useful as a treatment
for cocaine addiction and toxicity.
The endogenous activity of butyrylcholinesterase (BChE;
E.C.3.1.1.8)1 in plasma substantially influences the rate at which
cocaine is metabolized (Carmona et al., 1996). It has been demonstrated that inhibition of endogenous BChE activity by tetraisopropyl
pyrophosphoramide (Iso-OMPA, a selective plasma cholinesterase
inhibitor), followed by a single bolus cocaine challenge, significantly
increases cocaine lethality in mice and rats (Hoffman et al., 1992b).
Limited circumstantial evidence suggests that endogenous BChE activity is inversely correlated with the severity of acute cocaine toxicity
in humans. Individuals experiencing more severe medical problems
after cocaine use tend to have lower plasma BChE activity than those
experiencing less severe problems (Devenyi, 1989; Hoffman et al.,
1992a; Om et al., 1993). These results together suggest that alterations
in BChE activity can affect cocaine metabolism to a physiologically
significant degree. In particular, increasing endogenous BChE should
decrease plasma cocaine concentrations by accelerating the benzoyl-
ester hydrolysis of cocaine in plasma. If cocaine metabolism were
altered sufficiently, then the amount of cocaine entering the brain
might be decreased, resulting in a consequential decrease in cocaineinduced behavioral and toxic effects.
Systemic administration of BChE, at a dose sufficient to increase
plasma BChE levels 400-fold (5000 I.U.; i.v.), has been shown to
significantly decrease cocaine-induced locomotor activity in rats over
a 120-min session (Carmona et al., 1998). This dose of BChE alone
did not produce changes in locomotor behavior when compared with
saline controls. When added to rat plasma in vitro, BChE significantly
accelerated the metabolism of cocaine and shifted the primary metabolite from benzoylecgonine (BE) to ecgonine methyl ester (EME).
Likewise, exogenously-administered BChE (7.8 mg/kg i.v., a dose
sufficient to increase plasma enzyme levels as much as 800-fold), has
been shown to successfully protect against cocaine-induced hypertension and cardiac arrhythmias in the rat (Lynch et al., 1997), whereas
somewhat greater doses of BChE (13.7 or 27.4 mg/kg) provided
protection against seizures and death in mice (Hoffman et al., 1996).
Thus, increasing BChE levels may be a useful approach for treating
cocaine abuse (Gorelick, 1997).
The current study sought to further characterize the ability of BChE
to accelerate the metabolism of cocaine. First, we sought to determine
whether the addition of BChE to monkey plasma in vitro would
produce changes in metabolism similar to those observed in rats.
Monkeys have different basal BChE activity levels than do rats
(Carmona et al., 1996, 1998), which might result in a different overall
metabolic effect. We also evaluated whether BChE administration
could alter cocaine metabolism in vivo in monkeys. Although added
BChE has been shown to accelerate cocaine metabolism in vitro,
similar effects have not been reported after in vivo administration. The
Supported by National Institute on Drug Abuse Intramural Research Funds.
Portions of the data were presented in abstract form at the College on Problems
of Drug Dependence 57th Annual Scientific Meeting, Scottsdale, Arizona, 1995,
and the meeting Peripheral Blockers as Treatments for Substance Abuse and
Dependence. National Institutes of Health, National Institute on Drug Abuse,
Rockville, Maryland, 1998.
1
Abbreviations used are: BChE, butyrylcholinesterase; BE, benzoylecgonine;
EME, ecgonine methyl ester; Iso-OMPA, tetraisopropyl pyrophosphoramide; GCMS, gas chromatography-mass spectrometry.
Send reprint requests to: Gilberto N. Carmona, Behavioral Neuroscience
Branch, Preclinical Pharmacology Section, National Institutes of Health/National
Institute on Drug Abuse Intramural Research Program, 5500 Nathan Shock Dr.,
Baltimore, MD 21224. E-mail: [email protected]
367
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Butyrylcholinesterase (BChE) is known to metabolize cocaine in
humans. In the present study, three different experiments were
performed to determine whether the addition of horse serumderived BChE would accelerate the metabolism of cocaine. In the
first experiment, the addition of BChE to squirrel monkey plasma in
vitro reduced the half-life of cocaine by over 80%, decreased the
production of the metabolic product benzoylecgonine, and increased ecgonine methyl ester formation. The effect of BChE on
cocaine metabolism was reversed by a specific BChE inhibitor. In
the second, in vivo, experiment, exogenously administered BChE
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CARMONA ET AL.
presence of additional metabolic pathways in the intact animal may
alter the overall metabolic effect of added BChE. This study also
provides a direct comparison of the in vitro/in vivo action of BChE on
cocaine metabolism. Finally, we evaluated the effects of BChE addition on the in vitro metabolism of cocaine in human plasma. For this
experiment, the amount of BChE was varied to look for a possible
concentration-dependent metabolite profile.
Materials and Methods
Results
Squirrel Monkey In Vitro. Minimal spontaneous hydrolysis of
cocaine to BE was observed for the saline control conditions. The
cocaine concentration remained above 1000 ng/ml under saline control conditions throughout the entire 240-min sampling period (Fig.
1a). Cocaine metabolism was observed in the plasma-alone condition,
with a cocaine half-life of 43 min. The addition of BChE dramatically
accelerated cocaine metabolism, with the cocaine half-life decreasing
to 5.4 min. The addition of the BChE inhibitor completely blocked the
effect of the added and endogenous BChE. Cocaine concentrations in
the plasma sample with added inhibitor approximated those of the
saline samples.
For the saline control condition, EME was not detected at any time
point (Fig. 1b); however, there was a gradual increase in BE, with a
peak concentration of approximately 150 ng/ml observed during the
final sampling period (Fig. 1c). In plasma alone, there were gradual
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Squirrel Monkey (In Vitro). The plasma from six male squirrel monkeys
with no recent drug exposure was pooled and used for the in vitro assay. The
following conditions were investigated: 1) saline alone (1.65 ml); 2) monkey
plasma alone (1.6 ml); 3) monkey plasma (1.6 ml) ⫹ BChE (100.0 ␮l); and 4)
monkey plasma (1.6 ml) ⫹ BChE (100.0 ␮l) ⫹ BChE inhibitor (50.0 ␮l). The
added BChE inhibitor consisted of 27 mM physostigmine plus a saturated
solution of Iso-OMPA. Each sample received 100.0 ␮l of cocaine solution (for
a final calculated concentration of 1297 ng/ml), and the final volume was
adjusted to 1.85 ml with the addition of appropriate amounts of phosphate
buffer and saline. BChE solution was prepared in phosphate buffer (0.1 M, pH
7.4) to yield a final concentration of approximately 1000 U/ml (100 U/100 ␮l).
Thus, the final BChE concentration per sample was 54 I.U./ml. The pH of
plasma-containing samples was measured to be 7.81; nonplasma 7.40. Each
sample was placed in a 37°C water bath. A zero-timepoint aliquot (100 ␮l) was
removed from each sample just before the addition of BChE. Aliquots (100 ␮l)
for cocaine analysis were then removed from each sample at 5, 15, 30, 60, 90,
120, and 240 min. Aliquots were collected into tubes containing a saturated
solution of sodium fluoride in 10% acetic acid. Aliquots were immediately
placed on dry ice followed by storage at ⫺30°C until analysis by solid phase
extraction followed by gas chromatography-mass spectrometry (GC-MS).
Squirrel Monkey (In Vivo). Male monkeys (n ⫽ 4) were housed individually in light-, temperature-, and humidity-controlled rooms. Fresh drinking
water was available ad libitum. Daily food intake was controlled to maintain
a steady body weight (750 –1250 g). All monkeys had been in previous drug
studies (primarily cocaine), and were clinically healthy and drug-free for at
least 1 month at the time of experiment. Monkeys were anesthetized with
ketamine (5.0 –10.0 mg/kg; i.m.) for the insertion of catheters and administration of BChE and cocaine. Each monkey had two femoral vein catheters, one
for the administration of BChE and cocaine (left catheter), and the other for
time-dependent blood sampling (right catheter). BChE (5000 I.U.) or saline
was administered as a 30-min pretreatment before cocaine (3.0 mg/kg). This
dose, chosen to be comparable with the dose used in our prior studies
(Carmona et al., 1998), should yield an approximate final BChE activity of 140
U/ml of plasma (based on squirrel monkey blood volume of approximately 70
ml/kg, and plasma volume about half blood volume). BChE has a disappearance half-life of approximately 72 h in rhesus monkeys (Broomfield et al.,
1991), suggesting that BChE activity would have remained stable over the 2-h
sample collection period. Three animals were tested under both conditions,
whereas the fourth was tested only with BChE ⫹ cocaine due to catheter
difficulties. Both enzyme and cocaine were administered as single bolus i.v.
injections over 5 s. Equal volumes of saline were immediately injected after the
administration of BChE or cocaine to flush residuals from the catheter. Blood
samples were drawn (from the right catheter) 5 min before and 1, 2.5, 5, 10,
15, 30, 60, and 120 min after cocaine administration for assessment of plasma
cocaine and metabolite concentrations. Catheters were kept patent after each
sampling with infusions of heparin (0.3 ␮l). Samples were transferred into
heparinized collection tubes containing a saturated sodium fluoride solution (in
10% acetic acid). The plasma was separated by centrifugation at 3000 RPM for
10 min, and then stored at ⫺90°C until GC-MS analysis.
Human (In Vitro). Pooled human plasma (obtained from drug-naive volunteers) was used for the assay. The following conditions were investigated: 1)
saline (1.0 ml), 2) plasma (1.0 ml), 3) plasma (1.0 ml) ⫹ BChE (25 ␮l), 4)
plasma (1.0 ml) ⫹ BChE (50 ␮l), and 5) plasma (1.0 ml) ⫹ BChE (100 ␮l).
Cocaine solution (100.0 ␮l) was added to each sample and the final volume
was brought to 1.85 ml with equivalent amounts of phosphate buffer and saline
to maintain a consistent pH. The concentration of the added BChE solution was
1000 U/ml, yielding added doses of 13.5, 27, and 54 U/ml of BChE. The final
concentration of cocaine in the sample was 1143 ng/ml. Each sample was
placed in a 37°C water bath. A zero-timepoint aliquot (100 ␮l) was removed
from each sample just before BChE addition. Similar aliquots (100 ␮l) were
then removed from each sample at 5, 15, 30, 60, and 90 min. Aliquots were
immediately placed on dry ice followed by storage at ⫺30°C until GC-MS
analysis. A saturated sodium fluoride (in 10% acetic acid) solution was added
to each tube as a preservative before collection.
Chemicals and Materials. Drug standards and materials for use in the
analytical assay were obtained from the following sources: cocaine hydrochloride (National Institute on Drug Abuse Intramural Research Program, Baltimore, MD; Mallinckrodt, St. Louis, MO); BE tetrahydrate (Research Biochemicals International, Natick, MA); horse serum-derived BChE, EME
hydrochloride, [2H3]cocaine HCl, [2H3]BE tetrahydrate, [2H3]EME hydrochloride and Iso-OMPA (Sigma Chemical Company, St. Louis, MO), N,O-bis
(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (Pierce
Chemical Co, Rockford, IL), Clean Screen solid phase extraction columns
(ZSDAU020; United Chemical Technologies, Bristol, PA). Methanol, methylene chloride, 2-propanol, and acetonitrile were HPLC grade and all other
chemicals were reagent grade. Horse serum-derived BChE was provided as
1000 U/mg protein, and may have contained an unknown amount of protein
without BChE activity (personal communication, Sigma Chemical Co.).
Analytical Procedure for Cocaine and Metabolites. In vitro mixtures and
plasma specimens were analyzed for cocaine, BE, and EME by a modified
procedure (Cone et al., 1994). Briefly, plasma specimens were mixed with
deuterated internal standard solution and acidified with sodium acetate buffer
(2 M; pH 4.0) followed by centrifugation (3000 rpm for 10 min) and solid
phase extraction. Cocaine analytes were eluted with freshly prepared elution
solvent (methylene chloride/2-propanol/ammonium hydroxide; 80:20:2, v/v/v)
and the eluent was evaporated under nitrogen in a 40°C water bath and
reconstituted in 20 ␮l of acetonitrile. The samples were then transferred to
autosampler vials and combined with 20 ␮l of derivatizing reagent (N,O-bis
(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane). The vials
were sealed and incubated at 80°C for 30 min. Duplicate matrix-matched
calibration curves across the concentration range of 3.1 to 1000 ng/ml for
cocaine, BE, and EME were included in each batch of specimens. The limit of
detection of the assay was approximately 1 ng/ml for all analytes. Control
samples containing all analytes at concentrations of 100 and 500 ng/ml were
processed in duplicate with each run. Accuracy of control measurements was
within 20% for all analytes. Gas chromatography-mass selective detection was
performed with a Hewlett-Packard (Wilmington, DE) 5971 mass selective
detector interfaced to a Hewlett-Packard 5890A gas chromatograph with an
autosampler (HP7673A). A 1-␮l aliquot of the derivatized sample was injected
in the splitless mode onto an HP-1 fused silica capillary column (12 m ⫻ 0.2
mm i.d., 0.33 ␮m film thickness). The MS was operated in the selected ion
monitoring mode.
Data Analysis. Plasma elimination half-lives for cocaine were determined
from log cocaine concentration versus time plots. The data were fitted by linear
regression. The half-lives were then determined by the following relationship:
kel ⫽ ⫺2.203 ⫻ slope; t1/2 ⫽ 0.693/kel, where kel is the cocaine elimination rate
constant and t1/2 is the cocaine elimination half-life
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BUTYRYLCHOLINESTERASE AND COCAINE
Discussion
FIG. 1. Effect of added BChE on cocaine metabolism in squirrel monkey plasma.
Cocaine was added to all samples. Concentrations (in nanograms per milliliter)
of cocaine (a), EME (b), and BE (c) are shown for the 240-min time period after
cocaine administration. When compared with plasma and saline, the half-life of
cocaine was reduced significantly after the addition of BChE. EME concentrations
were increased, whereas BE concentrations were decreased. The BChE inhibitor
Iso-OMPA reversed these effects.
increases in both EME and BE. The addition of BChE reduced the
peak BE concentration by nearly 3-fold, and nearly doubled EME
concentrations (Fig. 1b). The addition of the BChE inhibitor reduced
Each of the in vitro experiments demonstrates that the addition of
BChE to plasma enhances cocaine metabolism. Humans and monkeys
tend to have differing baseline BChE activity, with a range of 1.27 to
3.66 U/liter in squirrel monkeys and 2.11 to 7.13 U/liter in humans
(Carmona et al., 1996; Washington et al., 1996). Despite these differences in endogenous activity, relatively comparable decreases in
cocaine half-life were observed for each species in vitro. The addition
of 100 U of BChE to each plasma type led to an 86.9% reduction in
the human and an 86.5% reduction in the monkey half-life of cocaine.
Additionally, the effect of added BChE was dose-related in human
plasma, with the greatest decrease in cocaine half-life occurring at the
highest dose. This degree of consistency and dose dependence suggests that the added BChE is solely responsible for accelerating the
metabolism of cocaine in plasma in vitro. Any reduction in the plasma
concentration of cocaine would be expected to decrease the amount of
cocaine in systemic circulation and thus, available to enter the brain,
thereby potentially reducing the behavioral and toxic effects of cocaine.
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the formation of EME, as EME was not detected throughout the
sampling period (assay limit of detection ⫽ 1 ng/ml). BE concentrations for the inhibitor condition approximated those of the saline
condition.
Squirrel Monkey In Vivo. Pretreatment with BChE (30 min before
cocaine administration) produced an immediate and sustained reduction in plasma cocaine concentration (when compared with controls).
Peak cocaine concentrations occurred at 1 min and were reduced
nearly 3-fold in the four monkeys receiving BChE pretreatment compared with controls (Fig. 2a). The reduction in plasma cocaine concentration was paralleled by an increased formation of EME, with
higher peak concentrations observed in monkeys pretreated with
BChE (146.2 ⫾ 32.8 ng/ml; mean ⫾ S.E.) compared with controls
(66.3 ⫾ 6.7 ng/ml; mean ⫾ S.E.; Fig. 2b). Concentrations of BE were
not significantly different for the two conditions (Fig. 2c). Monkeys
receiving BChE did not show changes in either physiological (i.e.,
eating, water consumption, gastrointestinal disturbances) or behavioral functioning (lowered behavioral activity or hyperactivity) after
recovery from anesthesia.
Human In Vitro. Cocaine concentration remained fairly stable in
the saline control condition, as only small amounts of BE were
detected. Cocaine metabolism was observed in the plasma-alone condition, with concentrations decreasing at a half-life of 100.3 min. The
addition of 25, 50, and 100 U of BChE accelerated cocaine metabolism in an activity-related manner, and dramatically reduced the
half-life of cocaine to 37.6 min (63% reduction), 21.5 min (79%
reduction), and 13.1 min (87% reduction), respectively. The elimination rate constant for the 0, 25, 50, and 100 U BChE conditions were
0.41, 1.11, 1.93, and 3.18 h⫺1, respectively.
The addition of BChE also changed the concentrations of cocaine
metabolites in an activity-related manner. In the saline sample, no
EME was detected at any time point, but there was a progressive
increase in BE concentrations, with the mean peak concentration (83.4
ng/ml) observed at the final sampling time point. In plasma alone,
EME and BE concentrations increased over time. Peak EME concentrations (485.5 ng/ml) and peak BE concentrations (80.9 ng/ml) were
observed at the end of the 90-min sampling period. The addition of 25,
50, and 100 U of BChE dose dependently lowered peak BE concentrations (from 80.9 to 48.7, 28.6 ng/ml, and to levels less than the limit
of quantitation), and increased EME concentrations (from 485.5 to
744, 945.9, and 921.4 ng/ml), respectively.
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CARMONA ET AL.
Doses are given in the text. Concentrations ⫾ S.E.M. (in nanograms per milliliter) of cocaine (a), EME (b), and BE (c) are shown for the 120-min time period
after cocaine administration. BChE reduced the peak cocaine concentration immediately after administration of cocaine. EME concentrations were increased,
whereas BE concentrations were unchanged.
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