Carboxylesterase-mediated transesterification of meperidine (Demerol) and methylphenidate (Ritalin) in the presence of [2H6]ethanol: Preliminary in vitro findings using a rat liver preparation

Full text of DOI:10.1021/js970072x

Carboxylesterase-Mediated Transesterification of Meperidine (Demerol) and
2
Methylphenidate (Ritalin) in the Presence of [ H ]Ethanol: Preliminary in Vitro
6
Findings Using a Rat Liver Preparation
To the Editor:
Carboxylesterase enzymes hydrolyze many ester-containing
1
xenobiotics to yield carboxylic acids. Certain forms of these
enzymes catalyze ethanolic transesterification reactions. A
widely publicized and significant transesterification reaction
is the conversion of cocaine to cocaethylene (ethylcocaine) in
the presence of ethyl alcohol.2
-4
Cocaethylene possesses
pharmacological activity nearly identical with cocaine and a
longer half-life and greater toxicity than cocaine.5 This new
metabolite provides additional concern about ethanol and
cocaine coabuse. We have recently shown the ethyl ester
exchange between deuterated ethanol and unlabeled cocaeth-
ylene.15
-7
Meperidine (Demerol) and methylphenidate (Ritalin) are
two commonly prescribed compounds, with potential for abuse
and coabuse with ethanol, and both are extensively hydrolyzed
to their corresponding carboxylic acids, meperidinic and
ritalinic acids, respectively.8
-10
To investigate whether or not
meperidine and methylphenidate, like cocaine, undergo car-
boxylesterase-mediated transesterification in the presence of
ethanol, an in vitro experimental design was employed.
Excised livers from male Sprague-Dawley rats (250-275
g) were homogenized and the supernatant 9000 (S9) fraction
collected. Total protein concentration was determined (BCA
Protein Assay, Pierce, Rockford, IL), as was esterase activity
by the method of Dean et al.2
Meperidine and methylphenidate (50 µM) were separately
Figure 1
produced from an extraction of rat S9 (1 mL) containing methylphenidate (50
M) and ethanol (50 mM) incubated at 37 C for 30 min. The bottom spectrum
is an injection (2 L) of ethylphenidate standard (100 ng/mL) in methanol. The
sUnderivatized ethylphenidate mass spectra (MS). The top MS was
incubated, in triplicate, with S9 at 37 °C for 4 h with and
2
µ
°
without [ H
6
]ethanol (51.3 mM). The experiments were
µ
repeated in buffer and S9 in the presence or absence of specific
and nonspecific esterase inhibitors. Samples (100 µL) were
collected and extracted using a modified solid-phase extraction
procedure designed for cocaine and metabolite extraction.11
Tropacocaine (25 µM) was used as an internal standard for
both assays. The above experiment was repeated using
meperidine (50 µM) and unlabeled ethanol (50 mM).
Parent drugs and predicted ethyl ester formation products
were assayed via gas chromatography/mass spectrometry (GC/
MS) in the selected ion monitoring (SIM) mode. The SIM
qualifier ions were chosen after examining full scan mass
spectra of each analyte. The major ion peaks selected were
retention times for both mass spectra were identical.
activity (∼10%) occurred after 4 h (272 ( 5 nmol/min/mg; p
<
0.05, Bonferroni’s t-test).
Ethylphenidate formation was confirmed by full scan mass
spectrometry following the 30 min incubation of methylpheni-
date and ethanol in rat S9 (Figure 1). This mass spectrum
was matched visually and by retention time to a standard of
ethylphenidate, conclusively showing the transesterifcation
2
of methylphenidate to ethylphenidate in vitro. [ H
5
]Ethyl-
phenidate was formed in vitro when methylphenidate was
2
incubated with [ H
6
]ethanol in the rat S9. The concentration-
tropacocaine (m/z 82, 124, 245), meperidine (m/z 71, 172, 247),
time profiles of unlabeled methylphenidate in the presence
2
[
H
5
]meperidine (m/z 71, 172, 252), methylphenidate (m/z 84,
2
2
of [ H
6 5
]ethanol and the formed [ H ]ethylphenidate are il-
2
9
5
1,150), and [ H ]ethylphenidate (m/z 84, 91, 169) with
lustrated in Figure 2. No change in the methylphenidate
retention times of 3.5, 2.0, 2.0, 1.9, and 2.1 min, respectively.
Quantitation was accomplished by calculating ion abundance
ratios of analyte to internal standard compared to a standard
curve of concentrations of meperidine or methylphenidate
2
6
disappearance rate was seen following addition of [ H ]ethanol.
The methylphenidate profile under control conditions (i.e., no
ethanol, Figure 2, open circles) was virtually identical with
2
the profile in the presence of [ H
6
]ethanol (Figure 2; solid
(
3.125, 6.25, 12.5, 25, 50 µM). The quantitation ions selected
circles). There were no statistical differences in t1/2 (179.9 (
2
were meperidine/tropacocaine (m/z 247/245), [ H
5
]meperidine/
1
8.6 min vs 178.1 ( 12.1 min).
tropacocaine (m/z 252/245), methylphenidate/tropacocaine
The concentration-time profiles of unlabeled meperidine
2
(
m/z 84/82), and [ H
Pharmacokinetic parameters were calculated using Win-
Nonlin.12 Statistical analysis of the half-life (t1/2) was ac-
5
]ethylphenidate/tropacocaine (m/z 84/82).
2
in the absence and presence of [ H
6
]ethanol and the formed
2
[ H ]meperidine are illustrated in Figure 3. These profiles
5
illustrate ethyl ester exchange between unlabeled meperidine
1
3
2
2
complished using Bonferroni’s t-test. The harmonic mean
and “pseudo” standard deviation of t1/2 values were calcu-
lated.14
6 6
and [ H ]ethanol and that the presence of [ H ]ethanol does
not influence the loss of meperidine. The exchange process
2
5
begins rapidly ([ H ]meperidine seen within 5 min; inset).
The protein concentration of rat S9 was ∼40 mg/mL.
Esterase activity from freshly thawed rat S9 was 305 ( 4
nmol/min per mg of protein. Esterase activity did not change
significantly after 1 and 2 h of incubation (303 ( 14 and 293
Figure 3 also illustrates (dashed line) total meperidine
concentrations (i.e., the sum of labeled and unlabeled mep-
eridine concentrations). That profile is virtually identical with
the meperidine profile in the presence of unlabeled ethanol
(Figure 4; solid squares in both cases). Similarly, the unla-
(
13 nmol/min/mg, respectively), but a significant loss of
1
494 / Journal of Pharmaceutical Sciences
S0022-3549(97)00072-5 CCC: $14.00
© 1997, American Chemical Society and
American Pharmaceutical Association
Vol. 86, No. 12, December 1997

2
Figure 2
s
Methylphenidate and [ H
5
]ethylphenidate concentrations as a function
of time. Methylphenidate (50
S9) fraction for 4 h at 37 °C. Unlabeled methylphenidate concentrations as a
µM) was incubated in rat liver 9000g supernatant
(
2
5
function of time in the absence (
O) [control condition] and in the presence (
b) of
Figure 3
Meperidine (50
for 4 h at 37
s
Meperidine and [ H ]meperidine concentrations as a function of time.
2
2
[
H
6
]ethanol (51.3 mM) and the resulting [ H
5
]ethylphenidate concentrations (
2).
µM) was incubated in rat liver 9000g supernatant (S9) fraction
C. Unlabeled meperidine concentrations in the absence (
Each value is the mean of three experiments and the crosshatched vertical bars
represent the standard deviation of the mean.
°
O
) [control
2
condition] and in the presence (
b
) of [ H ]ethanol (51.3 mM). Total meperidine
6
2
2
concentrations (
9
) (meperidine
+
[ H
5
]meperidine) in the presence of [ H
6
]ethanol
2
are shown with the dashed line. Inset graph: [ H
5
]Meperidine concentrations (
2
)
beled form of meperidine under control conditions (i.e., no
2
2
formed in the presence of [ H
6
]ethanol (51.3 mM). Each value is the mean of
ethanol) or in the presence of [ H
6
]ethanol (Figure 3; open and
three experiments and the crosshatched vertical bars represent the standard
deviation of the mean.
solid circles, respectively) provides nearly identical concentra-
tion-time profiles. There were no significant differences
between t1/2 values (6.1 ( 3.8 min vs 12.5 ( 2.7 min).
Figure 4 illustrates the in vitro disposition of 50 µM
meperidine with and without 50 mM unlabeled ethanol. The
t
3
1/2 increased by ∼9-fold in the presence of ethanol (16.1 (
.8 vs 144.9 ( 41.3 min.). The significant increase in t1/2 is
explained by the ethyl ester formation of additional meperi-
dine. This is corroborated by the extent of formation of
2
2
[
H
5
]meperidine from [ H
change in meperidine kinetics in the presence of ethanol
Figure 4) is identical with the change noted when comparing
6
]ethanol (inset, Figure 3). The
(
unlabeled meperidine to total meperidine in the presence of
labeled ethanol (Figure 3). The t1/2 of unlabeled meperidine
(
12.5 ( 2.7 min) increased to 145.3 ( 19.9 min for total
meperidine (labeled + unlabeled) in the presence of
2
[
H
6
]ethanol, an ∼11-fold increase.
Esterase inhibitors added to the in vitro incubation experi-
ment resulted in an “all or nothing” effect, reported here
2
2
5 5
qualitatively. [ H ]Ethylphenidate and [ H ]meperidine for-
mation were completely inhibited by the addition of bis(4-
nitrophenyl) phosphate (BNPP) (100 µM), a specific carbox-
ylesterase inhibitor. A 10% solution of saturated NaF
completely blocked ethyl ester formation products, while
physostigmine (100 µM), a cholinesterase inhibitor, had no
effect on the ethanolic transesterification for both methyl-
phenidate and meperidine. No deuterated formation products
Figure 4
each) was incubated in 9000g supernatant rat liver homogenate preparations for
4 h at 37 C in the absence ( ) and presence of ethanol (50 mM) ( ). Each
value is the mean of three experiments and the crosshatched vertical bars represent
the standard deviation of the mean.
sMeperidine concentrations as a function of time. Meperidine (50 µM
°
b
9
phenomenon. In the methylphenidate plus ethanol experi-
ment, a new metabolite, ethylphenidate, is formed. Prelimi-
nary data in our laboratory show that this phenomenon also
occurs in vivo and that ethylphenidate is pharmacologically
active in rats. In a clinical setting, coingestion of meth-
ylphenidate along with ethanol could result in formation of
ethylphenidate. We are pursuing the existence of this phe-
nomenon in humans. Meperidine contains a carboxyl ethyl
ester moiety that, when combined with ethanol and carboxy-
were detected when samples were incubated in buffer only
2
(
no S9) with [ H
6
]ethanol.
Both meperidine and methylphenidate were shown to
2
6
undergo transesterification in the presence of [ H ]ethanol.
Furthermore, the reactions only took place in the presence of
viable carboxylesterase enzyme activity. The formation of
2
[
5
H ]ethylphenidate conclusively showed ethanolic transes-
terification of methylphenidate, a previously unreported
Journal of Pharmaceutical Sciences / 1495
Vol. 86, No. 12, December 1997

lesterase enzyme, will either form additional meperidine
before hydrolyzing to meperidinic acid or be hydrolyzed
immediately to meperidinic acid. The presence of ethanol
provides an alternate pathway and prolongs the ultimate
hydrolysis of meperidine, increasing its elimination time.
Deuterated ethanol was used to distinguish between trans-
esterified meperidine and meperidine present as starting
material. The prolonged t1/2 of meperidine in the presence of
ethanol is not an indication of altered hepatic clearance due
to ethanol interaction with metabolizing enzymes. If ethanol
is affecting the rate of carboxylesterase hydrolysis by directly
like actions in drug discrimination studies. Europ. J . Pharmacol.
991, 197, 235-236.
. Katz, J . L.; Terry, P.; Witkin, J . M. Comparative behavioral
pharmacology and toxicology of cocaine and its ethanol-derived
metabolite, cocaine ethyl-ester (cocaethylene). Life Sci. 1992, 50,
1351-1361.
8. Luttrel, W. E.; Castle, M. C. Species differences in the hydrolysis
of meperidine and its inhibition by organophosphate compounds.
Fundam. Appl. Toxicol. 1988, 11, 323-332.
. Srinivas, N. R.; Hubbard, J . W.; Midha, K. K. Enantioselective
gas chromatographic assay with electron-capture detection for
dl-ritalinic acid in plasma. J . Chromatogr. 1990, 530, 327-336.
1
7
9
10. Ridalieu, E.; Bartlett, M. F.; Waldes, L. M.; Darrow, W. R.;
Egger, H.; Wagner, W. E. A study of methylphenidate in man
with respect to its major metabolite. Drug Metab. Dispos. 1982,
altering the enzyme, then one would expect unlabeled mep-
2
eridine profiles to differ when [ H
6
]ethanol is added. This was
1
0, 708-709.
not the case; the profiles of meperidine control and unlabeled
1
1. Biochemical Diagnostics, Inc. Detectabuse extraction columns
2
meperidine in the presence of [ H
6
]ethanol were indistinguish-
type GV- 50. 180 Heartland Blvd., Edgewood, NY 11717.
able (t1/2, 16.1 and 12.5 min, respectively). The increased t1/2
of meperidine in the presence of ethanol is more likely
explained by a metabolic system with an excess of ethanol
providing a substrate pool of an exchangeable ethyl group.
Perhaps of more general importance, however, is the likely
possibility that carboxylesterase-mediated transesterification
might be a phenomenon applicable to a wide variety of ester-
containing compounds. One would expect prolonged elimina-
tion and reduced clearance with ethyl ester compounds and
newly formed metabolites, potentially active or toxic, with
methyl ester compounds. We are currently pursuing the
possible generality of this transesterification reaction.
12. WinNonlin, Scientific Consulting Inc., Apex, NC 1995.
13. SigmaStat, Statistical Software for Windows, J andel Scientific.
1
4. Lam, F. C.; Hung, C. T.; Perrier, D. G. Estimation of variance
for harmonic mean half-lives. J . Pharm. Sci. 1985, 74, 229-
2
31.
1
5. Bourland, J . A.; Martin, D. K.; Mayersohn, M. In Vitro Trans-
esterification of Cocaethylene (Ethylcocaine) in the Presence of
Ethanol: Esterase-Mediated Ethyl Ester Exchange. Drug Metab.
Dispos. In press.
Acknowledgments
The authors gratefully acknowledge support for this work from a
grant from the National Institute on Drug Abuse (DA08094) and the
synthesis of ethylphenidate by Gene Mash, Ph.D., Department of
Chemistry, The University or Arizona (Synthetic Chemistry Core
Laboratory, Southwest Environmental Health Sciences Center, sup-
ported by NIEHS P-30-ES-06694).
References and Notes
1
2
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zoylecgonine and the formation of cocaethylene from alcohol and
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terification of cocaine in the presence of ethanol: evidence for
participation of microsomal carboxylesterases. J . Pharmacol.
Exp. Ther. 1992, 260, 939-946.
. Hearn, W. L.; Flynn, D. D.; Hime, G. W.; Rose, S.; Cofino, J . C.;
Mantero-Atienza, E.; Wetli, C. V.; Mash, D. C. Cocaethylene:
A unique cocaine metabolite displays high affinity for the
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. Woodward, J . J .; Mansbach, R.; Caroll, F. I.; Balster, R. L.
Cocaethylene inhibits dopamine uptake and produces cocaine-
J
AMES A. BOURLAND,
D
EBRA K. MARTIN
,
AND
3
X
M
ICHAEL AYERSOHN
M
Department of Pharmacy Practice
and Science
The Center for Toxicology
College of Pharmacy
The University of Arizona
Tucson, AZ 85721
4
5
6
Received February 18, 1997.
Accepted for publication September 4, 1997.
JS970072X
1
496 / Journal of Pharmaceutical Sciences
Vol. 86, No. 12, December 1997