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ACTA FACULTATIS PHARMACEUTICAE UNIVERSITATIS COMENIANAE
Tomus LIX 2012
THE EFFECT OF METHAMPHETAMINE
ON BIOTRANSFORMATION OF ETHANOL: PILOT STUDY
1,2
Zendulka, O. – 1,2
Sabová, M. – 1,2
Juřica, J. – 3
Machalíček, M. – 3
Švéda, P. –
3
Farková, M. – 1
Šulcová, A.
1
Masaryk University in Brno, Central European Institute of Technology, Experimental
and Applied Neuropsychopharmacology research group,
2
Masaryk University in Brno, Faculty of Medicine, Department of Pharmacology,
3
Masaryk University in Brno, Faculty of Science, Department of Chemistry
Methamphetamine is one of the most popular recreational drugs in Central Europe and
is often combined with ethanol. Various interactions between these two substances have
been described including the influence of administered ethanol on biotransformation
of methamphetamine. The aim of the present study was to describe the opposite effect –
the influence of methamphetamine on biotransformation of ethanol in rats.
Methamphetamine was administered for 10 days (10 mg/kg/day) i.p. and ethanol was
delivered as an intragastric bolus (2 g/kg) on the10th
day of experiment to both
methamphetamine administered rats and control animals. The pharmacokinetic
experiment on the whole animal was performed and plasma samples were drawn
at the 40th
, 120th
, 210th
and 300th
minute after ethanol administration.
Ethanol plasmatic levels reached significantly lower values in the 40th
and 120th
interval
when compared to controls. Differences were insignificant in the last two intervals.
Our results suggest that chronic methamphetamine administration induces ethanol
biotransformation. We suppose that this effect is caused by induction of alcohol
dehydrogenase metabolic activity or by allosteric interaction of methamphetamine and
this enzyme. More studies have to be conducted to confirm or disprove our hypothesis.
Keywords: methamphetamine – ethanol – biotransformation – interaction – rat
INTRODUCTION
Substance abuse is as old as mankind, and brings together health and social problems
affecting both the abuser and the society as well. Drugs of abuse are miscellaneous
substances eliciting different biologic effects, and their misuse is usually region-specific
due to differences in their availability, legislation, and social conditions in the specific
area. One of the most popular recreational drugs besides cannabis in the Central Europe
DOI 10.2478/v10219-012-0026-4
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64
is methamphetamine (METH). METH abuse in the Czech Republic has a long tradition,
as amphetamines serving as a substrate for METH production were available in this
region till late 1980s whereas in other European countries were restricted in 1970s.
In fact, the Czech Republic is the leader in prevalence of METH users, and the number
of dependent individuals stays consistent for many years (EMCDDA 2010).
The combinations of recreational drugs are usual in drug abusers, and ethanol (ET)
as a legal substance is often consumed by addicted people. Combining ET with other
substances can lead to severe pharmacokinetic or pharmacodynamic interactions.
Actually, the most frequent combinations of drugs that lead to visit of the Emergency
Department in U.S. in 2008 were those with ET (SAMHSA 2008).
The interactions between METH and ET have been widely studied. The experiments
focused on the behavioural aspects of ET and METH combination documented, that ET
accentuates the METH neurotoxicity, and both of them are evoking anxiolytic effects
in rats (Chuang et al., 2011). The combination is also more potent in causing memory
impairment in rats than administration of single drugs (Yamamura et al., 1992).
The molecular mechanism of these behavioural effects is probably carried by different
changes on dopamine and serotonin release, which are dependent not only
on the substance, but may also vary between rat strains (Nishiguchi et al., 2002,
Yamauchi et al., 2000). The chronic administration of METH alone could increase
the ET intake in mice because of induction of long term dopaminergic toxicity, and
hypodopaminergic state is described to increase the voluntary ET consumption
(Gutierrez-Lopez et al., 2010).
Preclinical experiments aimed on pharmacokinetic interactions of ET and METH
describe the inhibition of METH metabolism when combined with ET (Liang et al.,
2012). Increase in the METH plasmatic levels is due to inhibition of METH Ndemethylation
and parahydroxylation (Yamada et al., 2001) caused by ET. The similar
result was confirmed in humans, too (Shimosato, 1988), whereas other clinical trial
with ET and METH combination did not discover any change in METH levels among
subjects (Mendelson et al., 1995). The design of the study can substantially influence
results, since it was described that coincidental administration of ET and METH results
in inhibition of METH metabolism, whereas the same substance is biotransformed faster
in METH addicts with chronic ET ingestion (Shimosato et al., 1988).
Papers describing pharmacokinetic interaction between METH and ET are exclusively
aimed on the influence of ET on the biotransformation of METH, while our recent study
is addressed oppositely. We have determined the effect of 10 day long METH
administration on the pharmacokinetics of ET in rats. We hypothesised that ET
plasmatic levels could be influenced due to inhibitory effect of METH on cytochrome
P450 2E1 enzyme, which was documented (Minarikova et al., 2006). Therefore we
decided to use a relatively high but equal dose of METH as was administered
in Minarikova’s study.
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65
MATERIAL AND METHODS
Animals
The study was carried out on male Wistar albino rats weighing 200±20 g. Animals were
housed under temperature (21-22°C) and humidity (50-60 %) controlled conditions.
Rats were housed singly to avoid injuries in METH administered individuals in standard
plastic cages with free access to water and pelleted diet.
Animals were randomly divided into two groups (n=10), one of them was administered
with intraperitoneal injections of METH (Sigma Chemical Co., St. Louis, USA) at one
daily dose of 10.0 mg/kg for 10 consecutive days. Second group – controls, received
equal volume of saline.
All procedures were approved by the Czech Central Commission for Animal Welfare.
Pharmacokinetic study
The pharmacokinetic study in the whole animal was performed on the 10th
day
of METH treatment. The animals were injected either with METH or saline, and
consecutively with ET intragastrically. ET (Lach-Ner s.r.o., Neratovice, CZ) was
administered at the dose of 2.0 g/kg as a 20% solution dissolved in 5% glucose, to limit
the gastric irritation and supply rats with additional nutrition for the kinetic experiment.
Animals were anesthetized with diethylether, and blood from plexus retrobulbaris was
sampled with heparinized capillary tube in the following time intervals from ET
administration: 40 min, 120 min, 210 min, and 300 min. Samples were allowed to clot
at room temperature and were centrifuged 10 minutes at 4°C at 3000 g to obtain serum,
which was frozen until analysis at - 75°C.
Sample analysis
Gas chromatography (GC) was used to evaluate the levels of ET in samples. Analysis
was performed on YL 6100GC (YL Instruments Co., Kyounggi-do, KOR) with Agilent
5062-3587 (Agilent Technologies Int., Santa Clara, USA) inlet, flame ionization
detector and SLB-5ms 30 m x 0.25 mm x 0.25 µm (Sigma Chemical Co., St. Louis,
USA) column. Analysis conditions were as follows: carrier gas nitrogen, inlet
temperature 160°C; flow 2 ml/min; split 1:30; detector temperature 200°C, gas flow –
air 300 ml, hydrogen 30 ml, make up 20 ml; total separation time 6.5 min. Limit
of detection and quantification was determined to be 0.006‰ and 0.009‰
of ET respectively. The samples were analysed in duplicates and the mean value was
used for the statistical analysis. The calibration line was transformed into log line
for the values close to the quantification limit.
Statistical evaluation
Obtained results were statistically analysed with Dixon’s Q test to exclude outliers.
Repeated measure ANOVA with Fisher post-hoc test for multiple comparisons was
used for the data analysis using software Statistica 8 for Windows. Data are expressed
as means ± SD. Values of p<0.05 were considered to be statistically significant.
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66
RESULTS
The levels of ET measured declined in both METH and saline treated groups
during the experiment (Figure 1), but there were statistically significant differences
between the values in the first two sampling intervals. The plasmatic levels of ET in rats
which received METH were lower than in controls. In the 40th
minute ET values
in METH animals were 0.594‰, SD ± 0.303‰, while in the control group animals
1.618‰, SD ± 0.696‰; p<0.001. In the 120th
min in METH animals were measured
values 0.189‰, SD ± 0.169‰, in comparison to controls 0.853‰, SD ± 0.665‰;
p=0.002. There were no statistically significant differences in the last two intervals
assessed. ET concentration in the 210th
were METH 0.010‰, SD ± 0.005‰, control
0.331‰, SD ± 0.447‰, p= 0.31. In METH animals 0.007‰, SD ± 0.001 ‰ and
in control rats 0.014‰, SD ± 0.014‰ was detected.
Figure 1. Ethanol concentrations [‰] in controls and rats administered
with methamphetamine (10 mg/kg/day) for 10 days after single dose
of ethanol (2 g/kg). Data represents means ± SD
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67
DISCUSSION
Our results did not confirm our original hypothesis of ET metabolism inhibition
due to influence of METH on 2E1 enzyme. Oppositely, obtained data suggests that
chronic administration of METH increased the rate of ET biotransformation. This was
apparent from the lower concentrations of ET in METH administered animals than
in controls. However, the METH influence was statistically significant only in the first
two sampling intervals. Thus, we suppose that insignificancy of difference between
groups in samples drawn on the 210th
and 300th
minutes rise from very low ET level
reaching almost the detection limit of method used. As far as we know, induction of ET
biotransformation by METH was not described previously unlike of opposite effect of
ET on METH metabolism (Liang et al., 2012, Mendelson et al., 1995, Shimosato, 1988,
Shimosato et al., 1988, Yamada et al., 2001).
We suggest that there may be considered three possible mechanisms explaining the
pharmacokinetic interaction described: 1. METH decreases the bioavailability of ET; 2.
METH enhances the excretion of unchanged ET; 3. METH induces enzymes of ET
biotransformation pathway.
Most of the interactions on the level of absorption are based in the change of specific
carrier system activity and thus occur only in drugs using this mechanism of absorption.
ET with small molecule and its physico-chemical properties is an easily diffusible
compound (Holt, 1981) crossing well all biological barriers in the body. Therefore the
first above-mentioned suggestion can be excluded and because of the same reasons
the second one as well. The portion of unchanged ET excreted by lungs and kidneys
varies from 2 to 10% (Lieber, 1997) and no specific carriers are involved.
The third possible explanation: it has to be considered that approximately 89%
of METH is metabolized in rats with major metabolites 4-hydroxymethamphetamine
and 4-hydroxynorephedrine (Caldwell et al., 1972). The enzymes involved in the
metabolism of METH are cytochromes P450 (CYP), especially CYP2D enzymes
participating in 4-hydroxylation of METH aromatic ring (Lin et al., 1995).
While the major metabolic pathway of ET in rats is via alcohol dehydrogenase (ADH)
with essential role of ADH-3 isoenzyme (Boleda et al., 1989), the ADH is not the only
enzyme metabolizing ET. The minor metabolic pathway of ET microsomal oxidizing
system represents CYP2E1 (Ronis et al., 1993). Further, it is known, that CYP2E1 is
inducible and the observed effect of METH could be explained by massive increase
of CYP2E1 metabolic rate. However, the induction of this enzyme is described
for ethanol (McGehee et al., 1994) but not for METH. The influence of METH on CYP
enzymes is disputable. There are works describing induction of CYP2D, CYP2C6 and
CYP3A by METH (Dostalek et al., 2005, 2007). Nevertheless we did not document this
effect in our previous studies (Zendulka et al., 2010). Same result is reported
by Minarikova; moreover she documented a weak inhibitory effect of METH
on CYP2E1 (Minarikova et al., 2006). This means that the induction of ET metabolism
is definitely not carried by the effect of METH on CYP and thus only the possibility
of ADH induction remains. The increase of ADH activity can be either in posttranslational
phase by allosteric activation by METH or by genetic transcription
stimulation. The question whether it is caused directly by METH or indirectly via
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68
dopaminergic activity of METH in the CNS and its peripheral consequences is still not
answered. More studies have to be conducted to confirm or disprove our hypothesis.
CONCLUSIONS
Taken together, our results suggest that repeated pre-treatment with METH led
to the decrease of ET levels in time points between minutes 0 and 120 after acute
alcohol administration. According to the best of our knowledge such effect was not
described before. Thus our next study will be focused on dose dependence in the actual
documented effect of METH on ET metabolism.
ACKNOWLEDGMENT
This work was supported by the project “CEITEC - Central European Institute
of Technology” (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.
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Holt S. Observations on the Relation between Alcohol Absorption and the Rate
of Gastric-Emptying. CMAJ. 1981;124:267-277.
Chuang JY, Chang WT, Cherng CFG, Kao GS, Yu L. Repeated co-administrations
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Lin LY, Kumagai Y, Hiratsuka A, et al. Cytochrome P4502D Isozymes Catalyze the 4Hydroxylation
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Mendelson J, Jones RT, Upton R, Jacob P 3rd. Methamphetamine and ethanol
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Minarikova V, Hadasova E, Möritz K, Scheuch E, Siegmund E. Comparison
of the influence of methamphetamine and phenobarbital on the activity of hepatic
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Registered: September 15, 2012 PharmDr. Ondřej Zendulka, PhD.
Accepted: November 7, 2012 Masaryk University in Brno
Faculty of Medicine
Department of Pharmacology
Czech Republic
zendulka@med.muni.cz
VLIV METAMFETAMINU NA BIOTRANSFORMACI ETHANOLU – PILOTNÍ STUDIE
1,2
Zendulka, O. – 1,2
Sabová, M. – 1,2
Juřica, J. – 3
Machalíček, M. – 3
Švéda, P. – 3
Farková, M. –
1
Šulcová, A.
1
Masarykova Univerzita Brno, Středoevropský technologický institute, Výzkumní skupina
experimentální a aplikované neuropsychofarmakologie,
2
Masarykova Univerzita Brno, Lékařská fakulta, Farmakologický ústav,
3
Masarykova univerzita Brno, Přírodovědecká fakulta, Ústav chemie
Metamfetamin je jednou z nejčastěji zneužívaných drog v regionu Střední Evropy a velmi často je
jeho užívání kombinováno s požíváním alkoholu. V minulosti byly popsány rozličné interakce
mezi těmito dvěma látkami včetně vlivu ethanolu na biotransformaci metamfetaminu. Cíl této
práce je přesně opačný, tedy popsat vliv metamfetaminu na biotransformaci etanolu u potkana.
Metamfetamin (10 mg/kg/den) byl zvířatům aplikován ve formě i.p. injekcí 10 dní. Ethanol (2
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71
g/kg intragastricky) byl podán kontrolním i metamfetaminem premedikovaným zvířatům společně
s poslední dávkou metamfetaminu. Následně byl proveden farmakokinetický experiment na celém
zvířeti s odběrem vzorků krve v následujících časových intervalech od aplikace ethanolu: 40, 120,
210 a 300 minut.
Hladiny ethanolu v plazmě stanovené metodou plynové chromatografie byly signifikantně nižší
u metamfetaminem premedikovaných zvířat než u kontrol ve 40. a 120. minutě experimentu,
zatímco v následujících dvou časových intervalech byl rozdíl nesignifikantní. Naše výsledky
naznačují, že chronicky aplikovaný metamfetamin zrychluje biotransformaci ethanolu.
Domníváme se, že tento efekt je zprostředkován prostřednictvím alkoholdehydrogenázy
buď zvýšení genové exprese nebo alosterickou modulací tohoto enzymu vlivem metamfetaminu.
K ověření nebo vyvrácení naší hypotézy je nutné provést další studie.
Acta Fac. Pharm. Univ. Comen. LIX, 2012, (2), p. 63-71.
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