b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 7 5 2 – 7 6 0
759
untreated male rat liver microsomes yielded monophasic
kinetics, CYP2C11 and CYP3A2, both of which have similar Km
values of around 400 mM, seem to be involved in this reaction.
These enzymes are known to be male-specific or much more
abundant in male rats than in females [20–22], causing the sex
difference (male > female) in the Vmax values for this reaction.
However, it remains obscure at present what kinds of CYP
enzymes are involved in the N-dealkylation of 5-MeO-DIPT in
female rat liver microsomes.
and humans. The present study indicates that O-demethyla-
tion is the major oxidation pathway of 5-MeO-DIPT while N-
deisopropylation is the minor pathway in both human and rat
liver microsomal fractions, which is in agreement with the
previous in vivo results [10,12].
In toxicological studies of methylenedioxymethampheta-
mine (MDMA), another drug of abuse, the research group of Dr.
Cho at UCLA suggested that demethylenation of MDMA via
hydroxylation of the methylene bridge by CYP2D enzymes
produces a chemically reactive o-quinone, which may cause
neurotoxicity in MDMA abusers [24]. Similarly, 6-OH-5-MeO-
DIPT can be converted to o-quinone via further O-demethyla-
tion of the aromatic ring. The possibility of the formation of
the quinone metabolite and its potential neurotoxicity should
be evaluated next.
5-OH-DIPT was identified as a major metabolite in urine
samples from 5-MeO-DIPT users [10]. Moreover, 6-OH-5-MeO-
DIPT was also found as another 5-MeO-DIPT metabolite in the
same samples [10]. In our previous in vitro study using pooled
human liver microsomes [11], we did not confirm the presence
of 6-OH-5-MeO-DIPT because of the lack of an authentic
standard. This time, the research group of Dr. Tsuchihashi of
Osaka Prefectural Police Headquarters supplied with an
authentic sample, so we identified the metabolite. Compar-
ison of the HPLC chromatograms between rats and humans,
suggested the unidentified metabolite, M-2, in our previous
study [11] to be 6-OH-5-MeO-DIPT. It is thus thought that a
trace amount of 6-OH-5-MeO-DIPT can be formed from 5-MeO-
DIPT in human liver microsomes.
In summary, 5-MeO-DIPT was biotransformed mainly into
5-MeO-IPT and partially into 5-OH-DIPT in liver microsomal
fractions from untreated rats of both sexes. Kinetic and
inhibition experiments indicated that the side-chain N-deal-
kylation is mediated by CYP2C11 and CYP3A2, whereas the
aromatic ring O-demethylation is mediated by CYP2D2 and
CYP2C6 in untreated male rats. Pretreatment of male rats with
BNF produced 6-OH-5-MeO-DIPT, the formation of which is
thought to be mediated by CYP1A1 induced to express by BNF.
These results provide valuable information on the metabolic
fate of 5-MeO-DIPT in rats that can be used in the toxicological
study of this designer drug.
In the present study, the pretreatment with BNF caused the
formation of 6-OH-5-MeO-DIPT in male rat liver microsomes,
while this metabolite was undetectable in liver microsomal
fractions from untreated rats of either sex. Because the
fluorescence intensity of the metabolite peak corresponding to
6-OH-5-MeO-DIPT on the chromatogram was quickly attenu-
ated, we added some ascorbic acid to the reaction mixture just
after incubation according to a previous in vivo study [10],
which was effective of protecting the peak, but not complete.
We thus employed the calibration curve for 5-MeO-IPT to
estimate the activity to form 6-OH-5-MeO-DIPT on the
assumption that the fluorescence intensity was similar
between 6-OH-5-MeO-DIPT and 5-MeO-IPT.
Acknowledgments
We would like to express our gratitude to Dr. M. Funada,
National Institute of Mental Health, National Center of
Neurology and Psychiatry, Kodaira, Japan. This study was
supported in part by Grants-in-Aid for Scientific Research
(17390035 and 18590116) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
The Km value for the formation of 6-OH-5-MeO-DIPT in liver
microsomes from BNF-pretreated male rats was about 15 mM,
which is close to that of recombinant rat CYP1A1, the only
enzyme mediating the 6-hydroxylation of 5-MeO-DIPT under
the conditions employed. In addition, the intrinsic clearance
value (96 ml/(min mg protein)) for 6-hydroxylation was much
higher than that for N-deisopropylation (4–11 ml/(min mg
protein)) in rat liver microsomal fractions, indicating that
the 6-hydroxylation, which is catalyzed by CYP1A1, is one of
the major pathways by which 5-MeO-DIPT is oxidized in BNF-
pretreated rats, particularly in a low substrate concentration
range.
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