Oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes

Article abstract of DOI:10.1016/j.bcp.2006.01.015

In vitro quantitative studies of the oxidative metabolism of (5-methoxy-N,N-diisopropyltryptamine, 5-MeO-DIPT, Foxy) were performed using human liver microsomal fractions and recombinant CYP enzymes and synthetic 5-MeO-DIPT metabolites. 5-MeO-DIPT was mainly oxidized to O-demethylated (5-OH-DIPT) and N-deisopropylated (5-MeO-IPT) metabolites in pooled human liver microsomes. In kinetic studies, 5-MeO-DIPT O-demethylation showed monophasic kinetics, whereas its N-deisopropylation showed triphasic kinetics. Among six recombinant CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) expressed in yeast or insect cells, only CYP2D6 exhibited 5-MeO-DIPT O-demethylase activity, while CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 showed 5-MeO-DIPT N-deisopropylase activities. The apparent K_m value of CYP2D6 was close to that for 5-MeO-DIPT O-demethylation, and the K_m values of other CYP enzymes were similar to those of the low-K_m (CYP2C19), intermediate-K_m (CYP1A2, CYP2C8 and CYP3A4) and high-K_m phases (CYP2C9), respectively, for N-deisopropylation in human liver microsomes. In inhibition studies, quinidine (1 μM), an inhibitor of CYP2D6, almost completely inhibited human liver microsomal 5-MeO-DIPT O-demethylation at a substrate concentration of 10 μM. Furafylline, a CYP1A2 inhibitor, quercetin, a CYP2C8 inhibitor, sulfaphenazole, a CYP2C9 inhibitor and ketoconazole, a CYP3A4 inihibitor (5 μM each) suppressed about 60%, 45%, 15% and 40%, respectively, of 5-MeO-DIPT N-deisopropylation at 50 μM substrate. In contrast, omeprazole (10 μM), a CYP2C19 inhibitor, suppressed only 10% of N-deisopropylation by human liver microsomes, whereas at the same concentration the inhibitor suppressed the reaction by recombinant CYP2C19 almost completely. These results indicate that CYP2D6 is the major 5-MeO-DIPT O-demethylase, and CYP1A2, CYP2C8 and CYP3A4 are the major 5-MeO-DIPT N-deisopropylase enzymes in the human liver.

Full text of DOI:10.1016/j.bcp.2006.01.015

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Oxidative metabolism of 5-methoxy-N,
N-diisopropyltryptamine (Foxy) by human liver
microsomes and recombinant cytochrome P450 enzymes
a,
a
a
b
Shizuo Narimatsu *, Rei Yonemoto , Keita Saito , Kazuo Takaya ,
b
b
c
Takuya Kumamoto , Tsutomu Ishikawa , Masato Asanuma ,
d
e
e
f
Masahiko Funada , Kimio Kiryu , Shinsaku Naito , Yuzo Yoshida ,
g
a
Shigeo Yamamoto , Nobumitsu Hanioka
a
Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University,
-1-1 Tsushima-naka, Okayama 700-8530, Japan
1
b
Laboratory of Medicinal Organic Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi,
Inage, Chiba 263-8522, Japan
c
Department of Brain Science, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
2
d
-5-1 Shikata, Okayama 700-8558, Japan
Division of Drug Dependence, National Institute of Mental Health, National Center of Neurology and Psychiatry,
-1-1 Ogawa-Higashi, Kodaira 187-8502, Japan
4
e
Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory Inc., Naruto, Tokushima 772-8601, Japan
f
School of Pharmaceutical Sciences and Institute for Bioscience, Mukogawa Women’s University, Nishinomiya, Hyogo 663-8179, Japan
Laboratory of Biomolecular Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
g
Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In vitro quantitative studies of the oxidative metabolism of (5-methoxy-N,N-diisopropyl-
tryptamine, 5-MeO-DIPT, Foxy) were performed using human liver microsomal fractions
and recombinant CYP enzymes and synthetic 5-MeO-DIPT metabolites. 5-MeO-DIPT was
mainly oxidized to O-demethylated (5-OH-DIPT) and N-deisopropylated (5-MeO-IPT) meta-
bolites in pooled human liver microsomes. In kinetic studies, 5-MeO-DIPT O-demethylation
showed monophasic kinetics, whereas its N-deisopropylation showed triphasic kinetics.
Among six recombinant CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and
CYP3A4) expressed in yeast or insect cells, only CYP2D6 exhibited 5-MeO-DIPT O-demethy-
lase activity, while CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 showed 5-MeO-DIPT N-
Received 7 December 2005
Accepted 24 January 2006
Keywords:
Foxy
5
5
5
-MeO-DIPT
-OH-DIPT
-MeO-IPT
deisopropylase activities. The apparent K
DIPT O-demethylation, and the K values of other CYP enzymes were similar to those of the
low-K (CYP2C19), intermediate-K (CYP1A2, CYP2C8 and CYP3A4) and high-K phases
m
value of CYP2D6 was close to that for 5-MeO-
CYP2D6
CYP1A2
CYP2C8
CYP3A4
m
m
m
m
(CYP2C9), respectively, for N-deisopropylation in human liver microsomes. In inhibition
studies, quinidine (1 mM), an inhibitor of CYP2D6, almost completely inhibited human
liver microsomal 5-MeO-DIPT O-demethylation at a substrate concentration of 10 mM.
*
Corresponding author. Tel.: +81 86 251 7942; fax: +81 86 251 7942.
E-mail address: shizuo@pharm.okayama-u.ac.jp (S. Narimatsu).
006-2952/$ – see front matter # 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2006.01.015
0

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b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 3 7 7 – 1 3 8 5
Abbreviations:
Furafylline, a CYP1A2 inhibitor, quercetin, a CYP2C8 inhibitor, sulfaphenazole, a CYP2C9
inhibitor and ketoconazole, a CYP3A4 inihibitor (5 mM each) suppressed about 60%, 45%, 15%
and 40%, respectively, of 5-MeO-DIPT N-deisopropylation at 50 mM substrate. In contrast,
omeprazole (10 mM), a CYP2C19 inhibitor, suppressed only 10% of N-deisopropylation by
human liver microsomes, whereas at the same concentration the inhibitor suppressed the
reaction by recombinant CYP2C19 almost completely. These results indicate that CYP2D6 is
the major 5-MeO-DIPT O-demethylase, and CYP1A2, CYP2C8 and CYP3A4 are the major 5-
MeO-DIPT N-deisopropylase enzymes in the human liver.
CYP, cytochrome P450
OR, NADPH-cytochrome
P450 reductase
5
-MeO-DIPT, 5-methoxy-N,N-
diisopropyltryptamine
-MeO-IPT, 5-methoxy-N-
ispropyltryptamine
-OH-DIPT, 5-hydroxy-N,N-
5
5
#
2006 Elsevier Inc. All rights reserved.
diisopropyltryptamine
G-6-P, glucose 6-phosphate
HPLC, high-performance liquid
chromatography
LC/MS, liquid chromatography–
mass spectrometry
PCR, polymerase chain reaction
DIPT) and 5-methoxy-N-isopropyltryptamine (5-MeO-IPT) were
synthesized as described below. Furafylline, quercetin, sulfa-
phenazole, omeprazole and quinidine were purchased from
Sigma–Aldrich (St. Louis, MO); glucose 6-phosphate (G-6-P) and
NADPH were from Oriental Yeast Co. (Tokyo, Japan). Ketoco-
nazole was supplied by Dr. Y. Yoshida, School of Pharmaceu-
tical Sciences, Mukogawa Women’s University. Pooled human
liver microsomal fractions were obtained from BD Biosciences
Discovery Labware (Bedford, MA). Recombinant CYP2D6 [6] was
expressed in yeast cells according to the published methods.
Recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4
were expressed in yeast cells as described below. Insect cell
microsomal fractions (Supersomes) expressing CYP3A4, cyto-
1
.
Introduction
Various kinds natural and synthetic hallucinogenic indolethy-
lamines have been produced and abused worldwide, including
in Japan [1,2]. Among the many hallucinogenic indolethyla-
mines, the following five compounds are legally controlled in
Japan: N,N-dimethyltryptamine, N,N-diethyltryptamine, a-
ethyltryptamine, psilocine and psilocybin [2]. In addition, a-
methyltryptamine and 5-methoxy-N,N-diisopropyltrypta-
mine (5-MeO-DIPT, Foxy) have newly come under legal control
as of 2005.
5
-MeO-DIPT is one of the designer drugs, and its properties
such as pharmacological activities and toxicities have not
been fully elucidated [3]. 5-MeO-DIPT is taken orally because it
is resistant to the degradation by monoamine oxidase [3]. It is
thought that 5-MeO-DIPT undergoes oxidative metabolism in
the liver after oral intake. In fact, there are two case reports in
which several oxidative metabolites were detected in human
serum and urine samples [4,5] using gas chromatography–
mass spectrometry (GC–MS) techniques. However, most of the
oxidative metabolites of 5-MeO-DIPT were only speculated to
be produced on the basis of fragment ions in the GC–MS
analysis.
5
chrome b and NADPH-cytochrome P450 reductase (OR), and
5
expressing CYP3A4 and OR (without cytochrome b ) were
purchased from Gentest (Woburn, MA).
2.2.
Chemical synthesis of 5-OH-DIPT and 5-MeO-IPT
To a solution of 5-MeO-DIPT (50 mg, 0.18 mmol) in CH
2 2
Cl
(2.0 ml), 1.0 M BBr in CH Cl (0.92 ml, 0.92 mmol) was added at
3
2
2
ꢀ12 8C and the whole was stirred at ꢀ12 8C for 3 h. After cooling
the reaction mixture with an ice bath, 30% aq. NaOH was added
to pH 9–10 and the whole was extracted with a mixture of CHCl
and MeOH(9:1, 1 ꢁ 10 ml, 3 ꢁ 5 ml). The combinedorganic layer
was washed with brine (10 ml) and was dried over Na SO . The
solventwasevaporatedinvacuoand theresiduewaspurifiedby
column chromatography (NH silica, CHCl :MeO-
H:Et N = 20:1:0.1) to give pale yellow crystals (21 mg, 44%);
m.p. 81–83 8C; IR (ATR, cm ) 3330; H NMR (400 MHz, CDCl
(ppm): 1.12 (total 12H, d, J = 6.3 Hz, 4 ꢁ CH ), 2.76, 2.84 (each 2H,
m, 2 ꢁ CH ), 3.17 (total 2H, m, 2 ꢁ CH), 6.77 (1H, dd, J = 8.8, 2.4 Hz,
H-6), 7.01 (1H, d, J = 2.2 Hz, H-4), 7.03 (1H, d, J = 2.2 Hz, H-2), 7.21
(1H, d, J = 8.8 Hz, H-7), 7.83 (1H, s, NH, exchangeable with D O);
) d (ppm): 20.3, 27.5, 47.0, 49.7, 50.6,
103.2, 111.8, 112.0, 122.3, 128.2, 131.3, 150.1; HRFABMS found:
61.1982 (calculated for C16 O:261.1967). These analytical
3
There have thus far been no quantitative reports describing
detailed in vitro studies on the oxidative metabolism of 5-
MeO-DIPT. In the present study, we therefore investigated the
in vitro oxidative metabolism of 5-MeO-DIPT using human
liver microsomal fractions and recombinant cytochrome P450
enzymes as enzyme sources, and synthetic metabolites of 5-
MeO-DIPT as standards for analysis.
2
4
3
3
ꢀ1
1
3
) d
3
2
2.
Materials and methods
2
13
C NMR (125 MHz, CDCl
3
2
.1.
Materials
2
25 2
H N
5
-MeO-DIPT was supplied by Dr. M. Funada, National Institute
of Mental Health, National Center of Neurology and Psychiatry
Kodaira, Japan). Because this compound showed the purity
data supported the conclusion that the synthesized compound
was 3-(2-diisopropylaminoethyl)-1H-indol-5-ol (5-hydroxy-
N,N-diisopropyltryptamine, 5-OH-DIPT) (Fig. 1).
(
1
of above 99% in H NMR and HPLC, it was used without further
To a solution of 5-methoxytryptamine (93 mg, 0.49 mmol)
in MeOH (1.0 ml), 36% HCl (0.13 ml, 1.31 mmol), acetone
purification. 5-Hydroxy-N,N-diisopropyltryptamine (5-OH-

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 3 7 7 – 1 3 8 5
1379
0
3
(1.08 ml, 14.7 mmol), and NaBH CN (90%, 117 mg, 1.68 mmol)
were 5 -CCCAAGCTTAAAAAAATGGAACCTTTTGTGGTCCTGG-
0
0
were added at 0 8C and the mixture was stirred at room
3
and 5 -TTCAAGCTTCTCGAGTTCAGACAGGGATGAAGCA-
0
0
temperature for 73 h. Ten percent aqueous KOH (2.0 ml) was
GAT-3 for CYP2C8, and 5 -AAGCTTAAAAAAATGGATTCTC-
0
0
0
added to pH 8–9 and the whole was extracted with CH
1 ꢁ 5 ml, 1 ꢁ 3 ml, 2 ꢁ 2 ml). The combined organic layer was
washed with brine and was dried over K CO . The solvent was
2
Cl
2
TTGTGGTC-3 and 5 -AAGCTTTCAGACAGGAATGAAGCACA-3
for CYP2C9. The PCR products were directly introduced into
pGEM-T vector (Promega, Madison, WI) using the TA cloning
system, resulting in pGEM-T/CYP1A2, pGEM-T/CYP2C8 and
pGEM-T/CYP2C9. CYP2C19 cDNA cloned into pBluescript SK (ꢂ)
(pBluescript/CYP2C19) was supplied by Dr. J. Goldstein (NIEHS,
Research Triangle, NC). The cDNA containing the HindIII sites
and Kozak sequence was amplified by PCR from pcDNA3.1/
(
2
3
evaporated in vacuo and the residue was purified by column
chromatography (NH silica, benzene:AcOEt:MeOH = 20:2:0.75)
ꢀ1
1
to give a reddish brown oil (86 mg, 76%); IR (ATR, cm ) 3200; H
NMR (400 MHz, CDCl ),
), 3.85 (3H, s,
), 6.85 (1H, dd, J = 8.8, 2.4 Hz, H-6), 6.98 (1H, d, J = 2.4 Hz, H-
3
) d (ppm): 1.05 (6H, d, J = 6.2 Hz, 2 ꢁ CH
3
2
.82 (1H, quint. J = 6.2 Hz, CH), 2.94 (4H, s, 2 ꢁ CH
2
0
OCH
3
CYP2C19 as a template using theforward primer 5 -CCCAAGCT-
0
4
), 7.06 (1H, d, J = 2.4 Hz, H-2), 7.13 (1H, d, J = 8.8 Hz, H-7), 8.51
TAAAAAAATGGATCCTTTTGTGGTCC-3 and thereverse primer
13
0
0
(
1H, s, NH, exchangeable with D
d (ppm): 22.8, 25.9, 47.4, 48.5, 55.9, 100.7, 111.8, 112.0, 113.4,
22.8, 129.8, 131.6, 153.7; HREIMS found: 232.1555 (calculated
for C13 O:232.1575). These analytical data supported the
conclusion that the synthesized compound was isopropyl-[2-
5-methoxy-1H-indol-3-yl)ethyl]amine (5-methoy-N-isopro-
pyltryptamine, 5-MeO-IPT) (Fig. 1).
2
O); C NMR (125 MHz, CDCl
3
)
5 -GGAAAGCTTAGGAGCAGCCAGACCATCTGT-3 . The PCR pro-
duct was digested with HindIII and ligated into the same
restriction enzyme site of pcDNA3.1 (+), resulting in pcDNA3.1/
CYP2C19. pGEM-T/CYP1A2, pGEM-T/CYP2C8, pGEM-T/CYP2C9
and pcDNA3.1/CYP2C19 plasmids were sequenced in both the
forward and reverse directions using ABI BigDye terminator
cycle sequencing reaction kit v3.1 (Applied Biosystems, Pisc-
away, NJ) to confirm that there were no PCR errors. The DNA
fragments corresponding to CYP1A2, CYP2C8, CYP2C9 and
CYP2C19 were cut out with HindIII from the pGEM-T or
pcDNA3.1 (+) plasmid and were subsequently subcloned into
the pGYR1 yeast expression vector digested with HindIII. The
expression plasmids were sequenced to verify the correct
orientation with respect to the promoter for pGYR1. The
construction of CYP2D6 expression plasmid (pGYR1/CYP2D6)
was described previously [8]. CYP3A4 expression plasmid
(pGYR1/CYP3A4) was supplied by Dr. Y. Saito (National Institute
of Health Sciences, Tokyo, Japan).
1
18 2
H N
(
2
.3.
Construction of CYP expression plasmids
CYP1A2 cDNA for subcloning in expression vector pGYR1 was
prepared by polymerase chain reaction (PCR) from pcDNA3.1/
0
CYP1A2 plasmid [7] as a template using the forward primer, 5 -
0
AAGCTTAAAAAAATGGCATTGTCCCAGTCT-3 , and the reverse
0
0
primer, 5 -AAGCTTTCAGTTGATGGAGAAGCGCA-3 . The HindIII
0
sites (marked with the solid lines) were introduced to the 5 -end
0
of the start codon and the 3 -end of the stop codon to facilitate
subcloning into pGYR1. A Kozak sequence (marked in italics)
was also introduced upstream of the start codon to achieve high
expression of the protein in yeast cells. CYP2C8 and CYP2C9
cDNAs were amplified from human adult normal liver Quick-
Clone cDNA (BD Biosciences Clontech, Mountain View, CA). The
nucleotide sequences used for the forward and reverse primers
2.4.
Expression of CYP enzymes
The pGYR1 vectors containing CYP cDNAs were used to
transform Saccharomyces cerevisiae AH22 by the lithium acetate
Fig. 1 – Major metabolic pathways of 5-MeO-DIPT.

1380
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 3 7 7 – 1 3 8 5
method, and the cultivation of yeast transformants was
performed as described [9]. Microsomes from yeast were
prepared as described previously [10]. The yeast cell micro-
somal content of each recombinant CYP enzyme was as
follows: CYP1A2 (13.5 pmol/mg protein), CYP2C8 (95.3 pmol/
mg protein), CYP2C9 (76.0 pmol/mg protein), CYP2C19
280/340 nm. MS conditions were: ionization mode, ESI(+);
needle voltage, 2.0 kV; ring voltage, 45 V; orifice voltage, 0 V;
the temperatures of the orifice and desolvating plate were 80
and 220 8C; the resolution of the mass spectrometer was set at
1000 or 3000; collision gas, He.
(41.8 pmol/mg protein), CYP2D6 (65.0 pmol/mg protein) and
2.8.
Others
CYP3A4 (49.1 pmol/mg protein).
Total holo-CYP contents in yeast cell microsomal fractions
were spectrophotometrically measured by assessing the
reduced carbon monoxide spectra according to the method
2
.5.
Measurement of oxidation activities of 5-MeO-DIPT
ꢀ1
ꢀ1
A typical reaction mixture consisted of G-6-P (10 mM, final
concentration), NADPH (1 mM), MgCl (10 mM), EDTA (0.2 mM),
of Omura and Sato [11] using 91 mM cm as the absorption
coefficient. Protein concentration was determined by the
2
microsomal fraction from human liver or yeast cells expressing
CYP enzyme (0.08–0.12 mg protein) and the substrate (0.1–
method of Lowry et al. [12]. Kinetic parameters (apparent K
m
and Vmax values) were estimated by analyzing Michaelis–
Menten plots or Eadie–Hofstee plots using the computer
program Prism ver. 4.0 software (GraphPad Software, San
Diego, CA).
1
000 mM) in 50 mM potassium phosphate buffer (pH 7.4) in a 1.5-
ml Eppendorf-type tube (a final volume of 200 ml). Following
preincubation at 37 8C for 5 min, the reaction was started by
adding microsomal fractions from human livers or yeast cells
expressing CYP enzymes, continued for 5–10 min, and stopped
by adding aqueous 2 M phosphoric acid (10 ml) and 20 mM
ascorbic acid (20 ml), vigorously mixing with a Vortexmixer, and
chilling in an ice bath for 10 min. The tube was then centrifuged
at 14,000 ꢁ g at 4 8C for 10 min, and the supernatant was passed
through a 0.45-mm membrane filter (Millipore, Billerica, MA). An
aliquot (20 ml) was subjected to high-performance liquid
chromatography (HPLC) under the conditions described below.
Calibration curves of 5-OH-DIPT and 5-MeO-IPT were made by
spiking ice-cold reaction medium with known amounts of the
synthetic compounds, followed by the addition of aqueous 2 M
phosphoric acid and 20 mM ascorbic acid and treatment as
describedabove.Thedetectionlimitsfor5-OH-DIPTand5-MeO-
IPT were 0.5 and 1.0 pmol/ml, with a signal-to-noise ratio of 3 in
both cases. The intra- and inter-day coefficients of variation did
not exceed 10% for any assay.
3.
Results
First, we examined the in vitro oxidative metabolism of 5-
MeO-DIPT using pooled human liver microsomes from
Caucasians. When 50 mM 5-MeO-DIPT was used as the
substrate, two major metabolite peaks [M ꢀ 1 (retention time
of 10.3 min) and M ꢀ 3 (15.1 min)] were observed on the HPLC
chromatogram (Fig. 2). Because the retention times and
fragmentation profiles of M ꢀ 1 and M ꢀ 3 coincided with
those of 5-OH-DIPT and 5-MeOH-IPT synthetic standards in
LC/MS analysis, M ꢀ 1 and M ꢀ 3 were identified as 5-OH-DIPT
and 5-MeOH-IPT, respectively (Fig. 3).
Furthermore, two metabolite peaks [M ꢀ 2 (retention time
12.0 min) and M ꢀ 4 (17.7 min) in HPLC] were analyzed by
LC/MS. The fragment ions were as follows: M ꢀ 2; m/z 291
(
M + 1, 100%), 181 (42%), 140 (85%), 124 (18%): M ꢀ 4; m/z 305
2
.6.
HPLC conditions
(M + 1, 64%), 278 (18%), 204 (55%), 144 (100%). From the
molecular ion (m/z 291), M ꢀ 2 is thought to be a monohy-
The HPLC apparatus consisted of a Hitachi L-2130 pump, an L-
480 fluorescence detector, an L-2300 column oven, a D-2000
2
system manager (version 1.1) and a Rheodyne type 7725i
injector. Other conditions were as follows: column, Inertsil C8
(
150 mm ꢁ 4.6 mm i.d., GL Sciences, Co. Ltd., Tokyo, Japan);
column temperature, 40 8C; detection, fluorescence excita-
tion/emission wavelength, 280/340 nm. The mobile phase
used was a linear gradient system consisting of (A) 20 mM
ammonium acetate (pH 4.0)/acetonitrile (92:8 v/v) and (B)
2
0 mM ammonium acetate (pH 4.0)/acetonitrile (80:20) as
follows: 0–3 min, (A) 100%; 3–15 min, from (A) 100% to (B) 100%;
1
4
5–25 min, (B) 100%; 25–30 min, from (B) 100% to (A) 100%; 30–
0 min, (A) 100% at a flow rate of 0.9 ml/min.
2
.7.
LC/MS conditions
LC/MS analysis was performed using a JMS-700 MStation
JEOL, Tokyo, Japan). HPLC conditions were: column, Inertsil
Fig. 2 – A typical HPLC chromatogram of 5-MeO-DIPT and
its metabolites. The reaction mixture containing human
liver microsomes and 5-MeO-DIPT (50 mM) was incubated
in the presence of an NADPH-generating system and the
metabolites formed were examined by HPLC under the
conditions described in Section 2.
(
ODS-3 (150 mm ꢁ 2.1 mm i.d., GL Science); mobile phase,
aqueous 0.1% TFA/acetonitrile (84:16 v/v); column tempera-
ture, 408; flow rate, 0.2 ml/min; injection volume, 20 ml;
detection, fluorescence excitation/emission wavelength,

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 3 7 7 – 1 3 8 5
1381
Table 1 – Kinetic parameters for 5-MeO-DIPT oxidation
by human liver microsomes
K
m
(mM)
Vmax (pmol/
Vmax/K
m
min/mg
(ml/min/mg
protein)
protein)
O-demethylation
5.0
140
27.9
N-deisopropylation
Low-K
m
phase
24
25
1.03
0.69
Intermediate-K
m
257
178
phase
High-K
m
phase
1201
409
0.34
Fig. 3 – Mass fragment ions of M S 1 and M S 3. LC/MS
Each value represents the mean of two determinations.
conditions are given in Section 2.
MeO-DIPT N-deisopropylase activitiy. The activities were
ranked as CYP2C19 > CYP1A2 > CYP3A4 > CYP2C8 > CYP2C9
= CYP2D6.
droxylated 5-MeO-DIPT. It is feasible that M ꢀ 4 is a dehy-
drogenated product of dihydroxylated 5-MeO-DIPT.
We then performed kinetic analysis using substrate
concentrations ranging from 0.1 to 1000 mM and the pooled
human microsomal fraction and the yeast cell microsomal
fractions expressing recombinant CYP enzymes as enzyme
sources. Only for CYP3A4, the Supersomes expressing
It is reasonable to think that the formation of these
metabolites was catalyzed by CYP enzymes in the liver
microsomal fractions. We therefore examined what kinds of
CYP enzymes were involved in the formation of M ꢀ 1 and
M ꢀ 3 from 5-MeO-DIPT in human liver microsomes in the
second step of this study. For this, we used six human
recombinant CYP enzymes expressed in yeast cells: CYP1A2,
CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4.
5
CYP3A4, OR and cytochrome b was employed. 5-MeO-DIPT
O-demethylation by the pooled human liver microsomal
fraction exhibited monophasic kinetics (Fig. 5A), whereas
human liver microsomal N-deisopropylation showed triphasic
kinetics (Fig. 5B). Table 1 summarizes the kinetic parameters.
In this experiment, we employed two substrate concentra-
tions (1 and 50 mM). All of the CYP enzymes except for CYP3A4
exhibited the capacity to oxidize 5-MeO-DIPT (Fig. 4). CYP3A4
expressed in yeast cells did not produce any metabolites in
detectable amounts even at 50 mM substrate under the
conditions used. We further examined the metabolic capacity
of commercially available insect cell microsomal fractions
The apparent
demethylation was calculated to be 5 mM, while the low-,
intermediate- and high-K values for triphasic N-deisopro-
m
K value for monophasic 5-MeO-DIPT O-
m
pylation were calculated to be 24, 260 and 1200 mM, respec-
tively.
For 5-MeO-DIPT O-demethylation, recombinant CYP2D6
(
Supersomes) expressing CYP3A4, OR and cytochrome b
Interestingly, Supersomes co-expressing CYP3A4 with cyto-
chrome b exhibited considerable 5-MeO-DIPT N-deisopropy-
lase but not O-demethylase activity, whereas Supersomes
without cytochrome b did not show any detectable activity
Fig. 4). As a result, among the six CYP enzymes tested, only
5
.
m
yielded monophasic kinetics and an apparent K value of
2 mM. For 5-MeO-DIPT N-deisopropylation, CYP2C19, CYP3A4,
5
CYP1A2, CYP2C8 and CYP2C9 showed monophasic kinetics,
and gave K
respectively (Table 2). These K
of the low-, intermediate- and high-K
the human liver microsomal fraction (Table 1). In the case of N-
m
values of 35, 180, 260, 290 and 1660 mM,
values are similar to the values
phases, respectively, of
5
m
(
m
CYP2D6 exhibited 5-MeO-DIPT O-demethylase activity,
whereas all of the six recombinant enzymes showed 5-
Fig. 4 – Comparison of 5-MeO-DIPT oxidation activities of recombinant CYP enzymes expressed in yeast or insect cells.
For incubation, 5 pmol of each CYP enzyme was employed: (A) 5-MeO-DIPT O-demethylation; (B) 5-MeO-DIPT N-
deisopropylation. Open columns, 5-MeO-DIPT 1 mM; closed columns, 5-MeO-DIPT 50 mM. Each value represents the mean
5
of two determinations. 3A4 & b , Supersomes; N.D., not detectable.

1382
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 3 7 7 – 1 3 8 5
Fig. 5 – Kinetic analysis of 5-MeO-DIPT oxidation by human liver microsomes. (A) and (B) Eadie–Hofstee plots for 5-MeO-DIPT
O-demethylation and N-deisopropylation, respectively.
deisopropylation by CYP2D6, precise kinetic parameters were
not calculated because of the low activities.
CYP2D6 and CYP3A4, respectively. For 5-MeO-DIPT O-
demethylation by human liver microsomes, quinidine showed
a concentration-dependent inhibition. Over 95% of the activity
was suppressed by the inhibitor even at 5 mM (Fig. 6A).
Furafylline and quercetin inhibited human liver micro-
somal 5-MeO-DIPT N-deisopropylation in a concentration-
dependent manner, but about 30–40% of the activity remained
even at the highest concentration of the inhibitors (Fig. 6B and
C). Sulfaphenazole also caused a concentration-dependent
In the third step of the present study, we examined the
effects of inhibitors of the CYP enzymes on the oxidative
metabolism of 5-MeO-DIPT in human liver microsomes to
estimate the contribution of the CYP enzymes. For this, we
employed furafylline [13], quercetin [14], sulfaphenazole [15],
omeprazole [16], quinidine [17] and ketoconazole [18] as
specific inhibitors of CYP1A2, CYP2C8, CYP2C9, CYP2C19,
Fig. 6 – Effects of various CYP inhibitors on 5-MeO-DIPT oxidation by human liver microsomes: (A) quinidine (0, 0.5, 5 and
0 mM) as CYP2D6 inhibitor; (B) furafylline (0, 5, 10 and 50 mM) as CYP1A2 inhibitor; (C) quercetin (0, 5, 20 and 50 mM); (D)
2
sulfaphenazole (0, 5, 20 and 50 mM) as CYP2C9 inhibitor; (E) ketoconazole (0, 1, 5 and 20 mM) as CYP3A4 inhibitor; (F)
omeprazole (10 mM) as CYP2C19 inhibitor. (A) 5-MeO-DIPT O-demethylation; (B), (C), (D), (E) and (F) 5-MeO-DIPT N-
deisopropylation. The substrate concentrations used were 10 mM for (A) and 50 mM for (B), (C), (D), (E) and (F). Each value
represents the mean of two determinations. HLM, human liver microsomes.

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 3 7 7 – 1 3 8 5
1383
Table 2 – Kinetic parameters for 5-MeO-DIPT oxidation
by recombinant CYP enzymes expressed in yeast cells
and CYP2D6 [20,21]. Using the yeast cell expression systems of
CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4
constructed so far in this laboratory, we examined the
metabolic capacities of these recombinant enzymes for the
oxidation of 5-MeO-DIPT.
K
m
(mM)
V
max (pmol/
Vmax/K
m
min/pmol
CYP)
(ml/min/
pmol CYP)
Among the recombinant enzymes, only CYP2D6 showed
considerable 5-MeO-DIPT O-demethylase activity. This reac-
tion in human liver microsomes yielded monophasic kinetics,
and was almost completely suppressed by quinidine as a
O-demethylation
CYP2D6
2.0
29.8
14.8
N-deisopropylation
CYP1A2
CYP2C8
CYP2C9
CYP2C19
263
291
1663
35
9.4
1.7
4.2
6.9
4.4
0.04
specific inhibitor of CYP2D6. The apparent K
m
value (2 mM) for
0.006
0.003
0.19
the recombinant CYP2D6 was similar to that (5 mM) for the
human liver microsomes. These results indicate that 5-MeO-
DIPT O-demethylation was mainly mediated by CYP2D6 in
human livers.
a
CYP3A4
184
0.02
Each value represents the mean of two determinations.
a
Data from Supersomes in which CYP3A4 and cytochrome b
5
In contrast, the two CYP enzymes (CYP1A2 and CYP2C19) in
the yeast cell expression system exhibited considerable 5-
MeO-DIPT N-deisopropylase activities. The activities of
CYP2C8, CYP2C9 and CYP2D6 were much lower than those
of CYP1A2 and CYP2C19. Interestingly, yeast cell microsomal
CYP3A4 did not show any detectable activity for either O-
demethylation or N-deispropylation. Before coming to a
conclusion, we further examined the metabolic capacity of
were co-expressed.
inhibition, but suppressed only 25% of human liver micro-
somal 5-MeO-DIPT N-deisopropylation at the highest con-
centration of 50 mM (Fig. 6D). Ketoconazole suppressed 40%
and 65% of the N-deisopropylase activity at final concentra-
tions of 5 and 20 mM, respectively (Fig. 6E). Omeprazole (10 mM)
suppressed only 10% of 5-MeO-DIPT N-deisopropylation by
pooled human liver microsomes, though this inhibitor at the
same concentration suppressed the 5-MeO-DIPT N-deisopro-
pylation by recombinant CYP2C19 almost completely (Fig. 6F).
Supersomes expressing CYP3A4, OR and cytochrome b
because it is well known that co-existence of cytochrome b
5
,
5
increases the oxidation capacity of CYP3A4 for its substrates
[22,23]. As expected, Supersomes co-expressing CYP3A4 with
cytochrome
demethylase activity, whereas Supersomes without cyto-
chrome b did not.
The apparent K
b
5
exhibited considerable 5-MeO-DIPT N-
4.
Discussion
5
m
values for CYP2C19 (35 mM), CYP3A4
There have hitherto been no reports of the quantitative assays
using authentic samples of 5-MeO-DIPT metabolites to study
the formation of 5-MeO-DIPT metabolites. We therefore
chemically synthesized the two compounds, 5-OH-DIPT and
(180 mM), CYP1A2 (260 mM), CYP2C8 (290 mM) and CYP2C9
(1700 mM) seem to correspond to those for human liver
microsomal low- (24 mM), intermediate- (260 mM) and high-
m
K (1200 mM) phases, respectively. To confirm the involve-
5
-MeO-IPT, and conducted a quantitative analysis of the
ment of these CYP enzymes in human liver microsomal 5-
MeO-DIPT N-deisopropylation, the effects of furafylline,
quercetin, sulfaphenazole, omeprazole and ketoconazole
were examined as specific inhibitors for CYP1A2, CYP2C8,
CYP2C9, CYP2C19 and CYP3A4, respectively. Among them,
furafylline, quercetin and ketoconazole exerted considerable
inhibitory effects at relatively low concentrations (several mM).
We employed quercetin as the inhibitor of CYP2C8 in the
present study, however, this compound was reported to
inhibit the metabolic activities of CYP1A2, CYP2C19 and
CYP3A4 as well [24]. Therefore, quercetin could suppress
the activities not only of CYP2C8 but also of CYP1A2 and
CYP3A4 in 5-MeO-DIPT N-deisopropylation by human liver
microsomal fraction in this study. Inhibitory effect of
sulfaphenazole was found to be weak as compared to those
of furafylline and ketoconazole. Interestingly, the inhibitory
effect of omeprazole was very weak under the conditions
used. In this case, we employed an inhibitor concentration of
formation of these metabolites. Using these synthetic sam-
ples, we examined the in vitro oxidative metabolism of 5-MeO-
DIPT by human liver microsomes and recombinant CYP
enzymes. As expected, 5-MeO-DIPT was biotransformed into
5
-OH-DIPT and 5-MeO-IPT as major metabolites by human
liver microsomes under the conditions used. Two other
metabolites were also tentatively identified as monohydroxy-
lated 5-MeO-DIPT and a dehydrogenated product of dihy-
droxylated 5-MeO-DIPT on the basis of the data from LC/MS
analysis.
In a preliminary HPLC experiment, we compared the
amounts of 5-OH-DIPT and 5-MeO-IPT formed with the
amount of 5-MeO-DIPT consumed during the incubation of
the substrate (10 mM) under similar conditions to those
employed here. The results indicated that at least 95% of
substrate consumption was explained by the formation of the
two major metabolites (data not shown). Therefore, it is
reasonable to think that 5-OH-DIPT and 5-MeO-IPT are the
major metabolites in human liver microsomes at around
00
10 mM, which was sufficient to suppress the bufuralol 1 -
hydroxylase activities of CYP2C19 in our previous studies [25].
In fact, 10 mM omeprazole completely suppressed 5-MeO-DIPT
N-deisopropylation by recombinant CYP2C19 in the present
study.
1
0 mM substrate concentrations (Fig. 1).
Human liver microsomal 5-MeO-DIPT O-demethylation
and N-deisopropylation exhibited monophasic and triphasic
kinetics, respectively. We have been studying the relation-
ships between protein structures and enzymatic functions of
major drug-metabolizing CYP enzymes such as CYP1A2 [19]
Other drug-metabolizing-type CYP enzymes such as
CYP2E1, CYP2A6 and CYP2B6 could also be involved in the
oxidation of 5-MeO-DIPT in the human liver. In another

1384
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 1 3 7 7 – 1 3 8 5
preliminary experiment, we found that diethyldithiocarba-
mate (5, 20 and 100 mM final concentrations) as CYP2E1
inhibitor [26] did not affect 5-MeO-DIPT oxidation by the
pooled human liver microsomal fraction (data not shown).
Ono et al. [27] reported that diethyldithiocarbamate inhibits
the metabolic activities of CYP2A6 and CYP2C19 in addition to
that of CYP2E1. Furthermore, furafylline and ketoconazole
K
m
(CYP2C19), intermediate-K
m
(CYP1A2, CYP2C8 and CYP3A4)
m
and high-K (CYP2C9) phases, respectively, for N-deisopro-
pylation in human liver microsomes. These results together
with the results of the inhibitory studies indicate that CYP2D6
is the major 5-MeO-DIPT O-demethylase and CYP1A2, CYP2C8
and CYP3A4 are the major N-deisopropylase enzymes in the
human liver.
(5 mM each) suppressed 60% and 40%, respectively, of human
liver microsomal 5-MeO-DIPT N-deisopropylation under the
conditions employed. CYP3A4 is the most abundant CYP
enzyme followed by CYP2C and CYP1A2 in the human liver
Acknowledgments
[
28]. These results indicate that CYP1A2, CYP2C8 and CYP3A4
We would like to express our gratitude to Dr. Joyce A. Goldstein,
National Institutes of Environmental Health Sciences, Research
Triangle Park, NC, for her kind gift of CYP2C19 cDNA. This study
was supported in part by a grant from the Japan Research
Foundation for Clinical Pharmacology.
are the major 5-MeO-DIPT N-deisopropylases in the human
liver at substrate concentrations around 50 mM or less.
The present results of the measurement of enzyme
activities and the effects of inhibitors indicated that human
liver microsomal 5-MeO-DIPT O-demethylation is mainly
mediated by CYP2D6, whereas N-deisopropylation is mediated
mainly by CYP1A2 and CYP3A4, and also by CYP2C8, CYP2C9
and CYP2C19, at least in part. The major contribution of
CYP2D6 to 5-MeO-DIPT O-demethylation is predictable from
the previous findings of Yu et al. [29,30] on the role of CYP2D6
in 5-MeO-tryptamine metabolism. It should be noted that
though CYP2C19 exhibited the highest activity as 5-MeO-DIPT
N-deisopropylase among the six recombinant CYP enzymes
examined, its contribution was thought to be relatively low in
the reaction by the pooled human liver microsomal fractions
used in the present study.
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