Metabolism of anethole dithiolethione by rat and human liver microsomes: Formation of various products deriving from its o-demethylation and s-oxidation. Involvement of cytochromes P450 and flavin monooxygenases in these pathways

Article abstract of DOI:10.1124/dmd.118.082545

A study of the metabolism of anethole dithiolethione (ADT, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione) by rat and human liver microsomes showed the formation of the corresponding S-oxide and the S-oxide of desmethyl-ADT (dmADT, 5-(p-hydroxyphenyl)-3H-1,

Full text of DOI:10.1124/dmd.118.082545

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Metabolism of anethole dithiolethione by rat and human liver microsomes: formation of
various products deriving from its O-demethylation and S-oxidation. Involvement of
cytochromes P450 and flavin monooxygenases in these pathways.
Martin Dulac, Amor Sassi, Citra Nagarathinan, Marie-Odile Christen, Patrick Dansette,
Daniel Mansuy, Jean-Luc Boucher
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601,
University Paris Descartes, 45 rue des Saints-Pères, 75270 Paris, France (M.D., A.S., C.N.,
P.D., D.M., J-L.B.); Marie-Odile Christen Behavior, 27 rue Marceau 75116 Paris, France (M-
O.C.)
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Running title: Metabolism of anethole dithiolthione (Sulfarlem)
Correspondence to :
Jean-Luc Boucher, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
UMR 8601 CNRS, University Paris Descartes, 45 rue des Saints Pères, 75006 Paris, France.
Telephone : 33 (0)1 42 8621 91
Fax : 33 (0)1 42 86 83 87
Email: jean-luc.boucher@parisdescartes.fr
Text pages:
Tables:
25
2
Figures:
5
References:
Abstract:
35
137 words
478 words
Introduction:
Results and Discussion:
1284 words
Abbreviations
ADT, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione; ADO, 5-(p-methoxyphenyl)-3H-1,2-
dithiole-3-one; ADTSO, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione sulfoxide; Bz-ImH,
benzyl-imidazole; dmADT, 5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-thione; dmADTSO, 5-(p-
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hydroxyphenyl)-3H-1,2-dithiole-3-thione sulfoxide; dmADO, 5-(p-hydroxyphenyl)-3H-1,2-
dithiole-3-one; CYP, cytochrome P450; DEXA, dexamethasone; FMO, flavin monoxygenase;
HLM, human liver microsomes, HPLC-MS, high performance liquid chromatography-tandem
mass spectrometry, β-NF, β-naphthoflavone; PB, Phenobarbital; PDA, photodiode array;
pMA, 4-methoxy-acetophenone; pHA, 4-hydroxy-acetophenone; RLM, rat liver microsomes.
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ABSTRACT
A study of the metabolism of anethole dithiolethione (ADT) by rat and human liver
microsomes showed the formation of the corresponding S-oxide and the S-oxide of
desmethyl-ADT (dmADT), and of p-methoxy-acetophenone (pMA) and p-hydroxy-
acetophenone (pHA), in addition to the previously described metabolites, dmADT,
anethole dithiolone (ADO) and its demethylated derivative, dmADO. The microsomal
metabolism of ADO under identical conditions led to dmADO and to pMA and pHA.
The metabolites of ADT derive from two competing oxidative pathways: an O-
demethylation catalyzed by cytochromes P450 (CYPs) and an S-oxidation mainly
catalyzed by flavin-dependent monooxygenases (FMOs) and, to a minor extent, by
CYPs. The most active human CYPs for ADT demethylation appeared to be CYP1A1,
1A2, 1B1, 2C9, 2C19 and 2E1. ADT S-oxidation is catalyzed by FMOs 1 and 3, and, to
a minor extent, by CYPs such as CYP3A4.
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Introduction
Anethole dithiolethione (ADT, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione, formula
in Figure 1) is a drug that has been marketed in many countries for many years and used
in human therapy for its choleretic and sialogogic properties (Christen, 1995; Nagano et
al., 2001). ADT also exhibits chemoprotective effects against cancer and various kinds
of toxicity caused by some drugs and xenobiotics (Mansuy et al., 1986; Warnet et al.,
1989; Reddy et al., 1993; Christen, 1995; Dollo et al., 1999; Lam et al. 2002; Kwak et
al., 2003; Pouzaud et al., 2004; Zhang and Munday, 2008; Brooks et al., 2009). These
chemoprotective effects appear to be mainly due to its antioxidant properties (Mansuy et
al., 1986; Christen, 1995; Khanna et al., 1998) and its activity as a Nrf2 inducer,
resulting in an increase of the GSH level and of the Phase II detoxifying enzymes (Lee
and Surh, 2005; Giustarini et al., 2014).
ADT and its O-demethylated derivative, dmADT, were also extensively used as H2S
donors, and coupling of the OH group of dmADT with numerous anti-inflammatory
drugs have led to a variety of compounds described for their H2S-donor properties and
their therapeutic effects (Chen et al., 2010; Sparatore et al., 2011; Kashfi and Olson,
2013; Couto et al., 2015). However, despite the use of ADT as a choleretic and
sialologic drug for more than 50 years and the other promising protective and
therapeutic effects of ADT and ADT linked to various drugs as H2S donor hybrids, only
few data have been published on the metabolism of ADT in mammals. It has been
shown that it was O-demethylated to dmADT then conjugated into the corresponding
glucuronide or sulfate in vivo in animals (rats, mice, rabbits and dogs) and humans
(Gmelin and Lagler, 1963; Masoud and Bueding 1983; Sassi, 1986; Hayashi et al.,
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1990). The formation of 5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-one, ADO, and its O-
demethylated derivative, dmADO (Figure 1) was also observed in rats and dogs
(Hayashi et al., 1990). A more recent study of the metabolism of ADT by rat liver
microsomes also showed the rapid formation of dmADT, and indicated the formation of
metabolites resulting from the incorporation of an oxygen atom into ADT and dmADT.
However the complete structure of those oxygenated metabolites was not determined
(Chen et al., 2013).
The present article reports a study of the metabolism of ADT and ADO by human and
rat liver microsomes and shows the formation of new metabolites, ADT and dmADT S-
oxides (ADTSO and dmADTSO), p-methoxy-acetophenone (pMA) and p-hydroxy-
acetophenone (pHA), in addition to dmADT, ADO and dmADO. It also shows that
these metabolites derive from two competing oxidative pathways occurring on ADT: an
O-demethylation catalyzed by cytochromes P450 (CYPs) and an S-oxidation catalyzed
by flavin-dependent monooxygenases (FMOs) and CYPs. Finally, a study of the
oxidation of ADT by recombinant human liver microsomal CYPs and FMOs allowed
one to compare the abilities of those enzymes to catalyze ADT oxidation.
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MATERIALS and METHODS
General reagents
ADT was purchased from APIChem Technology (Hangzhou, Zhejiang, PR China).
NADPH, NADP, G6P, G6PDH, miconazole, N-benzyl-imidazole (Bz-ImH), pHA,
pMA, and all other chemicals were purchased from Sigma-Aldrich (Saint-Quentin
Fallavier, France) and were of the highest commercially available purity. HPLC-grade
acetonitrile and methanol were obtained from SDS (Peypin, France).
Synthesis of authentic samples
DmADT was prepared by heating ADT in the presence of pyridine hydrochloride for 30
min at 215°C, as previously described (Chen et al., 2010). ADO and dmADO were
prepared by treatment of ADT and dmADT, respectively, in the presence of mercuric
acetate as previously described (Klingsberg, 1972). ADTSO and dmADTSO were
prepared from ADT and dmADT, respectively, by treatment with 3-chloroperbenzoïc
acid at 0°C for 1h in dichloromethane, as previously described (Perez and Kresze, 1981;
1
Biart et al., 1992). All derivatives displayed H NMR (Supporting Information Table
S1), UV-visible (Supporting Information Table S2) and high resolution mass spectra
(Supporting Information Figure S1) in accordance with their structures.
Origin of microsomes and recombinant enzymes
Rat liver microsomes (RLM) were prepared as previously reported (Kremers et al.,
1981) from male Sprague-Dawley rats (Charles River, L’Arbresle, France) treated either
with phenobarbital (PB) (20 mg.kg-1, in 0.9% NaCl, i.p. for 4 days), β-naphthoflavone
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(β-NF) or dexamethasone (DEXA) (each 50 mg.kg-1, in corn oil, i.p. for 4 days).
Recombinant CYP1A1, 1A2, 1B1, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4, co-expressed
in baculovirus-transfected insect cells with human cytochrome P450 oxido-reductase
and cytochrome b5 (Supersomes) were from Corning Inc (Corning, Amsterdam, The
Netherlands). Human liver microsomes (HLM) and recombinant FMO1 and FMO3
were also from Corning Inc. Protein concentrations were determined by the Bradford
assay with bovine serum albumin as a standard (Bradford, 1976). Cytochrome P450
contents were determined by the method of Omura and Sato (Omura and Sato, 1964).
Typical microsomal incubation procedures
The standard oxidation reactions were conducted in 50 mM potassium phosphate buffer
(pH 7.4) containing 0.1 mM EDTA, microsomal proteins (usual concentration 3 µM
CYP), and 0.1 mM substrate. The mixtures were incubated for 2 min at 37°C and the
reactions (final volumes 200 µL) were started by the addition of NADPH (1 mM) and a
NADPH regenerating system (0.5 mM NADP + 5 mM G6P + 1 U/mL G6PDH). Usual
incubations were carried out for 0-120 min at 37°C and were quenched by the addition
of 100 µl of a cold acetonitrile-acetic acid (10:1 v/v) mixture. Reaction mixtures were
centrifuged (10 000 g, 10 min) to sediment the precipitated proteins and aliquots of the
supernatants were injected onto the HPLC-MS system. Incubations of ADT and ADO
with recombinant CYPs or FMOs were performed under identical conditions except that
the incubation mixtures contained 20 pmol of recombinant CYP or 0.2 mg/mL FMO.
Control experiments were carried out under identical conditions but without protein or
without NADPH and NADPH regenerating system.
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Large scale (100 mL) incubations of ADT in the presence of liver microsomes from PB-
pretreated rats were performed as above and were extracted with ethyl acetate. After
evaporation of solvent, ADT and its metabolites were separated on a Hypersil C18
column (250 x 3/8”, 5 µm; SFCC, Eragny, France). Elution was performed under
isocratic condition using a mixture of water-methanol-acetic acid (60/40/1, v/v) at a
flow rate of 3 mL/min and UV detection was performed at 310 nm.
HPLC-MS-MS analyses of metabolites
The supernatants were analyzed on a Surveyor HPLC system including a PDA detector
and coupled to a LCQ Advantage ion trap mass spectrometer (Thermo, Les Ulis,
France). HPLC separations were achieved on a Gemini C18 column (100 x 2 mm, 3
µm; Phenomenex, Le Pecq, France). Elution (flow rate 250 µL/min) was performed
using a mixture of solvent A (water + 0.1% formic acid) and B (acetonitrile + 0.1%
formic acid) with the following gradient conditions: 10% B for 1 min, linear gradient to
80% B in 18 min, linear gradient to 100% B in 1 min, holding at 100% B for 3 min,
returning to 10 % B in 0.1 min, and re-equilibration at 10% B for 5 min. The column
effluent was directed to the ion source from 3 to 23 min after injection to reduce
contamination. Mass spectra were obtained by electrospray ionisation in the positive
detection mode under the following conditions: source parameters: capillary
temperature, 250°C; capillary voltage, 6 V; spray voltage, 4.5 kV; primary gas flow, 20
a.u.; auxillary gas flow, 5 a.u. Mass spectra were recorded with a resolution of 1 a.m.u.,
with a frequency of 50 ms. The range of masses scanned for the total-ion chromatogram
was m/z 120-600. For all products, the indicated molecular ions corresponded to M+H+
(i.e. ADT, m/z 241, ADO, m/z 225). After data acquisition, HPLC-MS chromatograms
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and UV-Vis spectra (230-550 nm) were analyzed with Excalibur LC Device 2.2.0
software (Thermo). Quantitation of ADT and of its metabolites was performed by
integration of UV-visible spectra using calibration curves obtained from incubations of
authentic compounds incubated, worked-up and analyzed in an identical fashion to that
described above, except that NADPH and the NADPH-regenerating system were
omitted.
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RESULTS and DISCUSSION
Metabolism of ADT by rat and human liver microsomes
The HPLC-MS chromatogram of incubates of 100 µM ADT with liver microsomes
from rats pretreated with β-NF in the presence of an NADPH generating system (Figure
2) showed the formation of the previously described metabolites (Gmelin and Lagler,
1963; Masoud and Bueding, 1983; Hayashi et al., 1990; Chen et al., 2013): dmADT as a
major metabolite and ADO and dmADO in smaller amounts. It also showed the
formation of four other metabolites: ADTSO and dmADTSO, resulting from an S-
oxidation of the C=S moiety of ADT and dmADT, respectively, and pMA and pHA,
two compounds resulting from the cleavage of the dithiolthione ring with loss of a
carbon atom. All these metabolites were obtained from semi-preparative HPLC and
completely identified by comparison of their HPLC retention times, UV-visible, MS
and 1H NMR spectra with those of authentic samples that were synthesized as indicated
in Materials and Methods or commercially obtained in the case of pMA and pHA. It is
likely that the S-oxide ADTSO is the oxygenated metabolite of ADT mentioned by
Chen et al. (Chen et al., 2013). Figure 3A shows a typical kinetic study of the
metabolism of ADT in the presence of liver microsomes from rats pretreated with β-NF.
First, ADT was predominantly metabolized into dmADT and small amounts of
ADTSO, ADO, dmADO and pMA. DmADT, ADTSO and ADO then decreased with a
concomitant increase in dmADO and pMA formation. Identical incubations of ADT but
in the absence of microsomes or NADPH did not lead to any formation of those
metabolites (data not shown). Table 1 compares the amounts of the ADT metabolites
formed upon 2 h incubations of ADT with a NADPH regenerating system and liver
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microsomes from rats pretreated by several inducing agents, β-NF, PB, or DEXA, or
with HLM. It shows that the same metabolites are formed with the three types of RLM.
However, it is noteworthy that liver microsomes from DEXA-pretreated rat are less
active for ADT oxidation (25% residual ADT instead of 6-7% in the case of β-NF- or
PB-treated rats after 2 h). Moreover, they led to much less metabolites derived from an
ADT O-demethylation (22% compared to 75% total metabolites instead of about 60%
compared to 94% total metabolites in the case of the two other RLM) (Table 1). HLM
predominantly catalyzed the O-demethylation of ADT (65% of all metabolites), but
with a lower activity than the β-NF and PB-pretreated RLM (30% residual ADT after 2
h) (Table 1). Interestingly, incubation of ADT with liver microsomes from β-NF-treated
rats in the presence of a NADPH-regenerating system and of 5 mM glutathione almost
exclusively led to dmADT (Figure 3B). Identical results were observed with HLM (data
not shown). The lack of formation of ADTSO and metabolites deriving from ADTSO,
such as ADO and pMA, in the presence of GSH could be explained by the reduction of
the sulfoxide function of ADTSO by GSH back to ADT. Actually, reaction of 100 µM
ADTSO with 1mM GSH in 50 mM potassium phosphate buffer (pH 7.4) containing 0.1
mM EDTA for 30 min at 37°C almost quantitatively led to ADT. In a similar manner,
reaction of dmADTSO with GSH under identical conditions almost exclusively led to
dmADT (data not shown).
Metabolism of ADO by liver microsomes
To assess the sequential metabolism of ADT, incubations were conducted using ADO
as the starting compound. Metabolism of ADO by rat or human liver microsomes under
conditions identical to those used previously for ADT led to the three metabolites
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already observed in the case of ADT, dmADO, pMA and pHA. Figure 4 shows the
kinetics of formation of those metabolites in incubations of ADO with β-NF-treated rat
liver microsomes and HLM (Figure 4A and 4B, respectively). These data showed that
pMA is a metabolite of ADO and suggested that pHA derives from dmADO.
Accordingly, incubation of dmADO with the same microsomes led to pHA as a major
metabolite (data not shown).
The above data about ADT and ADO microsomal metabolism indicated that ADT
metabolites derive from two pathways for oxidation of the molecule: an O-
demethylation and an S-oxidation leading respectively to dmADT and ADTSO as
primary metabolites (Figure 5). ADTSO should then lead to ADO by oxidative
desulfuration in a manner similar to what was reported for the oxidative desulfuration of
compounds containing a C=S moiety (Hanzlik and Cashman, 1983) and to pMA
deriving from ADO as shown in Figure 5. The further microsomal metabolism of the
other primary metabolite dmADT should be similar to that of ADT, with the formation
of metabolites deriving from an S-oxidation, dmADTSO, dmADO and pHA (Figure 5).
Accordingly, metabolism of dmADT with the same microsomes led to dmADTSO,
dmADO and pHA as major metabolites (data not shown).
Nature of the microsomal enzymes involved in the metabolism of ADT
In order to determine the enzymes involved in the human liver metabolism of the drug
ADT, incubations of ADT with several recombinant human CYPs and FMOs involved
in xenobiotics metabolism were performed. Table 2 shows that CYPs of family 1
(CYP1A1, 1A2 and 1B1) and CYP2C19 are very good catalysts for ADT oxidation (O-
demethylation + S-oxidation). CYP2C9, 2D6, 2E1, and 3A4 are also active for ADT
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oxidation, even though less than those of family 1, whereas CYP2A6 is much less
active. Moreover, the observed O-demethylation/S-oxidation ratio very much varied as
a function of the involved CYPs. This ratio was much in favor of O-demethylation in
the case of CYP1B1, 2C19, 1A2, 2C9, 1A1, and 2E1 (values from 25 to 4), whereas it
was in favor of S-oxidation for CYP2D6 and 3A4 (values of about 0.5 and close to 0,
respectively). Interestingly, recombinant FMOs 1 and 3 failed to catalyze ADT O-
demethylation, as expected (Cashman, 1995; Cashman and Zhang, 2006), but were
active for ADT S-oxidation (Table 2). Actually, inactivation of microsomal FMOs by
preheating of HLM at 45°C for 10 min (Cashman and Zhang, 2006) led to a 60%
decrease of the formation of ADTSO and ADO whereas it led to an increase of the
formation of the O-demethylated product dmADT (data not shown). Moreover,
incubation of ADT with HLM in the presence of an usual CYP inhibitor, N-benzyl-
imidazole (1mM) (Testa and Jenner, 1981; Correia and Ortiz de Montellano, 2005), led
to a 60% inhibition of dmADT formation and only led to a 20% decrease of the ADTSO
formation (data not shown). These data indicated that ADT S-oxidation was mainly
catalyzed by FMOs and to a minor extent by CYPs. The results of Table 2 also show
that ADO was formed upon oxidation of ADT by most recombinant human CYPs and
FMOs, indicating that both enzymes are also able to catalyze the oxidation of ADTSO
to ADO. We have confirmed this point in the particular case of recombinant CYP3A4.
Actually, incubations of 100 µM ADTSO for 1 h at 37°C in the presence of 0.2 µM
recombinant CYP3A4 and 1 mM NADPH led to ADO as the major product (data not
shown).
The above data indicate that the products formed upon metabolism of ADT by HLM
derive from two pathways, an oxidative CYP-catalyzed O-demethylation (CYPs of
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family 1 and CYP2C19, 2C9 and 2E1 being the most active ones) and an S-oxidation of
the C=S group of ADT catalyzed by FMOs and by some CYPs such as CYP3A4. This
should also be true for ADT metabolism by RLM as the greatest O-demethylation/S-
oxidation ratio was observed in the case of microsomes of rats pretreated with β-NF, an
inducer of P450s from family 1, the lowest O-demethylation/S-oxidation ratio being
observed in the case of microsomes of rats pretreated with DEXA, an usual inducer of
P450s from the 3A subfamily (Williams et al., 2005).
CONCLUSION
The above described reinvestigation of ADT metabolism and study of ADO metabolism
by rat and human liver microsomes clearly established the formation of new
metabolites, ADTSO, dmADTSO, pMA and pHA, in addition to the previously
described metabolites, dmADT, ADO and dmADO (Gmelin and Lagler; 1963; Hayashi
et al., 1990; Chen et al., 2013). They also showed that all these metabolites derive from
two primary pathways, a CYP-dependent O-demethylation of ADT, and an S-oxidation
of the C=S group of ADT that is catalyzed both by FMOs and CYPs. The use of
recombinant human P450s showed that CYPs of family 1 and CYP2C19, 2C9, and 2E1
are the most active ones for ADT O-demethylation. ADO and the opened products
having lost one carbon and the three sulfur atoms of ADT, pMA and pHA, are derived
from the ADT S-oxidation pathway that can be catalyzed by FMOs and some CYPs
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such as CYP3A4. The reactions involved in the passage from ADO to pMA are under
study.
Acknowledgments
The authors thank A. Hessani for her help in HPLC-MS experiments, and M. Jaouen
and B. Ramassamy for their technical assistance.
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Authorship contributions
Participated in research design: Dansette, Boucher, Mansuy, Christen.
Conducted experiments: Dulac, Sassi, Nagarathinan, Boucher.
Contributed new reagents or analysis tools: Dulac, Sassi, Boucher, Dansette.
Performed data analysis: Dulac, Sassi, Boucher, Dansette, Mansuy.
Wrote or contributing to the writing of the manuscript: Mansuy, Boucher, Dansette.
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FIGURE LEGENDS
Figure 1: Formula of ADT and some of its metabolites.
Figure 2: HPLC chromatogram with PDA-detection from representative 2h incubation
at 37°C of 100 µM ADT with liver microsomes from β-NF-pretreated rats (3.0 µM
P450) in the presence of a NADPH-generating system. The incubation was conducted,
treated and analyzed by HPLC-MS as described in Materials and Methods. The little
blue squares appearing along the baseline are integration points.
Figure 3: (A) Representative kinetics of ADT metabolism by liver microsomes from β-
NF-pretreated rats (3.0 µM P450) under conditions indicated in Materials and Methods.
(B) Kinetics of an identical incubation containing 5 mM GSH. ADT, black squares;
dmADT, red empty triangles; dmADO, blue diamonds; ADTSO, red filled triangles;
ADO, blue filled circles; pMA, green empty circles.
Figure 4: Kinetics of ADO metabolism by liver microsomes from β-NF-pretreated rats
(3.0 µM P450) (A) or by HLM (2.2 µM P450) (B). Conditions indicated in Materials
and Methods. ADO, black squares; dmADO, red triangles; pMA, blue circles; pHA,
green diamonds.
Figure 5: Reactions involved in the metabolism of ADT and ADO by HLM and RLM.
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Table 1. Metabolism of ADT by liver microsomes from rats pretreated with different
inducing agents and by HLMa.
metabolites b
% Total
Compounds
PB
DEXA
HLM
β-NF
7 + 2
ADT
6 + 2
45 + 4
9 + 2
2 + 1
4 + 2
6 + 2
25 + 3
25 + 4
12 + 3
18 + 3
15 + 3
4 + 2
30 + 5
46 + 5
5 + 2
5 + 3
n.d. c
dmADT
ADTSO
ADO
53 + 5
4 + 2
4 + 1
dmADTSO
dmADO
pMA
3 + 2
11 + 1
16 + 2
6 + 2
n.d. c
18 + 2
12 + 3
a The amounts of pHA were too low to be accurately quantified. b Data are expressed as
% metabolites formed after 2 h incubation of 100 µM ADT in the presence of β-NF-,
PB- and DEXA-pretreated rat liver microsomes, or HLM, as indicated in Materials and
Methods. Data are means + s.d. from 2-5 experiments. c Not detected.
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Table 2. Metabolism of ADT with microsomes from insect cells expressing various
recombinant human CYPs and FMOs (Supersomes)a.
ADT
36 + 2
18 + 2
22 + 4
93 + 6
73 + 6
21 + 4
48 + 5
44 + 5
47 + 5
72 + 6
93 + 5
dmADT ADTSO
ADO
8 + 2
3 + 2
3 + 2
6 + 2
3 + 2
3 + 1
28 + 4
7 + 2
40 + 5
16 + 4
3 + 1
dmADO
2 + 1
4 + 2
1 + 1
n.d.
pMA
1 + 1
1 + 1
n.d.
58 + 5
75 + 5
74 + 5
n.d.
n.d.b
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4 + 2
4 + 2
CYP1A1
CYP1A2
CYP1B1
CYP2A6
CYP2C9
CYP2C19
CYP2D6
CYP2E1
CYP3A4
FMO1
n.d.
23 + 4
75 + 6
16 + 4
35 + 5
n.d.
1 + 1
1 + 1
n.d.
n.d.
n.d.
6 + 2
4 + 2
13 + 4
6 + 2
n.d.
8 + 3
n.d.
n.d.
n.d.
n.d.
n.d.
FMO3
a % ADT or metabolite yields + s. d. from 2-4 experiments, from 100 µM ADT after 2 h
incubation in the presence of 0.2 µM recombinant CYPs or from 1 h incubations
containing 1 mg/mL recombinant FMOs. Quantitation of ADT and of its metabolites
was performed by integration of UV-visible spectra using calibration curves as
described in Materials and Methods. b Not detected
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Figure 1
RO
X
S
S
ADT
X = S
X = S
R = CH3
R = H
dmADT
ADTSO
dmADTSO
ADO
X = SO
X = SO
X = O
R = CH3
R = H
R = CH3
R = H
dmADO
X = O
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Figure 2
RT: 6.0 - 18.0
70000
dmADT
NL:
7.17E4
Total Scan
PDA
ADT250215
06
65000
60000
55000
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
dmADO
ADT
ADTSO
ADO
pMA
dmADTSO
0
pHA
-5000
6
8
10
12
Time (min)
14
16
18
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Figure 3
100
80
60
40
20
A
0
0
20
40
60
80
100
120
140
Incubation time (min)
100
80
B
60
40
20
0
0
20
40
60
80
100
120
140
Incubation time (min)
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Figure 4
100
80
60
40
20
A
0
0
20
40
60
80
100
120
140
Incubation time (min)
100
80
60
40
20
B
0
0
20
40
60
80
100
120
140
Incubation time (min)
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Figure 5
HO
H3CO
P450
P450
S
S
S
S
S
S
S
S
S
S
ADT
dmADT
FMO
+ P450
FMO
+ P450
+ GSH
+ GSH
H3CO
HO
O
S
O
S
S
S
ADTSO
dmADTSO
FMO
+ P450
FMO
+ P450
HO
H3CO
P450
P450
O
O
S
S
ADO
dmADO
O
O
H3CO
HO
CH3
CH3
pMA
pHA
30