Stereoselective S-oxidation and reduction of flosequinan in rat

Article abstract of DOI:10.1080/004982599238263

1. The stereoselective S-oxidation and reduction pathways of flosequinan [(±)-7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone] in rat were investigated in vitro. 2. Cytosol from both the liver and kidney catalysed the reduction of R(+)-flosequinan (R-FSO) and S(-)-flosequinan (S-FSO) to flosequinan sulphide (FS, 7-fluoro-1-methyl-3-methylthio-4-quinolone). Flosequinan sulphone (FSO₂₂, 7-fluoro-1-methyl-3-methyl-sulphonyl-4-quinolone) was not reduced to R-FSO or S-FSO. 3. Liver microsomes catalysed four different S-oxidation pathways in the presence of NADPH, namely oxidation of FS to R-FSO and S-FSO and from R-FSO and S-FSO to FSO₂. The formation of R-FSO and S-FSO from FS each eshibited a biphasic kinetic pattern, indicating that at least two distinct enzymes were involved. The pathway from FS to R-FSO appeared mainly catalysed by flavin-containing monooxygenases (FMO). 4. S-oxidation of FS to R-FSO was more rapid than that of FS to S-FSO. S-oxidation of FS to either R-FSO or S-FSO in liver microsomes was more rapid than that of either R-FSO or S-FSO to FSO₂. 5. Microsomes from both the kidney and lung catalysed the stereoselective S-oxidation of FS to R-FSO, and FMO was likely to have participated in these reactions.

Full text of DOI:10.1080/004982599238263

xenobiotica , 1999, vol. 29, no. 8, 815±826
Stereoselective S-oxidation and reduction of
¯osequinan in rat
E. KASHIYAMA‹*, T. YOKOI‹, M. ODOMIŒ
and T. KAMATAKI‹
‹ Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido
University, Sapporo 060±0812, Japan
ŒDepartment of Drug Metabolism, Tokushima Research Institute, Otsuka
Pharmaceutical Co., Ltd, Tokushima 771±0192, Japan
Received 18 January 1999
1. The stereoselective S-oxidation and reduction pathways of ¯osequinan [( )-7-
6
¯uoro-1-methyl-3-methylsulphinyl-4-quinolone] in rat were investigated in vitro.
2. Cytosol from both the liver and kidney catalysed the reduction of R( )-¯osequinan
2
1
(R-FSO) and S( )-¯osequinan (S-FSO) to ¯osequinan sulphide (FS, 7-¯uoro-1-methyl-
3-methylthio-4-quinolone). Flosequinan sulphone (FSO#, 7-¯uoro-1-methyl-3-methyl-
sulphonyl-4-quinolone) was not reduced to R-FSO or S-FSO.
3. Liver microsomes catalysed four diåerent S-oxidation pathways in the presence of
NADPH, namely oxidation of FS to R-FSO and S-FSO and from R-FSO and S-FSO to
FSO . The formation of R-FSO and S-FSO from FS each exhibited a biphasic kinetic
patte#rn, indicating that at least two distinct enzymes were involved. The pathway from FS
to R-FSO appeared mainly catalysed by ¯avin-containing monooxygenases (FMO).
4. S-oxidation of FS to R-FSO was more rapid than that of FS to S-FSO. S-oxidation
of FS to either R-FSO or S-FSO in liver microsomes was more rapid than that of either R-
FSO or S-FSO to FSO#.
5. Microsomes from both the kidney and lung catalysed the stereoselectiveS-oxidation
of FS to R-FSO, and FMO was likely to have participated in these reactions.
Introduction
Flosequinan [rac-FSO, ( )-7-¯uoro-1-methyl-3-methylsulphinyl-4-quinol-
6
one] is a peripheral vasodilator with therapeutic eåects on both arterial and venous
vascular beds (Cowley et al. 1984, Yates 1991). The compound has a chiral sulphur
atom, resulting in two stereo-isomers, ( )-(R)- and ( )-(S)-¯osequinan (R-FSO
1
2
and S-FSO). The stereoselective pharmacokineticsin rat have been investigatedand
reported by Kashiyama et al. (1994b). Each enantiomer showed a diåerent in vivo
pharmacokinetic behaviour. Chiral interconversion appeared to occur through the
formation of ¯osequinan sulphide (FS, 7-¯uoro-1-methyl-3-methylthio-4-quinol-
one), since R-FSO and S-FSO were produced when FS was intravenously
administered to rat. The proposed in vivo metabolic pathways are shown in ®gure 1.
While FS is formed by the reduction of both enantiomers, R-FSO and S-FSO are
formed by the S-oxidation of FS. When each enantiomeris administered in vivo, the
main metabolite in both cases is ¯osequinan sulphone (FSO#, 7-¯uoro-1-methyl-3-
methylsulphonyl-4-quinolone). FSO# seems to be the terminal metabolite, since
* Author for correspondence at: Department of Drug Metabolism, Tokushima Research
Institute, Otsuka Pharmaceutical Co., Ltd, Tokushima 771±0192, Japan; e-mail:
j
e kashiyama!research.otsuka.co.jp
 Present address: Division of Drug Metabolism, Faculty of Pharmaceutical Science, Kanazawa
University, Kanazawa 920±0934, Japan.
}
’
http: www.tandf.co.uk JNLS xen.htm http: www.taylorandfrancis.com JNLS xen.htm
Xenobiotica ISSN 0049±8254 print ISSN 1366±5928 online
}} }}
1999 Taylor & Francis Ltd
}
}
}
}

816
E. Kashiyama et al.
only FSO was observed when FSO was intravenously administered to rat.
#
#
Therefore, there are four S-oxidation and two reduction pathways for ¯osequinan
metabolism in rat in vivo.
Sulphur-containing drugs are known to be metabolized via S-oxidation and
reduction pathways. The oxidation of sulphur compounds is catalysed almost
exclusively by cytochrome P450-dependent monooxygenases (P450) and ¯avin-
containing monooxygenases(FMO) in the liver. P450s are localized in many tissues
of mammals and consist of many isozymes (Nelson et al. 1996). P450s play an
essential role in the oxidative metabolism of endogenouscompoundsand of foreign
chemicals, including drugs (Kulkarni and Hodgson 1980). FMOs catalyse the
NADPH-dependentoxidativemetabolism of sulphur-,nitrogen-and phosphorous-
containingdrugs (Ziegler 1980, 1988). Distinct FMO proteinsand cDNA have been
puri®ed and isolated from mouse, rat, rabbit, guinea pig, pig and human (Lawton
and Philpot1993, Ziegler 1993). Northern and Western blot analyses have indicated
that FMO are mainly expressed in the liver, lung and kidney (Tynes and Philpot
1987, Itoh et al. 1993). Despite detailed studies on the oxidation of drugs, the
mechanisms responsible for the reduction of S-oxides have been reported for only
a few chemicals. For example, it has been reported that thioredoxin-linkedenzyme
}
systems and or aldehyde oxidase are capable of reducing sulphoxide-containing
compounds in the liver and kidney (Aymard et al. 1979, Fukazawa et al. 1987,
Anders et al. 1981, Yoshihara and Tatsumi 1985).
To clarify the mechanisms responsible for the chiral interconversion of
¯osequinan, the present study was conducted to investigate which enzymes are
involved in the stereoselective S-oxidation and reduction of ¯osequinan in vitro.
Materials and methods
Chemicals
rac-FSO, FS and FSO# were supplied by Boots Co. (Nottingham,UK). R-FSO
and S-FSO were prepared (Morita et al. 1994) by the TokushimaResearch Institute
of Otsuka Pharmaceutical Co. (Tokushima, Japan). The optical purity of R-FSO
"
and S-FSO was 99% as determined by hplc. Other chemicals were obtained from
the followingsources: glucose oxidase, dithiothreitol(DTT) and n-octylamine from
Wako Pure Chemical Industries (Osaka, Japan), 2-hydroxypyrimidine(2-OH-PM)
and 1-(1-naphthyl)-2-thiourea (naphthylthiourea) from Tokyo Chemical Industry
(Tokyo, Japan), SKF-525A hydrochloride from Funakoshi (Tokyo, Japan),
NADP+ , glucose 6-phosphate and glucose 6-phosphate dehydrogenase from
Oriental Yeast (Tokyo, Japan). All other chemicals used were of the highest grade
commercially available.
Preparation of microsomes and cytosol
Male Sprague±Dawley rats (6±7 weeks of age) were purchased from Japan SLC
(Shizuoka, Japan). Rats were decapitated and exsanguinated, and the liver, kidneys
and lungs immediately removed. Microsomes and cytosol were prepared as
described previously (Kamataki and Kitagawa 1973). Brie¯y, tissues were hom-
}
ogenized in 1.15% (w v) KCl solution using a Te¯on homogenizerand centrifuged
at 9000 g for 20 min. The supernatant fraction was then centrifuged at 105000 g for
1 h to yield both the microsomal pellet and cytosol. The microsomal pellet was

Stereoselective S-oxidation and reduction in rat
817
}
washed once and re-suspended in ice-cold 1.15% (w v) KCl solution. Protein
content was determined by the method of Lowry et al. (1951) using bovine serum
albumin as a standard. The tissue homogenates were frozen in liquid nitrogen and
stored at 80 C.
2
FMOs are unstable to heat treatment. Thus, to assess the possibility of FMO
Ê
being involved in the oxidation reactions, liver microsomes were heat-treated at
45 C for 5 min as described by Kashiyama et al. (1994c).
Ê
Analytical procedures
Reduction of ¯osequinan. The reductase activities for R-FSO, S-FSO and FSO
#
were determined by hplc as the production of FS, R-FSO and S-FSO respectively.
A typical reaction mixture (500 ll) consisted of 1 mm substrate, 0.1 m Na,K-
}
phosphate (pH 7.4), 0.1 mm EDTA and a cytosolic fraction (3±5 mg protein ml).
The mixture was pre-incubated at 37 C for 10 min, and the reaction was then
initiated by the addition of an NADPH-generating system (5.0 mm glucose 6-
Ê
+
}
phosphate, 0.5 mm NADP , 1 unit ml glucose 6-phosphate dehydrogenase, 5 mm
MgCl ), 2-OH-PM (2 mm) or DTT (10 mm) as an electron donor. When necessary,
#
the atmosphere in the reaction tube was replaced with nitrogen, and glucose oxidase
}
(10 units ml) and 50 mm glucose were also added to the reaction mixture to remove
oxygen. The reaction mixture was incubated at 37 C for 30 min, and then
Ê
chloroform (3 ml) was added to terminate the reaction, followed by the addition of
0.5 lg ( )-7-chloro-1-methyl-3-methylsulphinyl-4-quinolone as an internal stan-
6
dard. Metabolites were extracted with chloroform. The organic layer (2 ml) was
transferred to another tube and evaporated to dryness under a gentle stream of
nitrogen at 40 C. The residue was dissolved in the solvent used as the mobile phase
for hplc as described below and then injected into the hplc system.
Ê
S-oxidation. S-oxidase activities for FS, R-FSO and S-FSO were determined by
hplc as the production of R-FSO, S-FSO and FSO respectively. A typical reaction
#
mixture (200 ll) consisted of 0.1 m phosphatebuåer (pH 7.4), 0.1 mm EDTA, 1 mm
}
substrate and the microsomal fraction (0.5±1 mg protein ml). The mixture was pre-
incubated at 37 C for 3 min, and reactions were then initiated by the addition of the
Ê
NADPH-generating system. When appropriate, SKF-525A, n-octylamine or
naphthylthioureawas added at a respective ®nal concentration of 1, 3 or 1 mm. The
reaction mixture was incubated at 37 C for 10 min. All other experimental
Ê
conditions were the same as for the reductase assay.
Determination of metabolites
The determination of metabolites was performed using the hplc method
previously reported by our laboratory (Kashiyama et al. 1994a). The hplc system
consisted of an HLC-803D system (Tosoh, Tokyo, Japan) and a C-R6A integrator
(Shimadzu, Kyoto, Japan). The determination of R-FSO and S-FSO was
performed using a chiral-phase column (Chiralcel OD, 4.6 mm i.d. 250 mm;
3
Daicel Chemical Industries, Tokyo, Japan). Metabolites were eluted with an
}
}
ethanol±methanol (50:50 v v) mixture at 0.7 ml min. A UV-visible absorbance
detector (UV-8000; Tosoh) monitored the eluent at 320 nm. Determination of FS
and FSO was performed using a reversed-phase column (Inertsil ODS-2, 4.6 mm
#
i.d. 250 mm; GL Sciences, Tokyo, Japan) equilibrated and delivered with 25%
3

818
E. Kashiyama et al.
}
}
(v v) acetonitrile at 1 ml min. Elution of FSO was detected by the absorbance at
#
320 nm. FS was detected using a ¯uorescence detector (RF-530; Shimadzu) with an
excitation wavelength of 370 nm and an emission wavelength of 430 nm. The
calibration curve was generated for 10±1000 ng per reaction mixture by processing
the authentic standard substrates through the entire procedure. The linearity of
formation of S-oxidized metabolites was con®rmed for up to 30 min in the presence
}
of 0.05 and 1 mm substrate and 1 mg protein ml incubation mixture (Kashiyama et
al. 1997); while the reduction of R-FSO and S-FSO was linear for up to 60 min in
}
the presence of 1 mm substrate and 5 mg of cytosolic protein ml.
Kinetic analysis
Enzyme kinetic parameters were estimated by the non-linear least-squares
regression analysis of Eadie±Hofstee plots. Substrate concentration varied from 0.02
to 1 mm.
Inhibition of ¯osequinan metabolism by antibodies to rat NADPH-cytochrome P450
reductase
Antiserum to rat NADPH cytochrome P450 reductase was raised in rabbit as
described by Kamataki et al. (1976). Immuno-inhibition was measured in the
presence of 0, 2.5, 6, 10, 25 or 60 mg antiserum protein per mg of liver microsome
protein. The mixture of antiserum and microsomes was incubated for 60 min at 4 C
before incubation. All subsequent reaction conditions as described above.
Ê
Results
Enzyme(s) responsible for the reduction of ¯osequinan
To con®rm the proposed metabolic pathways of ¯osequinan, the metabolism of
¯osequinan was investigated in vitro. Reductase activities in rat cytosol from the
liver and from the kidney were examined (table 1). NADPH is known to be an
electron donorfor reductases. Aldehydeoxidase reduces the sulphoxidegroup in the
presence of 2-OH-PM underan anaerobic condition(Yoshiharaand Tatsumi 1985).
At a high concentration, DTT can be an electron donor for thioredoxin-dependent
sulphoxide reductase (Fukazawa et al. 1987). In the present study, under an
anaerobic condition using 2-OH-PM as an electron donor, liver cytosol supported
higher activity for reduction of R-FSO to FS than for reduction from S-FSO to FS.
2-OH-PM-dependent reduction was also observed in kidney cytosol, but the
activity was lower than that in liver cytosol. When DTT was used as an electron
donor, the reduction in kidney cytosol was higher than that in liver cytosol (table 1).
In kidney cytosol, the reduction of S-FSO to FS was twice as high as that of R-FSO
to FS. No activity for reduction of FSO to R-FSO or S-FSO was not detected (data
not shown).
#
Enzyme(s) responsible for S-oxidation of FS and ¯osequinan
As described above, it was demonstrated that FS was produced in vitro by
cytosolic enzymes. Since, as reported by Kashiyama et al. (1994b), FS is not
excreted in urine, FS must be the substance oxidized to produce the major ®nal
metabolite FSO (®gure 1). Thus, to clarify the rate of each S-oxidation pathway in
rat organs, the NADPH-dependent S-oxidase activities in microsomes from the
liver, kidney and lung were measured (table 2). Liver microsomes catalysed all four
#

Stereoselective S-oxidation and reduction in rat
819
Table 1. Electron donors for the stereoselective reduction of ¯osequinan in rat cytosol from both the
liver and kidney.
} }
Reduction (nmol h mg protein)
Electron
donor
!
R-FSO FS
!
S-FSO FS
Atmosphere
Liver
NADPH
air
0.08 0.01
0.11 0.02
6
6
NADPH
2-OH-PM
2-ON-PM
DTT
nitrogena
air
0.11 0.02
0.08 0.01
6
6
0.04 0.02
0.02 0.00
6
6
nitrogena
air
4.6
1.9
0.27 0.07
6
6
0.15 0.03
0.18 0.03
6
6
Kidney
NADPH
NADPH
2-OH-PM
2-OH-PM
DTT
air
0.13 0.01
0.02 0.00
6
6
nitrogena
air
0.13 0.01
0.01 0.00
6
6
n.d.
0.74 0.42
n.d.
0.09 0.04
nitrogena
air
6
6
6
0.49 0.08
1.0
0.1
6
a Reaction mixture contained 10 units glucose oxidase and 50 mm glucose.
Each value represents the mean SD from four individual determinations.
6
NADPH was generated in the mixture of an NADPH-generating system as described in the Material
and methods.
2-OH-PM, 2-hydroxypyrimidine; DTT, dithiothreitol; n.d., not detectable ( 0.01 nmol h mg
protein).
!
} }
Figure 1. Proposed metabolic pathways for ¯osequinan in rat.
S-oxidation reactions. The rates of formation of R-FSO and S-FSO from FS were
7±10 times higher than those from R-FSO and S-FSO to FSO . On the other hand,
#
microsomes from both the kidney and lung catalysed only the S-oxidation of FS to
R-FSO, and this stereoselective oxidation activity of microsomes from both the
kidney and lung was respectively 26 and 15% of the activity of liver microsomes.
To elucidate the catalytic properties of the S-oxidase, the kinetics of these S-
oxidation reactions were investigated. Typical Eadie±Hofstee plots of the four
diåerent S-oxidation reactions in liver microsomes are shown in ®gure 2. The plots
of the S-oxidation reactions of FS to R-FSO and to S-FSO in liver microsomes each
showed a biphasic kinetic behaviour. The S-oxidation of R-FSO and S-FSO to

820
E. Kashiyama et al.
Table 2. Stereoselective S-oxidation of ¯osequinan by microsomes from rat liver, kidney and lung.
}
}
S-oxidation (nmol min mg protein)
Liver Kidney Lung
3.44 0.25
Reaction
!
FS
FS
R-FSO
S-FSO
0.88 0.15
0.50 0.07
6
6
6
!
2.89 0.65
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
6
!
R-FSO FSO#
0.35 0.05
6
!
S-FSO FSO#
0.45 0.06
6
Each value represents the mean SD from six individual determinations.
6
!
}
}
n.d., Not detectable ( 0.01 nmol min mg protein).
Figure 2. Eadie±Hofstee plots for the S-oxidation of ¯osequinan in rat liver microsomes. Assays were
}
performed as described in the Material and methods. Protein concentration was 0.5 mg ml for
}
assayof the S-oxidation of FS and 1 mg ml for the assay of the S-oxidation of R-FSO and S-FSO.
Substrate concentration of FS, R-FSO and S-FSO ranged from 0.02 to 1 mm. v, Velocity of
}
product formation (nmol min mg protein).
}
FSO in liver microsomes exhibited a monophasic kinetic behaviour. The kinetic
parametersusingthetwo-enzymekineticapproach(Km , Km , V , V ), together
#
max
max
"
#
Km , V
"
#
Km ) for S-
}
oxidation of FS to R-FSO and to S-FSO, and the one-enzyme kinetic approach for
}
with the respective ratios or intrinsic clearance (V
max
max
#
"
"
#
}
was three times lower than V , whereas Km was 60 times lower than Km
S-oxidation of R-FSO and S-FSO to FSO (Km , Vmax , V
3. V
Km ) are shown in table
max
#
max
max
#
"
"
#
}
for the S-oxidation of FS to R-FSO. Thus, V
Km was calculated as 20 times
"
max
"
}
for S-oxidation mainly catalysed S-oxidation. Comparing the intrinsic clearance (V
higher than V
Km , indicating that an enzyme with a high-aænity component
#
max
#
}
Km ) for FS to R-FSO and to S-FSO, the former was nine times higher than the
latter, indicating that the S-oxidation of FS to R-FSO was a major pathway of FS
max
"

Stereoselective S-oxidation and reduction in rat
821
Table 3. Kinetic parameters for the S-oxidation of ¯osequinan in microsomes from rat liver,
kidney and lung.
}
Km
" "
or
Km
or"
Km
Vmax
or
Vmax
}
V
max
"
}
Vmax Km
(ml min
}
Km
Kmax
Vmax
}
V
max
#
#
#
#
}
}
}
}
} }
(mm) (nmol min
(mm) (nmol min (ml min
mg protein) mg protein)
Reaction
mg protein) mg protein)
Control
Liver
!
FS
FS
R-FSO 0.021
S-FSO 0.12
0.40
0.44
R-FSO 0.015
R-FSO 0.021
2.5
1.6
0.40
0.63
0.94
0.42
0.12
1.2
1.2
7.4
5.9
0.0062
0.0049
!
0.013
0.0010
0.0014
0.063
0.020
!
R-FSO FSO#
!
S-FSO FSO#
!
!
Kidney FS
Lung FS
Heat treatment
!
!
Liver
FS
FS
R-FSO
S-FSO 0.14
0.97
0.80
6.3
4.9
0.0065
0.0061
1.6
0.29
0.48
0.011
0.00066
0.0011
!
R-FSO FSO#
0.44
0.42
!
S-FSO FSO#
Figure 3. Eåects of various modi®ers of S-oxidase activity in rat liver microsomes. `-NADPH GS’ is
de®ned as the absence of an NADPH-generating system in the reaction mixture. Heat treatment
of the microsomes was performed at 45 C for 5 min. The concentrations of SKF-525A, n-
octylamine and naphthylthiourea added were respectively 1, 3 and 1 mm. Experimental details are
Ê
described in the text. Values are plotted as the mean SD for three-to-sixdeterminations. Results
6
!
signi®cantly diåerent from the control: **p 0.01.
S-oxidation. The kinetic parameters for the S-oxidation of R-FSO or S-FSO to
FSO were similar. The intrinsic clearance for the S-oxidation of R-FSO and S-
#
FSO to FSO were respectively 100 and 10 times lower than those for the S-
C
oxidation of FS to R-FSO and of FS to S-FSO.
C
#

822
E. Kashiyama et al.
Figure 4. Eåects of various modi®ers of the S-oxidation of FS to R-FSO in rat microsomes from both
the kidney and lung. Experimental details are the same as for ®gure 3. Values are plotted as the
mean SD for three-to-six determinations. Results signi®cantly diåerent from the control: **p
!
6
0.01.
On the other hand, the S-oxidation of FS to R-FSO in microsomes from both the
kidney and lung also showed monophasic kinetics, and the kinetic parameters were
calculated using the one-enzyme kinetic approach (table 3). Km for the S-oxidation
of FS to R-FSO in microsomes from both the kidney and lung were similar to the
Km seen with liver microsomes.
"
To clarify further which enzymes are involved in the metabolic pathways, the
eåects of inhibitors, activators and heat treatment of the microsomes on the S-
oxidationpathways in liver microsomes were investigated (®gure 3). All S-oxidation
reactions required an NADPH-generating system. The enzyme catalysing the S-
oxidation of FS to R-FSO was inhibited 30 and 70% by SKF-525A and
naphthylthiourearespectively. All S-oxidase activities were inhibitedby both SKF-
"
525A and n-octylamine ( 70%), but except for the S-oxidation of FS to R-FSO,
none of the activities was aåected by naphthylthiourea. In addition, heat treatment
of the microsomes resulted in a signi®cant decrease (65%) of S-oxidation of FS to
R-FSO, whereas the same heat treatment did not aåect any other S-oxidase
activities.
To analyse further the eåect of heat treatment of the liver microsomes on S-
oxidase activities, the kinetic parameters of the S-oxidation in the heat-treated
microsomes were calculated (table 3). The S-oxidation of FS to R-FSO in the heat-
treated microsomes showed a monophasic kinetic pattern. The high-aænity
component seen with untreated microsomes disappeared on heat treatment. The
kinetic parameters of other S-oxidation pathways were unaåected.
The FS S-oxidation to R-FSO in microsomes from both the kidney and lung
"
were decreased by 80% with heat treatment (®gure 4). The FS S-oxidation to R-
FSO was signi®cantly inhibited by naphthylthiourea but not by SKF-525A. n-
Octylamine enhanced the S-oxidase activities in microsomes from both the kidney
and lung.
The eåects of antibodies to rat NADPH-cytochrome P450 reductase on the S-
oxidation of ¯osequinan in microsomes from the liver, kidney and lung were also
C
investigated (®gure 5). S-oxidase activities in liver microsomes were inhibited
70%, except for the S-oxidation of FS to R-FSO (30%). The antibody did not
inhibit the S-oxidation of FS to R-FSO in microsomes from both the kidney and
lung.

Stereoselective S-oxidation and reduction in rat
823
Figure 5. Immuno-inhibition of S-oxidation by antiserum to NADPH-cytochrome P450 reductase.
Formation of R-FSO from FS (E ), S-FSO from FS (D ), FSO# from R-FSO (+ ) and FSO# from
S-FSO (* ) by liver microsomes and formation of R-FSO from FS by kidney (^) and lung (_ )
microsomes from rat are shown. Results are the amount of activity remaining relative to the
untreatedcontrol.Activities in the absence of antibody are the same as thoseshown in table 2. Data
are the mean of duplicate determinations.
Discussion
The reduction of drugs has not been studied as much as their S-oxidation. It has
been suggested that enzymes such as aldehyde oxidase (Tatsumi et al. 1983) and
thioredoxin-linked enzyme systems (Aymard et al. 1979, Fukazawa et al. 1987,
Anders et al. 1981) are involved in the reduction of sulphoxides to sulphides.
Although these enzymes have been puri®ed from liver and kidney cytosol, no
information is yet available on stereoselectivity in the metabolism of foreign
compounds. To our knowledge, the present paper is the ®rst to describe the
stereoselective reduction of sulphoxides.
Aldehyde oxidase usually reduces heterocyclic xenobiotics, such as 2-OH-PM
and N-methylnicotinamide, in the presence of an electron donor under anaerobic
conditions (Yoshihara and Tatsumi 1985). In the present study, it is possible that
aldehyde oxidase is involved in the reduction of R-FSO and S-FSO to FS, since
those activities were detectable when cytosol from both the liver and kidney was
used in the presence of 2-OH-PM. The reduction of R-FSO to FS was 17 times
higher than that of S-FSO to FS in liver microsomes, suggesting that aldehyde
oxidase predominantly reduced R-FSO to FS.
In the case of the thioredoxin-linked enzyme system, NADPH serves to
maintain thioredoxin in its reduced form, and thioredoxin, in turn, acts as a
hydrogen donor to a sulphoxide reductase (Anders et al. 1981). This thioredoxin
system can be eæciently replaced by a high concentration of DTT (Anders et al.
1980). In the present study, it is possible that the thioredoxin-linkedenzyme system
is also involved in the reduction of R-FSO and S-FSO to FS in the kidney.
The S-reduction of R-FSO and S-FSO to FS was con®rmed in vitro, even
although the level of reductase activity was relatively low in comparison with S-
oxidase activity. For example, sulindac reductase activity of aldehyde oxidase in the
guinea pig liver and that of the thioredoxin-linked enzyme system in rat kidney

824
E. Kashiyama et al.
"
cytosol (Anders et al. 1981, Tatsumi et al. 1983) were respectively
100 and 20
times higher than the ¯osequinan reductase activity seen in this study. It has been
reported that sulindac is mainly reduced in the organs, whereas sulphinpyrazone is
mainly reduced by gut ¯ora (Renwick et al. 1986). Flosequinan would likely be
mainly reduced by gut ¯ora rather than enzymes in the organs since the extent of
chiral interconversion when ¯osequinan was given to rat orally was greater than
when it was given intravenously (Kashiyama et al. 1994b, d). No reduction of FSO
to R-FSO or S-FSO was observed in the present study, supporting the ®nding that
#
FSO was not reduced when it was given to rat intravenously (Kashiyama et al.
1994b).
#
Liver microsomes catalysed four S-oxidation pathways in ¯osequinan metab-
olism. The results of the present study suggest that the S-oxidation of FS to R-FSO
is catalysed by both P450 and FMO, and that the other three S-oxidation reactions
are catalysed by P450 but not by FMO. Interestingly, microsomes from both the
kidney and lung exclusively catalysed the S-oxidation of FS to R-FSO. The
involvement of FMO was strongly implicated in the present study.
Eadie±Hofstee plot analysis indicated that the S-oxidation of FS to R-FSO in
liver microsomes was catalysed by at least two distinct enzymes. One is perhaps an
FMO and the other a P450. It is suggested that an FMO was responsible for the
high-aænity enzyme in S-oxidation since heat treatment caused the disappearance
}
of one of the two enzymes with a low Km in Eadie±Hofstee plots. V
Km for
"
max
"
}
to catalyse the S-oxidation of FS to R-FSO in the liver in vivo. This S-oxidation in
FMO was 20 times higher than V
Km . Therefore, FMO are considered mainly
#
max
#
kidney and lung microsomes is also suggested to be catalysed by FMO.
The present authors previously reported that rat hepatic FMO1A1 expressed in
yeast only catalysed the S-oxidation of FS to R-FSO (Kashiyama et al. 1994c). Km
for the reaction of FS to R-FSO seen with microsomes from the liver, kidney and
lung were all similar to that seen using rat FMO1A1 expressed in yeast (33 lm). The
presence of multiple FMO isoforms with diåerent catalytic properties has been
reported (Lawton and Philpot 1993, Ziegler 1993), and immunoblotand Northern
blot analyzes have suggested that one or more similarly structured FMO(s) are
expressed in microsomes from rat liver, kidney and lung (Itoh et al. 1993, Sausen et
al. 1993). Considering these ®ndings, one or more FMO(s) with the same or similar
catalytic properties are likely expressed in microsomes in rat liver, kidney and lung,
and they might catalyse the stereoselective S-oxidation of RS to R-FSO in these
organs. The S-oxidation pathway of FS to S-FSO in liver microsomes was also
shown to be catalysed by at least two distinct enzymes, indicating that isozymes of
P450 were responsible for the reaction of FS to S-FSO.
It has previously been reported that the plasma concentration of R-FSO was
higher than that of S-FSO when FS was given to rat either orally and intravenously
}
than that for S-oxidation of FS to S-FSO, indicating that FS was preferentially
(Kashiyamaet al. 1994b).V
Km for the S-oxidation of FS to R-FSO was higher
"
max
"
}
similar but lower than the V
oxidized to R-FSO. V
}
Km for the S-oxidation of FS to R-FSO and S-
FSO. These ®ndings suggest that the metabolism of R-FSO and S-FSO to FSO is
Km for S-oxidation of R-FSO and S-FSO to FSO were
#
max
max
"
"
#
the rate-limiting step. The results of the present in vitro study are consistentwith the
pharmacokinetic behaviour of ¯osequinan in vivo. Thus, the kinetic analysis of the
metabolism of drugs in vitro is expected to be useful in understanding in vivo
metabolism.

Stereoselective S-oxidation and reduction in rat
Acknowledgments
825
Part of the study was supported by a Grant-in-Aid from the Ministry of
Education, Science, and Culture of Japan.
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