Accepted Manuscript

Role of cytochrome P450 enzymes in fimasartan metabolism in vitro

Young Jae Choi, Ji-Yoon Lee, Chang Seon Ryu, Yong Ha Chi, Soo Heui Paik, Sang
Kyum Kim

PII: S0278-6915(18)30185-6
DOI: 10.1016/j.fct.2018.03.036
Reference: FCT 9672

To appear in: Food and Chemical Toxicology

Received Date: 13 October 2017

Revised Date: 14 March 2018
Accepted Date: 24 March 2018

Please cite this article as: Choi, Y.J., Lee, J.-Y., Ryu, C.S., Chi, Y.H., Paik, S.H., Kim, S.K., Role of
cytochrome P450 enzymes in fimasartan metabolism in vitro, Food and Chemical Toxicology (2018), doi:
10.1016/j.fct.2018.03.036.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT

Role of cytochrome P450 enzymes in fimasartan metabolism in vitro

Young Jae Choi1*, Ji-Yoon Lee1*, Chang Seon Ryu1, Yong Ha Chi2, Soo Heui Paik3, and Sang

Kyum Kim1

PT
1
College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea
2
Central Research Institute, Boryung Pharm. co., Ltd. Ansan, Gyeonggi 425-839, Republic of

RI
Korea
3
College of Pharmacy, Sunchon National University, Suncheon-si, Republic of Korea

SC
1

*
These authors contributed equally to this work.

Correspondence
U
AN
Sang Kyum Kim, Ph.D.: College of Pharmacy, Chungnam National University, 220 Gung-

dong, Yuseong-gu, Daejeon 305-764, Republic of Korea

M

Tel.: +82-42-821-5930. Fax: +82-42-823-6566. E-mail: sangkim@cnu.ac.kr.

D
TE
C EP
AC

1
Abbreviations: FMS, fimasartan; ARB, angiotensin II receptor blocker; HLM, human liver
microsomes; UGT, UDP-glucuronosyltransferase; CYP, cytochrome P450; FMO, flavin-
containing monooxygenase; Km, Michaelis–Menten constant; kcat, turnover number; MRM,
multiple reaction monitoring
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

1. Introduction

Fimasartan (FMS), the ninth angiotensin II receptor blocker (ARB), has been used for the

treatment of hypertension (Kim et al., 2012). Several clinical studies have been conducted on

the efficacy and safety of FMS (Lee et al., 2012; Lee et al., 2016a; Park et al., 2013), and to

PT
determine interactions with other concomitant drugs (Gu et al., 2012; Jeon et al., 2012). FMS

has been demonstrated to effectively and safely control hypertension in clinical studies.

RI
Recently, it has been suggested that FMS ameliorates nonalcoholic fatty liver disease in an

SC
animal model (Lee et al., 2017) and suppresses inflammatory response in astrocytes

stimulated by hemolysate (Yang et al., 2016).

U
In metabolism and pharmacokinetic studies using rats treated with radiolabeled FMS,
AN
oxidative desulfurization, N-glucuronidation, mono-oxygenation of pyrimidinone,

hydroxylation of the n-butyl group, and de-N-dimethylation were the major metabolic
M

pathways of FMS (Lee et al., 2011; Kim et al., 2014). In our previous study using authentic
D

metabolite standards, S-oxidation and N-glucuronidation were identified as the main

metabolic products of FMS in humans (Lee et al., 2016b). In addition, FMS S-oxide was
TE

proposed as a metabolic intermediate of BR-A-557 produced by oxidative desulfurization,

EP

and BR-A-535 produced by further metabolism of de-N-dimethylation, based on the results

of an in vitro kinetic study (Lee et al., 2016b). Moreover, 1-, 2-/ 3-, and 4-hydroxy-n-butyl
C

FMS was observed in pooled human liver microsomes (HLM). Recently, it was reported that
AC

UDP-glucuronosyltransferase (UGT) 1A3 was the major UGT isoform responsible for N-

glucuronidation of FMS (Jeong et al., 2015). However, characterization of the enzymes

responsible for the NADPH-dependent formation of FMS metabolites remains to be

characterized. Although cytochrome P450 (CYP) enzymes play a major role in xenobiotic

metabolism, heteroatom oxidation such as S-oxidation can be catalyzed by flavin-containing

1
 ACCEPTED MANUSCRIPT
monooxygenase (FMO) enzymes (Cashman, 2008).

The purpose of this study was to characterize enzymes involved in the NADPH-

dependent metabolism of FMS using in vitro systems. To identify the specific enzymes

responsible for FMS metabolism, metabolic screening was performed using recombinant

human CYP and FMO enzymes, and an inhibition study was conducted using pooled HLM

PT
pretreated with selective chemical inhibitors. The concentration–time profiles of FMS and its

RI
metabolites were determined for evaluation of the rates of FMS elimination and metabolite

formation. Moreover, the kinetic parameters of enzymes responsible for FMS metabolism

SC
were calculated to predict FMS metabolism in vivo using the Michaelis-Menten model.

U
AN
M
D
TE
C EP
AC

2
 ACCEPTED MANUSCRIPT

2. Materials and Methods

2.1. Chemicals and reagents

FMS potassium trihydrate (2-n-butyl-5-dimethylaminothiocarbonylmethyl-6-methyl-3-{[2-

PT
(1H-tetrazole-5-yl)biphenyl-4-yl]methyl}pyrimidine-4(3H)-one potassium salt trihydrate),

oxidative desulfurized metabolite of FMS (BR-A-557), 1-hydroxy-n-butyl FMS (1-OH butyl

RI
FMS), 2-hydroxy-n-butyl FMS (2-OH butyl FMS), 3-hydroxy-n-butyl FMS (3-OH butyl

SC
FMS), 4-hydroxy-n-butyl FMS (4-OH butyl-FMS), oxidative desulfurization and de-N-

dimethylation metabolite of FMS (BR-A-535), FMS S-oxide, and BR-A-563 (internal

U
standard) were supplied by Boryung Pharm. Co., Ltd. (Seoul, Korea). The nuclear magnetic
AN
resonance data for FMS and its metabolites were reported in our previous study (Lee et al.,

2016b). Glucose 6-phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PDH),

M

β-nicotinamide adenine dinucleotide phosphate hydrate (NADP+), formic acid, furafylline,

D

triethylenethiophosphoramide, sulfaphenazole, N-3-benzylnivanol, quinidine,

TE

diethyldithiocarbamate, ketoconazole, and MgCl2 were purchased from Sigma-Aldrich (St.

Louis, MO, USA). Pooled human liver microsomes (150 donors, mix; Catalog No. 452117),
EP

CYP supersomes 1A2 (Catalog No. 456203), 2A6 (Catalog No. 456254), 2B6 (Catalog No.

456255), 2C8 (Catalog No. 456252), 2C9*1(Arg144, Catalog No. 456218), 2C19 (Catalog
C

No. 456259), 2D6*1 (Catalog No. 456217), 2E1 (Catalog No. 456206), 3A4 (Catalog No.
AC

456202) 3A5 (Catalog No. 456256), FMO1 (Catalog No. 409901), FMO3 (Catalog No.

4168003), FMO5 (Catalog No. 4014002) and Mock (Catalog No. 456200), were purchased

from Corning Inc. (Corning, NY, USA). Twenty-donor pooled human hepatocytes (Catalog

No. HPCH20/1110111) and human CYP supersomes 1A1 (Catalog No. CYP/EZ014) were

purchased from Sekisui XenoTech (XenoTech, KS, USA). Tranylcypromine and montelukast

3
 ACCEPTED MANUSCRIPT
were purchased from SantaCruz (Dallas, USA). All other reagents and chemicals were of

analytical or high performance liquid chromatography (HPLC) grade.

2.2. Preparation of stock and working solutions

Stock solutions (10 mM) of FMS and its metabolites were prepared by dissolving in

PT
dimethyl sulfoxide (DMSO). Stock standard solutions were stored at -20°C. Mixed working

RI
standard solutions were prepared by dilution of the stock standards with a mixture of

acetonitrile and water (1:1, v/v). The working standard solution was serially diluted to

SC
prepare a concentration of 0.975, 3.9, 15.6, 62.5, 250, 1,000 and 4,000 nM in 0.1 M

potassium phosphate buffer (pH 7.4). The final concentrations of DMSO in the reaction

U
mixture were less than 0.3% except for experiments for determination of Km and kcat of FMS.
AN
In these experiments, the concentration of DMSO was approximately 0.725%.
M

2.3. Metabolism of FMS in pooled human hepatocytes

D

Reactions were conducted in triplicate in pooled human hepatocytes treated with 10 µM

TE

FMS for 120 min. FMS was incubated with 0.5 x 106 cells of pooled human hepatocytes in a

final volume of 200 µL. The procedure for thawing pooled human hepatocytes followed the
EP

protocol provided by the manufacturer. The treated cells were incubated at 37°C in a CO2
C

incubator. Reactions were initiated by addition of FMS and were then quenched by adding
AC

400 µL of ice-cold acetonitrile containing 100 nM BR-A-563 as an internal standard. The

samples were centrifuged at 1,000 × g for 20 min at 4 °C and the supernatants were subjected

to liquid chromatography–tandem mass spectrometry (LC-MS/MS).

2.4. Metabolism of FMS in recombinant CYP and FMO enzymes

4
 ACCEPTED MANUSCRIPT
Reactions were conducted in triplicate in 0.1 M potassium phosphate buffer (pH 7.4) in

eight-well tube strips in an 8 × 12 rack (1.2 mL; VWR, Emeryville, CA, USA). To perform

metabolic screening, 1 µM FMS was incubated with 50 pmol/mL of each recombinant human

enzyme (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, FMO1, FMO3, FMO5,

and mock) in the presence of an NADPH regenerating system (1 mM NADP+, 5 mM G6P,

PT
1 U/mL G6PDH) in a final volume of 200 µL for 120 min. The mixture was pre-incubated at

RI
37°C in a shaking incubator at approximately 100 rpm for 5 min. Reactions were initiated by

addition of the NADPH-regenerating system and were then quenched by adding 200 µL of

SC
ice-cold acetonitrile containing 100 nM BR-A-563 as an internal standard. The samples were

centrifuged at 1,000 × g for 20 min at 4 °C and the supernatants were subjected to LC-

U
AN
MS/MS.
M

2.5. Inhibition of CYP and FMO enzymes in HLM

Additional experiments were designed to compare the contribution of CYP and FMO
D

enzymes to FMS metabolism in HLM. Reactions were conducted in triplicate in 0.1 M

TE

potassium phosphate buffer (pH 7.4). To inhibit FMO enzymes, HLM were preheated at

45 °C for 5 min. The preheated 1 mg/mL HLM were incubated with 1 µM FMS for 10 min in
EP

the presence of 1 mM NADPH. To inhibit CYP enzymes, HLM were pretreated with 1-
C

aminobenzotriazole, a pan CYP inhibitor, for 30 min at 37 °C. The pretreated 1 mg/mL HLM
AC

were incubated with 1 µM FMS for 10 min in the presence of 1 mM NADPH. The reaction

was terminated and centrifuged using the same method described above.

2.6. Chemical inhibition studies

Reactions were conducted in triplicate in 0.1 M potassium phosphate buffer (pH 7.4) in

eight-well tube strips in an 8 × 12 rack (1.2 mL; VWR). Ten micromoles of FMS were
5
 ACCEPTED MANUSCRIPT
incubated with 1 mg/mL HLM, 1 mM NADPH, and each chemical inhibitor in a final volume

of 200 µL for 10 min. Specific CYP inhibitors were furafylline (10 µM; CYP1A2),

tranylcypromine (0.2 µM; CYP2A6), triethylenethiophosphoramide (50 µM; CYP2B6),

montelukast (0.1 µM; CYP2C8), sulfaphenazole (10 µM; CYP2C9), N-3-benzylnirvanol (2

µM; CYP2C19), quinidine (2 µM; CYP2D6), diethyldithiocarbamate (50 µM; CYP2E1), and

PT
ketoconazole (2 µM; CYP3A4). The specific CYP inhibitors and their concentrations were

RI
decided based on previous studies (Kim et al., 2006; Vermeir et al., 2009; Wójcikowski et al.,

2010). Reactions were initiated by addition of NADPH. After 10 min incubation, the reaction

SC
was terminated and centrifuged using the same method described above.

U
2.7. Determination of time profiles and kinetic parameters of FMS metabolites in
AN
recombinant CYP isoforms
M

To determination the time profile of FMS metabolism, 1 µM FMS was incubated with 50

pmol/mL of each recombinant CYP2C9, 3A4, and 3A5, in the presence of an NADPH
D

regenerating system in a final volume of 200 µL, for 0, 5, 10, 15, 30, or 60 min. To determine
TE

kinetic parameters, various concentrations (0, 0.11, 0.33, 1, 3, 9, 27, 81, 243, and 729 µM) of

FMS were incubated with 50 pmol/mL of each recombinant CYP2C9, 3A4, or 3A5 enzyme.
EP

FMS and recombinant CYP were added to 0.1 M potassium phosphate buffer (pH 7.4) and
C

the mixture was pre-incubated at 37°C in a shaking incubator at approximately 100 rpm for 5
AC

min. Reactions were initiated by the addition of an NADPH-regenerating system. The

reaction was terminated and centrifuged using the same method described above.

2.8. LC-MS/MS analysis

To identify and quantify FMS and its metabolites, the samples were analyzed using a

Prominence ultrafast liquid chromatography (UFLC) system with a parallel LC-20ADXR

6
 ACCEPTED MANUSCRIPT
pump, autosampler, and column oven (Shimadzu, Kyoto, Japan). The sample injection

volume was 10 µL, and separation was performed on an Acquity UPLC HSS dC18 column

(2.1 × 100 mm i.d., 1.8 µm; Waters, Milford, MA, USA) with a SecurityGuard C18 guard

column (2.0 × 4.0 mm i.d.; Phenomenex, Torrance, CA, USA) maintained at 30°C. The

HPLC flow rate was set at 0.3 mL/min. HPLC mobile phases consisted of A (deionized water

PT
containing 0.1% (v/v) formic acid) and B (acetonitrile containing 0.1% (v/v) formic acid). A

RI
linear gradient of the two solvents was used (0 to 1 min, 100% A; 1.1 min, 50% A; 4 min,

50% A; 4.1 min, 5% A; 6.0 min, 5% A; 6.01 min, 100% A). Among the hydroxylated

SC
metabolites in the n-butyl group, 2-OH butyl and 3-OH butyl FMS were not separated on the

LC and produced the same major fragment ions in tandem mass spectra. Thus, 2-OH and 3-

U
OH butyl FMS were not distinguishable with the analytical methods used in this study. The
AN
peak intensities of 2-OH butyl FMS and 3-OH butyl FMS determined using the Multiple
M

reaction monitoring (MRM) mode was comparable, and the same amount of 2-OH and 3-OH

butyl FMS mixture was used as a standard for quantification of 2- and 3-OH butyl FMS,
D

respectively.
TE

LC-MS/MS data were acquired with an Applied Biosystems SCIEX 3200 QTRAP hybrid

triple quadrupole-linear ion trap mass spectrometer (Foster City, CA, USA) equipped with a
EP

TurboIonSpray interface operating in the positive electrospray ionization mode. Acquisition

and analysis of data were performed with AnalystTM software (ver. 1.5.2; Applied
C
AC

Biosystems). The 3200 QTRAP was calibrated semiannually using a polypropylene glycol

solution for mass accuracy. To optimize source parameters, such as the declustering potential

and collision energy, FMS was used. Calibration standards (1 to 4000 nM) were prepared in

blank matrices pre-treated with ice-cold acetonitrile containing 100 nM BR-A-563.

Calibration curves constructed using linear-squares regression were linear over the

concentration range of the standards used (r2 >0.999). The relative standard deviation (RSD)

7
 ACCEPTED MANUSCRIPT
of the measured concentrations was used to assess precision. The mean measured

concentration was compared with the corresponding nominal concentration to assess

accuracy. Both the accuracy (80–120%) and precision (RSD <20%) of the assay were

acceptable. The estimated limit of quantification was less than 0.75 nM.

PT
2.9. Data analysis

RI
For all LC-MS/MS analyses, the peak areas of the parents and metabolites were expressed

as a ratio to the internal standard peak area for each concentration of test substance. Data

SC
were expressed as the mean ± SD for triplicate samples. The apparent kinetic parameters of

FMS metabolism were determined by fitting a one-enzyme Michaelis–Menten equation. The

U
kinetic parameters were estimated by plotting the activities over the logarithm of FMS
AN
concentration with GraphPad Prism 5 for Windows, version 5.01 (GraphPad Software Inc.,
M

San Diego, CA, USA).

D
TE
C EP
AC

8
 ACCEPTED MANUSCRIPT

3. Results

3.1. Metabolism of FMS in recombinant human CYP and FMO enzymes.

In our previous study using authentic standards, FMS S-oxide, BR-A-557, BR-A-535, 1-OH

butyl FMS, 2- or 3-OH butyl FMS and FMS N-glucuronide were observed in pooled human

PT
plasma (Lee et al., 2016b). To confirm metabolism of FMS, its metabolites were determined

in pooled human hepatocytes treated with 10 µM FMS for 120 min. FMS S-oxide (41.2 nM)

RI
was the most abundant metabolite in pooled human hepatocytes followed in decreasing order

SC
by FMS N-glucuronide (20.4 nM), 1-OH butyl FMS (5.5 nM), BR-A-557 (4.5 nM), 2- or 3-

OH butyl FMS (2.0 nM), 4-OH butyl FMS (1.8 nM) and BR-A-535 (1.0 nM). The results are

U
generally consistent with our previous results obtained from HLM and pooled human plasma
AN
(Lee et al., 2016b).

To determine the enzymes involved in NADPH-dependent FMS metabolism, each human

M

recombinant CYP and FMO enzyme was incubated with 1 µM FMS for 120 min and
D

analyzed using LC-MS/MS in MRM mode (Figure 1). The concentration of FMS observed in
TE

recombinant CYP2C9, CYP3A4, and CYP3A5 was decreased to 30.2%, 10.7%, and 26.8%,

respectively, relative to that of control (Figure 1A). The incubation of FMS with the other
EP

CYP isoforms and FMO enzymes resulted in a less than 10% decrease, indicating that these

enzymes were not significantly involved in FMS metabolism. FMS S-oxide was the major
C

metabolite in CYP2C9 (339 ± 1 nM), CYP3A4 (434 ± 35 nM), and CYP3A5 (582 ± 19 nM)
AC

(Figure 1B). CYP2C8, CYP2C19, CYP2D6, and FMOs exhibited marginal activity for S-

oxidation. BR-A-557, an oxidative desulfurized metabolite, was produced in CYP3A4 (360 ±

27 nM), followed in decreasing order by CYP3A5 (96 ± 5 nM) and CYP2C9 (12 ± 1 nM)

(Figure 1C). CYP3A4 was exclusively involved in the formation of BR-A-535, a metabolite

produced by oxidative desulfurization and de-N-dimethylation (Figure 1D). The formation of

9
 ACCEPTED MANUSCRIPT
1-OH butyl FMS was mainly mediated by CYP2C9, and to a lesser extent, CYP3A4 (Figure

1E). Hydroxylation of the 2- or 3- and 4-n-butyl groups was exclusively mediated by

CYP2C9 (Figure 1F and G), although other CYP2C subfamily enzymes, including CYP2C8

and CYP2C19, had marginal activity for the formation of 2- or 3-OH butyl FMS.

To confirm the results using recombinant enzymes, additional experiments were performed

PT
using HLM pretreated with 2.5 mM 1-aminobenzotriazole, a pan-CYP inhibitor, and HLM

RI
heated at 45°C for 5 min to inhibit FMO (Figure 2). FMS disappearance and FMS S-oxide

formation were not significantly different between HLM heated at 45°C for 5 min and HLM

SC
heated at 37°C for 5 min (control group). In contrast, the addition of 1-aminobenzotriazole

markedly inhibited both FMS disappearance and FMS S-oxide formation in HLM. 1-

U
minobenzotriazole at 2.5 mM decreased the activity of nine CYPs, including CYP1A1, 2A6,
AN
2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4, to less than 20% of control (data not shown).
M

3.2. Effects of selective CYP inhibitors on the metabolism of FMS in HLM

D

The contribution of individual CYP isoforms to FMS biotransformation in HLM was

TE

evaluated using selective chemical inhibitors (Figure 3). The formation of the metabolites

from FMS in HLM increased in a protein concentration (0.25 to 1 mg/ml) and incubation
EP

time (5 to 30 min) dependent manner (data not shown). The formation of FMS S-oxide
C

(Figure 3A) and BR-A-557 (Figure 3B) was markedly decreased, and BR-A-535 was not
AC

detectable in HLM pretreated with 2 µM ketoconazole, a selective CYP3A4/5 inhibitor

(Figure 3C). All n-butyl hydroxylated metabolites were decreased to less than half of control

in HLM pretreated with 10 µM sulfaphenazole, a selective CYP2C9 inhibitor (Figure 3D, E,

and F). Among the n-butyl hydroxylated metabolites, 1-OH butyl FMS was inhibited by

ketoconazole to 60% of control activity. These results demonstrate the major role of

CYP3A4/5 and CYP2C9 in S-oxidation and n-butyl hydroxylation, respectively. All other

10
 ACCEPTED MANUSCRIPT
inhibitors decreased the metabolite formation to less than 25%, except for

diethyldithiocarbamate, a selective CYP2E1. The formation of BR-A-557 was reduced to

68.7% in HLM pretreated with 50 µM diethyldithiocarbamate relative to control activity.

3.3. Time profiles of FMS metabolism in recombinants CYP 2C9, 3A4, and 3A5

PT
To determine time profiles of FMS metabolism, FMS and its metabolites were measured in

RI
50 pmol/mL recombinant CYP2C9, 3A4, and 3A5 incubated with 1 µM FMS for 0, 5, 10, 15,

30, or 60 min (Figure 4). The formation of FMS S-oxide linearly increased with increasing

SC
protein concentration of recombinant CYP2C9, 3A4 and 3A5 (12.5 to 50 pmol/ml) (data not

shown).

U
The half-life (95% confidence interval) of FMS in recombinant CYP2C9, CYP3A4, and
AN
CYP3A5 was 26.5 (20.9 to 36.1), 13.7 (11.1 to 17.6), and 31.3 (26.3 to 38.6) min,
M

respectively. FMS S-oxide was the major metabolite for all recombinant CYP enzymes. The

formation rate of FMS S-oxide by recombinant CYP2C9, CYP3A4, and CYP3A5 determined
D

at 5 min was 0.25, 0.98, and 0.47 min-1, respectively. The formation rate of FMS S-oxide was
TE

decreased as the incubation time was increased. The decrease in the FMS S-oxide formation

rate may be attributed to the formation of BR-A-557 from FMS S-oxide, as well as substrate
EP

depletion. The metabolic rates of the other metabolites were calculated from the slopes of the
C

regression lines. The metabolic rate of BR-A-557, the second major metabolite in CYP3A4

and CYP3A5 recombinants, was 0.107 ± 0.008 and 0.016 ± 0.001 min-1, respectively. The
AC

metabolic rate of BR-A-535, produced only by CYP3A4, was 0.016 ± 0.001 min-1. The 1-OH

butyl FMS was the metabolite with the lowest level in CYP2C9 and CYP3A4. The formation

rate of 1-OH butyl FMS by CYP2C9 was 0.007 ± 0.001 min-1, and its formation rate by

CYP3A4 was not calculated due to the absence of a linear portion of the graph. The

formation rate of 2- or 3-OH butyl FMS and 4-OH butyl FMS produced by CYP2C9 was

11
 ACCEPTED MANUSCRIPT
0.065 ± 0.003 and 0.048 ± 0.001 min-1, respectively.

In additional experiments, the time profiles of FMS S-oxide metabolism were determined in

1 mg/mL HLM incubated with 1 µM FMS S-oxide for 0, 5, 10, 30, 60, and 120 min. More

than 72% of FMS S-oxide remained after the 120-min incubation relative to control at zero

time, showing that FMS S-oxide is more metabolically stable than FMS in HLM (data not

PT
shown). BR-A-557 and BR-A-535 had formation rates of 1.031 ± 0.049 and 0.189 ± 0.011

RI
pmol/min/mg protein, respectively (data not shown).

SC
3.4. Kinetic parameters of FMS metabolism in recombinant CYP 2C9, 3A4 and 3A5

Enzyme kinetic analysis was performed in 50 pmol/mL recombinant CYP2C9, CYP3A4,

U
and 3A5 with various concentrations of FMS for 10 min (Figure 5 and Table 1). The
AN
formation of metabolites, except for FMS S-oxide and 1-OH butyl FMS in recombinant
M

CYP2C9, increased hyperbolically as the FMS concentration increased. FMS S-oxidation by

CYP2C9 exhibited substrate inhibition behavior when the FMS concentration was more than
D

10-fold of its Michaelis–Menten constant (Km), and hydroxylation of the 1-n-butyl group may
TE

be saturated at an FMS concentration of 729 µM (Figure 5A). The value of the specificity

constant (turnover number/Michaelis–Menten constant; kcat/Km) of S-oxidation was more

EP

than four-fold higher than the other reactions in all recombinant enzymes tested in this study
C

(Table 1). The kcat/Km values of 2- or 3-butyl and 4-butyl hydroxylation in recombinant
AC

CYP2C9 were comparable with that of 1-OH butyl FMS, and less than 22% relative to the

other hydroxylation reactions. The kcat/Km value of BR-A-557 determined in recombinant

CYP3A4 was approximately ten-fold relative to BR-A-535.

12
 ACCEPTED MANUSCRIPT

4. Discussion

Reaction phenotyping studies to identify specific enzymes involved in drug metabolism

are critical for the prediction of drug-drug interactions that can result in altered levels of

exposure to a substrate drug. In addition, reaction phenotyping can provide useful

PT
information for predicting pharmacokinetic and pharmacodynamic inter-individual variability.

In this study, using recombinant enzymes, CYP but not FMO enzymes were found to play a

RI
major role in FMS elimination and the formation of metabolites, including the major

SC
metabolite, FMS S-oxide. The role of CYP enzymes in FMS metabolism was confirmed in

additional experiments using HLM pretreated with a pan-CYP inhibitor, 1-

U
aminobenzotriazole, and HLM heated to inhibit FMO. FMO enzymes reportedly play an
AN
important role in heteroatom oxidation (Cashman, 2008). S-Oxidation of xenobiotics,

including ranitidine (Chung et al., 2000) and sulindac sulfide (Hamman et al., 2000), is also
M

catalyzed by FMO. In contrast, the S-oxidation of clindamycin (Wynalda et al., 2003) and
D

flosequinan (Kashiyama et al., 1997) is mediated by CYP3A. Both CYP and FMO enzymes

are involved in the S-oxidation of albendazole (Rawden et al., 2000) and tazarotenic acid
TE

(Attar et al., 2003). Thus, the physicochemical properties of drugs may be one of the
EP

determinants of their metabolizing enzymes.

Hepatic metabolism of ARB drugs may produce pharmacologically more active metabolites
C

than parent drug. For example, the carboxylic acid metabolite (E 3174) of losartan is more
AC

than 10-fold potent than parent compound (Sica et al., 2005). Inhibitory activities of FMS,

FMS S-oxide and BR-A-557 against angiotensin II-induced contraction in rabbit aorta were

determined. The IC50 values of FMS, FMS S-oxide and BR-A-557 were 0.18, 8.9 and 4.4 nM,

respectively. These results show that CYP-mediated metabolism of FMS is metabolic

inactivation.

13
 ACCEPTED MANUSCRIPT
In experiments using recombinant enzymes and selective chemical inhibitors, CYP2C9,

CYP3A4, and CYP3A5 were found to be highly involved in FMS metabolism. CYP2C9,

CYP3A4, and CYP3A5 played a role in the elimination of FMS, with half-lives of 26.5, 13.7,

and 31.3 min, respectively, measured at 50 pmol/mL concentration. Recombinant CYP2C9

enzyme catalyzed the formation of FMS S-oxide, 1-, 2- or 3-, and 4-OH butyl FMS with

PT
kcat/Km values of 0.21, 0.0076, 0.041, and 0.035 µM-1·min-1, respectively. FMS S-oxide and

RI
BR-A-557 were produced by recombinant CYP3A4 and CYP3A5 with kcat/Km values of 0.34

and 0.048, and 0.19 and 0.015 µM-1·min-1, respectively. Recombinant CYP3A4 played an

SC
exclusive role in the formation of BR-A-535, with a kcat/Km value of 0.0048 µM-1·min-1. In

vitro experiments to determine kinetic parameters, especially Km and Vmax or kcat, provide

U
important information for evaluating the drug-drug interaction and drug disposition. Various
AN
modeling studies can predict potential clinical drug-drug interactions and human exposure
M

using these kinetic parameters. In additional experiments, BR-A-557 and BR-A-535 were

produced in HLM incubated with FMS S-oxide, and the formation rates of these metabolites
D

were approximately two-fold of those in HLM incubated with FMS (Lee et al., 2016b). These
TE

results do not rule out the possibility that FMS can be directly metabolized to BR-A-557 or

BR-A-535; however, these data demonstrate that FMS S-oxide is an intermediate of these
EP

metabolites.
C

It is noteworthy that out of all the CYP and FMO enzymes tested, CYP2C9 is exclusively
AC

involved in the hydroxylation of FMS 2- or 3- and 4-n-butyl groups. This suggests that the

hydroxylation of FMS may be used in selective reactions for evaluating CYP2C9 activity.

The kcat/Km values of these reactions were compared with those of the other CYP2C9

selective reactions measured in a previous study (Kimar et al., 2006). Based on specificity

constant (kcat/Km) values, the hydroxylation of 2- or 3- and 4-n-butyl FMS groups are

comparable to tolbutamide 4-hydroxylation and S-warfarin 7-hdyroxylation, and are lower

14
 ACCEPTED MANUSCRIPT
than diclofenac 4'-hydroxylation and S-flurbiprofen 4-hydroxylation. Moreover, the

simultaneous determination of FMS S-oxide, BR-A-557, BR-A-535, and n-butyl

hydroxylated metabolites may provide further information on the activities of CYP3A4/5, as

well as CYP2C9.

The contribution of CYP2C9 and CYP3A4, the major CYPs, to the total hepatic CYP pools

PT
is comparable, as determined by LC-MS/MS (Kawakami et al, 2011; Ohtsuki et al., 2012;

RI
Gröer et al., 2014). CYP2C9 and CYP3A4 values ranged from 37.3 to 80.2 and from 32.6 to

64.0 pmol/mg in pooled HLM, respectively. CYP3A5 is present in concentrations equal to or

SC
less than 5% of CYP3A4, and ranged from 1.96 to 3.86 pmol/mg in pooled HLM. The

contribution of CYP2C9, CYP3A4, and CYP3A5 to the formation of FMS metabolites in

U
HLM was simulated using the Michaelis-Menten equation and the absolute protein levels.
AN
The contribution of CYP2C9 and CYP3A5 to the formation of FMS S-oxide was 63.0 to
M

56.6% and 2.8 to 3.0%, respectively, of that of CYP3A4, as the FMS concentration was

increased from 0 to 10 µM. The contribution of CYP3A5 to the formation of BR-A-557 is

D

less than 2% of that of CYP3A4.

TE

The CYP2C9 enzyme plays a major role in the hydroxylation of FMS n-butyl groups, and is

also involved in the formation of FMS S-oxide. CYP3A4 is mainly involved in the
EP

elimination of FMS and formation of FMS S-oxide, BR-A-557, and BR-A-535. CYP3A5
C

may play a minor role in FMS metabolism due to low protein expression in the liver,
AC

although the contribution of CYP3A5 may be substantial in patients with low expression of

CYP3A4 (Ragia et al., 2016). CYP2C9, CYP3A4, and CYP3A5 are frequently co-regulated

by ligands of nuclear receptors, including constitutive androstane receptor, pregnane X

receptor, glucocorticoid receptor, and bile acid receptor (Wallace and Redinbo, 2013; Zanger

and Schwab, 2013). Thus, these CYP enzymes can be induced by the ligands of these

receptors, such as barbiturates, carbamazepine, rifampicin, and atorvastatin (Wallace and

15
 ACCEPTED MANUSCRIPT
Redinbo, 2013). In contrast, CYP2C9 and CYP3A4/5 are inhibited by a number of

xenobiotics, including naturally occurring compounds as well as medicines (Liu et al., 2007;

Zanger and Schwab, 2013). Moreover, the gene coding for CYP2C9 is highly polymorphic;

therefore, changes in metabolic activity due to genetic variation in CYP2C9 play an

important role in adverse drug reactions, especially for CYP2C9-selective drugs with a low

PT
therapeutic index (Hiratsuka, 2016). In addition, the expression and activity of CYP3A4

RI
shows wide inter-individual variation (Kawakami et al, 2011; Ohtsuki et al., 2012; Gröer et

al., 2014), influencing both the pharmacological and toxicological effects of CYP3A4-

SC
substrate drugs.

In summary, these results suggest a major role for CYP, but not FMO, enzymes in FMS

U
metabolism. This was also confirmed in additional experiments using HLM with either CYP
AN
or FMO inhibition. The results from the experiments using recombinant enzymes and
M

selective chemical inhibitors showed that CYP2C9, CYP3A4, and CYP3A5 were involved in

FMS elimination and the formation of FMS S-oxide, the major metabolite of FMS. CYP2C9
D

played an exclusive role in the hydroxylation of FMS n-butyl groups. FMS S-oxide is an
TE

intermediate of BR-A-557, and BR-A-537 is produced by CYP3A4/5. These results suggest

that further research is needed on the influence of co-administered drugs on FMS metabolism
EP

due to inhibition or stimulation of CYP3A4/5 and 2C9 enzymes. Moreover, the kinetic
C

parameters determined in this study, such as kcat and Km, can be used to predict FMS
AC

metabolism in vivo. In addition, simultaneous determination of FMS S-oxide, BR-A-557, BR-

A-535, and n-butyl hydroxylated metabolites may be useful to evaluate CYP2C9 and

CYP3A4/5 activities.

16
 ACCEPTED MANUSCRIPT

Acknowledgments

This work was supported by the Ministry of Education of the Republic of Korea and the

National Research Foundation of Korea (2016R1A2B4008382).

PT
RI
U SC
AN
M
D
TE
C EP
AC

17
 ACCEPTED MANUSCRIPT

5. Reference

Attar, M., Dong, D., Ling, K.H., Tang-Liu, D.D., 2003. Cytochrome P450 2C8 and flavin-

containing monooxygenases are involved in the metabolism of tazarotenic acid in humans,

Drug Metab. Dispos. 31(4), 476-481.

PT
Cashman, JR., 2008. Role of flavin-containing monooxygenase in drug development. Expert

RI
Opin. Drug Metab. Toxicol. 4(12), 1507-21.

SC
Chung, W.G., Park, C.S., Roh, H.K., Lee, W.K., Cha, Y.N., 2000. Oxidation of ranitidine by

U
isozymes of flavin-containing monooxygenase and cytochrome P450. Jpn. J. Pharmacol.
AN
84(2), 213-20.
M

Gröer, C., Busch, D., Patrzyk, M., Beyer, K., Busemann, A., Heidecke, CD., Drozdzik, M.,
D

Siegmund, W., Oswald, S., 2014. Absolute protein quantification of clinically relevant

cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based

TE

targeted proteomics. J. Pharm. Biomed. Anal. 100, 393-401.

EP

Gu, N., Kim, B.H., Lim, K.S., Kim, S.E., Nam, W.S., Yoon, S.H., Cho, J.Y., Shin, S.G., Jang,
C

I.J., Yu, K.S., 2012. The effect of fimasartan, an angiotensin receptor type 1 blocker, on the
AC

pharmacokinetics and pharmacodynamics of warfarin in healthy Korean male volunteers: a

one-sequence, two-period crossover clinical trial. Clin. Ther. 34, 1592–1600.

Hamman, M.A., Haehner-Daniels, B.D., Wrighton, S.A., Rettie, A.E., Hall, S.D., 2000.

Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases.

18
 ACCEPTED MANUSCRIPT

Comparison of human liver and kidney microsomes and mammalian enzymes. Biochem.

Pharmacol. 60(1), 7-17.

Hiratsuka, M., 2016. Genetic Polymorphisms and in Vitro Functional Characterization of

PT
CYP2C8, CYP2C9, and CYP2C19 Allelic Variants. Biol. Pharm. Bull. 39(11), 1748-1759.

RI
Jeon, H., Lim, K.S., Shin, K.H., Kim, J., Yoon, S.H., Cho, J.Y., Shin, S.G., Jang, I.J., Yu, K.S.,

SC
2012. Assessment of the drug-drug interactions between fimasartan and hydrochlorothiazide

in healthy volunteers. J. Cardiovasc. Pharmacol. 59, 84–91.

U
AN
Jeong, E.S., Kim, Y.W., Kim, H.J., Shin, H.J., Shin, J.G., Kim, K.H., Chi, Y.H., Paik, S.H.,

Kim, D.H., 2015. Glucuronidation of fimasartan, a new angiotensin receptor antagonist, is

M

mainly mediated by UGT1A3. Xenobiotica 45, 10-18.

D

Kashiyama, E., Yokoi, T., Odomi, M., Funae, Y., Inoue, K., Kamataki, T., 1997. Cytochrome
TE

P450 responsible for the stereoselective S-oxidation of flosequinan in hepatic microsomes

EP

from rats and humans. Drug Metab. Dispos. 25(6), 716-24.

C

Kawakami, H., Ohtsuki, S., Kamiie, J., Suzuki, T., Abe, T., Terasaki, T., 2011. Simultaneous
AC

absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid

chromatography tandem mass spectrometry with in silico target peptide selection. J. Pharm.

Sci. 100(1), 341-52.

19
 ACCEPTED MANUSCRIPT

Kim, H.J., Yoon, Y.J., Kim, H.M., Kang, S.I., Cheon, H.G., Yoo, S.E., Shin, J.G., Liu, K.H.,

2006. Characterization of the cytochrome P450 enzymes involved in the metabolism of a new

cardioprotective agent KR-33028. Toxicol. Lett. 166(2), 105-114.

PT
Kim, J.H., Lee, J.H., Paik, S.H., Kim, J.H., Chi, Y.H., 2012. Fimasartan, a novel angiotensin

II receptor antagonist. Arch. Pharm. Res. 35, 1123–6.

RI
SC
Kim, T.H., Shin, S., Bashir, M., Chi, Y.H., Paik, S.H., Lee, J.H., Choi, H.J., Choi, J.H., Yoo,

S.D., Bulitta, J.B., Ma, E., Joo, S.H., Shin, B.S., 2014. Pharmacokinetics and metabolite

U
profiling of fimasartan, a novel antihypertensive agent, in rats. Xenobiotica 44, 913-925.
AN
Lee, H.W., Lim, M.S., Seong, S.J., Lee, J., Park, J., Seo, J.J., Cho, J.Y., Yu, K.S., Yoon, Y.R.,
M

2011. Effect of age on the pharmacokinetics of fimasartan (BR-A-657). Expert Opin. Drug
D

Metab. Toxicol. 7(11), 1337-1344.

TE

Lee, J.H., Yang, D.H., Hwang, J.Y., Hur, S.H., Cha, T.J., Kim, K.S., Kim, M.H., Chun, K.J.,
EP

Cha, G.S., Hong, G.R., Lee, S.G., Kim, D.S., Kim, D.I., Chae, S.C., 2016a. A Randomized,

Double-blind, Candesartan-controlled, Parallel Group Comparison Clinical Trial to Evaluate

C

the Antihypertensive Efficacy and Safety of Fimasartan in Patients with Mild to Moderate
AC

Essential Hypertension. Clin. Ther. 38(6), 1485-1497.

Lee, J.Y., Choi, Y.J., Oh, S.J., Chi, Y.H., Paik, S.H., Lee, K.H., Jung, J.K., Ryu, C.S., Kim,

K.B., Kim, D.H., Yoon, Y.R., Kim, S.K., 2016b. Characterization of fimasartan metabolites

in human liver microsomes and human plasma. Xenobiotica 46(1), 40-51.

20
 ACCEPTED MANUSCRIPT

Lee, S.E., Kim, Y.J., Lee, H.Y., Yang, H.M., Park, C.G., Kim, J.J., Kim, S.K., Rhee, M.Y., Oh,

B.H., Investigators., 2012. Efficacy and tolerability of fimasartan, a new angiotensin receptor

blocker, compared with losartan (50/100 mg): a 12-week, phase III, multicenter, prospective,

PT
randomized, double-blind, parallel-group, dose escalation clinical trial with an optional 12-

week extension phase in adult Korean patients with mild-to-moderate hypertension. Clin.

RI
Ther. 34, 552–568, 568. e1–568.e9.

SC
Lee, Y.J., Jang, Y.N., Han, Y.M., Kim, H.M., Jeong, J.M., Seo, H.S., 2017. Fimasartan

U
Ameliorates Nonalcoholic Fatty Liver Disease through PPARRδ Regulation in
AN
Hyperlipidemic and Hypertensive Conditions. PPAR Res. 2017, 8048720.
M

Liu, Y.T., Hao, H.P., Liu, C.X., Wang, G.J., Xie, H.G., 2007. Drugs as CYP3A probes,
D

inducers, and inhibitors. Drug. Metab. Rev. 39(4), 699-721.

TE

Ohtsuki, S., Schaefer, O., Kawakami, H., Inoue, T., Liehner, S., Saito, A., Ishiguro, N.,
EP

Kishimoto, W., Ludwig-Schwellinger, E., Ebner, T., Terasaki, T., 2012. Simultaneous

absolute protein quantification of transporters, cytochromes P450, and UDP-

C

glucuronosyltransferases as a novel approach for the characterization of individual human

AC

liver: comparison with mRNA levels and activities. Drug Metab. Dispos. 40(1), 83-92.

Park, J.B., Sung, K.C., Kang, S.M., Cho, E.J., 2013. Safety and efficacy of fimasartan in

patients with arterial hypertension (Safe-KanArb study): an open-label observational study.

Am. J. Cardiovasc. Drugs 13(1), 47–56.

21
 ACCEPTED MANUSCRIPT

Rawden, H.C., Kokwaro, G.O., Ward, S.A., Edwards, G., 2000. Relative contribution of

cytochromes P-450 and flavin-containing monoxygenases to the metabolism of albendazole

by human liver microsomes. Br. J. Clin. Pharmacol. 49(4), 313-322.

PT
Sica, D.A., Gehr, T.W., Ghosh, S., 2005. Clinical pharmacokinetics of losartan. Clin.

RI
Pharmacokinet. 44(8), 797-814.

SC
Vermeir, M., Hemeryck, A., Cuyckens, F., Francesch, A., Bockx, M., Van, Houdt, J.,

U
Steemans, K., Mannens, G., Avilés, P., De, Coster, R., 2009. In vitro studies on the
AN
metabolism of trabectedin (YONDELIS) in monkey and man, including human CYP reaction

phenotyping. Biochem. Pharmacol. 77(10), 1642-1654.

M
D

Wallace, B.D., Redinbo, M.R., 2013. Xenobiotic-sensing nuclear receptors involved in drug

metabolism: a structural perspective. Drug Metab. Rev. 45(1), 79-100.

TE
EP

Wójcikowski, J., Boksa, J., Daniel, W.A., 2010. Main contribution of the cytochrome P450

isoenzyme 1A2 (CYP1A2) to N-demethylation and 5-sulfoxidation of the phenothiazine

C

neuroleptic chlorpromazine in human liver--A comparison with other phenothiazines.

AC

Biochem. Pharmacol. 80(8), 1252-1259.

Wynalda, M.A., Hutzler, J.M., Koets, M.D., Podoll, T., Wienkers, L.C., 2003. In vitro

metabolism of clindamycin in human liver and intestinal microsomes. Drug Metab. Dispos.

31(7), 878-87.

22
 ACCEPTED MANUSCRIPT

Yang, X.L., Kim, C.K., Kim, T.J., Sun, J., Rim, D., Kim, Y.J., Ko, S.B., Jang, H., Yoon,

B.W., 2016. Anti-inflammatory effects of fimasartan via Akt, ERK, and NFκB pathways on

astrocytes stimulated by hemolysate. Inflamm. Res. 65(2), 115-23.

PT
Zanger, U.M., Schwab, M., 2013. Cytochrome P450 enzymes in drug metabolism: regulation

RI
of gene expression, enzyme activities, and impact of genetic variation, Pharmacol. Ther.

SC
138(1), 103-141.

U
AN
M
D
TE
C EP
AC

23
 ACCEPTED MANUSCRIPT

Figure legends

Figure 1. Metabolism of fimasartan (FMS) in recombinant human cytochrome P450 (CYP)

and flavin-containing monooxygenase (FMO) enzymes. FMS (1 µM) was incubated with 50

pmol/mL of each recombinant enzyme in the presence of an NADPH generating system for

PT
120 min. Control values were determined in a mock recombinant. Concentrations of FMS (A)

RI
and its metabolites, including FMS S-oxide (B), BR-A-557 (C), BR-A-535 (D), 1-OH butyl

FMS (E), 2- or 3-OH butyl FMS (F), and 4-OH butyl FMS (G) are expressed as means ± SD

SC
for three independent samples.

U
AN
Figure 2. Effects of heat-inactivation and 1-aminobenzotriazole on the disappearance of FMS
M

and formation of FMS S-oxide in human liver microsomes (HLM). HLM were preheated at

45°C for 5 min to inhibit FMO enzymes, or pre-incubated with 1-aminobenzotriazole (2.5
D

mM) for 30 min to inhibit CYP enzymes. One micromole of FMS was incubated with 1
TE

mg/mL HLM for 10 min in the presence of NADPH. Data are expressed as means ± SD for

three independent samples.

C EP
AC

Figure 3. Effects of selective CYP inhibitors on the metabolism of FMS in HLM. Each

selective CYP inhibitor was pre-incubated with 1 mg/mL HLM for 15 min, and FMS (10

µM) was added for 10 min in the presence of 1 mM NADPH. Control values were

determined by the incubation of FMS in the absence of CYP inhibitors. The concentrations of

FMS S-oxide (A), BR-A-557 (B), BR-A-535 (C), 1-OH butyl FMS (D), 2- or 3-OH butyl

FMS (E), and 4-OH FMS (F) are expressed as means ± SD for three independent samples.

24
 ACCEPTED MANUSCRIPT

FURA = furafylline (10 µM; CYP1A2 inhibitor). TRAN = tranylcypromine (0.2 µM;

CYP2A6 inhibitor). TEPA = triethylenethiophosphoramide (50 µM; CYP2B6 inhibitor).

MONT = montelukast (0.1 µM; CYP2C8 inhibitor). SULF = sulfaphenazole (10 µM;

CYP2C9 inhibitor). N-BEN = N-3-benzylnirvanol (2 µM; CYP2C19 inhibitor). QUIN =

PT
quinidine (2 µM; CYP2D6 inhibitor). DETC = diethyldithiocarbamate (50 µM; CYP2E1

inhibitor). KETO = ketoconazole (2 µM; CYP3A4 inhibitor). N/D = not detected.

RI
SC
Figure 4. Time profiles of FMS metabolism in recombinant CYP2C9 (A), 3A4 (B), and 3A5

U
(C). FMS (1 µM) was incubated with 50 pmol/mL recombinant CYP2C9, CYP3A4, or
AN
CYP3A5 enzyme in the presence of an NADPH generating system for 0, 5, 10, 15, 30, or 60

min. Concentrations of remaining FMS and its metabolites are expressed as means ± SD for
M

three independent samples.

D
TE

Figure 5. Kinetics of FMS metabolism in recombinant CYP2C9 (A), 3A4 (B), and 3A5 (C).
EP

FMS (0, 0.11, 0.33, 1.0, 3.0, 9.0, 27, 81, 243, or 729 µM) was incubated with 50 pmol/mL of

each recombinant CYP enzyme in the presence of an NADPH generating system for 10 min.
C

Data are expressed as means ± SD for three independent samples.

AC

Figure 6. Proposed CYP-mediated metabolic pathways of FMS.

25
 ACCEPTED MANUSCRIPT

Table 1. Kinetic parameters for the reactions of FMS metabolism determined using recombinant CYP2C9, 3A4, and 3A5
Metabolites
Kinetic parameters
FMS S-oxide BR-A-557 BR-A-535 1-OH butyl FMS 2-OH or 3-OH butyl FMS 4-OH butyl FMS

PT
Km 22.0 771 12.2 24.1
N/A N/A
(µM) (14.1 to 30.0) (712 to 831) (7.8 to 16.5) (17.7 to 30.6)

RI
2C9 kcat 4.7 5.9 0.50 0.84
N/A N/A
(min-1) (4.2 to 5.2) (5.6 to 6.2) (0.47 to 0.54) (0.79 to 0.89)
kcat/Km

SC
2.1 x 10-1 N/A N/A 7.6 x 10-3 4.1 x 10-2 3.5 x 10-2
(µM-1·min-1)
Km 32.8 45.1 74.6
N/A N/A N/A

U
(µM) (26.1 to 39.5) (33.2 to 57.1) (55.4 to 93.8)
3A4 kcat 11.1 2.1 0.36
N/A N/A N/A

AN
(min-1) (10.5 to 11.6) (2.0 to 2.3) (0.33 to 0.38)
kcat/Km
3.4 x 10-1 4.8 x 10-2 4.8 x 10-3 N/A N/A N/A
(µM-1·min-1)

M
Km 46.1 193
N/A N/A N/A N/A
(µM) (36.9 to 55.4) (134 to 253)

D
3A5 kcat 8.6 2.8
N/A N/A N/A N/A
(min-1) (8.1 to 9.0) (2.5 to 3.2)

TE
kcat/Km
1.9 x 10-1 1.5 x 10-2 N/A N/A N/A N/A
(µM-1·min-1)
EP
FMS (0, 0.11, 0.33, 1.0, 3.0, 9.0, 27, 81, 243, or 729 µM) was incubated with each recombinant CYP enzyme in the presence of an NADPH
C

generating system for 10 min. The apparent Km and Kcat values were calculated by non-linear regression using the Michaelis-Menten equation.
AC

Data are expressed as means ± SD for three independent samples. Values in parentheses represent 95% confidential intervals. N/A = not
available

26
 ACCEPTED MANUSCRIPT

The English in this document has been checked by at least two professional editors, both
native speakers of English. For a certificate, please see:

http://www.textcheck.com/certificate/7DuPY6

PT
RI
U SC
AN
M
D
TE
C EP
AC

27
 4-OH butyl FMS 1-OH butyl FMS BR-A-557 Remaining FMS
(A)

(E)

(G)
(C)
(nM) (nM) (nM) (nM)
C C

10
15

0
5
C

0
50
100
150
0
100
200
300
400
on on on C
tr

0
200
400
600
800
1000
1200

tr o tr on
o 1A l o tr
1A l 1A l o
1A1 1A l
1A1 1A1
2A2 1A1
2A2 2A2
AC
2B6
2B6 6 2A2
2C6
2B 6
2C6 2B
2 8 2C6
2C8 2C6
2C 9
1
C 2CC9
1
2C8
2C 9 2C8
2D9
2D9 1 2C 9

Isoform
1

Isoform
2D9

Isoform
2E6
6

Isoform
2E6 2D9
3A1 2E 6
3A1 4 2E
4 FM3A5 3A1
FM3A5 3A1
3 4
EP
FMO1
FMO1 FM A5 3 4
FMO3 FMA5
FMO3 FMO1
O
O 5 FMO1
5 FMO3
O FMO3
5 O
5
TE
D
Figure 1.

M
2- or 3-OH butyl FMS BR-A-535 FMS S-oxide

(F)
(D)
(B)

(nM) (nM) (nM)

C
0
10
20
30
40
50

C on C
0
200
400
600
800

50

0
100
150
on tr on
tr o tr
o 1A l o
1A l 1A l
1A1
AN
1A1 1A1
2A2 2A2 2A2
6
ACCEPTED MANUSCRIPT

2B6 2B 2B6
2C6
2C8
U 2C6
2C8
2C6
2C8
2C 9 2C 9 2C 9
1 1 1
2D9
Isoform

2D9 2D9
Isoform

Isoform
2E6 6 2E6
2E
3A1 3A1
SC
4 3A1 4
FM3A5 3 4 FM3A5
FMA5 FMO1
FMO1
FMO3 FMO1 FMO3
O FMO3 O
5 O 5
5
RI
PT
 ACCEPTED MANUSCRIPT

Figure 2.

(A) (B)
125 150

FMS S-oxide formation

Remaining FMS (% )

100
100
75

(nM)

PT
50
50
25

RI
0 0
in in T in in T
5m , 5m AB 5m 5m AB
o C, o C o C, o C,
37 45 37 45

U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

(E)

(C)
FMS S-oxide

(A)
2- or 3-OH butyl FMS (nM)
BR-A-535
(nM)

1000

2000

3000

4000
(nM)

20

40

60

80
0

0
PT
Control Control Control

FURA FURA FURA

RI
TRAN TRAN TRAN

TEPA TEPA TEPA

Inhibitors
Inhibitors

Inhibitors

SC
MONT MONT MONT

SULF SULF SULF

N-BEN
N-BEN N-BEN

U
QUIN
QUIN QUIN

AN
DETC
DETC DETC
KETO

N/D
KETO KETO

Figure 3.
M
(F)

(D)

(B)
D
4-OH butyl FMS 1-OH butyl FMS BR-A-557
(nM) (nM) (nM)

TE
100
20

40

60

80

10

20

30

40

10

20

30

40

50
0

0
Control Control Control

FURA FURA FURA

EP
TRAN TRAN TRAN
TEPA TEPA TEPA
Inhi bitors

Inhi bitors

Inhibitors
C

MONT MONT MONT

SULF SULF
AC

SULF
N-BEN N-BEN N-BEN
QUIN QUIN QUIN
DETC DETC DETC
KETO KETO
KETO
 ACCEPTED MANUSCRIPT

Figure 4.

(A)
1200 360

1000 300 FMS

Remaining FMS

FMS S-oxide

Metabolites
800 240
1-OH butyl FMS

(nM)
(nM)

600 180 2- or 3-OH butyl FMS

PT
400 120 4-OH butyl FMS

200 60

RI
0 0
0 20 40 60 80
Time (min)

U SC
(B)
AN
1200 600

1000 500 FMS

Remaining FMS

FMS S-oxide
Metabolites

800 400
M

BR-A-557
(nM)
(nM)

600 300 BR-A-535

400 200 1-OH butyl FMS
D

200 100

0 0
TE

0 20 40 60 80
Time (min)
EP

(C)
C

1200 600
AC

1000 500
Remaining FMS

Metabolites

800 400 FMS

FMS S-oxide
(nM)
(nM)

600 300
BR-A-557
400 200

200 100

0 0
0 20 40 60 80
Time (min)
 ACCEPTED MANUSCRIPT

Figure 5.

(A)
50
FMS S-oxide
40 1-OH butyl FMS
2-OH or 3-OH butyl FMS
(pmol/min)

PT
Velocity

30 4-OH butyl FMS

20

RI
10

0
0 200 400 600 800

SC
Concentration(uM)

U
(B)
AN
150
FMS S-oxide
120 BR-A-557
BR-A-535
(pmol/min)
Velocity

90
M

60
D

30

0
TE

0 200 400 600 800

Concentration(uM)
EP

(C)
100
FMS S-oxide
C

80 BR-A-557
(pmol/min)
AC Velocity

60

40

20

0
0 200 400 600 800
Concentration(uM)
 ACCEPTED MANUSCRIPT

Figure 6.

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

Highlight

1. CYP but not FMO played the major role in the NADPH-dependent fimasartan (FMS)

metabolism.

2. CYP2C9, CYP3A4 and CYP3A5 were involved in the formation of FMS S-oxide, a

PT
major FMS metabolite.

RI
3. The value of the kcat/Km of S-oxidation by CYP2C9, CYP3A4 or CYP3A5 was 0.21,

0.34 or 0.19 µM-1·min-1, respectively.

SC
4. FMS S-oxide was further metabolized to oxidative desulfurated metabolite, BR-A-

U
557 by CYP3A4/5.
AN
CYP2C9 played an exclusive role in the n-butyl hydroxylations of FMS
M
D
TE
C EP
AC