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choi2018ENZYMEEE PDF
choi2018ENZYMEEE PDF
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
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.
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Young Jae Choi1*, Ji-Yoon Lee1*, Chang Seon Ryu1, Yong Ha Chi2, Soo Heui Paik3, and Sang
Kyum Kim1
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College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea
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Central Research Institute, Boryung Pharm. co., Ltd. Ansan, Gyeonggi 425-839, Republic of
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Korea
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College of Pharmacy, Sunchon National University, Suncheon-si, Republic of Korea
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*
These authors contributed equally to this work.
Correspondence
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Sang Kyum Kim, Ph.D.: College of Pharmacy, Chungnam National University, 220 Gung-
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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
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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
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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.
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Recently, it has been suggested that FMS ameliorates nonalcoholic fatty liver disease in an
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animal model (Lee et al., 2017) and suppresses inflammatory response in astrocytes
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In metabolism and pharmacokinetic studies using rats treated with radiolabeled FMS,
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oxidative desulfurization, N-glucuronidation, mono-oxygenation of pyrimidinone,
hydroxylation of the n-butyl group, and de-N-dimethylation were the major metabolic
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pathways of FMS (Lee et al., 2011; Kim et al., 2014). In our previous study using authentic
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metabolic products of FMS in humans (Lee et al., 2016b). In addition, FMS S-oxide was
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of an in vitro kinetic study (Lee et al., 2016b). Moreover, 1-, 2-/ 3-, and 4-hydroxy-n-butyl
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FMS was observed in pooled human liver microsomes (HLM). Recently, it was reported that
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UDP-glucuronosyltransferase (UGT) 1A3 was the major UGT isoform responsible for N-
characterized. Although cytochrome P450 (CYP) enzymes play a major role in xenobiotic
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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
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pretreated with selective chemical inhibitors. The concentration–time profiles of FMS and its
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metabolites were determined for evaluation of the rates of FMS elimination and metabolite
formation. Moreover, the kinetic parameters of enzymes responsible for FMS metabolism
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were calculated to predict FMS metabolism in vivo using the Michaelis-Menten model.
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(1H-tetrazole-5-yl)biphenyl-4-yl]methyl}pyrimidine-4(3H)-one potassium salt trihydrate),
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FMS), 2-hydroxy-n-butyl FMS (2-OH butyl FMS), 3-hydroxy-n-butyl FMS (3-OH butyl
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FMS), 4-hydroxy-n-butyl FMS (4-OH butyl-FMS), oxidative desulfurization and de-N-
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standard) were supplied by Boryung Pharm. Co., Ltd. (Seoul, Korea). The nuclear magnetic
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resonance data for FMS and its metabolites were reported in our previous study (Lee et al.,
Louis, MO, USA). Pooled human liver microsomes (150 donors, mix; Catalog No. 452117),
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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
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No. 456259), 2D6*1 (Catalog No. 456217), 2E1 (Catalog No. 456206), 3A4 (Catalog No.
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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
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were purchased from SantaCruz (Dallas, USA). All other reagents and chemicals were of
Stock solutions (10 mM) of FMS and its metabolites were prepared by dissolving in
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dimethyl sulfoxide (DMSO). Stock standard solutions were stored at -20°C. Mixed working
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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
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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
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mixture were less than 0.3% except for experiments for determination of Km and kcat of FMS.
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In these experiments, the concentration of DMSO was approximately 0.725%.
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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
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protocol provided by the manufacturer. The treated cells were incubated at 37°C in a CO2
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incubator. Reactions were initiated by addition of FMS and were then quenched by adding
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samples were centrifuged at 1,000 × g for 20 min at 4 °C and the supernatants were subjected
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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,
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1 U/mL G6PDH) in a final volume of 200 µL for 120 min. The mixture was pre-incubated at
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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
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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-
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MS/MS.
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Additional experiments were designed to compare the contribution of CYP and FMO
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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
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the presence of 1 mM NADPH. To inhibit CYP enzymes, HLM were pretreated with 1-
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aminobenzotriazole, a pan CYP inhibitor, for 30 min at 37 °C. The pretreated 1 mg/mL HLM
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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.
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
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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),
µM; CYP2C19), quinidine (2 µM; CYP2D6), diethyldithiocarbamate (50 µM; CYP2E1), and
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ketoconazole (2 µM; CYP3A4). The specific CYP inhibitors and their concentrations were
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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
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was terminated and centrifuged using the same method described above.
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2.7. Determination of time profiles and kinetic parameters of FMS metabolites in
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recombinant CYP isoforms
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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
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regenerating system in a final volume of 200 µL, for 0, 5, 10, 15, 30, or 60 min. To determine
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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.
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FMS and recombinant CYP were added to 0.1 M potassium phosphate buffer (pH 7.4) and
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the mixture was pre-incubated at 37°C in a shaking incubator at approximately 100 rpm for 5
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reaction was terminated and centrifuged using the same method described above.
To identify and quantify FMS and its metabolites, the samples were analyzed using a
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
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containing 0.1% (v/v) formic acid) and B (acetonitrile containing 0.1% (v/v) formic acid). A
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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
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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-
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OH butyl FMS were not distinguishable with the analytical methods used in this study. The
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peak intensities of 2-OH butyl FMS and 3-OH butyl FMS determined using the Multiple
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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,
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respectively.
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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
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and analysis of data were performed with AnalystTM software (ver. 1.5.2; Applied
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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
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)
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of the measured concentrations was used to assess precision. The mean measured
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.
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2.9. Data analysis
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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
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were expressed as the mean ± SD for triplicate samples. The apparent kinetic parameters of
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kinetic parameters were estimated by plotting the activities over the logarithm of FMS
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concentration with GraphPad Prism 5 for Windows, version 5.01 (GraphPad Software Inc.,
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3. Results
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
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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)
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was the most abundant metabolite in pooled human hepatocytes followed in decreasing order
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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
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generally consistent with our previous results obtained from HLM and pooled human plasma
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(Lee et al., 2016b).
recombinant CYP and FMO enzyme was incubated with 1 µM FMS for 120 min and
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analyzed using LC-MS/MS in MRM mode (Figure 1). The concentration of FMS observed in
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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
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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
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metabolite in CYP2C9 (339 ± 1 nM), CYP3A4 (434 ± 35 nM), and CYP3A5 (582 ± 19 nM)
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(Figure 1B). CYP2C8, CYP2C19, CYP2D6, and FMOs exhibited marginal activity for S-
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
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1-OH butyl FMS was mainly mediated by CYP2C9, and to a lesser extent, CYP3A4 (Figure
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
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using HLM pretreated with 2.5 mM 1-aminobenzotriazole, a pan-CYP inhibitor, and HLM
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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
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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-
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minobenzotriazole at 2.5 mM decreased the activity of nine CYPs, including CYP1A1, 2A6,
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2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4, to less than 20% of control (data not shown).
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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
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time (5 to 30 min) dependent manner (data not shown). The formation of FMS S-oxide
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(Figure 3A) and BR-A-557 (Figure 3B) was markedly decreased, and BR-A-535 was not
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(Figure 3C). All n-butyl hydroxylated metabolites were decreased to less than half of control
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
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inhibitors decreased the metabolite formation to less than 25%, except for
3.3. Time profiles of FMS metabolism in recombinants CYP 2C9, 3A4, and 3A5
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To determine time profiles of FMS metabolism, FMS and its metabolites were measured in
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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
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protein concentration of recombinant CYP2C9, 3A4 and 3A5 (12.5 to 50 pmol/ml) (data not
shown).
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The half-life (95% confidence interval) of FMS in recombinant CYP2C9, CYP3A4, and
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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,
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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
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at 5 min was 0.25, 0.98, and 0.47 min-1, respectively. The formation rate of FMS S-oxide was
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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
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depletion. The metabolic rates of the other metabolites were calculated from the slopes of the
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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
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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
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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
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shown). BR-A-557 and BR-A-535 had formation rates of 1.031 ± 0.049 and 0.189 ± 0.011
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pmol/min/mg protein, respectively (data not shown).
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3.4. Kinetic parameters of FMS metabolism in recombinant CYP 2C9, 3A4 and 3A5
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and 3A5 with various concentrations of FMS for 10 min (Figure 5 and Table 1). The
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formation of metabolites, except for FMS S-oxide and 1-OH butyl FMS in recombinant
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CYP2C9 exhibited substrate inhibition behavior when the FMS concentration was more than
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10-fold of its Michaelis–Menten constant (Km), and hydroxylation of the 1-n-butyl group may
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be saturated at an FMS concentration of 729 µM (Figure 5A). The value of the specificity
than four-fold higher than the other reactions in all recombinant enzymes tested in this study
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(Table 1). The kcat/Km values of 2- or 3-butyl and 4-butyl hydroxylation in recombinant
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CYP2C9 were comparable with that of 1-OH butyl FMS, and less than 22% relative to the
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4. Discussion
are critical for the prediction of drug-drug interactions that can result in altered levels of
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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
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major role in FMS elimination and the formation of metabolites, including the major
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metabolite, FMS S-oxide. The role of CYP enzymes in FMS metabolism was confirmed in
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aminobenzotriazole, and HLM heated to inhibit FMO. FMO enzymes reportedly play an
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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
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catalyzed by FMO. In contrast, the S-oxidation of clindamycin (Wynalda et al., 2003) and
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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
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(Attar et al., 2003). Thus, the physicochemical properties of drugs may be one of the
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Hepatic metabolism of ARB drugs may produce pharmacologically more active metabolites
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than parent drug. For example, the carboxylic acid metabolite (E 3174) of losartan is more
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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,
inactivation.
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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,
enzyme catalyzed the formation of FMS S-oxide, 1-, 2- or 3-, and 4-OH butyl FMS with
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kcat/Km values of 0.21, 0.0076, 0.041, and 0.035 µM-1·min-1, respectively. FMS S-oxide and
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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
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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
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important information for evaluating the drug-drug interaction and drug disposition. Various
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modeling studies can predict potential clinical drug-drug interactions and human exposure
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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
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were approximately two-fold of those in HLM incubated with FMS (Lee et al., 2016b). These
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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
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metabolites.
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It is noteworthy that out of all the CYP and FMO enzymes tested, CYP2C9 is exclusively
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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
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than diclofenac 4'-hydroxylation and S-flurbiprofen 4-hydroxylation. Moreover, the
well as CYP2C9.
The contribution of CYP2C9 and CYP3A4, the major CYPs, to the total hepatic CYP pools
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is comparable, as determined by LC-MS/MS (Kawakami et al, 2011; Ohtsuki et al., 2012;
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Gröer et al., 2014). CYP2C9 and CYP3A4 values ranged from 37.3 to 80.2 and from 32.6 to
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less than 5% of CYP3A4, and ranged from 1.96 to 3.86 pmol/mg in pooled HLM. The
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HLM was simulated using the Michaelis-Menten equation and the absolute protein levels.
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The contribution of CYP2C9 and CYP3A5 to the formation of FMS S-oxide was 63.0 to
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56.6% and 2.8 to 3.0%, respectively, of that of CYP3A4, as the FMS concentration was
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
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elimination of FMS and formation of FMS S-oxide, BR-A-557, and BR-A-535. CYP3A5
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may play a minor role in FMS metabolism due to low protein expression in the liver,
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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
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
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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;
important role in adverse drug reactions, especially for CYP2C9-selective drugs with a low
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therapeutic index (Hiratsuka, 2016). In addition, the expression and activity of CYP3A4
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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-
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substrate drugs.
In summary, these results suggest a major role for CYP, but not FMO, enzymes in FMS
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metabolism. This was also confirmed in additional experiments using HLM with either CYP
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or FMO inhibition. The results from the experiments using recombinant enzymes and
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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
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played an exclusive role in the hydroxylation of FMS n-butyl groups. FMS S-oxide is an
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that further research is needed on the influence of co-administered drugs on FMS metabolism
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due to inhibition or stimulation of CYP3A4/5 and 2C9 enzymes. Moreover, the kinetic
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parameters determined in this study, such as kcat and Km, can be used to predict FMS
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A-535, and n-butyl hydroxylated metabolites may be useful to evaluate CYP2C9 and
CYP3A4/5 activities.
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Acknowledgments
This work was supported by the Ministry of Education of the Republic of Korea and the
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5. Reference
Attar, M., Dong, D., Ling, K.H., Tang-Liu, D.D., 2003. Cytochrome P450 2C8 and flavin-
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Cashman, JR., 2008. Role of flavin-containing monooxygenase in drug development. Expert
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Figure legends
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
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120 min. Control values were determined in a mock recombinant. Concentrations of FMS (A)
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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
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for three independent samples.
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Figure 2. Effects of heat-inactivation and 1-aminobenzotriazole on the disappearance of FMS
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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
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mg/mL HLM for 10 min in the presence of NADPH. Data are expressed as means ± SD for
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.
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FURA = furafylline (10 µM; CYP1A2 inhibitor). TRAN = tranylcypromine (0.2 µM;
MONT = montelukast (0.1 µM; CYP2C8 inhibitor). SULF = sulfaphenazole (10 µM;
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quinidine (2 µM; CYP2D6 inhibitor). DETC = diethyldithiocarbamate (50 µM; CYP2E1
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Figure 4. Time profiles of FMS metabolism in recombinant CYP2C9 (A), 3A4 (B), and 3A5
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(C). FMS (1 µM) was incubated with 50 pmol/mL recombinant CYP2C9, CYP3A4, or
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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
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Figure 5. Kinetics of FMS metabolism in recombinant CYP2C9 (A), 3A4 (B), and 3A5 (C).
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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.
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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
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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)
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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
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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
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(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
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(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)
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Km 46.1 193
N/A N/A N/A N/A
(µM) (36.9 to 55.4) (134 to 253)
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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)
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kcat/Km
1.9 x 10-1 1.5 x 10-2 N/A N/A N/A N/A
(µM-1·min-1)
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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
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generating system for 10 min. The apparent Km and Kcat values were calculated by non-linear regression using the Michaelis-Menten equation.
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Data are expressed as means ± SD for three independent samples. Values in parentheses represent 95% confidential intervals. N/A = not
available
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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
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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
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2A2 1A1
2A2 2A2
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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
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FMO1 FM A5 3 4
FMO3 FMA5
FMO3 FMO1
O
O 5 FMO1
5 FMO3
O FMO3
5 O
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Figure 1.
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2- or 3-OH butyl FMS BR-A-535 FMS S-oxide
(F)
(D)
(B)
C on C
0
200
400
600
800
50
0
100
150
on tr on
tr o tr
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2B6 2B 2B6
2C6
2C8
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2C8
2C6
2C8
2C 9 2C 9 2C 9
1 1 1
2D9
Isoform
2D9 2D9
Isoform
Isoform
2E6 6 2E6
2E
3A1 3A1
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4 3A1 4
FM3A5 3 4 FM3A5
FMA5 FMO1
FMO1
FMO3 FMO1 FMO3
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Figure 2.
(A) (B)
125 150
100
100
75
(nM)
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50
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0 0
in in T in in T
5m , 5m AB 5m 5m AB
o C, o C o C, o C,
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(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
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Control Control Control
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TRAN TRAN TRAN
Inhibitors
Inhibitors
Inhibitors
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MONT MONT MONT
N-BEN
N-BEN N-BEN
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QUIN
QUIN QUIN
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DETC
DETC DETC
KETO
N/D
KETO KETO
Figure 3.
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(F)
(D)
(B)
D
4-OH butyl FMS 1-OH butyl FMS BR-A-557
(nM) (nM) (nM)
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20
40
60
80
10
20
30
40
10
20
30
40
50
0
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Control Control Control
Inhi bitors
Inhibitors
C
SULF
N-BEN N-BEN N-BEN
QUIN QUIN QUIN
DETC DETC DETC
KETO KETO
KETO
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Figure 4.
(A)
1200 360
FMS S-oxide
Metabolites
800 240
1-OH butyl FMS
(nM)
(nM)
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400 120 4-OH butyl FMS
200 60
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0 20 40 60 80
Time (min)
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(B)
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1200 600
FMS S-oxide
Metabolites
800 400
M
BR-A-557
(nM)
(nM)
200 100
0 0
TE
0 20 40 60 80
Time (min)
EP
(C)
C
1200 600
AC
1000 500
Remaining FMS
Metabolites
600 300
BR-A-557
400 200
200 100
0 0
0 20 40 60 80
Time (min)
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Figure 5.
(A)
50
FMS S-oxide
40 1-OH butyl FMS
2-OH or 3-OH butyl FMS
(pmol/min)
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Velocity
20
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10
0
0 200 400 600 800
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Concentration(uM)
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(B)
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150
FMS S-oxide
120 BR-A-557
BR-A-535
(pmol/min)
Velocity
90
M
60
D
30
0
TE
(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)
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Figure 6.
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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
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major FMS metabolite.
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3. The value of the kcat/Km of S-oxidation by CYP2C9, CYP3A4 or CYP3A5 was 0.21,
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4. FMS S-oxide was further metabolized to oxidative desulfurated metabolite, BR-A-
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557 by CYP3A4/5.
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CYP2C9 played an exclusive role in the n-butyl hydroxylations of FMS
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