研究者詳細 - 秋好　健志

論文 - 秋好　健志

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Stoichiometric transport of estrone 3-sulfate among genetic variants of OATP1A2 and OATP2B1 and structural analysis by molecular dynamics simulation: Impairment of gating mechanism in the unstable inward-open conformation of OATP2B1 (Asp215Val) significantly suppress the transport activity.

Akiyoshi T, Tonduru AK, Kataoka H, Morita T, Yajima K, Imaoka A, Katayama K, Medarametla P, Uchida Y, Poso A, Ohtani H, Terasaki T

Drug metabolism and disposition: the biological fate of chemicals 53 （ 12 ） 100198 2025年11月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り, ISSN 0090-9556

This study investigated the impact of genetic variations in organic anion transporting polypeptides (OATPs) 1A2 and 2B1 on their transport activity at pH 6.3 and 7.4 by using HEK293 cells expressing OATP variants, focusing on stoichiometric transport kinetic parameters corrected for the number of transporters on the plasma membrane. In the OATP2B1 Asp215Val, the maximal velocity per OATP molecule and intrinsic clearance at pH 6.3 were drastically reduced to 0.0648- and 0.0178-fold, respectively, compared with the wild type. All tested OATP1A2 variants exhibited increased transport activity at pH 6.3, suggesting that OATP1A2 is more sensitive to extracellular pH. Furthermore, we used the AlphaFold model to explain the observed differences in transport activity among genetic variants. In OATP1A2, the Glu172Asp mutation replaces a longer glutamate side chain with a shorter aspartate, which may enhance substrate interactions while weakening the salt-bridge interactions with neighboring residues, potentially compromising structural integrity. In OATP2B1, the Asp215Val variant was found to disrupt a key salt-bridge interaction with Lys595, which destabilizes the outward-open conformation. Moreover, the Val201Met mutation appears to lock the transporter in a single conformational state. Our findings underscore the importance of transmembrane helix 4 in maintaining functional conformational dynamics and suggest that mutations in this region can significantly alter substrate binding and transport efficiency in OATP1A2 and 2B1. Significance Statement: This study combined uptake assays using transporter-expressing cell lines, liquid chromatography–tandem mass spectrometry transporter quantification, and computer modeling to clarify the changes in transport activity per molecule, and these mechanisms caused by amino acid substitutions in organic anion transporting polypeptides 1A2 and 2B1.

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Inhibition kinetics of citrus jabara juice and its components on CYP2C9 activity.

Koinuma K, Morita T, Uekusa Y, Kikuchi H, Imaoka A, Akiyoshi T, Ohtani H

Journal of pharmaceutical sciences 114 （ 11 ） 103983 2025年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3549

Our previous studies demonstrated that the ethyl acetate extract of the juice of jabara (JEx), a Japanese citrus fruit, inhibited cytochrome P450 (CYP) 3A4 activity in a time-dependent manner, but does not inhibit CYP2C19 activity. Its components, 3,3′,4′,5,6,7,8-heptamethoxyflavone (HpMF) and 3,3′,4′,5,6,7-hexamethoxyflavone (HxMF), have been shown to be responsible for this CYP3A4 inhibition. This study aimed to investigate the nature of CYP2C9 inhibition by JEx, HpMF, and HxMF. The time-dependent inhibition kinetics of JEx against human recombinant CYP2C9 were investigated in vitro using warfarin as a substrate. The time-independent inhibition kinetics of JEx, HpMF, and HxMF against CYP2C9 were also determined. JEx did not cause time-dependent inhibition of CYP2C9, while JEx, HpMF, and HxMF inhibited CYP2C9 activity in a time-independent manner with IC<inf>50</inf> values of 2.65 % equivalent, 16.2 µM, and 57.8 µM, respectively. HpMF and HxMF were considered to be primarily responsible for CYP2C9 inhibition by JEx. The ingestion of 250 mL of jabara juice was estimated to reduce intestinal CYP2C9 activity to 3 % of the control value assuming of dilution with 210 mL of intestinal fluid, suggesting that jabara juice may cause food-drug interactions via intestinal CYP2C9 inhibition.

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Comparative analysis of the single-molecule transport kinetics of OATP1B1 genetic variants.

Tsujii K, Yajima K, Akiyoshi T, Sakamoto K, Suzuki Y, Oka T, Imaoka A, Yamamura H, Kurokawa J, Ohtani H

Journal of pharmacological sciences 158 （ 3 ） 166 - 171 2025年07月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1347-8613

Several genetic variants of OATP1B1, a hepatic uptake transporter, increase the blood concentrations of substrate drugs, e.g., ∗15 carriers exhibit higher blood substrate concentrations than ∗1b carriers. It remains unclear whether these differences are due to changes in expression or intrinsic activity (transport activity per OATP1B1 molecule). This study compared the intrinsic activity of four OATP1B1 variants, ∗1a, ∗1b, ∗5, and ∗15, using HEK293 cell lines that co-expressed large-conductance Ca2+-activated K+ (BK) channels and one of the OATP1B1 variants. To estimate the kinetic parameters K<inf>m</inf> and V<inf>max</inf>, 2′, 7′-dichlorofluorescein uptake was evaluated. The number of OATP molecules per cell (Q<inf>T</inf>) was calculated from BK channel-mediated whole-cell conductance and the OATP1B1/BK channel expression ratio (ρ) (determined by LC-MS/MS). V<inf>max,int</inf> (maximum intrinsic transport velocity) was obtained by dividing V<inf>max</inf> by Q<inf>T</inf>, and intrinsic clearance (CL<inf>int</inf>) was calculated as V<inf>max,int</inf>/K<inf>m</inf>. The K<inf>m</inf> values of ∗1a, ∗1b, ∗5, and ∗15 were 12.5, 9.19, 7.53, and 10.4 μM, and their V<inf>max,int</inf> values were 3.0, 7.0, 1.5, and 1.2 × 10−21 mol/OATP molecule/min, respectively. Accordingly, the CL<inf>int</inf> value for OATP1B1∗15 was 15 % lower than that for OATP1B1∗1b, suggesting that the increased blood substrate concentrations observed in OATP1B1∗15 carriers may be due to the decreased intrinsic activity of OATP1B1∗15.

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The effect of organic solvents on the in vitro transport activity of three OATP isoforms.

Saito R, Akiyoshi T, Tsujii K, Takahashi R, Kataoka H, Imaoka A, Ohtani H

Toxicology in vitro : an international journal published in association with BIBRA 107 106059 2025年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0887-2333

Purpose: In vitro studies of transporter activity often require the addition of organic solvents such as dimethyl sulfoxide (DMSO), methanol, and ethanol, to dissolve substrates and/or inhibitors. Although this may attenuate the transport activity, the influence of different types of organic solvents on transporter activity has not been elucidated. This study aimed to quantitatively assess the impact of organic solvents on the transport activity of organic anion transporting polypeptide (OATP) 1B1, 1A2, and 2B1. Methods: The concentration-dependent influence of DMSO, methanol, and ethanol on the uptake of [3H]estrone 3-sulfate mediated by OATP1A2, OATP2B1 or 2′,7′-dichlorofluorescein (DCF) mediated by OATP1B1 was assessed using HEK293 cells expressed the respective OATP protein and the concentration that reduced the uptake by 20 % (IC20) was calculated. Results and discussion: The uptake activities of OATPs were reduced by methanol and ethanol in a concentration-dependent manner, with IC20 values of 2.4–3.4 % and 0.51–3.8 % respectively. In contrast, DMSO did not affect the OATP2B1-mediated uptake at the maximum concentration (10 %) of DMSO whereas it exhibited concentration-dependent inhibition for OATP1B1 and 1A2 with IC20 values of approximately 3 % and 0.7 %, respectively. This study provides useful information regarding experimental conditions for evaluating OATPs activity in vitro.

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タイ王国での薬局薬剤師の役割　医療格差が生む薬局薬剤師の職能域の拡大とヘルスケア提供者としての可能性

秋好健志

在宅薬学（一般社団法人日本在宅薬学会） 12 （ 1 ） 41 - 47 2025年

研究論文（学術雑誌）, 単著, ISSN 2188658X

Kinetics of the Inhibition of CYP3A4 and CYP2C19 Activity by Jabara Juice and Identification of the Responsible Inhibitory Components.

Koinuma K, Noto K, Morita T, Uekusa Y, Kikuchi H, Shimoji M, Seki H, Yamazaki H, Guengerich FP, Nakamura K, Yamamoto K, Imaoka A, Akiyoshi T, Ohtani H

Journal of pharmaceutical sciences 114 （ 2 ） 849 - 856 2024年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3549

Some citrus fruits are known to cause clinically significant drug interactions by inhibiting intestinal cytochrome P450 (CYP) enzymes. This in vitro study aimed to investigate the kinetics of the inhibition of CYP3A4 and CYP2C19 by the juice of jabara, a Japanese citrus fruit that does not contain furanocoumarins such as 6′,7′-dihydroxybergamottin, and to identify the inhibitory compound(s). CYP3A4 and CYP2C19 activity levels were determined in vitro using recombinant CYP preparations and their respective substrates. The ethyl acetate extract (EAE) of jabara juice was separated to isolate and identify the compound(s) that inhibited CYP3A4. Then, the time-dependent kinetics of the inhibition of CYP3A4 and CYP2C19 by the EAE and its inhibitory compound(s) were analyzed. The EAE of jabara juice was found to inhibit CYP3A4 in a time-dependent manner. Two flavonoids, 3,3′,4′,5,6,7,8-heptamethoxyflavone (HpMF) and 3,3′,4′,5,6,7-hexamethoxyflavone (HxMF), were identified as the responsible compounds. HpMF and HxMF inhibited CYP3A4 activity in a concentration- and time-dependent manner, with inhibition constants (KI) of 10.0 and 7.90 µM and maximal inactivation rate constants (kinact,max) of 0.00856 and 0.0134 min−1, respectively. The EAE did not inhibit CYP2C19, even when preincubation was employed. These findings imply that jabara juice may cause food-drug interactions via time-dependent inhibition of intestinal CYP3A4.

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低頻度バリアント保有者における薬物相互作用リスクの予測精度を改善するためのin vitro & in silico研究

秋好健志

臨床薬理の進歩（(公財)臨床薬理研究振興財団）（ 45 ） 142 - 151 2024年06月

ISSN 0914-4366

CYP3A4に対する代表的なMBI阻害剤であるエリスロマイシンおよびクラリスロマイシンのMBIキネティクスに対する遺伝子変異の影響が、結合部位の異なる基質を用いて評価した際に異なって観測されるのか、その違いは低頻度バリアントにおける薬物相互作用にどの程度影響を及ぼすのかをin vitro試験により検討した。結果、エリスロマイシンとクラリスロマイシンはいずれも各CYP3A4 variantsのミダゾラム1-水酸化を濃度依存的およびpreincubation時間依存的に阻害し、そのMBIパラメータはvariants間で異なり、変動のパターンはテストステロンを基質とした場合と類似していた。これらのことから、MBI阻害剤の阻害キネティクスは遺伝子変異の影響を受けるが、その影響は結合部位が異なる基質間で類似している可能性が示唆された。

The Effects of N-Glycosylation on the Expression and Transport Activity of OATP1A2 and OATP2B1.

Kataoka H, Akiyoshi T, Uchida Y, Imaoka A, Terasaki T, Ohtani H

Journal of pharmaceutical sciences （Journal of Pharmaceutical Sciences） 113 （ 5 ） 1376 - 1384 2024年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0022-3549

Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N-glycosylation sites, but their influence remains unclear. This study aimed to identify the N-glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1. Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N-glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N-glycosylation sites were established, and the Western blotting and uptake study were repeated. Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively. In conclusion, the N-glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function.

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Determination of single-molecule transport activity of OATP2B1 by measuring the number of transporter molecules using electrophysiological approach

Yajima K., Akiyoshi T., Sakamoto K., Suzuki Y., Oka T., Imaoka A., Yamamura H., Kurokawa J., Ohtani H.

Journal of Pharmacological Sciences （Journal of Pharmacological Sciences） 153 （ 3 ） 153 - 160 2023年11月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 13478613

Transporter-mediated clearance is determined by two factors, its single-molecule clearance, and expression level. However, no reliable method has been developed to evaluate them separately. This study aimed to develop a reliable method for evaluating the single-molecule activity of membrane transporters, such as organic anion transporting polypeptide (OATP) 2B1. HEK293 cells that co-expressed large conductance calcium-activated potassium (BK) channel and OATP2B1 were established and used for the following experiments. i) BK channel-mediated whole-cell conductance was measured using patch-clamp technique and divided by its unitary conductance to estimate the number of channels on plasma membrane (QI). ii) Using plasma membrane fraction, quantitative targeted absolute proteomics determined the stoichiometric ratio (ρ) of OATP2B1 to BK channel. iii) The uptake of estrone 3-sulfate was evaluated to calculate the Michaelis constant and uptake clearance (CL) per cell. Single-molecule clearance (CLint) was calculated by dividing CL by QI·ρ. QI and ρ values were estimated to be 916 and 2.16, respectively, yielding CLint of 5.23 fL/min/molecule. We successfully developed a novel method to reliably measure the single-molecule activity of a transporter, which could be used to evaluate the influences of factors such as genetic variations and post-translational modifications on the intrinsic activity of transporters.

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The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro.

Kojima M, Machida K, Cho S, Watanabe D, Seki H, Shimoji M, Imaoka A, Yamazaki H, Guengerich FP, Nakamura K, Yamamoto K, Akiyoshi T, Ohtani H

Xenobiotica; the fate of foreign compounds in biological systems （Xenobiotica） 53 （ 5 ） 1 - 9 2023年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0049-8254

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyse warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C. 2. Compared with that seen at 37 °C, the intrinsic clearance rates (V max/K m) of CYP2C9.1 and.2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1,.2, and.3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and.10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and.23 were increased (130 ∼ 134%). At 40 °C, the clearance rates of CYP2C19.1A,.1B,.10, and.23 remained unaffected, while that of CYP2C19.8 was decreased (74%). At 34 °C, the clearance rates of CYP3A4.1 and.16 were decreased (79 ∼ 84%), those of CYP3A4.2 and.7 were unchanged, and that of CYP3A4.18 was slightly increased (112%). At 40 °C, the clearance rate of CYP3A4.1 remained unaffected, while those of CYP3A4.2,.7,.16, and.18 were decreased (58 ∼ 82%). 3. These findings may be clinically useful for dose optimisation in patients with hypothermia or hyperthermia.

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Irinotecan-induced gastrointestinal damage alters the expression of peptide transporter 1 and absorption of cephalexin in rats.

Imaoka A, Hattori T, Akiyoshi T, Ohtani H

Biopharmaceutics & drug disposition （Biopharmaceutics and Drug Disposition） 44 （ 5 ） 372 - 379 2023年07月

ISSN 0142-2782

Irinotecan causes severe gastrointestinal damage, which may affect the expression of intestinal transporters. However, neither the expression of peptide transporter 1 (Pept1) nor the pharmacokinetics of Pept1 substrate drugs has been investigated under irinotecan-induced gastrointestinal damage. Therefore, the present study quantitatively investigated the effects of irinotecan-induced gastrointestinal damage on the intestinal expression of Pept1 and absorption of cephalexin (CEX), a typical Pept1 substrate, in rats. Irinotecan was administered intravenously to rats for 4 days to induce gastrointestinal damage. The expression of Pept1 mRNA and the Pept1 protein in the upper, middle, and lower segments of the small intestine of irinotecan-treated rats was assessed by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. The pharmacokinetic profile of CEX was examined after its oral or intravenous administration (10 mg/kg). In irinotecan-treated rats, ∼2-fold increases in Pept1 protein levels were observed in all three segments, whereas mRNA levels remained unchanged. The oral bioavailability of CEX significantly decreased to 76% of that in control rats. The decrease in passive diffusion caused by intestinal damage may have overcome the increase in Pept1-mediated uptake. In conclusion, irinotecan may decrease the intestinal absorption of Pept1 substrate drugs; however, it increased the expression of intestinal Pept1.

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The Effects of Jabara Juice on the Intestinal Permeation of Fexofenadine.

Han H, Akiyoshi T, Morita T, Tsuchitani T, Nabeta M, Yajima K, Imaoka A, Ohtani H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin） 46 （ 12 ） 1745 - 1752 2023年

ISSN 0918-6158

Jabara juice and its component narirutin inhibit the activity of organic anion-transporting polypeptides (OATPs) 1A2 and OATP2B1, which are considered to play significant roles in the intestinal absorption of fexofenadine. In this study, we investigated the effects of jabara juice on the intestinal absorption of fexofenadine in mice and the inhibitory effects of jabara juice and narirutin on the permeation of fexofenadine using Caco-2 cell monolayers and LLC-GA5-COL300 cell monolayers. In the in vivo study, the area under the plasma concentration–time curve (AUC) of fexofenadine in mice was increased 1.8-fold by jabara juice. In the permeation study, 5% jabara juice significantly decreased the efflux ratio (ER) of fexofenadine for Caco-2 monolayers. Furthermore, the ERs of fexofenadine and digoxin, which is a typical substrate of P-glycoprotein (P-gp), for LLC-GA5-COL300 cell monolayers were decreased in a concentration-dependent manner by jabara juice extract, suggesting that jabara juice may increase the intestinal absorption of fexofenadine by inhibiting P-gp, rather than by narirutin inhibiting OATPs. The present study showed that jabara juice increases the intestinal absorption of fexofenadine both in vivo and in vitro. The intestinal absorption of fexofenadine may be altered by the co-administration of jabara juice in the clinical setting.

Comparison of the transport kinetics of fexofenadine and its pH dependency among OATP1A2 genetic variants.

Han H, Akiyoshi T, Morita T, Kataoka H, Katayama K, Yajima K, Imaoka A, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics） 47 100470 2022年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1347-4367

Little is known about the influence of non-synonymous genetic variations in the organic anion-transporting polypeptide (OATP) 1A2 on the transport kinetics of its substrate fexofenadine. Moreover, the pH-dependency of fexofenadine uptake also remains unclear. This study aimed to evaluate the effects of genetic variants (Ile13Thr, Asn128Tyr, Glu172Asp, Ala187Thr, and Thr668Ser) on the OATP1A2-mediated uptake of fexofenadine at pH 6.3 and 7.4 and compare the pH dependency of OATP1A2-mediated uptake of fexofenadine and estrone 3-sulfate. The uptake clearances of 0.3 μM and 300 μM fexofenadine were compared with those of 0.3 μM and 300 μM estrone 3-sulfate at pH 6.3 and 7.4. Among the six variants examined, the Thr668Ser variant showed the highest fexofenadine uptake clearance (Vmax/Km); i.e., 4.53- and 6.28-fold higher uptake clearance than the wild type at pH 6.3 and 7.4, respectively. All variants exhibited significantly higher fexofenadine uptake at pH 6.3 than at pH 7.4. Compared with estrone 3-sulfate uptake, the uptake of 0.3 μM fexofenadine was less sensitive to pH. Our findings suggest that genetic variations in OATP1A2 may lead to altered intestinal absorption of fexofenadine, such as increased absorption in subjects bearing the Thr668Ser variant, which showed higher uptake activity.

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Comparison of the inhibitory properties of the fruit component naringenin and its glycosides against OATP1A2 genetic variants.

Araki N, Morita T, Akiyoshi T, Kataoka H, Yajima K, Katayama K, Imaoka A, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics） 46 100464 2022年05月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1347-4367

Non-synonymous genetic variants of organic anion-transporting polypeptide (OATP) 1A2 with altered transport activity have been identified. Naringin and narirutin, which are found in grapefruit, and their aglycon naringenin inhibit OATP1A2. However, their inhibitory effects on OATP1A2 variants have not been investigated, nor has the influence of their molecular structure, such as the number of sugar moieties, on their inhibitory potency. This study aimed to investigate the inhibitory effects of naringenin, its monosaccharide glycoside prunin, and its disaccharide glycosides naringin and narirutin on fexofenadine (FEX) uptake by OATP1A2 variants (Ile13Thr, Asn128Tyr, Ala187Thr, and Thr668Ser). Naringin, narirutin, and prunin inhibited FEX (0.3 μM) uptake by all of the examined OATP1A2 variants in a concentration-dependent manner. Compared with those for the wild type, the inhibition constants (Ki) of naringin, narirutin, and prunin for the Ala187Thr variant were significantly increased by 3.36-fold, 7.55-fold, and 10.6-fold, respectively. Naringenin inhibited all of the OATP1A2 variants, except Ala187Thr, concentration-dependently. The order of inhibitory potency was as follows for all variants: aglycone > monosaccharide glycoside > disaccharide glycosides. These results suggest that the Ala187Thr variant is less vulnerable to inhibition by naringenin and its glycosides. Moreover, greater glycosylation of naringenin reduces its inhibitory potency against OATP1A2.

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Mechanistic bottom-up estimation of passive drug absorption from the gastrointestinal tract: Comparison among primary cultured human intestinal cells, Caco-2 cells, artificial membrane, and animal scale-up.

Tsuchitani T, Akiyoshi T, Imaoka A, Ohtani H

International journal of clinical pharmacology and therapeutics （International journal of clinical pharmacology and therapeutics） 60 （ 5 ） 217 - 224 2022年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0946-1965

OBJECTIVE: The fraction of drug absorbed (Fa) from the intestine is an important parameter to characterize the pharmacokinetics of a drug. We aimed to search for an experimental system that provides the best parameters for estimating the effective permeability (Peff) used for the bottom-up prediction of Fa. MATERIALS AND METHODS: The absorption kinetics of 12 passively absorbed drugs were simulated by a compartment absorption transit (CAT) model using absorption parameters from four different experimental systems: human intestinal epithelial cell (HIEC) monolayer, Caco-2 monolayer, parallel artificial membrane permeability assay (PAMPA), and in situ rat intestinal perfusion. All absorption parameters were obtained from the literature. The in vitro apparent permeability coefficient (Papp) and rat in situ Peff were converted to human Peff using a bottom-up approach for each region, based on the morphological features of the human intestine. The simulated Fa values were compared to the respective observed values. Furthermore, plasma concentration profiles of the drugs were simulated by convolution using the time-course of the absorption rate simulated using the Peff values calculated from the HIEC Papp. RESULTS: The Fa values were best predicted by using the Peff values calculated from HEIC, within a 1.3-fold range of observed Fa in 11 out of 12 drugs. The simulated Cmax values of pharmacokinetic simulation using HIEC Papp fell within a 1.5-fold range of observed values for all the drugs examined. CONCLUSION: The HIEC monolayer was identified as the most suitable permeation parameter for estimating Fa and Cmax using a morphological feature-based bottom-up approach.

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Inhibitory effects of cranberry juice and its components on intestinal OATP1A2 and OATP2B1: Identification of avicularin as a novel inhibitor

Morita T, Akiyoshi T, Tsuchitani T, Kataoka H, Araki N, Yajima K, Katayama K, Imaoka A, Ohtani H

J Agric Food Chem （Journal of Agricultural and Food Chemistry） In press （ 10 ） 3310 - 3320 2022年02月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0021-8561

Organic anion-transporting polypeptide (OATP) 1A2 and OATP2B1 mediate the intestinal absorption of drugs. This study aimed to identify fruit juices or fruit juice components that inhibit OATPs and assess the risk of associated food-drug interactions. Inhibitory potency was assessed by examining the uptake of [3H]estrone 3-sulfate and [3H]fexofenadine into HEK293 cells expressing OATP1A2 or OATP2B1. In vivo experiments were conducted using mice to evaluate the effects of cranberry juice on the pharmacokinetics of orally administered fexofenadine. Of eight examined fruit juices, cranberry juice inhibited the functions of both OATPs most potently. Avicularin, a component of cranberry juice, was identified as a novel OATP inhibitor. It exhibited IC50 values of 9.0 and 37 μM for the inhibition of estrone 3-sulfate uptake mediated by OATP1A2 and OATP2B1, respectively. A pharmacokinetic experiment revealed that fexofenadine exposure was significantly reduced (by 50%) by cranberry juice. Cranberry juice may cause drug interactions with OATP substrates.

Evaluation of hepatic CYP3A enzyme activity using endogenous markers in lung cancer patients treated with cisplatin, dexamethasone, and aprepitant.

Hibino H, Sakiyama N, Makino Y, Makihara-Ando R, Horinouchi H, Fujiwara Y, Kanda S, Goto Y, Yoshida T, Okuma Y, Shinno Y, Murakami S, Hashimoto H, Akiyoshi T, Imaoka A, Ohe Y, Yamaguchi M, Ohtani H

European journal of clinical pharmacology （European Journal of Clinical Pharmacology） Accepted （ 4 ） 613 - 621 2022年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0031-6970

Purpose: Aprepitant is used with dexamethasone and 5-HT3 receptor antagonists as an antiemetic treatment for chemotherapy, including cisplatin. Aprepitant is a substrate of cytochrome P450 (CYP) 3A4 and is known to cause its inhibition and induction. In addition, dexamethasone is a CYP3A4 substrate that induces CYP3A4 and CYP3A5 expression. In this study, we aimed to quantitatively evaluate the profile of CYP3A activity using its endogenous markers in non-small cell lung cancer patients receiving a standard cisplatin regimen with antiemetics, including aprepitant. Methods: Urinary 11β-hydroxytestosterone (11β-OHT)/testosterone concentration ratio and plasma 4β-hydroxycholesterol (4β-OHC) concentrations were measured before and after cisplatin treatment (days 1, 4, and 8). CYP3A5 was genotyped, and plasma aprepitant concentrations were measured on day 4 to examine its influence on CYP3A endogenous markers. Results: The urinary 11β-OHT/testosterone concentration ratio in the 35 patients included in this study increased by 2.65-fold and 1.21-fold on days 4 and 8 compared with day 1, respectively. Their plasma 4β-OHC concentration increased by 1.46-fold and 1.66-fold, respectively. The mean plasma aprepitant concentration on day 4 was 1,451 ng/mL, which is far lower than its inhibitory constant. The allele frequencies of CYP3A5*1 and CYP3A5*3 were 0.229 and 0.771, respectively. In patients with the CYP3A5*1 allele, the plasma 4β-OHC concentration was significantly lower at baseline but more potently increased with chemotherapy. Conclusion: CYP3A activity was significantly induced from day 4 to day 8 in patients receiving cisplatin and three antiemetic drugs.

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Analysis of inhibition kinetics of three beverage ingredients, bergamottin, dihydroxybergamottin and resveratrol, on CYP2C9 activity.

Akiyoshi T, Uchiyama M, Inada R, Imaoka A, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics） In press 100429 2021年10月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1347-4367

Some grapefruit juice (GFJ) ingredients and resveratrol, a fruit-derived phytoalexin, are known to inhibit cytochrome P450 (CYP) 2C9. However, their inhibition modes and detailed inhibition kinetics remain undetermined. This study aimed to investigate the inhibitory effects of two GFJ ingredients, bergamottin (BG) and dihydroxybergamottin (DHB), and resveratrol on CYP2C9 activity in vitro. DHB inhibited CYP2C9 activity, as assessed by warfarin 7-hydroxylation, in a preincubation time-dependent manner (i.e., mechanism-based inhibition; MBI), in the same manner as CYP2C19 and CYP3A4. The maximal inactivation rate (kinact,max) was 0.0638 min−1 and 0.12- and 0.26-fold of that for CYP2C19 and CYP3A4, respectively. BG showed both MBI and time-independent competitive inhibition. Resveratrol showed non-competitive inhibition with an inhibition constant (Ki) of 3.64 μM. Unlike the inhibition of CYP2C19 and CYP3A4, resveratrol did not induce MBI. These findings are important for estimating the risk of drug interactions between CYP2C9 substrates and some beverages. (146 words)

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Time-dependent inhibition of CYP3A4-mediated midazolam metabolism by macrolide antibiotics in CYP3A4 genetic variants: Comparison with testosterone metabolism.

Akiyoshi T, Naitou R, Imaoka A, Miyazaki M, Guengerich FP, Nakamura K, Yamamoto K, Ohtani H

International journal of clinical pharmacology and therapeutics （International journal of clinical pharmacology and therapeutics） 59 （ 12 ） 745 - 752 2021年09月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 0946-1965

OBJECTIVES: The present study aimed to evaluate the effects of CYP3A4 genetic variation on the kinetics of mechanism-based inhibition (MBI) of both inhibitors using midazolam as a substrate for comparison with our previous study, as midazolam and testosterone have different binding sites. BACKGROUND: The genetic variation of cytochrome P450 (CYP) 3A4 affects MBI, expressed as the maximum inactivation rate constant (kinact,max) and the inhibitor concentration required to achieve half-maximal inactivation (KI). We previously showed, using testosterone as a substrate, that the MBI kinetics of erythromycin and clarithromycin differ among CYP3A4 variants. MATERIALS AND METHODS: Midazolam 1'-hydroxylation inactivation profiles of erythromycin and clarithromycin were assessed using recombinant CYP3A4.1, .2, .7, .16, and .18 expressed in Escherichia coli. MBI parameters were calculated from changes in the inactivation rate constant (Δkobs) by the inhibitors. RESULTS: Both inhibitors increased Δkobs value in a concentration- and preincubation time-dependent manner, and MBI kinetics differed among variants. Trends of differences in MBI parameters among variants were similar to those assessed using testosterone as a substrate; KI decreased for CYP3A4.7, and kinact,max decreased for CYP3A4.2, .7, and .16. CONCLUSION: The genetic variation of recombinant CYP3A4 affects the MBI profile of CYP3A4 by erythromycin and clarithromycin, while the influence of genetic variation was similarly observed regardless of substrates. Our findings are of clinical relevance because the residual enzyme activity of CYP3A4 in the presence of inhibitor was estimated to vary among genetic variants.

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Dual kinetics of OATP2B1: Inhibitory potency and pH-dependence of OATP2B1 inhibitors.

Sato R, Akiyoshi T, Morita T, Katayama K, Yajima K, Kataoka H, Imaoka A, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics） 41 100416 2021年08月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 1347-4367

Organic anion transporting polypeptide (OATP) 2B1 is expressed in the intestine and liver, and OATP2B1-mediated transport of estrone 3-sulfate is pH-dependent and consists of: the high-affinity component (Hc) and low-affinity component (Lc). This study aimed to evaluate the influence of pH on the transport kinetics of each component, along with the inhibitory nature of ten OATP2B1 inhibitors. The Michaelis constants (Km) were 4-fold and 1.5-fold lower at pH 6.3 than at pH 7.4, for Hc and Lc respectively. The inhibitory potencies of diclofenac, indomethacin, and ibuprofen towards Hc were 1.5–4.3 fold lower at pH 6.3 than at pH 7.4. Contrastingly, inhibitory potencies towards Lc were 9.0–52 fold lower at pH 7.4. Similarly, the inhibitory effect of naproxen was stronger towards Hc at pH 6.3 and towards Lc at pH 7.4. On the other hand, celecoxib selectively inhibited Lc transport at pH 7.4. Rifampicin inhibited both components at pH 6.3 and 7.4 to a similar extent, while bromosulphophthalein, naringin, and gefitinib selectively inhibited Hc irrespective of pH. Fexofenadine inhibited neither component. In conclusion, the transport affinities of both Hc and Lc were enhanced under acidic conditions. The influence of pH on the inhibitory potency towards each component varied among the inhibitors.

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