論文 - 秋好　健志

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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

　概要を見る

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.

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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

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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

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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

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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

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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.

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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

　概要を見る

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

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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

　概要を見る

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

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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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Digoxin absorption decreased independently of P-gp activity in rats with irinotecan-induced gastrointestinal damage.

Tsuchitani T, Akiyoshi T, Imaoka A, Ohtani H

Journal of pharmaceutical health care and sciences （Journal of Pharmaceutical Health Care and Sciences）  7 （ 1 ） 24 2021年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2055-0294

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Background: Irinotecan (CPT-11) is clinically known to cause severe diarrhea and gastrointestinal damage. Recently, we have reported that CPT-11-induced gastrointestinal damage is associated with the upregulation of intestinal P-glycoprotein (P-gp) expression and decreased absorption of its substrate, dabigatran etexilate (DABE), using a rat model. However, the P-gp activity or its contribution to the decreased absorption remains unclear. The aim of this study was to quantitatively evaluate how P-gp activity changes in rats with CPT-11-induced gastrointestinal damage, as assessed by the absorption of digoxin (DGX), a typical P-gp substrate. Methods: Male Sprague-Dawley rats were intravenously administered CPT-11 at a dose of 60 mg/kg/day for 4 days to induce gastrointestinal damage. Then, the rats were administered DGX orally (40 μg/kg), after some of them were orally administered clarithromycin (CAM; 10 mg/kg), a P-gp inhibitor. DGX (30 μg/kg) was administered intravenously to determine the bioavailability (BA). The rats’ DGX plasma concentration profiles were determined using LC-MS/MS. Results: CPT-11 treatment decreased the maximum concentration (Cmax) and area under the plasma concentration-time curve (AUCpo) of DGX, which does not contradict to the DABE study. Although in the CPT-11-treated group the BA of DGX was significantly decreased to 40% of the control value, CAM did not affect the BA of DGX in the CPT-11-treated group. Conclusions: Increased P-gp expression in rats with CPT-11-induced gastrointestinal damage is not necessarily associated with increased P-gp activity or contribution to the drug absorption in vivo. The decreased DGX absorption observed in this study might be attributable to other factors, such as a reduction in the absorptive surface area of the gastrointestinal tract.

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Estimation of absolute oral bioavailability without performing an intravenous clinical study.

Ohtani H, Iwata R, Imaoka A, Akiyoshi T

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics）  38   100392 2021年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1347-4367

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The absolute oral bioavailability (BA) of drugs are yet to be determined, and intravenous pharmacokinetic studies are currently considered indispensable for determining the BA values of oral drugs. The aim of this study was to develop and validate a novel approach to estimating BA values without intravenous pharmacokinetic data. Based on the drug inclusion criteria, such as exhibiting a urinary recovery rate of (R<inf>u</inf>) of ≥20% in a clinical study, 13 drugs were included in the present study, and pharmacokinetic data for them were collected from the literature. The fraction excreted unchanged into urine (f<inf>e</inf>) was calculated for healthy subjects by dividing the R<inf>u</inf> value by the total recovery rate. The contribution of renal excretion to total clearance from the systemic circulation (R<inf>ren</inf>) was estimated by subjecting oral clearance and creatinine clearance to regression in subjects with normal and impaired renal function. BA was estimated as f<inf>e</inf>/R<inf>ren</inf> and compared with the observed BA (BA<inf>obs</inf>). The predicted BA values for 9 drugs fell within ±20% of their BA<inf>obs</inf>. The examined approach makes it possible to estimate BA values for drugs with mean renal excretion values in healthy subjects and oral clearance in subjects with various renal function, without intravenous pharmacokinetic data.

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Comparison of the inhibitory effects of azole antifungals on cytochrome P450 3A4 genetic variants.

Yamaguchi Y, Akiyoshi T, Kawamura G, Imaoka A, Miyazaki M, Guengerich FP, Nakamura K, Yamamoto K, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics）  38   100384 2021年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1347-4367

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Cytochrome P450 (CYP) 3A4 is one of the major drug-metabolizing enzymes. Genetic variants of CYP3A4 with altered activity are one of the factors responsible for interindividual differences in drug metabolism. Azole antifungals inhibit CYP3A4 to cause clinically significant drug-drug interactions. In the present quantitative study, we investigated the inhibitory effects of three azole antifungals (ketoconazole, voriconazole, and fluconazole) on testosterone metabolism by recombinant CYP3A4 genetic variants (CYP3A4.1 (WT), CYP3A4.2, CYP3A4.7, CYP3A4.16, and CYP3A4.18) and compared them with those previously reported for itraconazole. The inhibition constants (K ) of ketoconazole, voriconazole, and fluconazole for rCYP3A4.1 were 3.6 nM, 3.2 μM, and 16.1 μM, respectively. The K values of these azoles for rCYP3A4.16 were 13.9-, 13.6-, and 6.2-fold higher than those for rCYP3A4.1, respectively, whereas the K value of itraconazole for rCYP3A4.16 was 0.54-fold of that for rCYP3A4.1. The other genetic variants had similar effects on the K values of the three azoles, whereas a very different pattern was seen for itraconazole. In conclusion, itraconazole has unique characteristics that are distinct from those shared by the other azole anti-fungal drugs ketoconazole, voriconazole, and fluconazole with regard to the influence of genetic variations on the inhibition of CYP3A4. i i i i

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Exacerbation of atrioventricular block associated with concomitant use of amlodipine and aprepitant in a lung cancer patient: A case report.

Hibino H, Makino Y, Sakiyama N, Makihara-Ando R, Hashimoto H, Akiyoshi T, Imaoka A, Fujiwara Y, Ohe Y, Yamaguchi M, Ohtani H

International journal of clinical pharmacology and therapeutics （International Journal of Clinical Pharmacology and Therapeutics）  59 （ 4 ） 328 - 332 2020年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0946-1965

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Objective: To report a case of second-degree atrioventricular block associated with concomitant use of aprepitant and amlodipine. Case: A 73-year-old man with lung cancer was treated with aprepitant for prophylactic use for the prevention of nausea and vomiting, concomitantly with cisplatin, gemcitabine, and an investigational drug (anti-epidermal growth factor receptor monoclonal antibody). He was diagnosed with first-degree atrioventricular block and was taking amlodipine for hypertension. During the first cycle of chemotherapy, 5 days after the start of aprepitant, he experienced Wenckebach second-degree atrioventricular block (Mobitz type I), and amlodipine was discontinued. After day 6, the atrioventricular block was not shown. According to the Naranjo adverse drug reaction scale, a score of 7 was obtained (causality: probable). In addition, using the Drug Interaction Probability Scale, a score of 6 was obtained (causality: probable). Conclusion: The drug-drug interaction between aprepitant and amlodipine was considered to have deteriorated his atrioventricular block, conceivably due to the inhibition of cytochrome P450 (CYP) 3A-mediated metabolism of amlodipine by aprepitant.

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Citrus Fruit-Derived Flavanone Glycoside Narirutin is a Novel Potent Inhibitor of Organic Anion-Transporting Polypeptides.

Morita T, Akiyoshi T, Sato R, Uekusa Y, Katayama K, Yajima K, Imaoka A, Sugimoto Y, Kiuchi F, Ohtani H

Journal of agricultural and food chemistry （Journal of Agricultural and Food Chemistry）  68 （ 48 ） 14182 - 14191 2020年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021-8561

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Organic anion-transporting polypeptides (OATPs) 1A2 and OATP2B1 are expressed in the small intestine and are involved in drug absorption. We identified narirutin, which is present in grapefruit juice, as a novel OATP inhibitor. The citrus fruit jabara also contains high levels of narirutin; therefore, we investigated the inhibitory potency of jabara juice against OATPs. The inhibitory effects of various related compounds on the transport activity of OATPs were evaluated using OATP-expressing HEK293 cells. The IC50 values of narirutin for OATP1A2- and OATP2B1-mediated transport were 22.6 and 18.2 μM, respectively. Other flavanone derivatives from grapefruit juice also inhibited OATP1A2/OATP2B1-mediated transport (order of inhibitory potency: naringenin > narirutin > naringin). Five percent jabara juice significantly inhibited OATP1A2- and OATP2B1-mediated transport by 67 ± 11 and 81 ± 5.5%, respectively (p < 0.05). Based on their inhibitory potency and levels in grapefruit juice, the inhibition of OATPs by grapefruit juice is attributable to both naringin and narirutin. Citrus × jabara, which contains narirutin, potently inhibits OATP-mediated transport.

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Relative contributions of metabolic enzymes to systemic elimination can be estimated from clinical DDI studies: Validation using an in silico approach.

Koinuma K, Tsuchitani T, Imaoka A, Akiyoshi T, Ohtani H

International journal of clinical pharmacology and therapeutics （International Journal of Clinical Pharmacology and Therapeutics）  In press （ 3 ） 231 - 238 2020年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0946-1965

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Objective: The contribution ratios (CR) of metabolic enzymes to the systemic clearance of a drug can be estimated from in vitro studies. Another feasible approach is to calculate them based on the increase in the area under the time-concentration curve (AUC) caused by the co-administration of a potent and selective inhibitor in a clinical drug-drug interaction (DDI) study. However, some factors, such as the inhibitory potency of the inhibitor and the inhibition of first-pass metabolism, might affect the estimation of CR based on clinical DDI studies. We aimed to validate the accuracy of the DDI-based estimation of CR using an in silico approach. Materials and methods: An in silico DDI study was conducted using a population-based physiological pharmacokinetic simulator to estimate the CR of cytochrome P450 (CYP)3A4 for zolpidem, sildenafil, omeprazole, triazolam, and repaglinide. The ratio of the AUC value seen in the presence of an inhibitor (ketoconazole or itraconazole) to that observed in the absence of the inhibitor (AUC ratio) was also calculated. The CR for CYP3A4 obtained using the simulator (CR ) were compared with those calculated from the AUC ratio (CR ). Results: When ketoconazole was used, good correlations between the CR and CR were obtained for all examined substrates (inconsistencies were seen in < 10% of subjects). CR estimates derived from the AUC ratio were found to be accurate. Some underestimation was observed, possibly due to incomplete inhibition, and some overestimation caused by extensive first-pass metabolism was noted. Conclusion: This study verified that CR obtained from AUC ratios in DDI studies are quite reliable. def est est def

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Novel method to estimate the appropriate dosing interval for activated charcoal to avoid interaction with other drugs.

Ohtani H, Nakamura K, Imaoka A, Akiyoshi T

European journal of clinical pharmacology （European Journal of Clinical Pharmacology）  76 （ 11 ） 1529 - 1536 2020年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0031-6970

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© 2020, Springer-Verlag GmbH Germany, part of Springer Nature. Purpose: Activated charcoal is known to adsorb a variety of drugs concomitantly administered and reduce their intestinal absorption, and separating the dosing is considered a practical approach to avoid this drug interaction. The aim of the present study was to develop and validate a simple method to estimate the sufficient dosing interval to avoid drug interaction using the pharmacokinetic profile of the subject drugs administered alone and the amplitude of interaction upon simultaneous administration with activated charcoal. Methods: For each subject drug, the pharmacokinetic profile and the amplitude of interaction, as assessed by AUCR (the ratio of area under the plasma concentration-time curve (AUC) in the presence of activated charcoal to that in its absence), were collected from previous reports. The AUCR value was estimated based on the compartment model under the assumption that the subject drug in the first gastrointestinal compartment is immediately adsorbed to a certain extent upon the administration of activated charcoal. The estimated AUCR (AUCRe) for each drug with certain dosing interval was compared with the respective AUCR value reported previously (AUCRobs). Results: Among twenty concentration profiles for 14 subject drugs obtained from previous reports, 15 AUCRe values fell in the range of 80–120% of the respective AUCRobs values. Conclusion: The developed method enabled estimation of the amplitude of DDI by activated charcoal administered in a certain dosing interval, whereas overestimation of AUCRe was observed for drugs that undergo extensive enterohepatic circulation.

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Profile of the inhibitory effects of gefitinib on CYP2D6 variants in vitro.

Semba Y, Akiyoshi T, Hibino H, Imaoka A, Ohtani H

International journal of clinical pharmacology and therapeutics （International journal of clinical pharmacology and therapeutics）  58 （ 10 ） 539 - 542 2020年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0946-1965

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OBJECTIVE: CYP2D6 is a highly polymorphic metabolic enzyme with more than 100 genetic variants, some of which are associated with significantly altered enzyme activity, such as CYP2D6.2 (Arg296Cys, Ser486Thr), CYP2D6.10 (Pro34Ser, Ser486Thr), and CYP2D6.39 (Ser486Thr). Gefitinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) and is used to treat non-small-cell lung cancer with EGFR mutations. Gefitinib is known to competitively inhibit CYP2D6 activity. The aim of this study was to quantitatively compare the inhibitory effects of gefitinib on CYP2D6 enzyme kinetics among CYP2D6 variants. MATERIALS AND METHODS: The enzymatic activity of several CYP2D6 genetic variants; i.e., CYP2D6.1 (wild type), CYP2D6.2, CYP2D6.10, and CYP2D6.39, was assessed by examining the O-demethylation of dextromethorphan. RESULTS AND CONCLUSION: The intrinsic clearance of dextromethorphan (Vmax/Km) for CYP2D6.2, CYP2D6.10, and CYP2D6.39 were 0.565-, 0.0376-, and 0.470-fold of that for wild type, respectively. For all variants, the mixed inhibition model was better at explaining the nature of the inhibitory effects of gefitinib than the competitive inhibition model. However, the inhibitory potency of gefitinib varied among the CYP2D6 genetic variants. The Ki values for CYP2D6.2, CYP2D6.10, and CYP2D6.39 were 1.4-, 2.5- and 1.5-fold higher than that for wild type, respectively, implying that these variants are less susceptible to the inhibition by gefitinib. The genetic variations in CYP2D6 might be one of the factors responsible for inter-individual differences in the strength of CYP2D6-mediated drug interactions involving gefitinib.

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pH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2.

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

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics）  35 （ 2 ） 220 - 227 2020年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1347-4367

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© 2019 The Japanese Society for the Study of Xenobiotics Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the Km value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.

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医薬品の第三者への転売・譲渡の違法性に関する消費者の意識調査.

大谷壽一, 藤井萌未, 今岡鮎子, 望月真弓, 山浦克典, 秋好健志.

医薬品情報学 （(一社)日本医薬品情報学会）  22 （ 1 ） 30 - 34 2020年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1345-1464

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医薬品をインターネットオークションに出品することや第三者に譲渡することの違法性を一般消費者がどれくらい認識しているか調査した。処方箋の調剤待ち時間を利用して来局した20歳以上の患者またはその家族にアンケートの概要を説明し、151件の有効回答を得た。28.5%の患者が医薬品を他人から譲り受けた経験を有していることが明らかとなった。薬局で購入した湿布薬を他人から譲り受けるかインターネットオークションで買うことに対する違法性の認識はそれぞれ9.9%、24.5%と低く、転売や譲渡が必ずしも違法とはならない化粧品や健康食品の譲渡に対する違法性の認識とほぼ同程度であった。「違法である」との認識は睡眠薬が最も高く、ついで風邪薬、湿布薬の順であった。病院で処方された湿布薬を「インターネットオークションで買うこと」について違法性の認識は60代未満または女性で有意に高かった。

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Irinotecan-induced gastrointestinal damage impairs the absorption of dabigatran etexilate.

Hattori T, Imaoka A, Akiyoshi T, Ohtani H

Biopharmaceutics & drug disposition （Biopharmaceutics and Drug Disposition）  40 （ 9 ） 315 - 324 2019年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0142-2782

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© 2019 John Wiley & Sons, Ltd. Irinotecan causes serious gastrointestinal damage. Dabigatran etexilate (DABE), an oral anticoagulant and substrate of P-glycoprotein (P-gp), is poorly absorbed and exhibits low bioavailability in humans. The aim of this study was to evaluate the effects of irinotecan-induced gastrointestinal damage on the pharmacokinetics/pharmacodynamics (PK/PD) of DABE. Irinotecan was administered intravenously to rats for 4 days to induce gastrointestinal damage. To investigate the PK profile of dabigatran (DAB), an active moiety of DABE, DABE was administered orally on day 5, and then DAB was administered intravenously on day 6. To evaluate the PD profile of DAB, the activated partial thromboplastin time (APTT) was measured. The protein expression level of intestinal P-gp was evaluated. In the irinotecan-treated rats, the area under the concentration–time curve of DAB after the oral administration of DABE and the bioavailability of DABE were decreased significantly. The APTT ratio also decreased, suggesting that the impaired efficacy of DABE was attributable to a reduction in its bioavailability. The expression of intestinal P-gp was higher in the irinotecan-treated rats. Taking into consideration the histological damage caused to the intestinal epithelium, both the increased P-gp expression and the reduced passive diffusion were considered to be responsible for the reduction in the bioavailability of DABE.

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インターネットオークションにおける無承認無許可医薬品の出品実態.

大谷壽一*, 藤井萌未, 今岡鮎子, 秋好健志.

医療薬学 （(一社)日本医療薬学会）  45 （ 10 ） 591 - 595 2019年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1346-342X

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インターネットオークションサイト「ヤフオク!」における無承認無許可医薬品の取引実態調査を行うことで、一般消費者に対する医薬品の適正使用啓蒙活動に必要な基礎的情報を構築することを目的とした。調査は第1回を平成28年6月13日〜平成28年6月17日、第2回を平成28年7月28日〜平成28年7月31日の期間内にそれぞれ実施した。第1回調査ではキーワードにより3758件の商品が検索され、そのなかから571件の違法商品と48件のグレー商品が確認された。第2回調査では3808件の商品が検索され、そのうち581件の違法商品と40件のグレー商品が確認された。違法商品のうち入札のあった商品は第1回目の調査では19件(3.3%)、第2回目の調査では24件(4.1%)であった。製造国は第1回調査では日本が423件(74.1%)、外国が146件(25.6%)、第2回調査では日本が446件(76.8%)、外国が134件(23.1%)であり、少なからず外国製の商品も見出された。

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Inhibitory kinetics of fruit components on CYP2C19 activity

Seki H, Akiyoshi T, Imaoka A, Ohtani H.

Drug Metabol Pharmacokinet （Drug Metabolism and Pharmacokinetics）  34 （ 3 ） 181 - 186 2019年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  13474367

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© 2019 The Japanese Society for the Study of Xenobiotics It has been suggested that the fruit components resveratrol (RSV), 6′, 7′-dihydroxybergamottin (DHB), and bergamottin (BG) might inhibit cytochrome P450 2C19 (CYP2C19) activity, but the mode and potency of such inhibition are yet to be investigated. This study aimed to investigate the mode and kinetics of the inhibition of CYP2C19-based omeprazole metabolism by RSV or grapefruit juice components (DHB or BG). RSV and DHB reduced CYP2C19 activity in a preincubation time-dependent manner, suggesting that they inactivated CYP2C19 via mechanism-based inhibition (MBI). Although BG inactivated CYP2C19 in a preincubation time- and concentration-dependent manner, suggesting that both MBI and reversible inhibition contributed to these effects, the concentration required to achieve 50% inhibition was 26-fold higher for reversible inhibition than for MBI (0.859 and 0.0331 μM, respectively), indicating that the inhibition of CYP2C19 by BG is primarily attributable to MBI. Based on the estimated intestinal concentrations of these components, it is considered that >90% of CYP2C19 would be inactivated after the consumption of normal amounts of grapefruit juice or RSV-containing substances. In conclusion, these findings suggest that food containing these components has the potential to evoke drug-food interactions caused by the MBI of intestinal CYP2C19 activity in the clinical setting.

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Effects of food type on the extent of drug-drug interactions between activated charcoal and phenobarbital in rats

Ogata Y., Imaoka A., Akiyoshi T., Ohtani H.

Drug Metabolism and Pharmacokinetics （Drug Metabolism and Pharmacokinetics）  34 （ 4 ） 287 - 291 2019年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  13474367

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© 2019 Activated charcoal is known to decrease the intestinal absorption of co-administered drug by adsorption. The extent of this drug-drug interaction (DDI) is attenuated by food intake. The aim of this study was to quantitatively evaluate the effects of food type on the extent of DDI between phenobarbital and activated charcoal using a rat model. Phenobarbital was orally administered at a dose of 1.5 mg/kg with or without 33 mg/kg of activated charcoal under fasted or fed conditions, and the plasma concentration profile of phenobarbital was monitored. Several fed conditions, such as a standard breakfast, high-fat meal or enteral nutrient used in human studies, were examined. Under the fasted conditions, activated charcoal significantly decreased the area under the plasma concentration - time curve (AUC) of phenobarbital by 45.2%. When the standard breakfast or high-fat meal was fed, this DDI was reduced to 28.3 and 18.0%, respectively, as assessed by the reduction in the AUC. On the contrary, enteral nutrient did not significantly attenuate the DDI. In conclusion, the influence of food intake on the extent of DDI between phenobarbital and activated charcoal was found to differ among the types of food concomitantly ingested.

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5-Fluororacil-Induced Gastrointestinal Damage Impairs the Absorption and Anticoagulant Effects of Dabigatran Etexilate.

Tsujii K, Hattori T, Imaoka A, Akiyoshi T, Ohtani H

Journal of pharmaceutical sciences （Journal of Pharmaceutical Sciences）  107 （ 5 ） 1430 - 1433 2018年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00223549

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© 2018 American Pharmacists Association® Fluoropyrimidines, including 5-fluororacil (5-FU), cause gastrointestinal damage in the clinical setting and might affect the gastrointestinal absorption of concomitantly administered drugs. We aimed to evaluate the effects of fluoropyrimidine-induced gastrointestinal damage on the pharmacokinetics and pharmacodynamics of dabigatran etexilate (DABE), an anticoagulant, in rats with gastrointestinal damage induced by the repeated oral administration of 5-FU. Rats were administered DABE orally or dabigatran (DAB), an active moiety of DABE, intravenously. The plasma DAB concentration was determined using liquid chromatography-tandem mass spectrometry. The activated partial thromboplastin time (APTT) was measured before and 30 min after the administration of each drug, and the APTT ratio was calculated. In 5-FU-treated rats, the maximum plasma concentration, the area under the concentration-time curve of DAB after the oral administration of DABE, and the oral bioavailability of DABE were significantly decreased to 18.3%, 22.9%, and 16.3% of the respective control values. The 5-FU-treated rats’ APTT ratio was also significantly lower than the control value. Fluoropyrimidine-induced gastrointestinal damage might reduce the plasma concentration of DAB by impairing DABE absorption and might attenuate the anticoagulant effects of DABE in the clinical setting.

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Food intake attenuates the drug interaction between new quinolones and aluminum.

Imaoka A, Abiru K, Akiyoshi T, Ohtani H

Journal of pharmaceutical health care and sciences （Journal of Pharmaceutical Health Care and Sciences）  14 （ 1 ） 4 - 11 2018年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2055-0294

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© 2018 The Author(s). Background: Intestinal absorption of new quinolones is decreased by oral administration of polyvalent metal cations. Some clinical studies have demonstrated this drug - drug interaction is more prominent under fasted condition. However, the effect of food intake on the extent of drug - drug interaction between new quinolones and metal cations remains to be investigated quantitatively and systematically. The aim of this study was to develop an animal model that enables to evaluate the effect of food intake on the extent of drug - drug interaction in the gastrointestinal tract by chelation and to apply the model to evaluate quantitatively the effect of food intake on the drug - drug interaction between two new quinolones, ofloxacin or ciprofloxacin and sucralfate. Methods: The rats were orally administered new quinolones (5.3 mg/kg of ofloxacin or 10 mg/kg of ciprofloxacin) with or without 13.3 mg/kg of sucralfate under fasted or fed condition and plasma concentration profiles of new quinolones were monitored. To the fed group, standard breakfast used in human studies was pasted and administered at a dose of 8.8 g/kg. Results: The area under the plasma concentration - time curves (AUC0-6) of ofloxacin and ciprofloxacin under the fasted condition were significantly decreased to 28.8 and 17.1% by co-administration of sucralfate, respectively. On the contrary, sucralfate moderately decreased the AUC0-6 of ofloxacin and ciprofloxacin to 54.9 and 33.2%, respectively, under fed condition. The effects of sucralfate and food intake on the kinetics of ofloxacin in this study were well consistent with the results of previous clinical trial. Conclusions: The developed animal model quantitatively reproduced the effect of food intake on the drug - drug interaction between ofloxacin and sucralfate. The similar influences were observed for the drug - drug interaction between ciprofloxacin and sucralfate, suggesting that the extent of drug - drug interaction caused by chelation is generally attenuated by food intake.

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The extent of drug-drug interaction between amlodipine and activated charcoal is attenuated by food intake in rats

Imaoka A, Seki K, Akiyoshi T, Ohtani H.

Drug Metabol Pharmacokinet （Drug Metabolism and Pharmacokinetics）  34 （ 1 ） 108 - 110 2018年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  13474367

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© 2018 Activated charcoal decreases gastrointestinal absorption of concomitantly administered drugs. The absorption of amlodipine (AML) was reported as almost completely attenuated by 25 g of activated charcoal under a fasted condition, but not affected by 2 g of activated charcoal under a fed condition. However, it is not clear whether this difference resulted from the food intake or the dose of activated charcoal. The aim of this study was to quantitatively evaluate the effect of food intake on drug interactions caused by adsorption to activated charcoal in the gastrointestinal tract in rats. The rats were orally administered 0.08 mg/kg of AML, with or without 33 mg/kg of activated charcoal, under the fasted or fed condition and the plasma concentration profiles of AML were monitored. For the fed group, the standard breakfast used in clinical studies was smashed and administered at a dose of 11 g/kg. The AUC value of AML under the fasted condition was significantly decreased to 24.8% by coadministration of activated charcoal. On the other hand, activated charcoal moderately decreased the AUC value of AML to 74.8% under the fed condition. These results suggest that the extent of drug interactions caused by activated charcoal is attenuated by food intake.

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5-Fluorouracil treatment alters the expression of intestinal transporters in rats.

Yotsumoto K, Akiyoshi T, Wada N, Imaoka A, Ohtani H

Biopharmaceutics & drug disposition （Biopharmaceutics and Drug Disposition）  38 （ 8 ） 509 - 516 2017年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0142-2782

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Copyright © 2017 John Wiley & Sons, Ltd. 5-Fluorouracil (5-FU), an anticancer drug, causes severe gastrointestinal damage, which may affect the absorption of orally administered drugs including the substrates of intestinal uptake and efflux transporters. This study aimed to investigate quantitatively the effect of 5-FU-induced intestinal damage on the expression of intestinal transporters: P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and peptide transporter 1 (PEPT1) in rats. The rats were treated with 5-FU (30 mg/kg/day, p.o.) for 5 days to induce intestinal damage, and then the upper, middle and lower intestinal segments were removed. The mRNA and protein expression levels of these transporters in each segment were determined using quantitative real-time PCR and Western blotting, respectively. In the 5-FU-treated rats, the protein levels of P-gp and Bcrp in the upper segment were significantly increased to 15- and 2.6-fold of the control, respectively, while those in other segments were unaffected. Pept1 expression was increased by 5-FU in almost all segments. A remarkable increase in P-gp expression was shown, the uptake of digoxin, a P-gp substrate, in each intestinal segment was measured using a rat everted sac. As a result, the uptake of digoxin in the upper segments of 5-FU-treated rats was decreased compared with that of the control. In conclusion, 5-FU-induced intestinal damage was shown to alter the expression of these transporters, especially in the upper intestinal segment, while the characteristics of the influence varied among the transporters. The 5-FU-induced intestinal damage may affect transporter-mediated drug absorption of orally administered drugs in the clinical setting.

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Development and evaluation of an overseas clinical rotation program for undergraduate pharmacy students in Japan.

Ohtani H, Mitsui R, Akiyoshi T, Imaoka A, Abe Y, Kanke M, Nakamura N, Foster P, Mochizuki M

Currents in pharmacy teaching & learning （Currents in Pharmacy Teaching and Learning）  9 （ 3 ） 452 - 459 2017年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1877-1297

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© 2017 Elsevier Inc. Background and purpose Internationalization of pharmacists, as well as pharmacy students, in terms of both the knowledge to care for international patients and to have medical information literacy, is a current concern in Japan. Educational activity and setting Keio University Faculty of Pharmacy has developed an elective course for pharmacy students, based on written agreements with the United States and Thailand that establish a student clinical rotation exchange program. The exchange program lasts for four to six weeks and involves clinical rotations in hospitals abroad during the students’ sixth year. Rotations follow a four-week didactic preparatory course. The course objectives are to acquire the knowledge, skills, and attitude needed to function as leading pharmacists with an international perspective. Methods We asked students to complete a feedback survey inquiring about the usefulness of preparatory courses, self-evaluation pre- and post-rotation satisfaction with the program, and overall self-assessment. Findings Twenty-four out of 41, i.e., 58.5% of the students replied with feedback. All respondents replied that the preparatory course was useful. They also replied that, based on their self-evaluation, they were satisfied with their level of English language skill improvement after the rotation. Pharmaceutical knowledge satisfaction, however, was slightly decreased. All respondents replied that this program was of a satisfactory nature, with 71%, 63%, and 92% of the respondents replying that they could acquire the knowledge, skills, and attitude program objectives respectively. Summary It is possible to successfully develop an overseas clinical rotation program. Students were quite satisfied upon completion and achieved the expected objectives.

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Inactivation kinetics and residual activity of CYP3A4 after treatment with erythromycin.

Ishikawa Y, Akiyoshi T, Imaoka A, Ohtani H.

Biopharmaceutics & drug disposition （Biopharmaceutics and Drug Disposition）  38 （ 7 ） 420 - 425 2017年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0142-2782

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Copyright © 2017 John Wiley & Sons, Ltd. This study aimed to characterize the inactivation kinetics of cytochrome P450 3A4 (CYP3A4) by erythromycin, which involves mechanism-based inhibition (MBI), in detail. In addition to an MBI assay based on the conventional method in which erythromycin and recombinant CYP3A4 were pre-incubated for 15 min, the study also evaluated the long-term MBI kinetics of this reaction by pre-incubation for 120 min. Mechanism-based inhibition profiles were obtained using three typical substrates, testosterone, midazolam and nifedipine. In the long-term assay, erythromycin evoked a time-dependent biphasic reduction in enzyme activity, but some residual activity (α) was detected in the terminal phase. The inactivation rate constant obtained in the presence of 30 μm erythromycin using nifedipine as a substrate was 1.44-fold higher than that acquired using testosterone, while there was no difference among the α values obtained with the three substrates. In the short-term assay, time-dependent monophasic inactivation was observed. To extrapolate these data to in vivo, the extent of the increase in the area under the curve (AUC ratio) induced by erythromycin was estimated from the results of the conventional short-term experiment and the long-term experiment examining residual activity. The AUC ratio estimated from the long-term kinetics (2.92) was closer to the clinically reported values (3.3–4.42). In conclusion, the relatively long-term evaluation of the kinetics of CYP3A4 inactivation revealed that the enzyme was not fully inactivated by erythromycin. To improve the estimation of the extent of the drug–drug interactions induced by MBI from in vitro data, longer-term investigations of the target enzyme's inactivation profile might be necessary.

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In silico evaluation of warfarin-bucolome therapy.

Ohtani H, Hakoda R, Imaoka A, Akiyoshi T.

Biopharmaceutics & drug disposition （Biopharmaceutics and Drug Disposition）  37 （ 4 ） 233 - 242 2016年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0142-2782

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Copyright © 2016 John Wiley & Sons, Ltd. Purpose: Some reports have suggested that bucolome, an inhibitor of cytochrome P450 2C9, is useful for decreasing inter-patient variation in warfarin clearance. The purpose of the present study was to evaluate the utility of the concomitant administration of bucolome and warfarin using an in silico approach. Methods: In vitro data regarding the enzymatic kinetics of (S)-warfarin and bucolome were collected from the literature. As a validation study, the geometric mean (GM) of the oral unbound clearance of (S)-warfarin and its inter-patient variation (assessed using the standard deviation of its natural logarithm (σ)) were predicted using a physiologically based population pharmacokinetic simulator (SimcypTM) and compared with clinical data. The utility of the concomitant administration was evaluated by comparing the GM and σ values predicted under various conditions (the prediction study). Results and Discussion: The σ values in the presence and absence of bucolome were predicted to be 0.73 and 0.68, respectively, suggesting that bucolome might increase the inter-patient variation, as clinically observed. In the prediction study, the σ value of the bucolome co-administered group was greater in almost all of the examined conditions. In conclusion, the concomitant administration of bucolome might not be useful for reducing the inter-patient variation of (S)-warfarin pharmacokinetics. Copyright © 2016 John Wiley & Sons, Ltd.

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Variation in the inhibitory potency of terbinafine among genetic variants of CYP2D6.

Akiyoshi T, Ishiuchi M, Imaoka A, Ohtani H.

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics）  30 （ 4 ） 321 - 324 2015年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1347-4367

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© 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved. Cytochrome P450 2D6 (CYP2D6) is a highly polymorphic enzyme that is involved in the metabolism of many drugs. Terbinafine (TER) is a CYP2D6 inhibitor and causes persistent drug interactions in the clinical setting; however, its inhibitory mechanism and the differences in its inhibitory potency among genetic variants of CYP2D6 remain to be investigated. This study aimed to investigate the inhibitory mechanism of TER and the differences in its inhibitory potency among three CYP2D6 variants, CYP2D6.1, CYP2D6.2, and CYP2D6.10. In a competitive inhibition study, the metabolic activity of the CYP2D6 was assessed based on their demethylation of dextromethorphan in the presence or absence of TER, and the time-dependency of the inhibitory effects were examined by preincubating the enzymes with TER. TER had weaker inhibitory effects on CYP2D6.2 and CYP2D6.10 than on CYP2D6.1; i.e., TER exhibited Ki values (the concentration of inhibitor that results in half-maximal inhibition) of 0.0525, 0.355, and 1.85 μM for CYP2D6.1, CYP2D6.2, and CYP2D6.10, respectively. The inhibitory effects of TER were not time-dependent. Since TER's Ki value for CYP2D6.10 was 35.2-fold higher than its Ki value for CYP2D6.1, the CYP2D6 genotype of subjects should be taken into account when estimating the severity of drug interactions involving TER.

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インターネットオークションでの違法な医薬品取引と主催者による監視、削除の実態

大谷 壽一, 今岡鮎子, 秋好 健志

薬学雑誌 （日本薬学会）  135 （ 3 ） 529 - 534 2015年02月

研究論文（学術雑誌）, 共著, 査読有り

常用量以下の曝露量における全身性副作用の用量依存性の推定

三井梨恵子, 秋好 健志, 今岡鮎子, 望月眞弓, 大谷壽一.

医薬品情報学 17 （ 2 ） 77 - 86 2015年

研究論文（学術雑誌）, 共著, 査読有り

Decrease in ciprofloxacin absorption by polyvalent metal cations is not fully attributable to chelation or adsorption.

Imaoka A, Hattori M, Akiyoshi T, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics）  29 （ 5 ） 414 - 418 2014年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1347-4367

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Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX). The drug interaction between new quinolone antibiotics (NQs) and polyvalent metal cation products, leading to a significant decrease in the absorption of NQ, is considered to be attributable to the formation of poorly absorbable chelate and physicochemical adsorption of NQs to cation products. To clarify the mechanisms of this drug interaction in detail, we investigated the effects of Al3+ or Mg2+ on the membrane permeation profile of ciprofloxacin (CPFX) across human colon carcinoma cell lines (Caco-2) in monolayer culture, and characterized the adsorption nature of CPFX to polyvalent metal cation products under physiological conditions. As a result, Al3+ or Mg2+ partially but not fully impaired the permeation of CPFX across Caco-2 monolayer up to 30% or 60% of control, respectively. Physicochemical adsorption of CPFX to cation products was not observed under physiological pH. In conclusion, two possible mechanisms investigated, the decrease in the permeability of CPFX by chelate formation and adsorption of CPFX to polyvalent metal cation products, may partially but not fully explain the extent of the drug interaction clinically observed.

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Prediction of the extent and variation of grapefruit juice-drug interactions from the pharmacokinetic profile in the absence of grapefruit juice.

Kogure N, Akiyoshi T, Imaoka A, Ohtani H

Biopharmaceutics & drug disposition （Biopharmaceutics and Drug Disposition）  35 （ 7 ） 373 - 381 2014年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0142-2782

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Copyright © 2014 John Wiley & Sons, Ltd. The aim of this study was to develop a method for predicting the extent of grapefruit juice (GFJ)-drug interactions and their interindividual variations from the pharmacokinetic profile in the absence of GFJ. The pharmacokinetic profiles of 13 drugs after intravenous and oral administration were used to develop and validate the method. For each drug, the proportion absorbed into the intestine and the intestinal availability (Fg) were calculated from clinical data taken from the literature. Then, the AUC ratio (the ratio of the AUC with GFJ to that without GFJ) was predicted by assuming that Fg was 1.0 when GFJ was concomitantly ingested. According to the developed method, the AUC ratio of felodipine was 2.50 and its coefficient of variation (CV) was 45%, which agreed well with the observed AUC ratio of 2.48 and CV of 51%. Although the developed method overestimated the AUC ratios of some drugs such as nisoldipine, no underestimation occurred. The predicted CV values were consistent with those observed. The developed method might be useful to predict the AUC ratio, along with its interindividual variation, from the pharmacokinetic profile in the absence of grapefruit juice.

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Effect of aromatase inhibitors on bone mineral density in a Japanese breast cancer population.

Akiyoshi T, Shimomura Y, Masuda S, Okamoto Y, Hiura S, Kato H, Asayama T, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics）  25 （ 5 ） 446 - 450 2013年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1347-4367

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We retrospectively analyzed the bone mineral density (BMD) of postmenopausal Japanese women taking an aromatase inhibitor (AI), exemestane, anastrozole or letrozole, and calculated the decrease rate constant of BMD in each individual to compare the influence of the three AIs on BMD. We also aimed to evaluate the preventive effect of bisphosphonates (BPs) on the AI-induced decrease in BMD. The decrease rate constant of BMD (ke) in each individual was determined as a slope of linear regression of the relationship between time and logarithm of BMD value in each patient during the AI therapy. To compensate for the age-related change in BMD level, we estimated the age-related decrease rate constant of BMD (ke,0) in healthy Japanese postmenopausal women from the literature. AIs decreased BMD with a ke value of -0.0329 yr-1, which was 4.7-fold larger than the ke,0 value of -0.00699 yr-1. No significant difference was detected in the influence on BMD among AIs. Co-administration of BP ameliorated the ke value to -0.0117 yr-1, a value similar to ke,0. The influence of AIs on BMD was quantitatively evaluated by using the decrease rate constant of BMD (ke). The present study also suggests that BPs may be useful to prevent the decrease in BMD induced by AIs. © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX).

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Mechanism-based inhibition profiles of erythromycin and clarithromycin with cytochrome P450 3A4 genetic variants.

Akiyoshi T, Ito M, Murase S, Miyazaki M, Guengerich FP, Nakamura K, Yamamoto K, Ohtani H

Drug metabolism and pharmacokinetics （Drug Metabolism and Pharmacokinetics）  25 （ 5 ） 411 - 415 2013年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1347-4367

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Inhibition of cytochrome P450 (CYP) 3A4 is the major cause of drug-drug interactions (DDI). We have previously reported that the genetic variation of CYP3A4 significantly affected the inhibitory profiles of typical competitive inhibitors. In addition to competitive inhibition, some clinically significant DDI are attributable to mechanism-based inhibition (MBI). However, the differences in the MBI kinetics among CYP3A4 genetic variants remain to be characterized. In this study, we quantitatively investigated the inhibition kinetics of MBI inhibitors, erythromycin and clarithromycin, on the CYP3A4 variants CYP3A4.1, 4.2, 4.7, 4.16, and 4.18. The activity of CYP3A4 was assessed using testosterone 6β-hydroxylation with recombinant CYP3A4. Both erythromycin and clarithromycin decreased the activity of CYP3A4 in a timedependent manner. The maximum inactivation rate constants, kinact,max, of erythromycin for CYP3A4.2 and CYP3A4.7 were 0.5-fold that for CYP3A4.1, while that for CYP3A4.16 and CYP3A4.18 were similar to that for CYP3A4.1. The KI values of erythromycin for CYP3A4.2, 4.7, 4.16, and 4.18 were 1.2-, 0.4-, 2.2- and 0.72-fold those of CYP3A4.1, respectively. Similar results were obtained for clarithromycin. In conclusion, the inhibitory profiles of MBI inhibitors, as well as competitive inhibitors, may possibly differ among CYP3A4 variants. This difference may contribute to interindividual differences in the extent of DDI based on MBI. © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX).

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Stability of the admixture of trace elements and vitamin-containing TPN solution in a multi-chamber bag (Fulcaliq).

Okuyama Y, Akiyoshi T, Asayama T, Hirai M, Matsuyama K Ohtani H* .

The Journal of Asian Association of Schools of Pharmacy 1 （ 3 ） 152 - 158 2012年09月

研究論文（学術雑誌）, 共著, 査読有り

Comparison of the inhibitory profiles of itraconazole and cimetidine in cytochrome P450 3A4 genetic variants.

Akiyoshi T, Saito T, Murase S, Miyazaki M, Murayama N, Yamazaki H, Guengerich FP, Nakamura K, Yamamoto K, Ohtani H

Drug metabolism and disposition: the biological fate of chemicals （Drug Metabolism and Disposition）  39 （ 4 ） 724 - 728 2011年04月

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

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CYP3A4, an important drug-metabolizing enzyme, is known to have genetic variants. We have previously reported that CYP3A4 variants such as CYP3A4.2, 7, 16, and 18 show different enzymatic kinetics from CYP3A4.1 (wild type). In this study, we quantitatively investigated the inhibition kinetics of two typical inhibitors, itraconazole (ITCZ) and cimetidine (CMD), on CYP3A4 variants and evaluated whether the genetic variation leads to interindividual differences in the extent of CYP3A4-mediated drug interactions. The inhibitory profiles of ITCZ and CMD on the metabolism of testosterone (TST) were analyzed by using recombinant CYP3A4 variants. The genetic variation of CYP3A4 significantly affected the inhibition profiles of the two inhibitors. In CYP3A4.7, the Ki value for ITCZ was 2.4-fold higher than that for the wild-type enzyme, whereas the Ki value for CMD was 0.64-fold lower. In CYP3A4.16, the Ki value for ITCZ was 0.54-fold lower than that for wild-type CYP3A4, whereas the Ki value for CMD was 3.2-fold higher. The influence of other genetic variations also differed between the two inhibitors. Docking simulations could explain the changes in the Ki values, based on the accessibility of TST and inhibitors to the heme moiety of the CYP3A4 molecule. In conclusion, the inhibitory effects of an inhibitor differ among CYP3A4 variants, suggesting that the genetic variation of CYP3A4 may contribute, at least in part, to interindividual differences in drug interactions mediated by CYP3A4 inhibition, and the pattern of the influences of genetic variation differs among inhibitors as well as substrates. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.

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未来先導型の研究と教育を目指して.

大谷壽一, 秋好健志.

薬剤学 （薬剤学）  70(4)   218-223 2010年07月

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Stability of gabexate mesilate products: Influence of the addition of mannitol.

Sakurai M, Abe H, Okamura N, Inoue Y, Akiyoshi T, Matsuyama K, Uchida T, Otsuka M

Bio-medical materials and engineering （Bio-Medical Materials and Engineering）  20 （ 1 ） 13 - 20 2010年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0959-2989

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Gabexate mesilate is a non-peptide protease inhibitor, developed in Japan, which is used in the treatment of acute pancreatitis and disseminated intravascular coagulation. This compound is readily hydrolyzed as it has ester bonds in its structure. It is now out of patent in Japan and there are many generic versions on the market. The crystal structure and the hydrolysate content of the branded product and nine generic versions were evaluated by X-ray diffractometry, thermal analysis and HPLC. The results showed that generic products containing mannitol as an additive had a higher content of hydrolysate as an impurity than the branded product or generic products formulated without mannitol, suggesting that the crystal structure might be altered and stability impaired in mannitol-containing drug products. © 2010 - IOS Press and the authors. All rights reserved.

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The potential of vitamin K3 as an anticancer agent against breast cancer that acts via the mitochondria-related apoptotic pathway.

Akiyoshi T, Matzno S, Sakai M, Okamura N, Matsuyama K

Cancer chemotherapy and pharmacology （Cancer Chemotherapy and Pharmacology）  65 （ 1 ） 143 - 150 2009年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0344-5704

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Purpose: We tried to clarify the cytotoxic mechanism of VK3 using the breast cancer cell line MCF-7. Methods: Cytotoxicity was measured via intracellular esterase activity. DNA fragmentation was assessed by agarose gel electrophoresis. JC-1 staining was applied to measure mitochondrial dysfunction. Caspase activation and reactive oxidative species (ROS) generation were also measured. Results: VK3 exhibited cytotoxicity that caused DNA fragmentation in MCF-7 cells with an IC50 of 14.2 μM. JC-1 staining revealed that VK3 caused mitochondrial dysfunction including a disappearance of mitochondrial membrane potential. Additional investigation showed that the mitochondrial damage was induced by the generation of ROS and the subsequent activation of caspase-7 and -9. Conclusions: Our findings demonstrate that VK3-induced apoptosis is selectively initiated by the mitochondria-related pathway and might be useful in breast cancer chemotherapy. © 2009 Springer-Verlag.

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Synergistic action of statins and nitrogen-containing bisphosphonates in the development of rhabdomyolysis in L6 rat skeletal myoblasts.

Nishiguchi T, Akiyoshi T, Anami S, Nakabayashi T, Matsuyama K, Matzno S

The Journal of pharmacy and pharmacology （Journal of Pharmacy and Pharmacology）  61 （ 6 ） 781 - 788 2009年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0022-3573

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Objectives: Nitrogen-containing bisphosphonates, which are widely used to treat osteoporosis, act as inhibitors of farnesyl pyrophosphate synthase, one of the key enzymes of the mevalonate pathway, and thus may have the potential to enhance the effect of statins (inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase). In this study, we evaluated the synergistic effect of two nitrogen-containing bisphosphonates, alendronate and risedronate, in statin-induced apoptosis in rat skeletal L6 myoblasts. Methods: L6 rat myoblasts were differentiated with drugs. DNA fragmentation was measured and small GTPase was detected by immunoblotting. Key findings: Alendronate and risedronate caused dose-dependent apoptosis of L6 myoblasts. Risedronate induced detachment of rho GTPase from the cell membrane, followed by activation of the caspase-8-related cascade. Risedronate-induced apoptosis was synergistically enhanced with atorvastatin and significantly reduced by addition of geranylgeraniol. By contrast, alendronate did not reduce membrane GTPases and the apoptosis was caspase independent. Conclusions: These results suggest that risedronate-induced apoptosis is related to geranylgeranyl pyrophosphate depletion followed by rho detachment, whereas alendronate affects are independent of rho. Our results suggest a risk of synergistic action between nitrogen-containing bisphosphonates and statins in the development of rhabdomyolysis when treating osteoporosis in women with hyperlipidaemia. © 2009 The Authors.

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An attempt to evaluate the effect of vitamin K3 using as an enhancer of anticancer agents.

Matzno S, Yamaguchi Y, Akiyoshi T, Nakabayashi T, Matsuyama K

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  31 （ 6 ） 1270 - 1273 2008年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

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The possibility of vitamin K3 (VK3) as an anticancer agent was assessed. VK3 dose-dependently diminished the cell viability (measured as esterase activity) with IC50 of 13.7 μM and Hill coefficient of 3.1 in Hep G2 cells. It also decreased the population of S phase and arrested cell cycle in the G2/M phase in a dose-dependent manner. G2/M arrest was regulated by the increment of cyclin A/cdk1 and cyclin A/cdk2 complex, and contrasting cyclin B/cdk1 complex decrease. Finally, combined application demonstrated that VK3 significantly enhanced the cytotoxicity of etoposide, a G2 phase-dependent anticancer agent, whereas it reduced the cytotoxic activity of irinotecan, a S phase-dependent agent. These findings suggest that VK3 induces G 2/M arrest by inhibition of cyclin B/cdk1 complex formation, and is thus useful as an enhancer of G2 phase-dependent drugs in hepatic cancer chemotherapy. © 2008 Pharmaceutical Society of Japan.

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Effects of quinine on the intracellular calcium level and membrane potential of PC 12 cultures.

Akiyoshi T, Tanaka N, Nakamura T, Matzno S, Shinozuka K, Uchida T

The Journal of pharmacy and pharmacology （Journal of Pharmacy and Pharmacology）  59 （ 11 ） 1521 - 1526 2007年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0022-3573

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The mechanism for the perception of bitterness appears to be quite complicated, even for quinine, which is a model bitter substance, and thus has yet to be completely elucidated. To investigate the possibility of being able to predict the bitterness of quinine solutions, we examined the effects of quinine on intracellular calcium ion concentration ([Ca2+]i) and membrane potentials in PC 12 cultures. [Ca2+]i and membrane potentials were analysed by fluorescence confocal microscopic imaging using the Ca 2+-sensitive probe Calcium Green 1/AM and the membrane potential-sensitive probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)). Quinine elicited an increase in the membrane potential along with a concentration-dependent increase in [Ca2+]i. These increases were inhibited by extracellular Ca2+-free conditions, thapsigargin, which is a Ca2+-pump inhibitor, and U73122, which is a phospholipase C inhibitor. The quinine-induced increase in [Ca2+]i levels was inhibited by nifedipine, an L-type Ca2+-channel blocker, ufconotoxin, a T-type Ca2+-channel blocker, and BMI-40, which is a bitterness-masking substance. These results suggest that responses in PC 12 cultures may be used as a simple model of bitterness perception. © 2007 The Authors.

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Clofibrate-induced apoptosis is mediated by Ca2+-dependent caspase-12 activation.

Matzno S, Yasuda S, Kitada Y, Akiyoshi T, Tanaka N, Juman S, Shinozuka K, Nakabayashi T, Matsuyama K

Life sciences （Life Sciences）  78 （ 16 ） 1892 - 1899 2006年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0024-3205

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The mechanism of fibrate-induced myopathy was investigated in this report. When clofibrate (30 to 300 μM) was applied to L6 rat skeletal myoblasts, dose-dependently apoptosis was observed within 24 h. In the apoptotic myoblasts, a caspase-12 cleavage was observed at 2 h and with following caspases-9 and -3-related cascade activation. In contrast, the neutral protease calpain, that is a key enzyme in ER stress-related apoptosis via caspase-12 activation, was significantly decreased during apoptosis. Next, the authors evaluated a role of calcium-dependent signal(s). When clofibrate was added into medium, cytosolic calcium concentration was rapidly and persistently increased. On the other hand, an addition of 10 mM EGTA depressed sustained calcium phase, and concurrent myoblasts apoptosis was completely inhibited. Taken together, our findings indicate that the clofibrate-induced myopathy is triggered by Ca2+ influx, then activated cytosolic caspase-12 through calpain-independent cascade, and consequently caused apoptotic DNA fragmentation. © 2005 Elsevier Inc. All rights reserved.

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高速液体クロマトグラフィによる血清中アミオダロン濃度測定時における採血管分離ゲル剤からの溶出物質による妨害ピークの影響.

上野和行,秋好健志, 川井仁之, 今野秀樹, 桑原史郎, 米田孝司, 片山善章.

TDM研究 20(3)   274-9 2003年07月

研究論文（学術雑誌）, 共著, 査読有り

Effect of quinine solutions on intracellular Ca2+ levels in neuro-2a cells--conventional physiological method for the evaluation of bitterness.

Nakamura T, Akiyoshi T, Tanaka N, Shinozuka K, Matzno S, Nakabayashi T, Matsuyama K, Kashiwayanagi M, Uchida T

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  26 （ 11 ） 1637 - 40 2003年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

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The purpose of the present study was to examine the effect of quinine on intracellular Ca2+ ([Ca2+]i) levels in cultured neuro-2a cells, and to investigate the possibility of using [Ca 2+]i levels to predict the bitterness of quinine solutions. [Ca2+]i levels in neuro-2a cells increased following stimulation by quinine in a concentration-related manner. There was a good linear correlationship between the quinine-induced increase in [Ca 2+]i levels increase and the bitterness scores of the quinine solutions as assessed in human gustatory sensation tests (r 2=0.918). The quinine-induced increase in [Ca2+] i levels was inhibited by thapsigargin (an inhibitor of the Ca 2+ pump into intracellular stores), U73122 (an inhibitor of phospholipase C) and ω-conotoxin (an N-type Ca2+-channel blocker), but not by nifedipine (an L-type Ca2+-channel blocker). © 2003 Pharmaceutical Society of Japan.

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The effect of various substances on the suppression of the bitterness of quinine-human gustatory sensation, binding, and taste sensor studies.

Nakamura T, Tanigake A, Miyanaga Y, Ogawa T, Akiyoshi T, Matsuyama K, Uchida T.

Chemical & pharmaceutical bulletin 50 （ 12 ） 1589 - 1593 2002年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0009-2363

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