Published papers

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.

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.

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 IC50 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.

Organic anion transporting polypeptide (OATP) 1B1 is crucial for hepatic drug uptake. Three N-glycosylation sites, Asn134, Asn503, and Asn516, have been identified, and substituting the amino acid at these sites with glutamine reduces the expression of the OATP1B1 protein on the plasma membrane. However, the effects of disrupted N-glycosylation on the transport kinetics of OATP1B1 molecule remained uninvestigated. This study quantitatively analyzed the impact of N-glycosylation modifications on the intrinsic transport activity of OATP1B1. We established artificial mutant OATP1B1-expressing HEK293 cell lines with substitution of asparagine at one or multiple N-glycosylation sites with glutamine. The change in molecular weight of OATP1B1 protein in the plasma membrane fraction was assessed by Western blotting. OATP1B1 transport activity was evaluated by measuring 2',7'-dichlorofluorescein (DCF) uptake to determine the Michaelis constant (Km) and maximum transport rate (Vmax). The Vmax value per OATP1B1 molecule (Vmax,c) was calculated by dividing Vmax by the expression level of OATP1B1 in the plasma membrane fraction quantified by LC-MS/MS. Western blotting revealed that all mutant OATP1B1 proteins showed lower molecular weights than the wild-type. Among mutation sites, Asn134 was confirmed to be the primary glycosylation site, as reported previously. Regarding transport activity, the Km value of the Asn134/503/516Gln mutant decreased to 0.3-fold that of the wild-type. However, its Vmax,c value decreased to 0.2-fold that of the wild-type. Thus, N-glycosylation of OATP1B1 is not only important for the plasma membrane localization but also for transport activity.

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).

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.

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.

© 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.

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

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.

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 Km and Vmax, 2′, 7′-dichlorofluorescein uptake was evaluated. The number of OATP molecules per cell (QT) was calculated from BK channel-mediated whole-cell conductance and the OATP1B1/BK channel expression ratio (ρ) (determined by LC-MS/MS). Vmax,int (maximum intrinsic transport velocity) was obtained by dividing Vmax by QT, and intrinsic clearance (CLint) was calculated as Vmax,int/Km. The Km values of ∗1a, ∗1b, ∗5, and ∗15 were 12.5, 9.19, 7.53, and 10.4 μM, and their Vmax,int values were 3.0, 7.0, 1.5, and 1.2 × 10−21 mol/OATP molecule/min, respectively. Accordingly, the CLint 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.

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.

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.

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.

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.

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.