論文

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.

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.

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.

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 IC50values 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.

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.

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.

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.

Purpose: Bevacizumab (BV) is an effective therapeutic antibody utilized in various cancers. Serum BV concentration can be a factor that potentially affects its therapeutic efficacy. Although proteinuria could affect BV pharmacokinetics, its influence was not evaluated in the previous population pharmacokinetic (PopPK) studies. Because BV can cause proteinuria as an adverse event, the present study aimed to develop a PopPK model in patients with proteinuria and to evaluate the influence of proteinuria on BV pharmacokinetics. Methods: This prospective cohort study enrolled 70 Japanese cancer patients newly starting BV, and 368 concentration samples from these patients were analyzed. Serum BV concentrations were measured at several time points including at the onset of proteinuria. PopPK analysis was conducted using a non-linear mixed-effects modeling program. A two-compartment model was used to estimate total body clearance (CL). Results: Serum BV concentrations divided by the dose per body weight and dosing interval tended to be lower in patients with higher urinary protein to creatinine ratio (UPCR). The covariate analysis showed that increasing BV CL was associated with decreasing serum albumin concentration and increasing body weight and UPCR. The simulated median trough concentrations of BV in patients with Common Terminology Criteria for Adverse Events grades 1, 2, and 3 proteinuria were decreased by 12.0%, 20.6%, and 31.5%, respectively, compared to those in patients with grade 0. Conclusion: We successfully established a PopPK model incorporating UPCR to predict serum BV concentrations in patients with proteinuria. Our study provides additional insights to better understand BV pharmacokinetics.

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.