Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1.

Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively), compared to 19 nM for the wild-type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8,420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. Significance Statement Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting OATs. This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 was found to mediate hydrogen bonding.

Magnitude of Fruit Juice-Drug Interactions Due to Osmolality-Dependent Fluid Secretion: Differences among Apple, Orange, and Grapefruit Juices.

We recently reported that the gastrointestinal (GI) fluid volume is influenced by the solution osmolality, and proposed that this effect may play a role in beverage-drug interactions. Here, we investigated whether osmolality-dependent fluid secretion can explain the difference in the magnitudes of fruit juice-drug interactions depending on the type of fruit juice (grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ)). The osmolality of GFJ, OJ, and AJ used in this study was found to be 552, 686, and 749 mOsm/kg, respectively. Measurements of intestinal fluid movement following beverage administration by the in situ closed-loop technique revealed the following rank order for fluid volume in rat ileum: AJ > OJ > GFJ > purified water, suggesting that water movement is dependent on the osmolality of these beverages. Such changes in GI fluid volume are expected to alter the luminal drug concentration, potentially contributing to the magnitude of beverage-drug interactions. Indeed, in vivo pharmacokinetic study in rats revealed that the plasma concentration of atenolol, a low-permeability drug, was the highest after oral administration in purified water, followed by GFJ and OJ, and was the lowest after administration in AJ. In contrast, antipyrine, a high-permeability drug, showed no significant difference in plasma concentration after administration in purified water and fruit juices, suggesting that the absorption of high-permeability drugs is less affected by solution osmolality. Our findings indicate that differences in the magnitude of beverage-drug interactions can be at least partly explained by differences in the osmolality of the beverages ingested.

Quantitative analysis of the impact of membrane permeability on intestinal first-pass metabolism of CYP3A substrates.

The aim of this study was firstly to investigate the effect of membrane permeability on the intestinal availability (Fg ) of 10 cytochrome P450 3A4 substrates with differing permeability (Papp ) and metabolic activity (CLint ) using Madin-Darby canine kidney II (MDCKII) cells expressing human CYP3A4 (MDCKII/CYP3A4 cells), and secondly to confirm the essential factors by simulations. A membrane permeation assay using MDCKII/CYP3A4 cells showed a significant correlation between human intestinal extraction ratio (ER) (Eg (=1 - Fg )) and in vitro cellular ER (r = 0.834). This relationship afforded better predictability of Eg values than the relationship between Eg and CLint,HIM values obtained from human intestinal microsomes (r = 0.598). An even stronger correlation was observed between 1 - Fa ·Fg and ER (r = 0.874). Simulation with a cellular kinetic model indicated that ER is sensitive to changes of PSpassive and CLint values, but not to the intracellular unbound fraction (fu,cell ) or P-gp-mediated efflux (PSP - gp ). It may be concluded that, based on the concentration-time profile of drugs in epithelial cells, transmembrane permeability influences Fg (or ER) and drug exposure time to metabolizing enzymes for P450 substrate.

The Use of Carboxyfluorescein Reveals the Transport Function of MCT6/SLC16A5 Associated with CD147 as a Chloride-Sensitive Organic Anion Transporter in Mammalian Cells.

Oral drug absorption involves drug permeation across the apical and basolateral membranes of enterocytes. Although transporters mediating the influx of anionic drugs in the apical membranes have been identified, transporters responsible for efflux in the basolateral membranes remain unclear. Monocarboxylate transporter 6 (MCT6/SLC16A5) has been reported to localize to the apical and basolateral membranes of human enterocytes and to transport organic anions such as bumetanide and nateglinide in the Xenopus oocyte expression system; however, its transport functions have not been elucidated in detail. In this study, we characterized the function of MCT6 expressed in HEK293T cells and explored fluorescent probes to more easily evaluate MCT6 function. The results illustrated that MCT6 interacts with CD147 to localize at the plasma membrane. When the uptake of various fluorescein derivatives was examined in NaCl-free uptake buffer (pH 5.5), the uptake of 5-carboxyfluorescein (5-CF) was significantly greater in MCT6 and CD147-expressing cells. MCT6-mediated 5-CF uptake was saturable with a Km of 1.07 mM and inhibited by several substrates/inhibitors of organic anion transporters and extracellular Cl ion with an IC50 of 53.7 mM. These results suggest that MCT6 is a chloride-sensitive organic anion transporter that can be characterized using 5-CF as a fluorescent probe.

Macrolide and Ketolide Antibiotics Inhibit the Cytotoxic Effect of Trastuzumab Emtansine in HER2-Positive Breast Cancer Cells: Implication of a Potential Drug-ADC Interaction in Cancer Chemotherapy.

Macrolides are widely used for the long-term treatment of infections and chronic inflammatory diseases. The pharmacokinetic features of macrolides include extensive tissue distribution because of favorable membrane permeability and accumulation within lysosomes. Trastuzumab emtansine (T-DM1), a HER2-targeting antibody-drug conjugate (ADC), is catabolized in the lysosomes, where Lys-SMCC-DM1, a potent cytotoxic agent, is processed by proteinase degradation and subsequently released from the lysosomes to the cytoplasm through the lysosomal membrane transporter SLC46A3, resulting in an antitumor effect. We recently demonstrated that erythromycin and clarithromycin inhibit SLC46A3 and attenuate the cytotoxicity of T-DM1; however, the effect of other macrolides and ketolides has not been determined. In this study, we evaluated the effect of macrolide and ketolide antibiotics on T-DM1 cytotoxicity in a human breast cancer cell line, KPL-4. Macrolides used in the clinic, such as roxithromycin, azithromycin, and josamycin, as well as solithromycin, a ketolide under clinical development, significantly attenuated T-DM1 cytotoxicity in addition to erythromycin and clarithromycin. Of these, azithromycin was the most potent inhibitor of T-DM1 efficacy. These antibiotics significantly inhibited the transport function of SLC46A3 in a concentration-dependent manner. Moreover, these compounds extensively accumulated in the lysosomes at the levels estimated to be 0.41-13.6 mM when cells were incubated with them at a 2 µM concentration. The immunofluorescence staining of trastuzumab revealed that azithromycin and solithromycin inhibit the degradation of T-DM1 in the lysosomes. These results suggest that the attenuation of T-DM1 cytotoxicity by macrolide and ketolide antibiotics involves their lysosomal accumulation and results in their greater lysosomal concentrations to inhibit the SLC46A3 function and T-DM1 degradation. This suggests a potential drug-ADC interaction during cancer chemotherapy.

Quantitative analysis of gastrointestinal fluid absorption and secretion to estimate luminal fluid dynamics in rats.

The drug absorption profile is dependent on the luminal drug concentration, which in turn is influenced by the gastrointestinal (GI) fluid dynamics. In the present study, therefore, we aimed to examine the luminal fluid dynamics by kinetically analyzing fluid absorption and secretion along the GI tract in rats using the in situ closed-loop technique with non-absorbable fluorescein isothiocyanate-dextran 4000 (FD-4) and tritium water labeling ([3H]water) under different osmotic conditions. We found that the luminal fluid volume in the jejunum and ileum, but not the colon, gradually decreased and reached a steady state. In contrast, [3H]water almost completely disappeared in all intestinal regions. Kinetic analysis revealed the following rank order for the rate constant of fluid secretion: jejunum > ileum > colon, whereas a negligible regional difference was observed in the rate constant of fluid absorption. Fluid secretion under an isosmotic condition (300 mOsm/kg) was higher than that at 0 mOsm/kg in all intestinal regions, though no significant changes in fluid absorption were observed. Thus, the fluid secretion process appears to be the major determinant of the regional differences in GI fluid dynamics. Our findings indicate that the luminal fluid volume is altered as a result of water ingestion, absorption, and secretion, and finally reaches an apparent steady state, which is regulated mainly by the process of fluid secretion.

Potentiation of the Uricosuric Effect of Dotinurad by Trans-Inhibition of the Uric Acid Reabsorptive Transporter 1.

Urate transporter 1 (URAT1) is a transporter responsible for uric acid (UA) reabsorption by renal proximal tubules and a pharmacological target of uricosuric agents. Probenecid and benzbromarone have been used as uricosuric agents, while dotinurad was recently approved in Japan. Notably, the in vitro IC 50 of dotinurad on URAT1 is not strong enough to explain its in vivo uricosuric effect estimated based on clinical unbound plasma concentrations, suggesting the presence of mechanisms other than competition with UA uptake at the extracellular domain of URAT1 (cis-inhibition). In this study, trans-inhibition was hypothesized as the mechanism underlying URAT1 inhibition by dotinurad, wherein intracellularly accumulated dotinurad inactivates URAT1. In URAT1-expressing Madin-Darby Canine Kidney-II cells and Xenopus oocytes, pre-incubation with dotinurad potentiated the inhibitory effect more than co-incubation alone, but this effect was not observed with benzbromarone or probenecid. Under co-incubation, dotinurad inhibited UA uptake in a competitive manner (cis-inhibition). When we pre-injected dotinurad directly into oocytes and immediately measured [14C]UA uptake without coincubation (only trans-inhibition), dotinurad noncompetitively inhibited UA uptake. URAT1 is an exchange transporter for UA and monocarboxylates such as nicotinic acid (NA). Pre-injected dotinurad and extracellular UA attenuated and facilitated efflux of [3H]NA, respectively, whereas pre-injection of benzbromarone or probenecid did not affect it, suggesting that dotinurad exhibits trans-inhibition by attenuating URAT1-mediated efflux of monocarboxylates, which is a driving force for UA uptake by URAT1. Accordingly, dotinurad ameliorates URAT1-mediated UA reabsorption by both cis- and trans-inhibition, explaining its clinically stronger uricosuric effect than that estimated by the in vitro IC50 value. SIGNIFICANCE STATEMENT: The uricosuric agent dotinurad inhibits uric acid reabsorptive transporter (URAT) 1 with a clinical potency stronger than that estimated from IC 50 obtained by in vitro URAT1 inhibition. This in vivo-in vitro discrepancy was explained by the trans-inhibition effect of dotinurad on URAT1. Trans-inhibition was due to the attenuation of monocarboxylates efflux via URAT1, which is a driving force for URAT1-mediated exchange transport of uric acid. Overall, this is the first study to experimentally demonstrate trans-inhibition mechanism of URAT1.

Induction of open-form bile canaliculus formation by hepatocytes for evaluation of biliary drug excretion.

Biliary excretion is a major drug elimination pathway that affects their efficacy and safety. The currently available in vitro sandwich-cultured hepatocyte method is cumbersome because drugs accumulate in the closed bile canalicular lumen formed between hepatocytes and their amounts cannot be mealsured directly. This study proposes a hepatocyte culture model for the rapid evaluation of drug biliary excretion using permeation assays. When hepatocytes are cultured on a permeable support coated with the cell adhesion protein claudins, an open-form bile canalicular lumen is formed at the surface of the permeable support. Upon application to the basolateral (blood) side, drugs appear on the bile canalicular side. The biliary excretion clearance of several drugs, as estimated from the obtained permeabilities, correlates well with the reported in vivo biliary excretion clearance in humans. Thus, the established model is useful for applications in the efficient evaluation of biliary excretion during drug discovery and development.

Apple-derived extracellular vesicles modulate the expression of human intestinal bile acid transporter ASBT/SLC10A2 via downregulation of transcription factor RARα.

PURPOSE: Plant-derived extracellular vesicles (EVs) have been reported to exert biological activity on intestinal tissues by delivering their contents into intestinal cells. We previously reported that ASBT/SLC10A2 mRNA was downregulated by apple-derived extracellular vesicles (APEVs). ASBT downregulation is effective in the treatment of cholestasis and chronic constipation, similar to the beneficial effects of apples. Therefore, this study aimed to establish the mechanism of ASBT downregulation by APEVs, focusing on microRNAs present in APEVs. RESULTS: APEVs downregulated the expression of ASBT, but no significant effect on SLC10A2-3'UTR was observed. Proteomics revealed that APEVs decreased the expression of RARα/NR1B1. The binding of RARα to SLC10A2 promoter was also decreased by APEVs. The stability of NR1B1 mRNA was attenuated by APEVs and its 3'UTR was found to be a target for APEVs. Apple microRNAs that were predicted to interact with NR1B1-3'UTR were present in APEVs, and their mimics suppressed NR1B1 mRNA expression. CONCLUSIONS: Suppression of ASBT by APEVs was indirectly mediated by the downregulation of RARα, and its stability was lowered by microRNAs present in APEVs. This study suggested that macromolecules in food directly affect intestinal function by means of EVs that stabilize them and facilitate their cellular uptake.

Apple juice relieves loperamide-induced constipation in rats by downregulating the intestinal apical sodium-dependent bile acid transporter ASBT.

Apples are known to exhibit various beneficial effects on human health. In the present study, we investigated the effect of continuous intake of apple juice (AJ) on constipation status. A single dose of loperamide in rats as the constipation model markedly decreased the weight and number of fecal pellets compared to saline-administered rats as a control. After the administration of AJ twice a day for seven days, recovery of defecation close to that of the control was observed in loperamide-treated rats. In addition, the total bile acid content in the feces increased from day 4 after the administration of AJ. Among hepatic and intestinal transporters and enzymes that regulate bile acids, the mRNA expression of the apical sodium-dependent bile acid transporter (Asbt, slc10a2) was decreased by AJ in rats. Furthermore, the Asbt-mediated bile acid transport activity in the rat ileum decreased after AJ administration. Moreover, in human colonic cancer-derived Caco-2 cells, AJ exposure for 24 and 48 h decreased the expressions of ASBT mRNA and protein, and the uptake activity of taurocholic acid in both 7- and 21-d cultures. Several components of AJ, such as procyanidins, decreased the expression of ASBT in Caco-2 cells. In conclusion, ASBT downregulation is a possible mechanism responsible for the constipation-relieving effect of apples, and procyanidins may play a role in downregulating ASBT, which leads to the beneficial effects of apples against constipation. Although it is generally agreed that the common dietary compositions play a role in constipation relief, the novel specific mechanism of apples found in this study would facilitate understanding food functions.

Quantitative Analysis of Gastrointestinal Water Dynamics by Means of a Physiologically Based Fluid Kinetic Model.

Since the processes of dissolution and membrane permeation are affected by the water content in the gastrointestinal (GI) tract, the water dynamics in the GI tract is expected to have a significant impact on the absorption of orally administered drugs. Here, we aimed to develop a physiologically based fluid kinetic (PBFK) model using GI water kinetic parameters obtained from in situ closed-loop studies in rats in order to quantitatively predict GI water dynamics. By incorporating the experimentally measured site-specific parameters of GI water absorption and secretion into a GI compartment model, we developed a bottom-up PBFK model that successfully simulates the reported GI fluid dynamics in rats and humans observed using positron emission tomography and magnetic resonance imaging, respectively. The simulations indicate that the water volume in both the stomach and duodenum is transiently increased by water ingestion, while that in the intestine below the jejunum is unchanged and remains in a steady state in both rats and humans. Furthermore, sensitivity analysis of the effect of ingested water volume on the volume-time profiles of water in the GI tract indicated that the impact of ingested water is limited to the proximal part of the GI tract. Simulations indicated that changes in water kinetic parameters may alter the impact of the ingested water on GI fluid dynamics, especially in the proximal part. Incorporating this PBFK model into a physiologically based pharmacokinetic (PBPK) absorption model has the potential to predict oral drug absorption in a variety of GI water environments.

The Glycosylated N-Terminal Domain of MUC1 Is Involved in Chemoresistance by Modulating Drug Permeation Across the Plasma Membrane.

Mucin 1 (MUC1) is aberrantly expressed in various cancers and implicated in cancer progression and chemoresistance. Although the C-terminal cytoplasmic tail of MUC1 is involved in signal transduction, promoting chemoresistance, the role of the extracellular MUC1 domain [N-terminal glycosylated domain (NG)-MUC1] remains unclear. In this study, we generated stable MCF7 cell lines expressing MUC1 and cytoplasmic tail-deficient MUC1 (MUC1ΔCT) and show that NG-MUC1 is involved in drug resistance by modulating the transmembrane permeation of various compounds without cytoplasmic tail signaling. Heterologous expression of MUC1ΔCT increased cell survival in treating anticancer drugs (such as 5-fluorouracil, cisplatin, doxorubicin, and paclitaxel), in particular by causing an approximately 150-fold increase in the IC50 of paclitaxel, a lipophilic drug, compared with the control [5-fluorouracil (7-fold), cisplatin (3-fold), and doxorubicin (18-fold)]. The uptake studies revealed that accumulations of paclitaxel and Hoechst 33342, a membrane-permeable nuclear staining dye, were reduced to 51% and 45%, respectively, in cells expressing MUC1ΔCT via ABCB1/P-gp-independent mechanisms. Such alterations in chemoresistance and cellular accumulation were not observed in MUC13-expressing cells. Furthermore, we found that MUC1 and MUC1ΔCT increased the cell-adhered water volume by 2.6- and 2.7-fold, respectively, suggesting the presence of a water layer on the cell surface created by NG-MUC1. Taken together, these results suggest that NG-MUC1 acts as a hydrophilic barrier element against anticancer drugs and contributes to chemoresistance by limiting the membrane permeation of lipophilic drugs. Our findings could help better the understanding of the molecular basis of drug resistance in cancer chemotherapy. SIGNIFICANCE STATEMENT: Membrane-bound mucin (MUC1), aberrantly expressed in various cancers, is implicated in cancer progression and chemoresistance. Although the MUC1 cytoplasmic tail is involved in proliferation-promoting signal transduction thereby leading to chemoresistance, the significance of the extracellular domain remains unclear. This study clarifies the role of the glycosylated extracellular domain as a hydrophilic barrier element to limit the cellular uptake of lipophilic anticancer drugs. These findings could help better the understanding of the molecular basis of MUC1 and drug resistance in cancer chemotherapy.

Food effects on gastrointestinal physiology and drug absorption.

Food ingestion affects the oral absorption of many drugs in humans. In this review article, we summarize the physiological factors in the gastrointestinal (GI) tract that affect the in vivo performance of orally administered solid dosage forms in fasted and fed states in humans. In particular, we discuss the effects of food ingestion on fluid characteristics (pH, bile concentration, and volume) in the stomach and small intestine, GI transit of water and dosage forms, and microbiota. Additionally, case examples of food effects on GI physiology and subsequent changes in oral drug absorption are provided. Furthermore, the effects of food, especially fruit juices (e.g., grapefruit, orange, apple) and green tea, on transporter-mediated permeation and enzyme-catalyzed metabolism of drugs in intestinal epithelial cells are also summarized comprehensively.

Identification of 5-Carboxyfluorescein as a Probe Substrate of SLC46A3 and Its Application in a Fluorescence-Based In Vitro Assay Evaluating the Interaction with SLC46A3.

The therapeutic modalities that involve the endocytosis pathway, including antibody-drug conjugates (ADCs), have recently been developed. Since the drug escape from endosomes/lysosomes is a determinant of their efficacy, it is important to optimize the escape, and the cellular evaluation system is needed. SLC46A3, a lysosomal membrane protein, has been implicated in the pharmacological efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC used for the treatment of breast cancer, and the cellular uptake efficacy of lipid-based nanoparticles. Recently, we identified the SLC46A3 function as a proton-coupled steroid conjugate and bile acid transporter, which can directly transport active catabolites of T-DM1. Thus, the rapid and convenient assay systems for evaluating the SLC46A3 function may help to facilitate ADC development and to clarify the physiological roles in endocytosis. Here, we show that SLC46A3 dC, which localizes to the plasma membrane owing to lacking a lysosomal-sorting motif, has a great ability to transport 5-carboxyfluorescein (5-CF), a fluorescent probe, in a pH-dependent manner. 5-CF uptake mediated by SLC46A3 was significantly inhibited by compounds reported to be SLC46A3 substrates/inhibitors and competitively inhibited by estrone 3-sulfate, a typical SLC46A3 substrate. The inhibition assays followed by uptake studies revealed that SG3199, a pyrrolobenzodiazepine dimer, which has been used as an ADC payload, is a substrate of SLC46A3. Accordingly, the fluorescence-based assay system for the SLC46A3 function using 5-CF can provide a valuable tool to evaluate the interaction of drugs/drug candidates with SLC46A3.

Characterization of Aripiprazole Uptake Transporter in the Blood-Brain Barrier Model hCMEC/D3 Cells by Targeted siRNA Screening.

AIM: Identification of blood-brain barrier (BBB) uptake transporters is a major challenge in the research and development of central nervous system (CNS) drugs. However, conventional methods that consider known drug uptake characteristics have failed at identifying the responsible transporter molecule. The present study aimed at identifying aripiprazole uptake transporters in BBB model hCMEC/D3 cells using a knockdown screening study targeting various transporters, including uncharacterized ones. METHODS: We evaluated the effect of 214 types of siRNA targeting transporters on the uptake of aripiprazole, an atypical antipsychotic drug, in hCMEC/D3 cells. Aripiprazole uptake was determined using Xenopus oocytes expressing the candidate genes extracted from the siRNA screening assay. RESULTS: The estimated unbound brain to plasma concentration ratio (Kp,uu,brain) of aripiprazole was estimated as 0.67 in wild-type mice and 1.94 in abcb1a/1b/abcg2 knockout mice, suggesting the involvement of both uptake and efflux transporters in BBB permeation. According to siRNA knockdown screening studies, organic cation/carnitine transporter 2 (OCTN2) and long-chain fatty acid transporter 1 (FATP1) were identified as candidate genes. The uptake of aripiprazole by hCMEC/D3 cells was decreased by OCTN2 inhibitors, but not by FATP1 inhibitors. A partially increased uptake of aripiprazole was observed in OCTN2-expressing Xenopus oocytes. Finally, to evaluate transporter-mediated BBB permeation of drugs, the reported and estimated Kp,uu,brain values were summarized. CONCLUSIONS: A knockdown screening study in combination with Kp,uu,brain values showed that aripiprazole was a potential substrate of OCTN2. The technique described in this study can be applied to identifying novel BBB transporters for CNS drugs.

Evaluation of Platinum Anticancer Drug-Induced Kidney Injury in Primary Culture of Rat Kidney Tissue Slices by Using Gas-Permeable Plates.

The type of method adopted for the evaluation of drug-induced kidney injury (DIKI) plays an important role during the drug discovery process. In the present study, the usefulness of cultured rat kidney tissue slices maintained on gas-permeable poly(dimethylsiloxane) (PDMS) plates for DIKI was assessed by monitoring the ATP content as a marker of cell viability. The amount of ATP in the kidney slices cultured on the PDMS plates was higher than that in the slices cultured on gas-impermeable polystyrene plates. The protein expression of organic cation transporter-2 (Oct2) was maintained for 3 d. Cisplatin showed a time- and concentration-dependent reduction in ATP in the slices with a half-effective concentration value of 24 µM, which was alleviated by cimetidine, an Oct2 inhibitor, suggesting that cisplatin-induced kidney injury in the cultured slices was regulated by the basolateral uptake transporter Oct2. Furthermore, the intensity of platinum anticancer drug-induced nephrotoxicity in the cultured slices was consistent with that of the in vivo study. In conclusion, the primary culture of rat kidney tissue slices on gas-permeable plates is expected to aid in the prediction of the extent of nephrotoxicity of drugs, even when transporters are responsible for the accumulation of drugs in kidney tissues.

Effect of ingested fluid volume and solution osmolality on intestinal drug absorption: Impact on drug interaction with beverages.

It was recently shown that osmolality-dependent fluid movement is a significant factor causing the clinically observed apple juice (AJ)-atenolol interaction. Here we examined whether osmolality-dependent fluid movement may also explain the AJ volume dependence of the AJ-atenolol interaction. In Wistar rats, the luminal fluid volume after administration of different volumes of purified water (0.5 and 1.0 mL) gradually reduced to a similar steady-state level, while that after administration of different volumes of AJ (0.5 and 1.0 mL) increased and attained different apparent steady-state levels. It was hypothesized that osmolality-dependent fluid secretion would account for the volume dependence of the apparent steady-state. Indeed, the luminal concentration of FD-4, a non-permeable compound, after administration in AJ was attenuated depending upon the ingested volume, whereas that after administration in purified water was independent of the ingested fluid volume. An in vivo pharmacokinetic study in rats showed that co-administration of AJ and hyperosmotic solution (adjusted to the osmolality of AJ) with atenolol volume-dependently reduced the AUC and Cmax of atenolol significantly. These results show that osmolality-dependent variations in luminal fluid volume may indirectly influence the absorption characteristics of drugs, and can account for the observed volume dependence of beverage-drug interactions.

Multiple Transport Mechanisms Involved in the Intestinal Absorption of Metformin: Impact on the Nonlinear Absorption Kinetics.

The aim of this study was to investigate the contributions of multiple transport mechanisms to the intestinal absorption of metformin, focusing on OCT3, PMAT, THTR2, SERT and OCTN2. We also assessed the impact of these transporters on the nonlinear absorption of metformin. Uptake studies with MDCKII cells expressing OCT3, PMAT, THTR2 or SERT confirmed that metformin is a substrate of these transporters. Decynium22 strongly inhibited metformin uptake mediated by all the transporters. 7-Cyclopentyl inhibited OCT3- and THTR2-mediated uptake of metformin. AG835, thiamine and paroxetine specifically inhibited PMAT-, THTR2- and SERT-mediated uptake of metformin, respectively. Using these inhibitors, the relative contributions of OCT3, PMAT, THTR2, SERT, OCTN2 and others to the intestinal permeation of metformin across Caco-2 cells were estimated to be 9.77%, 9.68%, 22.2%, 1.52%, 0% and 0.66%, respectively. Concentration-dependent analysis of metformin uptake by Caco-2 cells revealed nonlinear kinetics with the similar Km(app) value to the value for THTR2. Further in situ absorption study demonstrated that rat intestinal permeability of metformin was significantly decreased in the presence of decynium22, 7-cyclopentyl and thiamine. The present study indicated that THTR2 is the major determinant of the nonlinear absorption of metformin, although multiple transport mechanisms contribute to its intestinal absorption.

SLC46A3 is a lysosomal proton-coupled steroid conjugate and bile acid transporter involved in transport of active catabolites of T-DM1.

Antibody-drug conjugates (ADCs) represent a new class of cancer therapeutics that enable targeted delivery of cytotoxic drugs to cancer cells. Although clinical efficacy has been demonstrated for ADC therapies, resistance to these conjugates may occur. Recently, SLC46A3, a lysosomal membrane protein, was revealed to regulate the efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC that has been widely used for treating breast cancer. However, the role of SLC46A3 in mediating T-DM1 cytotoxicity remains unclear. In this study, we discovered the function of SLC46A3 as a novel proton-coupled steroid conjugate and bile acid transporter. SLC46A3 preferentially recognized lipophilic steroid conjugates and bile acids as endogenous substrates. In addition, we found that SLC46A3 directly transports Lys-SMCC-DM1, a major catabolite of T-DM1, and potent SLC46A3 inhibitors attenuate the cytotoxic effects of T-DM1, suggesting a role in the escape of Lys-SMCC-DM1 from the lysosome into the cytoplasm. Our findings reveal the molecular mechanism by which T-DM1 kills cancer cells and may contribute to the rational development of ADCs that target SLC46A3.