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As an expansion investigation of drug-drug interaction (DDI) from previous clinical trials, additional plasma endogenous metabolites were quantitated in the same subjects to further identify the potential biomarkers of organic anion transporter (OAT) 1/3 inhibition. In the single dose, open label, three-phase with fixed order of treatments study, 14 healthy human volunteers orally received 1000 mg probenecid alone, or 40 mg furosemide alone, or 40 mg furosemide at 1 hour after receiving 1000 mg probenecid on days 1, 8, and 15, respectively. Endogenous metabolites including kynurenic acid, xanthurenic acid, indo-3-acetic acid, pantothenic acid, p-cresol sulfate, and bile acids in the plasma were measured by liquid chromatography–tandem mass spectrometry. The Cmax of kynurenic acids was significantly increased about 3.3- and 3.7-fold over the baseline values at predose followed by the treatment of probenecid alone or in combination with furosemide respectively. In comparison with the furosemide-alone group, the Cmax and area under the plasma concentration–time curve (AUC) up to 12 hours of kynurenic acid were significantly increased about 2.4- and 2.5-fold by probenecid alone, and 2.7- and 2.9-fold by probenecid plus furosemide, respectively. The increases in Cmax and AUC of plasma kynurenic acid by probenecid are comparable to the increases of furosemide Cmax and AUC reported previously. Additionally, the plasma concentrations of xanthurenic acid, indo-3-acetic acid, pantothenic acid, and p-cresol sulfate, but not bile acids, were also significantly elevated by probenecid treatments. The magnitude of effect size analysis for known potential endogenous biomarkers demonstrated that kynurenic acid in the plasma offers promise as a superior addition for early DDI assessment involving OAT1/3 inhibition. SIGNIFICANCE STATEMENT This article reports that probenecid, an organic anion transporter (OAT) 1 and OAT3 inhibitor, significantly increased the plasma concentrations of kynurenic acid and several uremic acids in human subjects. Of those, the increases of plasma kynurenic acid exposure are comparable to the increases of furosemide by OAT1/3 inhibition. Effect size analysis for known potential endogenous biomarkers revealed that plasma kynurenic acid is a superior addition for early drug-drug interaction assessment involving OAT1/3 inhibition.

Renal clearance of many drugs is mediated by renal organic anion transporters OAT1/3 and inhibition of these transporters may lead to drug‐drug interactions (DDIs). Pyridoxic acid (PDA) and homovanillic acid (HVA) were indicated as potential biomarkers of OAT1/3. The objective of this study was to develop a population pharmacokinetic model for PDA and HVA to support biomarker qualification. Simultaneous fitting of biomarker plasma and urine data in the presence and absence of potent OAT1/3 inhibitor (probenecid, 500 mg every 6 h) was performed. The impact of study design (multiple vs. single dose of OAT1/3 inhibitor) and ability to detect interactions in the presence of weak/moderate OAT1/3 inhibitors was investigated, together with corresponding power calculations. The population models developed successfully described biomarker baseline and PDA/HVA OAT1/3‐mediated interaction data. No prominent effect of circadian rhythm on PDA and HVA individual baseline levels was evident. Renal elimination contributed greater than 80% to total clearance of both endogenous biomarkers investigated. Estimated probenecid unbound in vivo OAT inhibitory constant was up to 6.4‐fold lower than in vitro values obtained with PDA as a probe. The PDA model was successfully verified against independent literature reported datasets. No significant difference in power of DDI detection was found between multiple and single dose study design when using the same total daily dose of 2000 mg probenecid. Model‐based simulations and power calculations confirmed sensitivity and robustness of plasma PDA data to identify weak, moderate, and strong OAT1/3 inhibitors in an adequately powered clinical study to support optimal design of prospective clinical OAT1/3 interaction studies.

BackgroundEndogenous biomarkers are promising tools to assess transporter-mediated drug–drug interactions early in humans.MethodsWe evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulphate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein, and assessed the in vivo biomarker sensitivity towards the strong organic anion transporter (OAT) inhibitor probenecid at 500 mg every 6 h to reach close to complete OAT inhibition.ResultsPDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and multidrug resistance-associated protein 4; GCDCA-S was more selective, having affinity only towards OAT3 and multidrug resistance-associated protein 2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to those of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter.ConclusionsThe current findings illustrate a clear benefit of measuring PDA plasma exposure during phase I studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data. Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended to tease out the involvement of OAT1/3 in the inhibition interaction.

The Wnt/β-catenin signaling pathway is dysregulated in diseases and Wnt inhibitors like PRI-724 are in clinical development. This study evaluated the regulatory actions of PRI-724 and other Wnt inhibitors on the transport activity of human renal Organic anion transporters (OATs) and Organic anion transporting polypeptides (OATPs). The substrate uptake by OAT4 and OATP2B1 was markedly decreased by PRI-724 (Vmax/Km: ∼26% and ∼17% of corresponding control), with less pronounced decreases in OAT1, OAT3 and OAT1A2. PRI-724 decreased the plasma membrane expression of inhibited OATs/OATPs but didn't affect their total cellular expression. Two model Wnt inhibitors - FH535 and 21H7 - were also tested in comparative studies. Like PRI-724, they also strongly decreased the activities and membrane expression of multiple OATs/OATPs. In contrast, FH535 didn't affect the substrate uptake by organic cation transporters. In control studies, the EGFR inhibitor lapatinib did not inhibit the function of some OATs/OATPs. Together these findings suggest that Wnt inhibitors selectively modulate the function of multiple organic anions transporters, so their clinical use may have unanticipated effects on drug entry into cells. These findings are pertinent to current clinical trials that have been designed to understand the safety and efficacy of new Wnt inhibitor drugs.

Context Relinqing granules (RLQ) are being used alone or in combination with antibacterial drugs to treat urological disorders. Objective This study investigates the pharmacokinetics of RLQ in humans and the potential for RLQ-perpetrated interactions on transporters. Materials and methods Twelve healthy subjects (six women and six men) participated to compare single- and multiple-dose pharmacokinetics of RLQ. In the single-dose study, all 12 subjects received 8 g of RLQ orally. After a 7-d washout period, the subjects received 8 g of RLQ for seven consecutive days (t.i.d.) and then a single dose. Gallic acid (GA) and protocatechuic acid (PCA) in plasma and urine samples were analysed using LC-MS/MS. The transfected cells were used to study the inhibitory effect of GA (50–5000 μg/L) and PCA (10–1000 μg/L) on transporters OAT1, OAT3, OCT2, OATP1B1, P-gp and BCRP. Results GA and PCA were absorbed into the blood within 1 h after administration and rapidly eliminated with a half-life of less than 2 h. The mean peak concentrations of GA (102 and 176 μg/L) and PCA (4.54 and 7.58 μg/L) were lower in males than females, respectively. The 24 h urine recovery rates of GA and PCA were about 10% and 5%, respectively. The steady-state was reached in 7 d without accumulation. GA was a potent inhibitor of OAT1 (IC50 = 3.73 μM) and OAT3 (IC50 = 29.41 μM), but not OCT2, OATP1B1, P-gp or BCRP. Discussion and conclusions GA and PCA are recommended as PK-markers in RLQ-related pharmacokinetic and drug interaction studies. We should pay more attention to the potential for RLQ-perpetrated interactions on transporters.

We previously reported that blue eggshell color in chickens is associated with a partial endogenous retroviral (EAV-HP) insertion in the promoter region of the solute carrier organic anion transporter family member 1B3 (SLCO1B3) gene. The EAV-HP sequence includes numerous regulatory elements, which may modulate the expression of adjacent genes. To determine whether this insertion influences the expression of neighboring genes, we screened the expression of solute carrier organic anion transporter family members 1C1, 1B1 (SLCO1C1, SLCO1B1), and SLCO1B3 in 13 and 10 tissues from female and male Yimeng chickens, respectively. We observed that the insertion only significantly modulated the expression of SLCO1B3 and did not majorly affect that of SLCO1C1 and SLCO1B1. High expression of SLCO1B3 was detected in the shell gland, magnum, isthmus, and vagina of the oviduct in female blue-eggshell chickens. We also observed ectopic expression of SLCO1B3 in the testes of male chickens. SLCO1B3 is typically highly expressed in the liver; however, the EAV-HP insertion significantly reduces SLCO1B3 expression. As a liver-specific transporter, a reduction in the expression of SLCO1B3 may affect liver metabolism, particularly that of bile acids. We also detected higher ectopic expression of SLCO1B3 in the lungs of birds heterozygous for the EAV-HP insertion than in homozygous genotypes. In conclusion, we confirmed that the EAV-HP insertion modifies SLCO1B3 expression, and showed, for the first time, similar expression profile of this gene in all parts of the oviduct in females and testis in males. We also observed different levels of SLCO1B3 expression in the liver, which were associated with the EAV-HP insertion, and significantly higher expression in the lungs of birds with heterozygous genotype. The effects of these changes in the SLCO1B3 expression pattern on the function of the tissues warrant further investigation.

Mercury is a widespread pollutant. Mercuric ions uptake into tubular cells is supported by the Organic anion transporter 1 (Oat1) and 3 (Oat3) and its elimination into urine is through the Multidrug resistance-associated protein 2 (Mrp2). We investigated the effect of recombinant human erythropoietin (Epo) on renal function and on renal expression of Oat1, Oat3, and Mrp2 in a model of mercuric chloride (HgCl2)-induced renal damage. Four experimental groups of adult male Wistar rats were used: Control, Epo, HgCl2, and Epo + HgCl2. Epo (3000 IU/kg, b.w., i.p.) was administered 24 h before HgCl2 (4 mg/kg, b.w., i.p.). Experiments were performed 18 h after the HgCl2 dose. Parameters of renal function and structure were evaluated. The protein expression of Oat1, Oat3 and Mrp2 in renal tissue was assessed by immunoblotting techniques. Mercury levels were determined by cold vapor atomic absorption spectrometry. Pretreatment with Epo ameliorated the HgCl2-induced tubular injury as assessed by histopathology and urinary biomarkers. Immunoblotting showed that pretreatment with Epo regulated the renal expression of mercury transporters in a way to decrease mercury content in the kidney. Epo pretreatment ameliorates HgCl2-induced renal tubular injury by modulation of mercury transporters expression in the kidneys.

Forsythin extracted from Forsythiae Fructus is widely used to treat fever caused by the common cold or influenza in China, Japan and Korea. The present study aimed to analyze the pharmacokinetics, metabolism and excretion routes of forsythin in humans and determine the major enzymes and transporters involved in these processes. After a single oral administration, forsythin underwent extensive metabolism via hydrolysis and further sulfation. In total, 3 of the 13 metabolites were confirmed by comparison to reference substances, i.e., aglycone M1, M1 sulfate (M2), and M1 glucuronide (M7). Hydrolysis was the initial and main metabolic pathway of the parent compound, followed by extensive sulfation to form M2 and a reduced level of glucuronidation to form M7. In addition, the plasma exposure of M2 and M7 were 86- and 4.2-fold higher than that of forsythin. Within 48 h, ~75.1% of the administered dose was found in urine, with M2 accounting for 71.6%. Further phenotyping experiments revealed that sulfotransferase 1A1 and UDP-glucuronosyltransferase 1A8 were the most active hepatic enzymes involved in the formation of M2 and M7, respectively. The in vitro kinetic study provided direct evidence that M1 showed a preference for sulfation. Sulfated conjugate M2 was identified as a specific substrate of organic anion transporter 3, which could facilitate the renal excretion of M2. Altogether, our study demonstrated that sulfation dominated the metabolism and pharmacokinetics of forsythin, while the sulfate conjugate was excreted mainly in the urine.

Methotrexate (MTX), an antifolate agent, is primarily eliminated by the kidney. Organic anion transporter 3 (OAT3) contributes to renal MTX clearance. Several studies have shown an association between co-administration of proton pump inhibitors (PPIs) and delayed elimination of MTX, but the findings are conflicting. In this study, we aimed to evaluate whether the differential inhibitory effects of PPIs on the OAT3-mediated transport of MTX are associated with the risks of delayed MTX elimination. We investigated the effects of PPIs on rat (r) OAT3-mediated MTX uptake using HEK293T cells expressing rOAT3. To examine whether PPIs could affect the pharmacokinetics of MTX, changes in plasma concentration–time profiles were assessed when MTX (50 mg/kg, ip) and a range of PPIs (2 mg/kg, iv) were administered to rats. In vitro studies demonstrated that PPIs inhibited rOAT3-mediated uptake of MTX, with estimated IC50 values of 2.1–5.2 μM, and a rank order of esomeprazole ≈ lansoprazole ≈ omeprazole > rabeprazole. When MTX and esomeprazole were co-administered to rats, the plasma concentration of MTX 6 h after administration and the t1/2 were significantly higher than those in the vehicle group. The effect of lansoprazole was not significant, but showed a tendency to prolong plasma MTX levels. Famotidine, a histamine H2-receptor antagonist, showed a weak inhibitory effect on rOAT3-mediated MTX uptake, although it did not affect plasma concentration–time profile of MTX in vivo. Esomeprazole increases the t1/2 of MTX in rats, which may be partially attributed to the inhibition of rOAT3.

Background and Objectives Citrus flavonoids are not only components of daily nutrition, they are also promoted as dietary supplements and are important ingredients in traditional medicines. Interactions of flavonoids with synthetic drugs represent an often neglected issue. We therefore investigated in vitro whether the polymethoxyflavones nobiletin, sinensetin, and tangeretin and the flavonoid rutinosides didymin, hesperidin, and narirutin can inhibit human organic anion transporting polypeptides (OATP) 1B1, 1B3, and 2B1, which are important transporters mediating drug-drug and food-drug interactions. Methods Inhibition was investigated by quantifying the decreased uptake of the fluorescent OATP1B1 and OATP1B3 substrate 8-fluorescein-cAMP in HEK293 cells overexpressing OATP1B1 or OATP1B3 and of the fluorescent OATP2B1 substrate 4′,5′-dibromofluorescein in HEK293 cells overexpressing OATP2B1. Results We demonstrate that all flavonoids investigated inhibit OATP2B1 in the lower micromolar range (IC50 between 1.6 and 14.2 µM), but only the polymethoxyflavones also inhibit OATP1B1 and 1B3 (IC50 between 2.1 and 21 µM). Conclusions All flavonoids investigated might contribute to the intestinal OATP2B1-based interactions with drugs observed with citrus juices or fruits. In contrast, the concentration of the polymethoxyflavones after consumption of citrus juices or fruits is most likely too low to reach relevant systemic concentrations and thus to inhibit hepatic OATP1B1 and OATP1B3, but there might be a risk when they are consumed as medicines or as dietary supplements.

Relation between the renal function and the membrane environment where the organic anion transporters Oat1 and Oat3 are localized is scarce. The aim of this study was to examine the Oat1 and Oat3 distribution in different cellular fractions under physiological conditions as well as the effects of extrahepatic cholestasis on membrane distribution of both proteins. Besides, the potential role of jaundice serum on the Oat1 and Oat3 expression in suspensions of renal tubular cells was evaluated. Cellular and membrane fractions of renal cortex were obtained from control rats to evaluate Oat1 and Oat3 protein expressions. Other rats were subjected to bile duct ligation (BDL) or Sham operation to determine the membrane distribution of Oat1 and Oat3 between lipid raft domains (LRD) and non-LRD. Incubation of renal cortical cells with serum from Sham and BDL were also performed to study Oat1 and Oat3 protein expressions. In physiological conditions, Oat1 and Oat3 were concentrated in LRD. The pathology induced a shift of Oat1 from LRD to non-LRD, while Oat3 showed no changes in its distribution. In cells exposed to BDL serum, Oat1 protein expression in membranes significantly increased. For Oat3, no difference between groups was observed. The Oat1 redistribution to non-LRD in BDL could be favoring the increase in renal transport of organic anions previously observed. This change was specific to Oat1. The in vitro experiment allows to conclude that some component present in BDL serum is responsible for the alterations observed in Oat1 expression in cortical membranes.

Introduction: Transporters and enzymes play an important role in absorption, distribution, clearance and elimination of drugs.Areas covered: This review provides an overview of the extended clearance concept and usefulness of extended clearance classification system (ECCS) in early identification of predominant clearance mechanisms. Clinical studies demonstrating transporter-enzyme interplay, challenges in scaling clearance from in vitro systems, utility of animal models and modeling approaches for evaluating hepatic clearance and drug-drug interactions are reviewed.Expert opinion: Clinical evidence exists supporting organic anion transporting peptide (OATP)1B and drug metabolizing enzymes involvement in clearance of ECCS class 1B drugs. Emerging evidence point toward contribution of organic cation transporter (OCT)1 to hepatic uptake of cationic drugs. Although, limited clinical evidence is presented, preclinical studies and modeling suggests organic anion transporter (OAT)2-enzyme interplay in clearance of class 1A drugs. Data from in vitro assays and preclinical models coupled with physiologically based modeling approaches are key for understanding transporter-enzyme interplay, enabling prediction of pharmacokinetics, tissue exposure and drug interactions. Current methodologies incur limitations and emphasis should be placed on the development of physiologically relevant in vitro models and characterize in vivo animal models to inform mechanistic modeling and improve confidence in prospective predictions.

Background and purpose Diclofenac is a widely used nonsteroidal anti-inflammatory drug. However, adverse effects in the kidney limit its clinical application. The present study was aimed to evaluate the potential effect of cilastatin on diclofenac-induced acute kidney injury and to clarify the potential roles of renal organic anion transporters (OATs) in the drug-drug interaction between cilastatin and diclofenac. Experimental approach The effect of cilastatin was evaluated in diclofenac-induced acute kidney injury in mice. Human OAT1/3-transfected HEK293 cells and renal primary proximal tubule cells (RPTCs) were used to investigate OAT1/3-mediated transport and the cytotoxicity of diclofenac. Key results Cilastatin treatment decreased the pathological changes, renal dysfunction and elevated renal levels of oxidation products, cytokine production and apoptosis induced by diclofenac in mice. Moreover, cilastatin increased the plasma concentration and decreased the renal distribution of diclofenac and its glucuronide metabolite, diclofenac acyl glucuronide (DLF-AG). Similarly, cilastatin inhibited cytotoxicity and mitochondrial damage in RPTCs but did not change the intracellular accumulation of diclofenac. DLF-AG but not diclofenac exhibited OAT-dependent cytotoxicity and was identified as an OAT1/3 substrate. Cilastatin inhibited the intracellular accumulation and decreased the cytotoxicity of DLF-AG in RPTCs. Conclusion and implications Cilastatin alleviated diclofenac-induced acute kidney injury in mice by restoring the redox balance, suppressing inflammation, and reducing apoptosis. Cilastatin inhibited OATs and decreased the renal distribution of diclofenac and DLF-AG, which further ameliorated the diclofenac-induced nephrotoxicity in mice. Cilastatin can be potentially used in the clinic as a therapeutic agent to alleviate the adverse renal reaction to diclofenac.

The multispecific organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), the main kidney elimination pathways for many common drugs, are often considered to have largely-redundant roles. However, whereas examination of metabolomics data from Oat-knockout mice (Oat1 and Oat3KO) revealed considerable overlap, over a hundred metabolites were increased in the plasma of one or the other of these knockout mice. Many of these relatively unique metabolites are components of distinct biochemical and signaling pathways, including those involving amino acids, lipids, bile acids, and uremic toxins. Cheminformatics, together with a “logical” statistical and machine learning-based approach, identified a number of molecular features distinguishing these unique endogenous substrates. Compared with OAT1, OAT3 tends to interact with more complex substrates possessing more rings and chiral centers. An independent “brute force” approach, analyzing all possible combinations of molecular features, supported the logical approach. Together, the results suggest the potential molecular basis by which OAT1 and OAT3 modulate distinct metabolic and signaling pathways in vivo. As suggested by the Remote Sensing and Signaling Theory, the analysis provides a potential mechanism by which “multispecific” kidney proximal tubule transporters exert distinct physiological effects. Furthermore, a strong metabolite-based machine-learning classifier was able to successfully predict unique OAT1 versus OAT3 drugs; this suggests the feasibility of drug design based on knockout metabolomics of drug transporters. The approach can be applied to other SLC and ATP-binding cassette drug transporters to define their nonredundant physiological roles and for analyzing the potential impact of drug–metabolite interactions.

Multiple doses of tenofovir disoproxil fumarate (TDF) together with emtricitabine is effective for HIV preexposure prophylaxis (PrEP). TDF is converted to tenofovir (TFV) in circulation, which is subsequently cleared via tubular secretion by organic ion transporters (OATs; OAT1 and OAT3). Using in vitro kinetic parameters for TFV and the OAT1 and OAT3 inhibitor probenecid, a bottom‐up physiologically‐based pharmacokinetic model was successfully developed for the first time that accurately describes the probenecid–TFV interaction. This model predicted an increase in TFV plasma exposure by 60%, which was within 15% of the observed clinical pharmacokinetic data, and a threefold decrease in renal cells exposure following coadministration of a 600 mg TDF dose with 2 g probenecid. When compared with multiple‐dose regimens, a single‐dose probenecid‐boosted TDF regimen may be effective for HIV PrEP and improve adherence and safety by minimizing TFV‐induced nephrotoxicity by reducing TFV accumulation in renal cells.

Organic anion transporter 3 (OAT3) plays a critical role in the renal excretion of many xenobiotics. Because steviol acyl glucuronide (SVAG), an OAT3 substrate, is the major circulating metabolite after oral ingestion of steviol glycosides and is excreted into the urine, inhibition of OAT3 activity may alter pharmacokinetic profiles of SVAG. The present study showed that drugs such as probenecid and glimepiride displayed potent inhibition toward the OAT3-mediated SVAG transport, with IC50 values of 4.9 and 0.8 μM, respectively. No species differences were observed. Probenecid and glimepiride could significantly elevate plasma concentrations of SVAG after oral administration of rebaudioside A, with significant increases in plasma maximum (Cmax) and area under the plasma time-concentration curve values. The inhibitory effect on the OAT3-mediated SVAG transport exemplified a unique case between drugs and the metabolite of a food additive. Our data suggest that caution should be exercised when giving steviol glycoside products to human subjects with compromised renal function.

Rivaroxaban is indicated for stroke prevention in nonvalvular atrial fibrillation (AF). Its elimination is mediated by both hepatic metabolism and renal excretion. Consequently, its clearance is susceptible to both intrinsic (pathophysiological) and extrinsic (concomitant drugs) variabilities that in turn implicate bleeding risks. Upon systematic model verification, physiologically based pharmacokinetic (PBPK) models are qualified for the quantitative rationalization of complex drug-drug-disease interactions (DDDIs). Hence, this study aimed to develop and verify a PBPK model of rivaroxaban systematically. Key parameters required to define rivaroxaban's disposition were either obtained from in vivo data or generated via in vitro metabolism and transport kinetic assays. Our developed PBPK model successfully predicted rivaroxaban's clinical pharmacokinetic parameters within predefined success metrics. Consideration of basolateral organic anion transporter 3 (OAT3)-mediated proximal tubular uptake in tandem with apical P-glycoprotein (P-gp)-mediated efflux facilitated mechanistic characterization of the renal elimination of rivaroxaban in both healthy and renal impaired patients. Retrospective drug-drug interaction (DDI) simulations, incorporating in vitro metabolic inhibitory parameters, accurately recapitulated clinically observed attenuation of rivaroxaban's hepatic clearance due to enzyme-mediated DDIs with CYP3A4/2J2 inhibitors (verapamil and ketoconazole). Notably, transporter-mediated DDI simulations between rivaroxaban and the P-gp inhibitor ketoconazole yielded minimal increases in rivaroxaban's systemic exposure when P-gp-mediated efflux was solely inhibited, but were successfully characterized when concomitant basolateral uptake inhibition was incorporated in the simulation. In conclusion, our developed PBPK model of rivaroxaban is systematically verified for prospective interrogation and management of untested yet clinically relevant DDDIs pertinent to AF management using rivaroxaban. SIGNIFICANCE STATEMENT: Rivaroxaban is susceptible to DDDIs comprising renal impairment and P-gp and CYP3A4/2J2 inhibition. Here, systematic construction and verification of a PBPK model of rivaroxaban, with the inclusion of a mechanistic kidney component, provided insight into the previously arcane role of OAT3-mediated basolateral uptake in influencing both clinically observed renal elimination of rivaroxaban and differential extents of transporter-mediated DDIs. The verified model holds potential for investigating clinically relevant DDDIs involving rivaroxaban and designing dosing adjustments to optimize its pharmacotherapy in atrial fibrillation.

Following oral administration, dabigatran etexilate (DABE) is rapidly hydrolyzed to its active form, dabigatran. DABE, but not dabigatran, presents as a P-glycoprotein (P-gp) substrate and has increasingly been used as a probe drug. Therefore, although dosed as DABE, a P-gp drug-drug interaction (DDI) is reported as a dabigatran plasma concentration ratio (perpetrator versus placebo). Because the majority of a DABE dose (80 to 85%) is recovered in urine as unchanged dabigatran (renal active secretion is ∼25% of total clearance), dabigatran was evaluated in vitro as a substrate of various human renal transporters. Active (pyrimethamine-sensitive) dabigatran uptake was observed with human embryonic kidney (HEK) 293 cells expressing multidrug and toxin extrusion protein 1 (MATE1) and 2K (MATE2K), with Michaelis-Menten constant (Km) values of 4.0 and 8.0 μM, respectively. By comparison, no uptake of 2 μM dabigatran (versus mock-transfected HEK293 cells) was evident with HEK293 cells transfected with organic cation transporters (OCT1 and OCT2) and organic anion transporters (OAT1, 2, 3, and 4). The efflux ratios of dabigatran across P-gp- and BCRP (breast cancer resistance protein)-MDCK (Madin-Darby canine kidney) cell monolayers were 1.5 and 2.0 (versus mock-MDCK cell monolayers), suggesting dabigatran is a relatively poor P-gp and BCRP substrate. Three of five drugs (verapamil, ketoconazole, and quinidine) known to interact clinically with dabigatran, as P-gp inhibitors, presented as MATE inhibitors in vitro (IC50 = 1.0 to 25.2 μM). Taken together, although no basolateral transporter was identified for dabigatran, the results suggest that apical MATE1 and MATE2K could play an important role in its renal clearance. MATE-mediated renal secretion of dabigatran needs to be considered when interpreting the results of P-gp DDI studies following DABE administration.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF’s active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5−100 μmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC 50 value of 4.91 μmol/L. TER (5 μmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells’ uptake of acyclovir and increased the plasma concentration.

A bioactivity guided program exploring the interaction of phytochemicals in the entire plant Primula macrocalyx with the organic anion transporters (OAT1 and OAT3) and microorganisms led to the elucidation of ten known flavones (1–4, 6–10, 12) and two previously undescribed flavones (5, 11). The structures of the compounds were determined by extensive analysis of spectroscopic data, as well as by comparison with data from previous reports. Two known flavones (9, 12) are reported for the first time from the family Primulaceae. All compounds were evaluated for inhibition of OAT1 and OAT3. Six flavones (2, 3, 6–8, 12) showed potent inhibitory activity on OAT1, while seven flavones (2, 3, 6–9, 12) showed marked inhibitory activity on OAT3, with IC50 ≤ 10.0 µM. Antimicrobial activities of crude fractions against sixteen microorganisms were tested to give a target yeast strain Candida rugosa for further evaluation of MICs on the isolates. Three flavones (7, 8, 12) showed marked antifungal activity with MIC µM. To our knowledge, this study is the first to evaluate these flavones as inhibitors of the OAT1 and OAT3, and as antifungal agents.

Small changes to molecules can have profound effects on their pharmacological activity as exemplified by the addition of the two-carbon acetyl group to make drugs more effective by enhancing their pharmacokinetic or pharmacodynamic properties. N-acetyl-d,l-leucine is approved in France for vertigo and its l-enantiomer is being developed as a drug for rare and common neurological disorders. However, the precise mechanistic details of how acetylation converts leucine into a drug are unknown. Here we show that acetylation of leucine switches its uptake into cells from the l-type amino acid transporter (LAT1) used by leucine to organic anion transporters (OAT1 and OAT3) and the monocarboxylate transporter type 1 (MCT1). Both the kinetics of MCT1 (lower affinity compared to LAT1) and the ubiquitous tissue expression of MCT1 make it well suited for uptake and distribution of N-acetyl-l-leucine. MCT1-mediated uptake of a N-acetyl-l-leucine as a prodrug of leucine bypasses LAT1, the rate-limiting step in activation of leucine-mediated signalling and metabolic process inside cells such as mTOR. Converting an amino acid into an anion through acetylation reveals a way for the rational design of drugs to target anion transporters.

Phellodendri Chinensis Cortex (PC) is a traditional medicinal material used to treat gout and hyperuricemia (HUA) in China. Berberine (BBR), the main component of PC, possesses anti-hyperuricemic and anti-gout effects. However, BBR exhibits low bioavailability due to its extensive metabolism and limited absorption. Thus, the metabolites of BBR are believed to be the potential active forms responsible for its in vivo biological activities. Berberrubine (BRB), one of the major metabolites of BBR, exhibits appreciable biological activities even superior to BBR. In this work, the anti-hyperuricemic efficacy of BRB was investigated in HUA model mice induced by co-administration with intraperitoneal potassium oxonate (PO) and oral hypoxanthine (HX) for 7 days. Results showed that administration with BRB (6.25, 12.5, and 25.0 mg/kg) significantly decreased the serum levels of uric acid (UA) by 49.70%, 75.35%, and 75.96% respectively, when compared to the HUA group. In addition, BRB sharply decreased the levels of serum creatinine (CRE) (by 19.62%, 28.98%, and 38.72%, respectively) and blood urea nitrogen (BUN) (by 16.19%, 25.07%, and 52.08%, respectively) and reversed the PO/HX-induced renal histopathological damage dose-dependently. Additionally, BRB lowered the hepatic XOD activity, downregulated the expressions of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), upregulated expressions of organic anion transporter 1/3 (OAT1/3) and ATP-binding cassette transporter subfamily G member 2 (ABCG2) at both protein and mRNA levels, and suppressed the activation of the JAK2/STAT3 signaling pathway. In addition, BRB significantly decreased the levels of inflammatory mediators (IL-1β, IL-6, and TNF-α). In conclusion, our study indicated that BRB exerted anti-hyperuricemic effect, at least in part, via regulating the urate transporter expressions and suppressing the JAK2/STAT3 signaling pathway. BRB was believed to be promising for further development into a potential therapeutic agent for HUA treatment.

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 are liver-specific transport proteins that express on the basolateral membrane of human hepatocytes and mediate hepatic uptake of many drugs, including statins. They are important determinants of transporter-mediated drug-drug interactions (DDIs). It has been reported that pre-incubation with some OATP1B1 and OATP1B3 inhibitors potentiates the inhibitory effects, yielding reduced IC50 values. The US FDA draft guidance has recently recommended to use the lower IC50 values after inhibitor-preincubation to assess OATP1B1 and OATP1B3-mediated DDIs. However, it remains unknown whether the potentiation effects of inhibitor-preincubation on IC50 values occur in a physiologically relevant cell model. The current study was designed to determine the IC50 values of OATP1B1 and OATP1B3 inhibitors everolimus (EVR), sirolimus (SIR), and dasatinib against OATP1B substrates in physiologically relevant primary human hepatocytes with or without inhibitor-preincubation and to compare the OATP-mediated DDI prediction using data from primary human hepatocytes and that reported previously in transporter-expressing cell lines. Primary human hepatocytes were cultured in a sandwich configuration. Accumulation of [3H]-CCK-8 (1 µM, 1.5 min), [3H]-rosuvastatin (0.5 µM, 2 min) and [3H]-pitavastatin (1 µM, 0.5 min) was determined in human sandwich-cultured hepatocytes (SCH) in the presence of vehicle control or an inhibitor with or without inhibitor-preincubation at designated concentrations, and was utilized to determine the IC50 values for these inhibitors. R-value models were used to predict OATP-mediated DDIs. Pre-incubation with EVR at a clinically relevant concentration of 0.2 µM significantly reduced accumulation of [3H]-CCK-8 and [3H]-rosuvastatin even after washing. Reduced IC50 values following inhibitor pre-incubation were observed for all three inhibitors using [3H]-CCK-8 and [3H]-rosuvastatin as substrates in human SCH. The IC50 values after inhibitor-preincubation were lower or comparable in transporter-expressing cell lines compared with that in human SCH. For dasatinib, R-values from both cell lines and human SCH were greater than the US FDA cut-off value of 1.1. For EVR, R values from cell lines were 1.23 and were lowered to near 1.1 (1.08–1.09) in human SCH. For SIR, R values from either cell type were less than the cut-off values of 1.1. In conclusion, the current study is the first to report that pre-incubation with OATP1B inhibitors potentiates inhibitory effects in physiologically relevant primary human hepatocytes, supporting the rationale of the current US FDA draft guidance of including an inhibitor-preincubation step when assessing OATP-mediated DDIs in vitro. IC50 values after inhibitor-preincubation in transporter-expressing cell lines may be used for DDI prediction for the purpose of mitigating false-negative OATP-mediated DDI prediction.

Organic anion transporter 3 (OAT3) plays an important role in the disposition of various anionic drugs which impacts the pharmacokinetics and pharmacodynamics of the therapeutics, thus influencing the pharmacological effects and toxicity of the drugs. In this study, we investigated the effect of insulin on the regulation of OAT3 function, expression, and SUMOylation. We demonstrated that insulin induced an increase in OAT3 transport activity through a dose- and time-dependent manner in COS-7 cells. The insulin-induced elevation in OAT3 function was blocked by PKA inhibitor H89, which correlated well with OAT3 protein expression. Moreover, both PKA activator Bt2-cAMP-induced increase and insulin-induced increase in OAT3 function were blocked by PKB inhibitor AKTi1/2. To further investigate the involvement of SUMOylation, we treated OAT3-expressing cells with insulin in presence or absence of H89 or AKTi1/2 followed by examining OAT3 SUMOylation. We showed that insulin enhanced OAT3 SUMOylation, and such enhancement was abrogated by H89 and AKTi1/2. Lastly, insulin increased OAT3 function and SUMOylation in rat kidney slice. In conclusion, our investigations demonstrated that insulin regulated OAT3 function, expression, and SUMOylation through PKA/PKB signaling pathway. Graphical abstract.

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell–derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

Human OAT1 and OAT3 play major roles in renal drug elimination and drug-drug interactions. However, there is little information on the interactions of drug metabolites with transporters. The goal of this study was to characterize the interactions of drug metabolites with OAT1 and OAT3 and compare their potencies of inhibition with those of their corresponding parent drugs. Using HEK293cells stably transfected with OAT1 and OAT3, 25 drug metabolites and their corresponding parent drugs were screened for inhibitory effects on OAT1-and OAT3-mediated 6-carboxyfluorescein uptake at a screening concentration of 200μM for all but 3 compounds. 20 and 24 drug metabolites were identified as inhibitors (inhibition>50%) of OAT1 and OAT3, respectively. Seven drug metabolites were potent inhibitors of either or both OAT1 and OAT3 with Ki values less than 1μM. 22 metabolites were more potent inhibitors of OAT3 than OAT1. Importantly, one drug and four metabolites were predicted to inhibit OAT3 at unbound plasma concentrations achieved clinically (Cmax,u/Ki values≥0.1). In conclusion, our study highlights the potential interactions of drug metabolites with OAT1 and OAT3 at clinically relevant concentrations, suggesting that drug metabolites may modulate therapeutic and adverse drug response by inhibiting renal drug transporters.

Objectives: To perform a genome-wide association study (GWAS) of gout cases vs. asymptomatic hyperuricaemia (AH) controls, and gout cases vs. normouricaemia controls, and to generate a polygenic risk score (PRS) to determine gout-case vs. AH-control status.Methods: Gout cases and AH controls (serum urate (SU) ?6mg/dL) from the UK Biobank were divided into discovery (4,934 cases, 56,948 controls) and replication (2,115 cases, 24,406 controls) cohorts. GWAS was conducted and PRS generated using summary statistics in discovery cohort as the base dataset and the replication cohort as the target dataset. The predictive ability of the model was evaluated. GWAS were performed to identify variants associated with gout compared to normouricaemic controls using SU ? 6mg/dL) from the UK Biobank were divided into discovery (4,934 cases, 56,948 controls) and replication (2,115 cases, 24,406 controls) cohorts. GWAS was conducted and PRS generated using summary statistics in discovery cohort as the base dataset and the replication cohort as the target dataset. The predictive ability of the model was evaluated. GWAS were performed to identify variants associated with gout compared to normouricaemic controls using SU < 6.0 mg/dL and < 7.0 mg/dL thresholds respectively.Results: Thirteen independent SNPs in ABCG2, SLC2A9, SLC22A11, GCKR, MEPE, PPM1K-DT, LOC105377323, and ADH1B reached genome-wide significance and replicated as predictors of AH to gout transition. Twelve of 13 associations were novel for this transition, and rs1229984 (ADH1B) was identified as GWAS locus for gout for the first time. The best PRS model was generated from association data of 17 SNPs; and had predictive ability of 58.5% that increased to 69.2% upon including demographic factors. Two novel SNPs rs760077(MTX1) and rs3800307(PRSS16) achieved GWAS significance for association with gout compared with normouricaemic controls using both SU thresholds. Conclusion: The association of urate transporters with gout supports the central role of hyperuricaemia in its pathogenesis. Larger GWAS are required to identify if variants in inflammatory pathways contribute to progression from AH to gout.

The aim of the present study was to investigate changes in plasma concentrations and tissue distribution of endogenous substrates of organic anion transporting polypeptide (OATP) 1B, hexadecanedioate (HDA), octadecanedioate (ODA), tetradecanedioate (TDA), and coproporphyrin-III, induced by its weak inhibitor, probenecid (PBD), in rats. PBD increased the plasma concentrations of these four compounds regardless of bile duct cannulation, whereas liver-to-plasma (Kp,liver) and kidney-to-plasma concentration ratios of HDA and TDA were reduced. Similar effects of PBD on plasma concentrations and Kp,liver of HDA, ODA, and TDA were observed in kidney-ligated rats, suggesting a minor contribution of renal disposition to the overall distribution of these three compounds. Tissue uptake clearance of deuterium-labeled HDA (d-HDA) in liver was 16-fold higher than that in kidney, and was reduced by 80% by PBD. This was compatible with inhibition by PBD of d-HDA uptake in isolated rat hepatocytes. Such inhibitory effects of PBD were also observed in the human OATP1B1-mediated uptake of d-HDA. Overall, the disposition of HDA is mainly determined by hepatic OATP-mediated uptake, which is inhibited by PBD. HDA might, thus, be a biomarker for OATPs minimally affected by urinary and biliary elimination in rats.

Gyps species have been previously shown to be highly sensitive to the toxic effects of diclofenac, when present in their food sources as drug residues following use as a veterinary medicine. Vultures exposed to diclofenac soon become depressed and die with signs of severe visceral gout and renal damage on necropsy. The molecular mechanism behind toxicity and renal excretion of uric acid is still poorly understood. With the clinical pictures suggesting renal uric acid excretion as the target site for toxicity, as a first step the following study was undertaken to determine the uric acid excretory pathways present in the African white-backed vulture (Gyps africanus) (AWB), one of the species susceptible to toxicity. Using transcriptome analysis, immunohistochemistry and functional predictions, we demonstrated that AWB makes use of the organic anion transporter 2 (OAT2) for their uric acid excretion. RT-qPCR analysis subsequently demonstrated relatively similar expression of the OAT2 transporter in the vulture and chicken. Lastly docking analysis, predicted that the non-steroidal drugs induce their toxicity through an allosteric binding.

Biapenem is a carbapenem antibiotic. It is excreted predominantly through the kidney as unchanged forms. However, the molecular mechanism of renal excretion of biapenem and potential drug-drug interactions (DDIs) were still unknown. In the present study, the role of organic anion transporters (OAT) 1/3 and organic cation transporters (OCT) 2 in the renal excretion of biapenem, and the potential DDIs between biapenem and six clinical commonly prescribed antibiotics and antiviral drugs that acted as substrates or inhibitors of OAT3 were evaluated in vitro. Further, the effect of probenecid on the pharmacokinetics of biapenem was explored in the rats. We observed that biapenem could not inhibit the transport activities of OAT1 or OCT2, while mildly inhibited OAT3 (IC50 >500 μM). Among the tested antibiotics and antiviral drugs, the relatively high DDI index values (maximal unbound plasma concentration over IC50, Imax,u/IC50) were found for piperacillin, linezolid and benzylpenicillin, which were 2.84, 1.7 and 0.62, respectively. Although probenecid had the highest DDI index (27.1) in vitro, no significant impact of it on the pharmacokinetics of biapenem was observed in the rats. Our results indicated that biapenem was primarily eliminated by the glomerular filtration, while OAT3-mediated renal tubular secretion was a minor route. Biapenem is not a clinically relevant substrate or inhibitor because of its low affinity to OAT3. According to current results, it would be safe to use biapenem with other antibiotics and antiviral drugs that acted as substrates or inhibitors of OAT3.

The central nervous system (CNS) is segregated from the circulating blood and peripheral tissues by endothelial and epithelial barriers. To overcome refractory CNS diseases, it is important to understand the membrane transport systems of drugs and the endogenous compounds that relate to the pathogenesis of CNS diseases at these barriers. The endothelial barrier in the brain is the blood-brain barrier (BBB). Our studies clarified the efflux transport of prostaglandin E2 (PGE2), a modulator of neural excitation and inflammatory responses, across the BBB via plasma membrane transporters such as organic anion transporter 3 (Oat3) and multidrug resistance-associated protein 4 (Mrp4). This efflux transport was attenuated by peripheral inflammation or cerebral treatment with neuroexcitatory l-glutamate, suggesting that BBB-mediated PGE2 elimination was altered under several pathological conditions. We also examined excitatory amino acid transporter (EAAT) 1 and 3 as l-glutamate efflux transporters of the inner blood-retinal barrier (BRB) and blood-cerebrospinal barrier. It was considered that these efflux membrane transporters participated in the homeostasis of neuroexcitatory and neuroinflammatory responses in the brain and retina. Moreover, we identified connexin 43 (Cx43) hemichannels as a new membrane transport system that is activated under pathological conditions and recognizes several monocarboxylate drugs, such as valproate. As it is expected that the action of these membrane transporters across the CNS barriers is of great importance in understanding the pathology of various neuroexcitatory diseases, our studies should contribute to the establishment of therapeutic strategies for refractory CNS diseases.

Cisplatin is an anticancer drug commonly used for solid tumors. However, it causes nephrotoxicity. OAT1 and OAT3 are organic anion transporters known to contribute to the uptake of cisplatin into renal tubular cells. The present study was designed to examine the protective role of ellagic acid nanoformulation (ellagic acid nano) on cisplatin-induced nephrotoxicity in rats, and the role of OAT1/OAT3 in this effect. Four groups of male Wistar rats were used (n = 6): (1) control, (2) cisplatin (7.5 mg/kg single dose, intraperitoneal), (3) cisplatin + ellagic acid nano (1 mg/kg), and (4) cisplatin + ellagic acid nano (2 mg/kg). Nephrotoxic rats treated with ellagic acid nano exhibited a significant reduction in elevated serum creatinine, urea, and oxidative stress marker, malondialdehyde (MDA). Additionally, ellagic acid nano restored renal glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Ellagic acid nano improved the histopathological changes induced by cisplatin, such as tubular dilatation, necrosis, and degeneration. Interestingly, OAT1 and OAT3 showed significantly lower expression at both mRNA and protein levels following ellagic acid nano treatment relative to the cisplatin-exposed group. These findings reveal a potential inhibitory role of ellagic acid antioxidant on OAT1 and OAT3 expression and thus explains its nephroprotective effect against cisplatin nephrotoxicity.

Organic anion transporters 1 (OAT1) and 3 (OAT3) play important roles in the renal elimination of a range of substrate molecules. Little is known about natural products that can modulate OAT1 and OAT3 activities. The medullae of Juncus effusus is often used for the treatment of dysuria in traditional Chinese medicine. To study the interactions of phytochemicals in J. effusus with human OAT1 and OAT3, a bioactivity guided phytochemical investigation led to seven new phenanthrenoids along with nine known compounds, including eight phenanthrenoids and a benzophenone from the dichloromethane soluble fraction of a methanol extract of the medullae of J. effusus. The structures were established by physical data analysis, including high-resolution electrospray ionization mass spectrometry and 1D and 2D NMR. The compounds were evaluated for inhibition of OAT1 and OAT3 in vitro. Compounds 10 and 16 were inhibitors for OAT1, and compounds 1-3, 10, and 16 were inhibitors for OAT3 with IC50 values less than 5.0 μM. Dihydrophenanthrene 1 markedly altered the pharmacokinetic parameters of the diuretic drug furosemide, a known substrate of both OAT1 and OAT3, in vivo.

The role of the renal organic anion transporters OAT1 (also known as SLC22A6, originally identified as NKT) and OAT3 (also known as SLC22A8) in chronic kidney disease (CKD) remains poorly understood. This is particularly so from the viewpoint of residual proximal tubular secretion, a key adaptive mechanism to deal with protein-bound uremic toxins in CKD. Using the subtotal nephrectomy (STN) model, plasma metabolites accumulating in STN rats treated with and without the OAT inhibitor, probenecid, were identified. Comparisons with metabolomics data from Oat1-KO and Oat3-KO mice support the centrality of the OATs in residual tubular secretion of uremic solutes, such as indoxyl sulfate, kynurenate, and anthranilate. Overlapping our data with those of published metabolomics data regarding gut microbiome-derived uremic solutes - which can have dual roles in signaling and toxicity - indicates that OATs play a critical role in determining their plasma levels in CKD. Thus, the OATs, along with other SLC and ABC drug transporters, are critical to the movement of uremic solutes across tissues and into various body fluids, consistent with the remote sensing and signaling theory. The data support a role for OATs in modulating remote interorganismal and interorgan communication (gut microbiota-blood-liver-kidney-urine). The results also have implications for understanding drug-metabolite interactions involving uremic toxins.

Over the course of more than 500 million years, the kidneys have undergone a remarkable evolution from primitive nephric tubes to intricate filtration-reabsorption systems that maintain homeostasis and remove metabolic end products from the body. The evolutionarily conserved solute carriers organic cation transporter 2 (OCT2) and organic anion transporters 1 and 3 (OAT1/3) coordinate the active secretion of a broad range of endogenous and exogenous substances, many of which accumulate in the blood of patients with kidney failure despite dialysis. Harnessing OCT2 and OAT1/3 through functional preservation or regeneration could alleviate the progression of kidney disease. Additionally, it would improve current in vitro test models that lose their expression in culture. With this review, we explore OCT2 and OAT1/3 regulation from different perspectives: phylogenetic, ontogenetic, and cell dynamic. Our aim is to identify possible molecular targets both to help prevent or compensate for the loss of transport activity in patients with kidney disease and to enable endogenous OCT2 and OAT1/3 induction in vitro in order to develop better models for drug development.

Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of organic anion transporter (OAT) 1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single-dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 hour after receiving PROB (40 and 1000 mg orally) on days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased the area under the plasma concentration-time curve (AUC) of PDA by 3.1-fold (dosed alone; P 0.05) and 2.1-fold (P

Accumulation of detrimental glutathione-conjugated metabolites in the brain potentially causes neurological disorders, and must therefore be exported from the brain. However, in vivo mechanisms of glutathione-conjugates efflux from the brain remain unknown. We investigated the involvement of transporters in glutathione-conjugates efflux using 6-bromo-7-[11C]methylpurine ([11C]1), which enters the brain and is converted into its glutathione conjugate, S-(7-[11C]methylpurin-6-yl)glutathione ([11C]2). In mice of control and knockout of P-glycoprotein/breast cancer resistance protein and multidrug resistance-associated protein 2 ([ Mrp2] −/−), [11C]2 formed in the brain was rapidly cleared, with no significant difference in efflux rate. In contrast, [11C]2 formed in the brain of Mrp1 −/− mice was slowly cleared, whereas [11C]2 microinjected into the brain of control and Mrp1 −/− mice was 75% cleared within 60 min, with no significant difference in efflux rate. These suggest that Mrp1 contributes to [11C]2 efflux across cell membranes, but not BBB. Efflux rate of [11C]2 formed in the brain was significantly lower in Mrp4 −/− and organic anion transporter 3 ( Oat3) −/− mice compared with control mice. In conclusion, Mrp1, Oat3, and Mrp4 mediate [11C]2 efflux from the brain. Mrp1 may contribute to [11C]2 efflux from brain parenchymal cells, while extracellular [11C]2 is likely cleared across the BBB, partly by Oat3 and Mrp4.

Phytochemical investigation of Camphorosma lessingii has resulted in the isolation of four previously unreported isoflavones (1–4) and eight known compounds (5–12). Nine of these compounds (1–6, 8–10) are reported for the first time from members of the family Amaranthaceae. The structures of all isolated compounds were determined by spectroscopic methods, primarily one-dimensional and two-dimensional nuclear magnetic resonance and mass spectrometry. The absolute configuration of 6 was confirmed by circular dichroism. Inhibition of the organic anion transporters, OAT1 and OAT3, by the isolated compounds was evaluated. Among them, 7, 2′-dihydroxy- 6,8-dimethoxyisoflavone (1), 2′-hydroxy-6,7,8-trimethoxyisoflavone (2), 6,2′-dihydroxy-7,8-dimethoxyisoflavone (3), and 7-methoxyflavone (5) showed a significant inhibitory effect on 6-carboxyfluorescein uptake mediated by OAT1 and OAT3.

Organic anion transporter (OAT) 2 and OAT7 were recently shown to be involved in the hepatic uptake of drugs; however, there is limited understanding of the population variability in the expression of these transporters in liver. There is also a need to derive relative expression-based scaling factors (REFs) that can be used to bridge in vitro functional data to the in vivo drug disposition. To this end, we quantified OAT2 and OAT7 surrogate peptide abundance in a large number of human liver tissue samples ( n = 52), as well as several single-donor cryopreserved human hepatocyte lots ( n = 30) by a novel, validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The average surrogate peptide expression of OAT2 and OAT7 in the liver samples was 1.52 ± 0.57 and 4.63 ± 1.58 fmol/μg membrane protein, respectively. While we noted statistically significant differences ( p < 0.05) in hepatocyte and liver tissue abundances for both OAT2 and OAT7, the differences were relatively small (1.8- and 1.5-fold difference in median values, respectively). Large interindividual variability was noted in the hepatic expression of OAT2 (16-fold in liver tissue and 23-fold in hepatocytes). OAT7, on the other hand, showed less interindividual variability (4-fold) in the livers, but high variability for the hepatocyte lots (27-fold). A significant positive correlation in OAT2 and OAT7 expression was observed, but expression levels were neither associated with age nor sex. In conclusion, our data suggest marked interindividual variability in the hepatic expression of OAT2/7, which may contribute to the pharmacokinetic variability of their substrates. Because both transporters were less abundant in hepatocytes than livers, a REF-based approach is recommended when scaling in vitro hepatocyte transport data to predict hepatic drug clearance and liver exposure of OAT2/7 substrates.

Organic anion transporter 2 (OAT2/SLC22A7) is an uptake transporter that plays an important role in drug disposition. Here, we investigate a potential role of liver receptor homolog-1 (Lrh-1) in regulation of Oat2 and docetaxel pharmacokinetics. Hepatoma cells (Hepa1-6 and HepG2 cells) were transfected with Lrh-1/LRH-1 expression vector or siRNA. The relative mRNA and protein levels of Oat2/OAT2 in the cells or livers of Lrh-1hep-/- mice were determined by qPCR and Western blotting, respectively. Transcriptional regulation of Oat2/OAT2 by Lrh-1/LRH-1 was investigated using luciferase reporter, mobility shift, and chromatin immunoprecipitation (ChIP) assays. Pharmacokinetic studies were performed with wild-type (Lrh-1fl/fl) and Lrh-1hep-/- mice after intraperitoneal injection of docetaxel. Overexpression of Lrh-1 in Hepa1-6 cells led to significant increases in Oat2 mRNA and protein. Consistently, Lrh-1 knockdown caused decreases in Oat2 mRNA and protein, as well as reduced cellular uptake of PGE2, a prototypical substrate of Oat2. Similarly, an activation effect of LRH-1 on OAT2 expression was observed in HepG2 cells. In addition, the levels of Oat2 mRNA and protein were markedly reduced in Lrh-1hep-/- mice. Lrh-1/LRH-1 induced the transcription of Oat2/OAT2 in luciferase reporter assays. Truncation analysis revealed a potential Lrh-1 response element (-716- to -702-bp) in Oat2 promoter. Direct binding of Lrh-1 to this response element was confirmed by mobility shift and ChIP assays. Furthermore, systemic exposure of docetaxel was upregulated in Lrh-1hep-/- mice due to reduced hepatic uptake. In conclusion, Lrh-1 transcriptionally regulates Oat2, thereby impacting tissue uptake and pharmacokinetics of Oat2 substrates.

The purpose of the present study was to clarify the molecular basis of zonated drug distributions in mouse liver based on the protein expression levels of transporters and metabolizing enzymes in periportal (PP) and pericentral (PC) vein regions of mouse hepatic lobules. The distributions of sulforhodamine 101 (SR-101), a substrate of organic anion transporting polypeptides (Oatps), and ribavirin, a substrate of equilibrative nucleoside transporter 1 (Ent1), were elucidated in frozen liver sections of mice, to which each compound had been intravenously administered. Regions strongly positive for SR-101 (SR-101+) and regions weakly positive or negative for SR-101 (SR-101-) were separated by laser microdissection. The zonated distribution of protein expression was quantified in terms of the liver zonation index. Quantitative targeted absolute proteomics revealed the selective expression of glutamine synthetase in the SR-101+ region, indicating predominant distribution of SR-101 in hepatocytes of the PC vein region. The protein levels of Oatp1a1, Oatp1b2, organic cation transporter 1 (Oct1), and cytochrome P450 (P450) 2e1 were greater in the PC vein regions, whereas the level of organic anion transporter 2 (Oat2) was greater in the PP vein regions. Mouse Oatp1a1 mediated SR-101 transport. On the other hand, there were no statistically significant differences in expression of Ent1, Na+-taurocholate cotransporting polypeptide, several canalicular transporters, P450 enzymes, and UDP-glucuronosyltransferases between the PP and PC vein regions. This is consistent with the almost uniform distribution of ribavirin in the liver. In conclusion, sinusoidal membrane transporters such as Oatp1a1, Oatp1b2, Oct1, and Oat2 appear to be determinants of the zonated distribution of drugs in the liver.

Human organic anion transporter-3 (hOAT3) is richly expressed in the kidney, where it plays critical roles in the secretion of clinically important drugs, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. In the current study, we examined the role of AG490, a specific inhibitor of the Janus tyrosine kinase 2 (JAK2), in hOAT3 transport activity in the kidney COS-7 cells. AG490 induced a time- and concentration-dependent inhibition of hOAT3-mediated uptake of estrone sulfate, a prototypical substrate for the transporter. The inhibitory effect of AG490 correlated with a reduced expression of hOAT3 at the cell surface. Our lab previously demonstrated that Nedd4-2, a ubiquitin ligase, down regulates OAT expression and transport activity by enhancing OAT ubiquitination, which leads to an internalization of OAT from cell surface to intracellular compartments and subsequent degradation. In the current study, we showed that treatment of hOAT3-expressing cells with AG490 resulted in an enhanced hOAT3 ubiquitination and degradation, which was accompanied by a strengthened association of Nedd4-2 with hOAT3 and a reduction in Nedd4-2 phosphorylation. SiRNA knockdown of endogenous Nedd4-2 abrogated the effects of AG490 on hOAT3. In summary, our study demonstrated that AG490 regulates hOAT3 expression and transport activity through the modulation of Nedd4-2. Keywords: Organic anion transporter, Drug transport, Regulation, Janus kinase 2 inhibitor, Ubiquitin ligase Nedd4-2

Although arachnoid mater epithelial cells form the blood-arachnoid barrier (BAB), acting as a blood-CSF interface, it has been generally considered that the BAB is impermeable to water-soluble substances and plays a largely passive role. Here, we aimed to clarify the function of transporters at the BAB in regulating CSF clearance of water-soluble organic anion drugs based on quantitative targeted absolute proteomics (QTAP) and in vivo analyses. Protein expression levels of 61 molecules, including 19 ATP-binding-cassette (ABC) transporters and 32 solute-carrier (SLC) transporters, were measured in plasma membrane fraction of rat leptomeninges using QTAP. Thirty-three proteins were detected; others were under the quantification limits. Expression levels of multidrug resistance protein 1 (Mdr1a/P-gp/Abcb1a) and breast cancer resistance protein (Bcrp/Abcg2) were 16.6 and 3.27 fmol/μg protein (51.9- and 9.82-fold greater than in choroid plexus, respectively). Among those organic anion transporters detected only at leptomeninges, not choroid plexus, organic anion transporter 1 (oat1/Slc22a6) showed the greatest expression (2.73 fmol/μg protein). On the other hand, the protein expression level of oat3 at leptomeninges was 6.65 fmol/μg protein, and the difference from choroid plexus was within two-fold. To investigate oat1's role, we injected para-aminohippuric acid (PAH) with or without oat1 inhibitors into cisterna magna (to minimize the contribution of choroid plexus function) of rats. A bulk flow marker, FITC-inulin, was not taken up from CSF up to 15 min, whereas uptake clearance of PAH was 26.5 μL/min. PAH uptake was completely blocked by 3 mM cephalothin (inhibits both oat1 and oat3), while 17% of PAH uptake was inhibited by 0.2 mM cephalothin (selectively inhibits oat3). These results indicate that oat1 and oat3 at the BAB provide a distinct clearance pathway of organic anion drugs from CSF independently of choroid plexus.

Interindividual variability in warfarin dose requirement demands personalized medicine approaches to balance its therapeutic benefits (anticoagulation) and bleeding risk. Cytochrome P450 2C9 ( CYP2C9) genotype-guided warfarin dosing is recommended in the clinic, given the more potent S-warfarin is primarily metabolized by CYP2C9. However, only about 20-30% of interpatient variability in S-warfarin clearance is associated with CYP2C9 genotype. We evaluated the role of hepatic uptake in the clearance of R- and S-warfarin. Using stably transfected HEK293 cells, both enantiomers were found to be substrates of organic anion transporter (OAT)2 with a Michaelis-Menten constant ( Km) of ∼7-12 μM but did not show substrate affinity for other major hepatic uptake transporters. Uptake of both enantiomers by primary human hepatocytes was saturable ( Km ≈ 7-10 μM) and inhibitable by OAT2 inhibitors (e.g., ketoprofen) but not by OATP1B1/1B3 inhibitors (e.g., cyclosporine). To further evaluate the potential role of hepatic uptake in R- and S-warfarin pharmacokinetics, mechanistic modeling and simulations were conducted. A "bottom-up" PBPK model, developed assuming that OAT2-CYPs interplay, well recovered clinical pharmacokinetics, drug-drug interactions, and CYP2C9 pharmacogenomics of R- and S-warfarin. Clinical data were not available to directly verify the impact of OAT2 modulation on warfarin pharmacokinetics; however, the bottom-up PBPK model simulations suggested a proportional change in clearance of both warfarin enantiomers with inhibition of OAT2 activity. These results suggest that variable hepatic OAT2 function, in conjunction with CYP2C, may contribute to the high population variability in warfarin pharmacokinetics and possibly anticoagulation end points and thus warrant further clinical investigation.

Tolbutamide is primarily metabolized by CYP2C9, and, thus, is frequently applied as a clinical probe substrate for CYP2C9 activity. However, there is a marked discrepancy in the in vitro-in vivo extrapolation of its metabolic clearance, implying a potential for additional clearance mechanisms. The goal of this study was to evaluate the role of hepatic uptake transport in the pharmacokinetics of tolbutamide and to identify the molecular mechanism thereof. Transport studies using singly transfected cells expressing six major hepatic uptake transporters showed that tolbutamide is a substrate to organic anion transporter 2 (OAT2) alone with transporter affinity [Michaelis-Menten constant (Km)] of 19.5 ± 4.3 µM. Additionally, OAT2-specific transport was inhibited by ketoprofen (an OAT2 inhibitor) and 1 mM rifamycin SV (pan inhibitor), but not by cyclosporine and rifampicin (OAT polypeptides/Na+-taurocholate cotransporting polypeptide inhibitors). Uptake studies in primary human hepatocytes confirmed the predominant role of OAT2 in the active uptake with significant inhibition by rifamycin SV and ketoprofen, but not by the other inhibitors. Concentration-dependent uptake was noted in human hepatocytes with active transport characterized by Km and Vmax values of 39.3 ± 6.6 µM and 426 ± 30 pmol/min per milligram protein, respectively. Bottom-up physiologically based pharmacokinetic modeling was employed to verify the proposed role of OAT2-mediated hepatic uptake. In contrast to the rapid equilibrium (CYP2C9-only) model, the permeability-limited (OAT2-CYP2C9 interplay) model better described the plasma concentration-time profiles of tolbutamide. Additionally, the latter well described tolbutamide pharmacokinetics in carriers of CYP2C9 genetic variants and quantitatively rationalized its known drug-drug interactions. Our results provide first-line evidence for the role of OAT2-mediated hepatic uptake in the pharmacokinetics of tolbutamide, and imply the need for additional clinical studies in this direction.

Lansoprazole, a proton pump inhibitor, potently inhibits human organic anion transporter, hOAT3 (SLC22A8). Lansoprazole has an asymmetric atom in its structure and is clinically administered as a racemic mixture of (R)-and (S)-enantiomers. However, little is known about the stereoselective inhibitory potencies of lansoprazole against hOAT3 and its homolog, hOAT1. In the present study, the stereoselective inhibitory effect of lansoprazole was evaluated using hOAT1-and hOAT3-expressing cultured cells. hOAT1 and hOAT3 transported [14C]p-aminohippurate and [3H]estrone-3-sulfate (ES) with Michaelis-Menten constants of 29.8 ± 4.0 and 30.1 ± 9.0 µmol/L respectively. Lansoprazole enantiomers inhibited hOAT1- and hOAT3-mediated transport of each substrate in a concentration-dependent manner. The IC50 value of (S)-lansoprazole against hOAT3-mediated transport of [3H]ES (0.61 ± 0.08 µmol/L) was significantly lower than that of (R)-lansoprazole (1.75 ± 0.31 µmol/L). In contrast, stereoselectivity was not demonstrated for the inhibition of hOAT1. Furthermore, (S)-lansoprazole inhibited hOAT3-mediated transport of pemetrexed and methotrexate (hOAT3 substrates) more strongly than the corresponding (R)-lansoprazole. This study is the first to demonstrate that the stereoselective inhibitory potency of (S)-lansoprazole against hOAT3 is greater than that of (R)-lansoprazole. The present findings provide novel information about the drug interactions associated with lansoprazole.

Carbapenem antibiotics are excreted preferentially in the urine after intravenous administration, with organic anion transporters (OATs) known to be involved in the renal tubular secretion of carbapenem antibiotics. Various uremic toxins (UTs) accumulate in the blood of patients with end-stage renal failure, and some UTs such as indoxyl sulfate (IS) and creatinine (Cr) are excreted in the urine via OATs. However, information about the possible interactions between these UTs and carbapenems in the renal secretion remains limited. In this study, we investigated the effects of IS and Cr on the renal transport of anionic meropenem and zwitterionic biapenem by using rat renal cortical slices. The uptake of meropenem and biapenem in the renal cortical slices was significantly decreased in the presence of 0.1 mM IS or 1 mM Cr. When biapenem and Cr were co-administered to rats intravenously, biapenem clearance from the plasma was clearly retarded, reflecting the current in vitro results. However, IS and Cr exerted no inhibitory effect on the uptake of metformin, a substrate of renal organic cation transporter (OCT) 2, in the renal cortical slices. Thus, our findings indicate that IS and Cr interfere with the renal secretion of carbapenem antibiotics by preferentially inhibiting OATs.

Perturbation of organic anion transporter (OAT) 1- and OAT3-mediated transport can alter the exposure, efficacy, and safety of drugs. Although there have been reports of the endogenous biomarkers for OAT1/3, none of these have all of the characteristics required for a clinical useful biomarker. Cynomolgus monkeys were treated with intravenous probenecid (PROB) at a dose of 40 mg/kg in this study. As expected, PROB increased the area under the plasma concentration-time curve (AUC) of coadministered furosemide, a known substrate of OAT1 and OAT3, by 4.1-fold, consistent with the values reported in humans (3.1- to 3.7-fold). Of the 233 plasma metabolites analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics method, 29 metabolites, including pyridoxic acid (PDA) and homovanillic acid (HVA), were significantly increased after either 1 or 3 hours in plasma from the monkeys pretreated with PROB compared with the treated animals. The plasma of animals was then subjected to targeted LC-MS/MS analysis, which confirmed that the PDA and HVA AUCs increased by approximately 2- to 3-fold by PROB pretreatments. PROB also increased the plasma concentrations of hexadecanedioic acid (HDA) and tetradecanedioic acid (TDA), although the increases were not statistically significant. Moreover, transporter profiling assessed using stable cell lines constitutively expressing transporters demonstrated that PDA and HVA are substrates for human OAT1, OAT3, OAT2 (HVA), and OAT4 (PDA), but not OCT2, MATE1, MATE2K, OATP1B1, OATP1B3, and sodium taurocholate cotransporting polypeptide. Collectively, these findings suggest that PDA and HVA might serve as blood-based endogenous probes of cynomolgus monkey OAT1 and OAT3, and investigation of PDA and HVA as circulating endogenous biomarkers of human OAT1 and OAT3 function is warranted.

Aim: Coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) in plasma and urine have been proposed as biomarkers for assessing drug–drug interactions involving hepatic drug transporters such as organic anion-transporting peptides (OATP), 1B1 and 1B3. Materials & methods: Plasma and urine extracts were analyzed for CP-I/CP-III using a TripleTOF API6600 mass spectrometer. Results: Previously unreported, CP-I/CP-III doubly charged ions (m/z 328.14) were used as precursor ions to improve the assay sensitivity and selectivity over the singly charged precursor ions (m/z 655.28). Levels of CP-I and CP-III measured ranged 0.45–1.1 and 0.050–0.50 ng/ml in plasma and 5–35 and 1–35 ng/ml in urine, respectively. Conclusion: The described highly selective and sensitive CP-I/CP-III LC–HRMS assay offers options for earlier characterization and clinical safety projections for OATP1B1/3-mediated drug–drug interactions along with pharmacokinetic analyses of a new chemical entity as part of first-in-human clinical studies.

d-Malate inhibits a Krebs cycle enzyme and the tubular transport of α-ketoglutarate, an intermediate of the Krebs cycle and the driving force for rat organic anion transporter 1 (rOAT1) and rOAT3 in the kidney. This study examined the effects of d-malate on the rat organic anion transport system. The uptake of 6-carboxyfluorescein by HEK293 cells expressing rOAT1 or rOAT3 was not affected by d-malate and l-malate. Up to 60 min after the intravenous injection of phenolsulfonphthalein (PSP), a typical substrate of the renal organic anion transporters, as a bolus to rats, 47.1% of the dose was recovered in the urine, and its renal clearance was estimated to be 8.60 ml/min/kg. d-Malate but not l-malate interfered with its renal excretion, resulting in the delayed elimination of PSP from plasma. No effect of d-malate was recognized on creatinine clearance or the expression level of rOAT3 in the kidney cortex. d-Malate increased the plasma concentration of α-ketoglutarate. In addition, the compound greatly stimulated the renal excretion of α-ketoglutarate, implying that d-malate inhibited its reabsorption. The content of α-ketoglutarate was significantly decreased in the kidney cortex of rats administered d-malate. Collectively, this study shows that d-malate abrogates the tubular secretion of PSP, and the reduction of the renal content of α-ketoglutarate was proposed to be one of the mechanisms. A relationship between the reabsorption of α-ketoglutarate and the basolateral uptake of organic anion in the kidney is suggested.