Blockade of Organic Anion Transport in Humans After Treatment With the Drug Probenecid Leads to Major Metabolic Alterations in Plasma and Urine.

Probenecid is used to treat gout and hyperuricemia as well as increase plasma levels of antiviral drugs and antibiotics. In vivo, probenecid mainly inhibits the renal SLC22 organic anion transporters OAT1 (SLC22A6), OAT3 (SLC22A8), and URAT1 (SLC22A12). To understand the endogenous role of these transporters in humans, we administered probenecid to 20 healthy participants and metabolically profiled the plasma and urine before and after dosage. Hundreds of metabolites were significantly altered, indicating numerous drug-metabolite interactions. We focused on potential OAT1 substrates by identifying 97 metabolites that were significantly elevated in the plasma and decreased in the urine, indicating OAT-mediated clearance. These included signaling molecules, antioxidants, and gut microbiome products. In contrast, urate was the only metabolite significantly decreased in the plasma and elevated in the urine, consistent with an effect on renal reuptake by URAT1. Additional support comes from metabolomics analyses of our Oat1 and Oat3 knockout mice, where over 50% of the metabolites that were likely OAT substrates in humans were elevated in the serum of the mice. Fifteen of these compounds were elevated in both knockout mice, whereas six were exclusive to the Oat1 knockout and 4 to the Oat3 knockout. These may be endogenous biomarkers of OAT function. We also propose a probenecid stress test to evaluate kidney proximal tubule organic anion transport function in kidney disease. Consistent with the Remote Sensing and Signaling Theory, the profound changes in metabolite levels following probenecid treatment support the view that SLC22 transporters are hubs in the regulation of systemic human metabolism.

Endogenous Plasma Kynurenic Acid in Human: A Newly Discovered Biomarker for Drug-Drug Interactions Involving Organic Anion Transporter 1 and 3 Inhibition.

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.

Hypouricemic agents reduce indoxyl sulfate excretion by inhibiting the renal transporters OAT1/3 and ABCG2.

Indoxyl sulfate (IS) accumulates in the body in chronic kidney disease (CKD). In the renal proximal tubules, IS excretion is mediated by OAT1/3 and ABCG2. These transporters are inhibited by some hypouricemic agents; OATs by probenecid and benzbromarone, ABCG2 by febuxostat and benzbromarone. Thus, we evaluated whether hypouricemic agents including dotinurad, a novel selective urate reabsorption inhibitor with minimal effect on OATs or ABCG2, affect IS clearance in rats. Intact and adenine-induced acute renal failure rats were orally administered hypouricemic agents, and both endogenous IS and exogenously administered stable isotope-labeled d4-IS in the plasma and kidney were measured. Our results demonstrated that OATs inhibitors, such as probenecid, suppress IS uptake into the kidney, leading to increased plasma IS concentration, whereas ABCG2 inhibitors, such as febuxostat, cause renal IS accumulation remarkably by suppressing its excretion in intact rats. The effects of these agents were reduced in adenine-induced acute renal failure rats, presumably due to substantial decrease in renal OAT1/3 and ABCG2 expression. Dotinurad did not significantly affected the clearance of IS under both conditions. Therefore, we suggest that hypouricemic agents that do not affect OATs and ABCG2 are effective therapeutic options for the treatment of hyperuricemia complicated by CKD.

Investigation of the arcane inhibition of human organic anion transporter 3 by benzofuran antiarrhythmic agents.

The combination of antiarrhythmic agents, amiodarone or dronedarone, with the anticoagulant rivaroxaban is used clinically in the management of atrial fibrillation for rhythm control and secondary stroke prevention respectively. Renal drug-drug interactions (DDIs) between amiodarone or dronedarone and rivaroxaban were previously ascribed to inhibition of rivaroxaban secretion by P-glycoprotein at the apical membrane of renal proximal tubular epithelial cells. Benzbromarone, a known inhibitor of organic anion transporter 3 (OAT3), shares a benzofuran scaffold with amiodarone and dronedarone. However, inhibitory activity of amiodarone and dronedarone against OAT3 remains arcane. Here, we conducted in vitro transporter inhibition assays in OAT3-transfected HEK293 cells which revealed amiodarone, dronedarone and their respective major pharmacologically-active metabolites N-desethylamiodarone and N-desbutyldronedarone possess inhibitory activity against OAT3, with corrected Ki values of 0.042, 0.019, 0.028 and 0.0046 µM respectively. Protein binding effects and probe substrate dependency were accounted for in our assays. Static modelling predicted 1.29-, 1.01-, 1.29- and 1.16-fold increase in rivaroxaban exposure, culminating in a predicted 1.29-, 1.01-, 1.28- and 1.15-fold increase in major bleeding risk respectively, suggesting potential OAT3-mediated DDI between amiodarone and rivaroxaban. Future work involving physiologically-based pharmacokinetic modelling is crucial in holistically predicting the complex DDIs between the benzofuran antiarrhythmic agents and rivaroxaban.

Upregulation of histone acetylation reverses organic anion transporter 2 repression and enhances 5-fluorouracil sensitivity in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The failure of chemotherapy in HCC patients is partly due to inadequate intracellular drug accumulation caused by abnormally expressed drug transporters. Human organic anion transporter 2 (hOAT2), a transporter mainly expressed in liver and kidney, is responsible for uptake of various antineoplastic drugs such as 5-fluorouracil (5-FU). Among 32 pairs of human HCC samples, we preliminarily found that OAT2 was suppressed in HCC tissues compared with matched tumor-adjacent tissues at both mRNA and protein levels, which resulted in 5-FU resistance in HCC. However, the epigenetic regulatory mechanisms of OAT2 downregulation have not been investigated. In this study, we first proved it was histone hypoacetylation rather than DNA hypermethylation that participated in transcriptional repression of OAT2 in two HCC cell lines (BEL-7402 and SMMC-7721). In general, there were two pathways confirmed using tissues and cells: 1) Increased histone deacetylase sirtuin 7 (SIRT7) mediated loss of histone 3 lysine 18 acetylation (H3K18ac) at the promoter of OAT2 and inhibited its transcription. 2) More histone deacetylase 7 (HDAC7) instead of lysine acetyltransferase 8 (KAT8) enrichment at the promoter of OAT2 led to low levels of histone 4 lysine 16 acetylation (H4K16ac). Further, we found that histone deacetylases inhibitor vorinostat (SAHA) could reverse histone hypoacetylation state to activate OAT2 transcription and enhance uptake of classic OAT2 substrate zidovudine. Therefore, we evaluated the effect of combining SAHA and 5-FU and the results demonstrated that SAHA could sensitize HCC cells to 5-FU. Collectively, we proposed such a combination treatment to overcome 5-FU resistance in HCC from the perspective of epigenetically restoring OAT2.

Population pharmacokinetic modeling and simulation to support qualification of pyridoxic acid as endogenous biomarker of OAT1/3 renal transporters.

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.

Regulatory guidelines do not accurately predict tolvaptan and metabolite interactions at BCRP, OATP1B1, and OAT3 transporters.

Tolvaptan (TLV) was US Food and Drug Administration (FDA)-approved for the indication to slow kidney function decline in adults at risk of rapidly progressing autosomal dominant polycystic kidney disease in 2018. In vitro, TLV was a breast cancer resistance protein (BCRP) inhibitor, whereas the oxobutyric acid metabolite of TLV (DM-4013) was an inhibitor of organic anion transport polypeptide (OATP)1B1 and organic anion transporter (OAT)3. Based on the 2017 FDA guidance, potential for clinically relevant inhibition at these transporters was indicated for the highest TLV regimen. Consequently, two postmarketing clinical trials in healthy subjects were required. In trial 1, 5 mg rosuvastatin calcium (BCRP and OATP1B1 substrate) was administered alone, with 90 mg TLV or 48 h following 7 days of once daily 300 mg TLV (i.e., in the presence of DM-4103). In trial 2, 40 mg furosemide (OAT3 substrate) was administered alone and in presence of DM-4103. For BCRP, rosuvastatin geometric mean ratios (90% confidence intervals [CIs]) for maximum plasma concentration (Cmax ) were 1.54 (90% CI 1.26-1.88) and for area under the concentration-time curve from time 0 to the time of the last measurable concentration (AUCt ) were 1.69 (90% CI 1.34-2.14), indicating no clinically significant interaction. DM-4103 produced no clinically meaningful changes in rosuvastatin or furosemide concentrations, indicating no inhibition at OATP1B1 or OAT3. The BCRP prediction assumed the drug dose is completely soluble in 250 ml; TLV has solubility of ~0.01 g/250 ml. For OATP1B1/OAT3, if fraction unbound for plasma protein binding (PPB) is less than 1%, then 1% is assumed. DM-4103 has PPB greater than 99.8%. Use of actual drug substance solubility and unbound fraction in plasma would have produced predictions consistent with the clinical results.

Hypoxia Active Platinum(IV) Prodrugs of Orotic Acid Selective to Liver Cancer Cells.

Platinum(IV) complexes of orotic acid selectively target liver cancer cells displaying enhanced activity and higher uptake in Hep G2. The comparatively higher expression of Organic Anion Transporter 2 (OAT2) in Hep G2 and decrease in toxicity in the presence of OAT2 inhibitor suggest its involvement in the uptake of the complexes. They are resistant to sequestration by the copper transporter ATP7B, unlike cisplatin and oxaliplatin.

Effects of rhein and Rheum palmatum L. extract on the pharmacokinetics and tissue distribution of aristolochic acid I and its demethylated metabolite in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acid nephropathy (AAN) is a kidney disease caused by the administration of plants containing aristolochic acids (AAs). Aristolochic acid I (AAI) is the main toxic component in AAs. Organic anion transporters (OATs) 1 and 3 mediate the renal uptake of AAI, which is related to AAN. In our previous study, we found that anthraquinones derived from the herbal medicine Rheum palmatum L. (RP) inhibited both OAT1 and OAT3, with rhein exhibiting the greatest potency among the components. AIM OF THE STUDY: This study aimed to investigate the effects of rhein and RP extract on the pharmacokinetics and tissue distribution of AAI and its demethylated metabolite (8-hydroxy-aristolochic acid I [AAIa]) in rats. MATERIALS AND METHODS: Rhein and RP extract were used as OAT inhibitors, and AAI was used as the toxic substrate. The pharmacokinetics and tissue distribution of AAI and AAIa in rats following the intravenous injection of AAI (10 mg/kg) in the presence and absence of rhein (100 mg/kg) or RP extract (5 g crude drug/kg) were investigated. RESULTS: Co-administration with rhein increased AUC0-∞ of AAI and AAIa by 39 and 44%, respectively. However, the renal level of AAI was decreased to 50, 42, and 58% of those in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively, and the renal level of AAIa was decreased to 58, 57, and 61% of the level in rats treated with AAI alone, respectively, at these time points. In the RP extract co-administration group, AAI and AAIa plasma exposure was not significantly increased, but renal accumulation of AAI was decreased to 63, 58, and 68% of that in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively. In addition, renal accumulation of AAIa was decreased to 74, 70, and 70% of that in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively. CONCLUSIONS: This study indicated that co-administration with rhein significantly increased the plasma exposure of AAI and AAIa while decreased their renal accumulation in rats. RP extract reduced the renal accumulation of AAI and AAIa, but have no significant effect on their plasma exposure levels in rats.

Inflammation-Induced Attenuation of Prostaglandin D2 Elimination across Rat Blood-Brain Barrier: Involvement of the Downregulation of Organic Anion Transporter 3 and Multidrug Resistance-Associated Protein 4.

Prostaglandin (PG) D2 is a lipid mediator, and in the brain, overproduction of PGD2 is reportedly involved in the progression and exacerbation of neuroinflammation. The objective of this study was to elucidate PGD2 efflux transport, under normal and inflammatory conditions, across the blood-brain barrier (BBB), which is formed by brain capillaries. Elimination of [3H]PGD2 across the BBB of normal and lipopolysaccharide (LPS)-induced inflammatory rats was examined by the intracerebral microinjection technique. After intracerebral injection, the percentage of [3H]PGD2 remaining in the ipsilateral cerebrum decreased with time, with a half-life of 13 min. This [3H]PGD2 elimination across the BBB was significantly inhibited by the co-administration of unlabeled PGD2, which suggests carrier-mediated PGD2 efflux transport at the BBB. In isolated rat brain capillaries, mRNA expression of organic anion transporter (Oat) 3, organic anion-transporting polypeptide (Oatp) 1a4, and multidrug resistance-associated protein (Mrp) 4 was observed. In addition, co-administration of substrates/inhibitors for Oat3, Oatp1a4, and/or Mrp4, such as benzylpenicillin and cefmetazole, reduced [3H]PGD2 elimination across the BBB. Data suggest that Oat3 and Mrp4, but not Oatp1a4 are involved in PGD2 elimination across the BBB, as Oatp1a4-expressing Xenopus (X.) oocytes did not show the significant [3H]PGD2 uptake compared with water-injected X. oocytes. In LPS-treated rats, [3H]PGD2 elimination across the BBB and mRNA expression levels of Oat3 and Mrp4 were significantly decreased. Our data suggest that Oat3- and Mrp4-mediated PGD2 elimination across the BBB is attenuated under inflammatory conditions.

Potential Risk of Food-Drug Interactions: Citrus Polymethoxyflavones and Flavanones as Inhibitors of the Organic Anion Transporting Polypeptides (OATP) 1B1, 1B3, and 2B1.

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.

Uric Acid Elevation by Favipiravir, an Antiviral Drug.

In light of the recent pandemic, favipiravir (Avigan®), a purine nucleic acid analog and antiviral agent approved for use in influenza in Japan, is being studied for the treatment of coronavirus disease 2019 (COVID-19). Increase in blood uric acid level is a frequent side effect of favipiravir. Here, we discussed the mechanism of blood uric acid elevation during favipiravir treatment. Favipiravir is metabolized to an inactive metabolite M1 by aldehyde oxidase and xanthine oxidase, and excreted into urine. In the kidney, uric acid handling is regulated by the balance of reabsorption and tubular secretion in the proximal tubules. Favipiravir and M1 act as moderate inhibitors of organic anion transporter 1 and 3 (OAT1 and OAT3), which are involved in uric acid excretion in the kidney. In addition, M1 enhances uric acid reuptake via urate transporter 1 (URAT1) in the renal proximal tubules. Thus, favipiravir is thought to decrease uric acid excretion into urine, resulting in elevation of uric acid levels in blood. Elevated uric acid levels were returned to normal after discontinuation of favipiravir, and favipiravir is not used for long periods of time for the treatment of viral infection. Thus, the effect on blood uric acid levels was subclinical in most studies. Nevertheless, the adverse effect of favipiravir might be clinically important in patients with a history of gout, hyperuricemia, kidney function impairment (in which blood concentration of M1 increases), and where there is concomitant use of other drugs affecting blood uric acid elevation.

Interactions between Oroxylin A with the solute carrier transporters and ATP-binding cassette transporters: Drug transporters profile for this flavonoid.

Oroxylin A is a flavonoid monomer extracted from Scutellaria baicalensis Georgi with neuroprotective, anti-tumor activity and many other biological functions. However, the interaction between Oroxylin A and the drug transporters has not been clearly reported. The purpose of this study is to investigate the interaction between Oroxylin A and the solute carrier transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1, and MATE2K), and ATP-binding cassette transporters (BCRP, MDR1). The HEK293 cell lines (HEK293-OATP1B1, HEK293-OATP1B3, HEK293-OAT1, HEK293-OAT3, HEK293-OCT2, HEK293-MATE1, and HEK293-MATE2K) that stably expressing previous listed human-derived transporters were employed to evaluate the solute carrier transporters. Vesicles expressing human BCRP and MDR1 transporters was employed to research ATP-binding cassette transporters. Our work suggested that Oroxylin A was a substrate of OATP1B1, OATP1B3, but not a substrate of the other transporters in the concentration range of our study. Oroxylin A shows concentration-dependent inhibition of OATP1B1, OAT1, OAT3 and BCRP transportation with the half-inhibitory concentration (IC50) of 7.03, 0.961, 0.112 µM, and 0.477 µM, respectively. No inhibitory effects on the transport activities of other transporters were observed for Oroxylin A. Drug transporters profile of Oroxylin A was first confirmed by our work, which provides important information for its pharmacokinetics, pharmacodynamics, and drug-drug interactions studies.

Differential interactions of the ß-lactam cloxacillin with human renal organic anion transporters (OATs).

The ß-lactam penicillin antibiotic cloxacillin (CLX) presents wide inter-individual pharmacokinetics variability. To better understand its molecular basis, the precise identification of the detoxifying actors involved in CLX disposition and elimination would be useful, notably with respect to renal secretion known to play a notable role in CLX elimination. The present study was consequently designed to analyze the interactions of CLX with the solute carrier transporters organic anion transporter (OAT) 1 and OAT3, implicated in tubular secretion through mediating drug entry at the basolateral pole of renal proximal cells. CLX was first shown to block OAT1 and OAT3 activity in cultured OAT-overexpressing HEK293 cells. Half maximal inhibitory concentration (IC50 ) value for OAT3 (13 µm) was however much lower than that for OAT1 (560 µm); clinical inhibition of OAT activity and drug-drug interactions may consequently be predicted for OAT3, but not OAT1. OAT3, unlike OAT1, was next shown to mediate CLX uptake in OAT-overexpressing HEK293 cells. Kinetic parameters for this OAT3-mediated transport of CLX (Km  = 10.7 µm) were consistent with a possible in vivo saturation of this process for high CLX plasma concentrations. OAT3 is consequently likely to play a pivotal role in renal CLX secretion and consequently in total renal CLX elimination, owing to the low plasma unbound fraction of the antibiotic. OAT3 genetic polymorphisms as well as co-administered drugs inhibiting in vivo OAT3 activity may therefore be considered as potential sources of CLX pharmacokinetics variability.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

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 IC50 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.

Probenecid-Boosted Tenofovir: A Physiologically-Based Pharmacokinetic Model-Informed Strategy for On-Demand HIV Preexposure Prophylaxis.

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.

Pharmacological Evaluation of Dotinurad, a Selective Urate Reabsorption Inhibitor.

The effect of dotinurad [(3,5-dichloro-4-hydroxyphenyl)(1,1-dioxo-1,2-dihydro-3H-1λ 6-1,3-benzothiazol-3-yl)methanone] was compared with that of commercially available uricosuric agents-namely, benzbromarone, lesinurad, and probenecid. Its effect on urate secretion transporters was evaluated using probe substrates for respective transporters. Dotinurad, benzbromarone, lesinurad, and probenecid inhibited urate transporter 1 (URAT1) with IC50 values of 0.0372, 0.190, 30.0, and 165 µM, respectively. Dotinurad weakly inhibited ATP-binding cassette subfamily G member 2 (ABCG2), organic anion transporter 1 (OAT1), and OAT3, with IC50 values of 4.16, 4.08, and 1.32 µM, respectively, indicating higher selectivity for URAT1. The hypouricemic effects of dotinurad and benzbromarone were evaluated in Cebus monkeys. Dotinurad, at doses of 1-30 mg/kg, concomitantly decreased plasma urate levels and increased fractional excretion of urate (FEUA) in a dose-dependent manner. On the contrary, benzbromarone, at a dose of 30 mg/kg, showed a modest effect on plasma urate levels. The inhibitory effect of dotinurad on urate secretion transporters was evaluated in Sprague-Dawley rats, with sulfasalazine and adefovir as probe substrates of ABCG2 and OAT1, respectively. Drugs, including febuxostat as a reference ABCG2 inhibitor, were administered orally before sulfasalazine or adefovir administration. Dotinurad had no effect on urate secretion transporters in vivo, whereas benzbromarone, lesinurad, probenecid, and febuxostat increased the plasma concentrations of probe substrates. These results suggested dotinurad is characterized as a selective urate reabsorption inhibitor (SURI), which is defined as a potent URAT1 inhibitor with minimal effect on urate secretion transporters, including ABCG2 and OAT1/3, because of its high efficacy in decreasing plasma urate levels compared with that of other uricosuric agents. SIGNIFICANCE STATEMENT: Our study on the inhibitory effects on urate transport showed that dotinurad had higher selectivity for urate transporter 1 (URAT1) versus ATP-binding cassette subfamily G member 2 (ABCG2) and organic anion transporter (OAT) 1/3 compared to other uricosuric agents. In Cebus monkeys, dotinurad decreased plasma urate levels and increased fractional excretion of urate in a dose-dependent manner. To determine the inhibitory effect of dotinurad on urate secretion transporters, we studied the movement of substrates of ABCG2 and OAT1 in rats. Dotinurad had no effect on these transporters, whereas the other uricosuric agents increased the plasma concentrations of the substrates. These results suggested dotinurad as a potent and selective urate reabsorption inhibitor is characterized by increased efficacy with decreasing plasma urate levels.

Zebrafish (Danio rerio) Oat1 and Oat3 transporters and their interaction with physiological compounds.

Organic anion transporters (OATs) are membrane proteins within the Solute carrier family 22 (SLC22). They play important roles in cellular uptake of various organic compounds, and due to their expression in barrier tissues of major excretory and non-excretory organs are considered as crucial elements in absorption and distribution of a wide range of endobiotic and xenobiotic compounds. Based on our previous work and initial insights on SLC22 members in zebrafish (Danio rerio), in this study we aimed at in vitro characterization of Oat1 and Oat3 transporters and understanding of their interaction with potential physiological substrates. We first performed synteny analysis to describe in more detail orthological relationship of zebrafish oat1 and oat3 genes. We then developed stable cell lines overexpressing Oat1 and Oat3, and identified Lucifer yellow as Oat1 model fluorescent substrate (Km = 11.4 µM) and 6-carboxyfluorescein as Oat3 model substrate (Km = 5.8 µM). Initial identification performed using the developed assays revealed Kreb's cycle intermediates, bilirubin, bile salts and steroid hormones as the most potent of Oat1 and Oat3 interactors, with IC50 values in micromolar range. Finally, we showed that bilirubin, deoxycholic acid, α-ketoglutarate, pregnenolone, estrone-3-sulfate and corticosterone are in vitro substrates of zebrafish Oat1, and bilirubin and deoxycholic acid are Oat3 substrates. In conclusion, using the approach described, structural and functional similarities of both transporters to human and mammalian orthologs are revealed, their broad ligand selectivity confirmed, potent interactors among endobiotic compounds identified, and first indications of their potential physiological role(s) in zebrafish obtained.

Unusual Flavones from Primula macrocalyx as Inhibitors of OAT1 and OAT3 and as Antifungal Agents against Candida rugosa.

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 < 2.0 µM. To our knowledge, this study is the first to evaluate these flavones as inhibitors of the OAT1 and OAT3, and as antifungal agents.

Roles of organic anion transporter 2 and equilibrative nucleoside transporter 1 in hepatic disposition and antiviral activity of entecavir during non-pregnancy and pregnancy.

BACKGROUND AND PURPOSE: Entecavir (ETV), a first-line antiviral drug against hepatitis B virus (HBV), has the possibility to be used to prevent mother-to-child transmission. The aim of present study was to clarify the mechanism of ETV uptake into hepatocytes and evaluate the alteration of ETV's hepatic distribution during pregnancy. EXPERIMENTAL APPROACH: The roles of equilibrative nucleotide transporter (ENT) 1 and organic anion transporter (OAT) 2 in ETV accumulation and anti-HBV efficacy were studied in human ENT1 or OAT2 overexpressed cell models and HepG2.2.15 cells, respectively; meanwhile, the liver-to-plasma ETV concentration ratios in non-pregnant and pregnant mice were measured to evaluate the effect of pregnancy on ETV hepatic distribution. KEY RESULTS: ETV was shown to be a substrate of ENT1 and OAT2. An ENT1 inhibitor significantly decreased the efficacy of ETV in HepG2.2.15 cells, while overexpression of OAT2 increased susceptibility of HBV to ETV. The liver-to-plasma ETV concentration ratios in pregnant mice were sharply reduced; whereas, the absolute concentration of ETV in the liver did not obviously alter in pregnancy. Although oestradiol and progesterone showed a concentration-dependent inhibition on ETV accumulation both in hepatic cell lines and in primary human hepatocytes, a physiologically relevant concentration of oestradiol and progesterone did not affect antiviral activity of ETV. CONCLUSIONS AND IMPLICATIONS: OAT2 and ENT1 are the main transporters involved in the hepatic uptake and anti-HBV efficacy of ETV. The concentration of ETV in the liver was not obviously altered during pregnancy, which indicates that dosage adjustment in pregnancy is not necessary.