Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

BACKGROUND: In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, a phenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. METHODS: The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). RESULTS: This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. CONCLUSIONS: We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Interplay of Ritonavir-Boosted Oral Cabazitaxel with the Organic Anion-Transporting Polypeptide (OATP) Uptake Transporters and Carboxylesterase 1 in Mice.

Intravenously administered chemotherapeutic cabazitaxel is used for palliative treatment of prostate cancer. An oral formulation would be more patient-friendly and reduce the need for hospitalization. We therefore study determinants of the oral pharmacokinetics of cabazitaxel in a ritonavir-boosted setting, which reduces the CYP3A-mediated first-pass metabolism of cabazitaxel. We here assessed the role of organic anion-transporting polypeptides (OATPs) in the disposition of orally boosted cabazitaxel and its active metabolites, using the Oatp1a/b-knockout and the OATP1B1/1B3-transgenic mice. These transporters may substantially affect plasma clearance and hepatic and intestinal drug disposition. The pharmacokinetics of cabazitaxel and DM2 were not significantly affected by Oatp1a/b and OATP1B1/1B3 activity. In contrast, the plasma AUC0-120 min of DM1 in Oatp1a/b-/- was 1.9-fold (p < 0.05) higher than that in wild-type mice, and that of docetaxel was 2.4-fold (p < 0.05) higher. We further observed impaired hepatic uptake and intestinal disposition for DM1 and docetaxel in the Oatp-ablated strains. None of these parameters showed rescue by the OATP1B1 or -1B3 transporters in the humanized mouse strains, suggesting a minimal role of OATP1B1/1B3. Ritonavir itself was also a potent substrate for mOatp1a/b, showing a 2.9-fold (p < 0.0001) increased plasma AUC0-120 min and 3.5-fold (p < 0.0001) decreased liver-to-plasma ratio in Oatp1a/b-/- compared to those in wild-type mice. Furthermore, we observed the tight binding of cabazitaxel and its active metabolites, including docetaxel, to plasma carboxylesterase (Ces1c) in mice, which may complicate the interpretation of pharmacokinetic and pharmacodynamic mouse studies. Collectively, these results will help to further optimize (pre)clinical research into the safety and efficacy of orally applied cabazitaxel.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

Microcystin-LR-induced epithelial-mesenchymal transition-like cells acquire resistance to multi-toxins.

The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation.

Inhibitory interaction of flavonoids with organic anion transporter 3 and their structure-activity relationships for predicting nephroprotective effects.

The organic anion transporter 3 (OAT3), an important renal uptake transporter, is associated with drug-induced acute kidney injury (AKI). Screening and identifying potent OAT3 inhibitors with little toxicity in natural products, especially flavonoids, in reducing OAT3-mediated AKI is of great value. The five strongest OAT3 inhibitors from the 97 flavonoids markedly decreased aristolochic acid I-induced cytotoxicity and alleviated methotrexate-induced nephrotoxicity. The pharmacophore model clarified hydrogen bond acceptors and hydrophobic groups are the critical pharmacophores. These findings would provide valuable information in predicting the potential risks of flavonoid-containing food/herb-drug interactions and optimizing flavonoid structure to alleviate OAT3-related AKI.

The fluorescence-based competitive counterflow assay developed for organic anion transporting polypeptides 1A2, 1B1, 1B3 and 2B1 identifies pentamidine as a selective OATP1A2 substrate.

Organic anion transporting polypeptides OATP1A2, OATP1B1, OATP1B3 and OATP2B1 are Na+ - and ATP-independent exchangers of large, organic compounds, encompassing structurally diverse xenobiotics, including various drugs. These OATPs influence intestinal absorption (OATP2B1), hepatic clearance (OATP1B1/3) and blood to brain penetration (OATP1A2, OATP2B1) of their drug substrates. Consequently, OATP-mediated drug or food interactions may lead to altered pharmacokinetics and toxicity. During drug development, investigation of hepatic OATP1B1 and OATP1B3 is recommended by international regulatory agencies. Most frequently, OATP-drug interactions are investigated in an indirect assay, i.e., by examining uptake inhibition of a radioactive or fluorescent probe. However, indirect assays do not distinguish between transported substrates and non-transported OATP inhibitors. To fill this hiatus, a novel assay, termed competitive counterflow (CCF) has been developed and has since been applied for several OATPs to differentiate between substrates and non-transported inhibitors. However, previous OATP CCF assays, with the exception of that for OATP1B1, used radioactive probes. In the current study, we demonstrate that sulforhodamine 101 or pyranine can be used as fluorescent probes in a CCF assay to identify transported substrates of OATP1A2, or OATPs 1B1, 1B3 and 2B1, respectively. With the help of the newly developed fluorescence-based CCF method, we identify the FDA-approved anti-protozoal drug, pentamidine as a unique substrate of OATP1A2. Furthermore, we confirm the selective, OATP1A2-mediated uptake of pentamidine in a cytotoxicity assay. Based on our results, OATP1A2 may be an important determinant of pentamidine transport through the blood-brain barrier.

Hyperoside ameliorates cisplatin-induced acute kidney injury by regulating the expression and function of Oat1.

Cisplatin is a widely used chemotherapeutic agent to treat solid tumours in clinics. However, cisplatin-induced acute kidney injury (AKI) limits its clinical application. This study investigated the effect of hyperoside (a flavonol glycoside compound) on regulating AKI.The model of cisplatin-induced AKI was established, and hyperoside was preadministered to investigate its effect on improving kidney injury.Hyperoside ameliorated renal pathological damage, reduced the accumulation of SCr, BUN, Kim-1 and indoxyl sulphate in vivo, increased the excretion of indoxyl sulphate into the urine, and upregulated the expression of renal organic anion transporter 1 (Oat1). Moreover, evaluation of rat kidney slices demonstrated that hyperoside promoted the uptake of PAH (p-aminohippurate, the Oat1 substrate), which was confirmed by transient over-expression of OAT1 in HEK-293T cells. Additionally, hyperoside upregulated the mRNA expression of Oat1 upstream regulators hepatocyte nuclear factor-1α (HNF-1α) and pregnane X receptor (PXR).These findings indicated hyperoside could protect against cisplatin-induced AKI by promoting indoxyl sulphate excretion through regulating the expression and function of Oat1, suggesting hyperoside may offer a potential tactic for cisplatin-induced AKI treatment.

Effect of probenecid on blood levels and renal elimination of furosemide and endogenous compounds in rats: Discovery of putative organic anion transporter biomarkers.

Transporter-mediated drug-drug interactions (DDIs) are assessed using probe drugs and in vitro and in vivo models during drug development. The utility of endogenous metabolites as transporter biomarkers is emerging for prediction of DDIs during early phases of clinical trials. Endogenous metabolites such as pyridoxic acid and kynurenic acid have shown potential to predict DDIs mediated by organic anion transporters (OAT1 and OAT3). However, these metabolites have not been assessed in rats as potential transporter biomarkers. We carried out a rat pharmacokinetic DDI study using probenecid and furosemide as OAT inhibitor and substrate, respectively. Probenecid administration led to a 3.8-fold increase in the blood concentrations and a 3-fold decrease in renal clearance of furosemide. High inter-individual and intra-day variability in pyridoxic acid and kynurenic acid, and no or moderate effect of probenecid administration on these metabolites suggest their limited utility for prediction of Oat-mediated DDI in rats. Therefore, rat blood and urine samples were further analysed using untargeted metabolomics. Twenty-one m/z features (out of >8000 detected features) were identified as putative biomarkers of rat Oat1 and Oat3 using a robust biomarker qualification approach. These m/z features belong to metabolic pathways such as fatty acid analogues, peptides, prostaglandin analogues, bile acid derivatives, flavonoids, phytoconstituents, and steroids, and can be used as a panel to decrease variability caused by processes other than Oats. When validated, these putative biomarkers will be useful in predicting DDIs caused by Oats in rats.

The regulation of human organic anion transporter 4 by insulin-like growth factor 1 and protein kinase B signaling.

Human organic anion transporter 4 (hOAT4), mainly expressed in the kidney and placenta, is essential for the disposition of numerous drugs, toxins, and endogenous substances. Insulin-like growth factor 1 (IGF-1) is a hormone generated in the liver and plays important roles in systemic growth, development, and metabolism. In the current study, we explored the regulatory effects of IGF-1 and downstream signaling on the transport activity, protein expression, and SUMOylation of hOAT4. We showed that IGF-1 significantly increased the transport activity, expression, and maximal transport velocity Vmax of hOAT4 in kidney-derived cells. This stimulatory effect of IGF-1 on hOAT4 activity was also confirmed in cells derived from the human placenta. The increased activity and expression were correlated well with the reduced degradation rate of hOAT4 at the cell surface. Furthermore, IGF-1 significantly increased hOAT4 SUMOylation, and protein kinase B (PKB)-specific inhibitors blocked the IGF-1-induced regulations on hOAT4. In conclusion, our study demonstrates that the hepatic hormone IGF-1 regulates hOAT4 expressed in the kidney and placenta through the PKB signaling pathway. Our results support the remote sensing and signaling theory, where OATs play a central role in the remote communications among distal tissues.

Relevance of the organic anion transporting polypeptide 1B3 (OATP1B3) in the personalized pharmacological treatment of hepatocellular carcinoma.

Although pharmacological treatment is the best option for most patients with advanced hepatocellular carcinoma (HCC), its success is very limited, partly due to reduced uptake and enhanced efflux of antitumor drugs. Here we have explored the usefulness of vectorizing drugs towards the organic anion transporting polypeptide 1B3 (OATP1B3) to enhance their efficacy against HCC cells. In silico studies (RNA-Seq data, 11 cohorts) and immunohistochemistry analyses revealed a marked interindividual variability, together with general downregulation but still expression of OATP1B3 in the plasma membrane of HCC cells. The measurement of mRNA variants in 20 HCC samples showed the almost absence of the cancer-type variant (Ct-OATP1B3) together with marked predominance of the liver-type variant (Lt-OATP1B3). In Lt-OATP1B3-expressing cells, the screening of 37 chemotherapeutical drugs and 17 tyrosine kinase receptors inhibitors (TKIs) revealed that 10 classical anticancer drugs and 12 TKIs were able to inhibit Lt-OATP1B3-mediated transport. Lt-OATP1B3-expressing cells were more sensitive than Mock parental cells (transduced with empty lentiviral vectors) to some Lt-OATP1B3 substrates (paclitaxel and the bile acid-cisplatin derivative Bamet-UD2), but not to cisplatin, which is not transported by Lt-OATP1B3. This enhanced response was abolished by competition with taurocholic acid, a known Lt-OATP1B3 substrate. Tumors subcutaneously generated in immunodeficient mice by Lt-OATP1B3-expressing HCC cells were more sensitive to Bamet-UD2 than those derived from Mock cells. In conclusion, Lt-OATP1B3 expression should be screened before deciding the use of anticancer drugs substrates of this carrier in the personalized treatment of HCC. Moreover, Lt-OATP1B3-mediated uptake must be considered when designing novel anti-HCC targeted drugs.

Characterization of Bile Acid Sulfate Conjugates as Substrates of Human Organic Anion Transporting Polypeptides.

Drug interactions involving the inhibition of hepatic organic anion transporting polypeptides (OATPs) 1B1 and OATP1B3 are considered important. Therefore, we sought to study various sulfated bile acids (BA-S) as potential clinical OATP1B1/3 biomarkers. It was determined that BA-S [e.g., glycochenodeoxycholic acid 3-O-sulfate (GCDCA-S) and glycodeoxycholic acid 3-O-sulfate (GDCA-S)] are substrates of OATP1B1, OATP1B3, and sodium-dependent taurocholic acid cotransporting polypeptide (NTCP) transfected into human embryonic kidney 293 cells, with minimal uptake evident for other solute carriers (SLCs) like OATP2B1, organic anion transporter 2, and organic cation transporter 1. It was also shown that BA-S uptake by plated human hepatocytes (PHH) was inhibited (≥96%) by a pan-SLC inhibitor (rifamycin SV), and there was greater inhibition (≥77% versus ≤12%) with rifampicin (OATP1B1/3-selective inhibitor) than a hepatitis B virus myristoylated-preS1 peptide (NTCP-selective inhibitor). Estrone 3-sulfate was also used as an OATP1B1-selective inhibitor. In this instance, greater inhibition was observed with GDCA-S (76%) than GCDCA-S (52%). The study was expanded to encompass the measurement of GCDCA-S and GDCA-S in plasma of SLCO1B1 genotyped subjects. The geometric mean GDCA-S concentration was 2.6-fold (90% confidence interval 1.6, 4.3; P = 2.1 × 10-4) and 1.3-fold (1.1, 1.7; P = 0.001) higher in individuals homozygous and heterozygous for the SLCO1B1 c.521T > C loss-of-function allele, respectively. For GCDCA-S, no significant difference was noted [1.2-fold (0.8, 1.7; P = 0.384) and 0.9-fold (0.8, 1.1; P = 0.190), respectively]. This supported the in vitro data indicating that GDCA-S is a more OATP1B1-selective substrate (versus GCDCA-S). It is concluded that GCDCA-S and GDCA-S are viable plasma-based OATP1B1/3 biomarkers, but they are both less OATP1B1-selective when compared to their corresponding 3-O-glucuronides (GCDCA-3G and GDCA-3G). Additional studies are needed to determine their utility versus more established biomarkers, such as coproporphyrin I, for assessing inhibitors with different OATP1B1 (versus OATP1B3) inhibition signatures.

Cellular uptake of rose bengal is mediated by OATP1B1/1B3 transporters.

Due to its fluorescent properties and high yield of singlet oxygen, rose bengal (RB) is one of the most promising photosensitizers for cancer treatment. However, the negative charge of RB molecule may significantly hamper its intracellular delivery by passive diffusion through the cell membrane. Thus, specific membrane protein transporters may be needed. The organic anion transporting polypeptides (OATPs) are a well-characterized group of membrane protein transporters, responsible for cellular uptake of a number of drugs. To our knowledge, this is the first study that evaluates cellular transport of RB mediated by the OATP transporter family. First, electrified liquid-liquid interface, together with biophysical analysis and molecular dynamics simulations were used to characterize the interaction of RB with several models of a cellular membranes. These experiments proved that RB interacts only with the membrane's surface, without spontaneously crossing the lipid bilayer. Evaluation of intracellular uptake of RB by flow cytometry and confocal microscopy showed significant differences in uptake between liver and intestinal cell line models differing in expression of OATP transporters. The use of specific pharmacological inhibitors of OATPs, together with Western blotting and in silico analysis, indicated that OATPs are crucial for cellular uptake of RB.

Characterization of Elimination Pathways and the Feasibility of Endogenous Metabolites as Biomarkers of Organic Anion Transporter 1/3 Inhibition in Cynomolgus Monkeys.

Organic anion transporters 1 and 3 (OAT1/3) occupy a key role in mediating renal elimination. Kynurenic acid (KYNA) was previously discovered as an effective endogenous biomarker to assess drug-drug interaction (DDI) for OAT inhibitors. Here, further in vitro and in vivo investigation was performed to characterize the elimination routes and feasibility of KYNA, along with other reported endogenous metabolites, as biomarkers of Oat1/3 inhibition in bile duct-cannulated (BDC) cynomolgus monkeys. Our results suggested that KYNA is a substrate of OAT1/3 and OAT2, but not OCT2, MATE1/2K, or NTCP, and that it shares comparable affinities between OAT1 and OAT3. Renal and biliary excretions and plasma concentration-time profiles of KYNA, pyridoxic acid (PDA), homovanillic acid (HVA), and coproporphyrin I (CP-I) were assessed in BDC monkeys dosed with either probenecid (PROB) at 100 mg/kg or the control vehicle. Renal excretion of KYNA, PDA, and HVA was determined to be the major elimination route. The maximum concentration and the area under the plasma concentration-time curve (Cmax and AUC0-24h) of KYNA were about 11.6- and 3.7-fold higher in the PROB group than in the vehicle group. Renal clearance of KYNA decreased by 3.2-fold, but biliary clearance (CLbile) was not altered after PROB administration. A similar trend was observed for PDA and HVA. Interestingly, an elevation of plasma concentration and reduction of CP-I CLbile were observed after PROB treatment, which suggested inhibition of the CP-I Oatp-Mrp2 transport axis by PROB. Overall, our results indicated that KYNA could potentially facilitate early and reliable assessment of DDI liabilities of Oat inhibition in monkeys. SIGNIFICANCE STATEMENT: This work reported renal excretion as the major elimination pathway for kynurenic acid, pyridoxic acid, and homovanillic acid. Administration of probenecid reduced renal clearance and increased plasma exposure of these biomarkers in monkeys, consistent with the observation in humans. These endogenous biomarkers discovered in monkeys could be potentially used to evaluate the clinical drug-drug interactions in the early phase of drug development.

Preclinical assessment of drug-drug interaction of fb-PMT, a novel anti-cancer thyrointegrin αvß3 antagonist.

The objective of the current study was to identify potential drug-drug interactions (DDIs) with the drug candidate fb-PMT, a novel anticancer thyrointegrin αvß3 antagonist. This was accomplished by using several in vitro assays to study interactions of fb-PMT with both cytochrome P450 (CYP) enzymes and drug transporters, two common mechanisms leading to adverse drug effects. In vitro experiments showed that fb-PMT exhibited weak reversible inhibition of CYP2C19 and CYP3A4. In addition, fb-PMT did not show time-dependent inhibition with any of the seven CYP isoforms tested, including 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. Human liver microsomal incubations demonstrated that fb-PMT is stable. Potential transporter-mediated DDIs with fb-PMT were assessed with two ATP binding cassette (ABC) family transporters (P-glycoprotein and breast cancer resistance protein) using Caco2 cells and seven solute carrier family (SLC) transporters (organic cation transporter OCT2, organic anion transporters OAT1 and OAT3, organic anion transporter peptides OATP1B1 and OATP1B3, and the multidrug and toxic extrusion proteins MATE1 and MATE2-K using transfected HEK293 cells). Fb-PMT was not a substrate for any of the nine transporters tested in this study, nor did it inhibit the activity of seven of the transporters tested. However, fb-PMT inhibited the uptake of rosuvastatin by both OATP1B1 and OATP1B3 with half-maximal inhibitory concentrations greater than 3 and less than 10 µM. In summary, data suggest that the systemic administration of fb-PMT is unlikely to lead to DDIs through CYP enzymes or ABC and SLC transporters in humans.

Lack of efflux of diglycolic acid from proximal tubule cells leads to its accumulation and to toxicity of diethylene glycol.

Diethylene glycol (DEG) mass poisonings have resulted from ingestion of adulterated pharmaceuticals, leading to proximal tubular necrosis and acute kidney injury. Diglycolic acid (DGA), one of the primary metabolites, accumulates greatly in kidney tissue and its direct administration results in toxicity identical to that in DEG-treated rats. DGA is a dicarboxylic acid, similar in structure to Krebs cycle intermediates such as succinate. Previous studies have shown that DGA is taken into kidney cells via the succinate-related dicarboxylate transporters. These studies have assessed whether the DGA that is taken up by primary cultures of human proximal tubule (HPT) cells is effluxed. In addition, a possible mechanism for efflux, via organic anion transporters (OATs) that exchange external organic anions for dicarboxylates inside the cell, was assessed using transformed cell lines that actively express OAT activities. When HPT cells were cultured on membrane inserts, then loaded with DGA and treated with the OAT4/5 substrate estrone sulfate or the OAT1/3 substrate para-aminohippurate, no DGA efflux was seen. A repeat of this experiment utilizing RPTEC/TERT1 cells with overexpressed OAT1 and OAT3 had similar results. In these cells, but not in HPT cells, co-incubation with succinate increased the uptake of PAH, confirming the presence of OAT activity in the RPTEC/TERT1 cells. Thus, despite OATs stimulation in cells with OAT activity, there was little to no efflux of DGA from the cells. This study concluded that DGA is poorly transported out of cells and that stimulation of OAT transporters is not a viable target for reducing DGA accumulation in cells.

OAT3 Participates in Drug-Drug Interaction between Bentysrepinine and Entecavir through Interactions with M8-A Metabolite of Bentysrepinine-In Rats and Humans In Vitro.

Bentysrepinine (Y101) is a novel phenylalanine dipeptide for the treatment of hepatitis B virus. Renal excretion played an important role in the elimination of Y101 and its metabolites, M8 and M9, in healthy Chinese subjects, although the molecular mechanisms of renal excretion and potential drug-drug interactions (DDIs) remain unclear. The present study aimed to determine the organic anion transporters (OATs) involved in the renal disposition of Y101 and to predict the potential DDI between Y101 and entecavir, the first-line agent against HBV and a substrate of OAT1/3. Pharmacokinetic studies and uptake assays using rat kidney slices, as well as hOAT1/3-HEK293 cells, were performed to evaluate potential DDI. The co-administration of probenecid (an inhibitor of OATs) significantly increased the plasma concentrations and area under the plasma concentration-time curves of M8 and M9 but not Y101, while reduced renal clearance and the cumulative urinary excretion of M8 were observed in rats. The time course of Y101 and M8 uptake via rat kidney slices was temperature-dependent. Moreover, the uptake of M8 was inhibited significantly by probenecid and benzylpenicillin, but not by p-aminohippurate or tetraethyl ammonium. M8 was found to be a substrate of hOAT3, but Y101 is not a substrate of either hOAT1 or hOAT3. Additionally, the entecavir inhibited the uptake of M8 in the hOAT3-transfected cells and rat kidney slices in vitro. Interestingly, no significant changes were observed in the pharmacokinetic parameters of Y101, M8 or entecavir, regardless of intravenous or oral co-administration of Y101 and entecavir in rats. In conclusion, M8 is a substrate of OAT3 in rats and humans. Furthermore, M8 also mediates the DDI between Y101 and entecavir in vitro, mediated by OAT3. We speculate that it would be safe to use Y101 with entecavir in clinical practice. Our results provide useful information with which to predict the DDIs between Y101 and other drugs that act as substrates of OAT3.

Pancreatic Hormone Insulin Modulates Organic Anion Transporter 1 in the Kidney: Regulation via Remote Sensing and Signaling Network.

Organic anion transporter 1 (OAT1) expressed in the kidney plays an important role in the elimination of numerous anionic drugs used in the clinic. We report here that insulin, a pancreas-secreted hormone, regulated the expression and activity of kidney-specific OAT1 both in cultured cells and in rats. We showed that treatment of OAT1-expressing cells with insulin led to an increase in OAT1 expression, transport activity, and SUMOylation. Such insulin-induced increase was blocked by afuresertib, a specific inhibitor for protein kinase B (PKB), suggesting insulin regulates OAT1 through PKB signaling pathway. Furthermore, insulin stimulated transport activity and SUMOylation of endogenously expressed OAT1 in rat kidneys. In conclusion, our data support a remote sensing and signaling model, in which OAT1 plays an essential role in intercellular and inter-organ communication and in maintaining local and whole-body homeostasis. Such complex and dedicated communication is carried out by insulin, and PKB signaling and membrane sorting.

Inhibition of proteasome, but not lysosome, upregulates organic anion transporter 3 in vitro and in vivo.

Organic anion transporter 3 (OAT3), an indispensable basolateral membrane transporter predominantly distributed in the kidney proximal tubules, mediated the systemic clearance of substrates including clinical drugs, nutrients, endogenous and exogenous metabolites, toxins, and critically sustains body homeostasis. Preliminary data in this study showed that classical proteasome inhibitors (e.g., MG132), but not lysosome inhibitors, significantly increased the OAT3 ubiquitination and OAT3-mediated transport of estrone sulfate (ES) in OAT3 stable expressing cells, indicating that proteasome rather than lysosome is involved in the intracellular fate of OAT3. Next, bortezomib and carfilzomib, two FDA-approved and widely applied anticancer agents through selective targeting proteasome, were further used to define the role of inhibiting proteasome in OAT3 regulation and related molecular mechanisms. The results showed that 20S proteasome activity in cell lysates was suppressed with bortezomib and carfilzomib treatment, leading to the increased OAT3 ubiquitination, stimulated transport activity of ES, enhanced OAT3 surface and total expression. The upregulated OAT3 function by proteasome inhibition was attributed to the augment in maximum transport velocity and stability of membrane OAT3. Lastly, in vivo study using Sprague Dawley rats validated that proteasome inhibition using bortezomib induced enhancement of OAT3 ubiquitination and membrane expression in kidney. These data suggest that activity of proteasome but not lysosome could have an impact on the physiological function of OAT3, and proteasome displayed a promising target for OAT3 regulation in vitro and in vivo, and could be used in restoring OAT3 impairment under pathological conditions, avoiding OAT3-associated toxicity and diseases, ensuring drug efficacy and safety.

The Effect of Original Russian Neurotropic Drugs on Organic Anion Transporting Polypeptides OATP1B1 and OATP1B3.

In experiments on HepG2 cells, we studied the effect of the original domestic neurotropic drugs omberacetam, fabomotizole, and ethylmethylhydroxypyridine succinate (EMHPS) (1-500 µM) on the activity and content of organic anion transporting polypeptides OATP1B1 and OATP1B3. It was shown that omberacetam (500 µM) increased the content of OATP1B1 and OATP1B3, fabomotizole did not affect the level of both transporters, and EMHPS (500 µM) increased the content of OATP1B1 compared to the control and did not affect the level of OATP1B3. The tested substances also reduced the OATP1B1/OATP1B3 ratio, as evidenced by a decrease in the penetration of atorvastatin, a substrate of the transporters, into HepG2 cells in the presence of omberacetam (100-500 µM), fabomotizole (500 µM), and EMHPS (10-500 µM). Evaluation of clinical significance of the obtained results, according to the FDA approach based on the calculation of the Cmax/IC50 ratio, showed that the effect of the tested substances on OATP1B1/OATP1B3 is clinically insignificant.