Preincubation Time-Dependent, Long-Lasting Inhibition of Drug Transporters and Impact on the Prediction of Drug-Drug Interactions.

Transporter-mediated drug-drug interaction (DDI) is of clinical concern, and the quantitative prediction of DDIs is an indispensable part of drug development. Cell-based inhibition assays, in which a representative probe substrate and a potential inhibitor are coincubated, are routinely performed to assess the inhibitory potential of new molecular entities on drug transporters. However, the inhibitory effect of cyclosporine A (CsA) on organic anion transporting polypeptide (OATP) 1B1 is substantially potentiated with CsA preincubation, and this effect is both long-lasting and dependent on the preincubation time. This phenomenon has also been reported with transporters other than OATP1Bs, but it is considered more prevalent among OATP1Bs and organic cation transporters. Regulatory agencies have also noted this preincubation effect and have recommended that pharmaceutical companies consider inhibitor preincubation when performing in vitro OATP1B1 and OATP1B3 inhibition studies. Although the underlying mechanisms responsible for the preincubation effect are not fully understood, a trans-inhibition mechanism was recently demonstrated for OATP1B1 inhibition by CsA, in which CsA inhibited OATP1B1 not only extracellularly (cis-inhibition) but also intracellularly (trans-inhibition). Furthermore, the trans-inhibition potency of CsA was much greater than that of cis-inhibition, suggesting that trans-inhibition might be a key driver of clinical DDIs of CsA with OATP1B substrate drugs. Although confidence in transporter-mediated DDI prediction is generally considered to be low, the predictability might be further improved by incorporating the trans-inhibition mechanism into static and dynamic models for preincubation-dependent inhibitors of OATP1Bs and perhaps other transporters. SIGNIFICANCE STATEMENT: Preincubation time-dependent, long-lasting inhibition has been observed for OATP1B1 and other solute carrier transporters in vitro. Recently, a trans-inhibition mechanism for the preincubation effect of CsA on OATP1B1 inhibition was identified, with the trans-inhibition potency being greater than that of cis-inhibition. The concept of trans-inhibition may allow us to further understand the mechanism of transporter-mediated DDIs not only for OATP1B1 but also for other transporters and to improve the accuracy and confidence of DDI predictions.

Experimental and Modeling Evidence Supporting the Trans-Inhibition Mechanism for Preincubation Time-Dependent, Long-Lasting Inhibition of Organic Anion Transporting Polypeptide 1B1 by Cyclosporine A.

Cyclosporine A (CsA) and rifampin are potent inhibitors of organic anion transporting polypeptide (OATP) 1B1 and are widely used to assess the risk for drug-drug interactions. CsA displays preincubation time-dependent, long-lasting inhibition of OATP1B1 in vitro and in rats in vivo, and a proposed mechanism is the trans-inhibition by which CsA inhibits OATP1B1 from the inside of cells. The current study aimed to experimentally validate the proposed mechanism using human embryonic kidney 293 cells stably expressing OATP1B1. The uptake of CsA reached a plateau following an approximate 60-minute incubation, with the cell-to-buffer concentration ratio of 3930, reflective of the high-affinity, high-capacity intracellular binding of CsA. The time course of CsA uptake was analyzed to estimate the kinetic parameters for permeability clearance and intracellular binding. When the OATP1B1-mediated uptake of [3H]estradiol-17ß-glucuronide was measured following preincubation with CsA for 5 to 120 minutes, apparent Ki values became lower with longer preincubation. Our kinetic modeling incorporated the two reversible inhibition constants [Ki,trans and Ki,cis for the inhibition from inside (trans-inhibition) and outside (cis-inhibition) of cells, respectively] and estimated Ki,trans value of CsA was smaller by 48-fold than the estimated Ki,cis value. Rifampin also displayed preincubation time-dependent inhibition of OATP1B1, albeit the extent of enhancement was only twofold. The current study provides experimental evidence for the preincubation time-dependent shift of apparent Ki values and a mechanistic basis for physiologically based pharmacokinetic modeling that incorporates permeability clearance, extensive intracellular binding, and asymmetry of Ki values between the inside and outside of cells. SIGNIFICANCE STATEMENT: In vitro data and kinetic modeling support that preincubation time-dependent, long-lasting inhibition of OATP1B1 by CsA can be explained by the extensive intracellular binding and reversible OATP1B1 inhibition intracellularly (trans-inhibition) as well as extracellularly (cis-inhibition). For inhibitors to display time-dependency, the following factors were found important: time to reach a steady-state cellular concentration, trans-inhibition potency relative to cis-inhibition, and the degree of cellular inhibitor accumulation. This study would aid in the accurate prediction of drug-drug interactions mediated by OATP1B1 inhibition.

Alteration in the Plasma Concentrations of Endogenous Organic Anion-Transporting Polypeptide 1B Biomarkers in Patients with Non-Small Cell Lung Cancer Treated with Paclitaxel.

Paclitaxel has been considered to cause OATP1B-mediated drug-drug interactions at therapeutic doses; however, its clinical relevance has not been demonstrated. This study aimed to elucidate in vivo inhibition potency of paclitaxel against OATP1B1 and OATP1B3 using endogenous OATP1B biomarkers. Paclitaxel is an inhibitor of OATP1B1 and OATP1B3, with Ki of 0.579 ± 0.107 and 5.29 ± 3.87 µM, respectively. Preincubation potentiated its inhibitory effect on both OATP1B1 and OATP1B3, with Ki of 0.154 ± 0.031 and 0.624 ± 0.183 µM, respectively. Ten patients with non-small cell lung cancer who received 200 mg/m2 of paclitaxel by a 3-hour infusion were recruited. Plasma concentrations of 10 endogenous OATP1B biomarkers-namely, coproporphyrin I, coproporphyrin III, glycochenodeoxycholate-3-sulfate, glycochenodeoxycholate-3-glucuronide, glycodeoxycholate-3-sulfate, glycodeoxycholate-3-glucuronide, lithocholate-3-sulfate, glycolithocholate-3-sulfate, taurolithocholate-3-sulfate, and chenodeoxycholate-24-glucuronide-were determined in the patients with non-small cell lung cancer on the day before paclitaxel administration and after the end of paclitaxel infusion for 7 hours. Paclitaxel increased the area under the plasma concentration-time curve (AUC) of the endogenous biomarkers 2- to 4-fold, although a few patients did not show any increment in the AUC ratios of lithocholate-3-sulfate, glycolithocholate-3-sulfate, and taurolithocholate-3-sulfate. Therapeutic doses of paclitaxel for the treatment of non-small cell lung cancer (200 mg/m2) will cause significant OATP1B1 inhibition during and at the end of the infusion. This is the first demonstration that endogenous OATP1B biomarkers could serve as surrogate biomarkers in patients. SIGNIFICANCE STATEMENT: Endogenous biomarkers can address practical and ethical issues in elucidating transporter-mediated drug-drug interaction (DDI) risks of anticancer drugs clinically. We could elucidate a significant increment of the plasma concentrations of endogenous OATP1B biomarkers after a 3-hour infusion (200 mg/m2) of paclitaxel, a time-dependent inhibitor of OATP1B, in patients with non-small cell lung cancer. The endogenous OATP1B biomarkers are useful to assess the possibility of OATP1B-mediated DDIs in patients and help in appropriately designing a dosing schedule to avoid the DDIs.

Recent advances in preclinical in vitro approaches towards quantitative prediction of hepatic clearance and drug-drug interactions involving organic anion transporting polypeptide (OATP) 1B transporters.

Hepatic uptake mediated by organic anion transporting polypeptide (OATP) 1B1 and 1B3 can serve as a major elimination pathway for various anionic drugs and as a site of drug-drug interactions (DDIs). This article provides an overview of the in vitro approaches used to predict human hepatic clearance (CLh) and the risk of DDIs involving OATP1Bs. On the basis of the so-called extended clearance concept, in vitro-in vivo extrapolation methods using human hepatocytes as in vitro systems have been used to predict the CLh involving OATP1B-mediated hepatic uptake. CLh can be quantitatively predicted using human donor lots possessing adequate OATP1B activities. The contribution of OATP1Bs to hepatic uptake can be estimated by the relative activity factor, the relative expression factor, or selective inhibitor approaches, which offer generally consistent outcomes. In OATP1B1 inhibition assays, substantial substrate dependency was observed. The time-dependent inhibition of OATP1B1 was also noted and may be a mechanism underlying the in vitro-in vivo differences in the inhibition constant of cyclosporine A. Although it is still challenging to quantitatively predict CLh and DDIs involving OATP1Bs from only preclinical data, understanding the utility and limitation of the current in vitro methods will pave the way for better prediction.

Relative Activity Factor (RAF)-Based Scaling of Uptake Clearance Mediated by Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 in Human Hepatocytes.

In vitro-in vivo extrapolation based on uptake clearance determined in human hepatocytes has been used to predict in vivo hepatic clearance of organic anion transporting polypeptide (OATP) substrates. This study evaluated the relative activity factor (RAF) approach to extrapolate active uptake clearance in transporter-transfected cell systems (CLuptake) to that in human hepatocyte suspensions (PSinf,act). RAF values for OATP1B1 and OATP1B3 were determined in two batches of cryopreserved human hepatocytes using estrone-3-sulfate and cholecystokinin octapeptide as reference substrates, respectively. Fourteen OATP1B substrate drugs selected (atorvastatin, bosentan, cerivastatin, fexofenadine, fluvastatin, glibenclamide, irbesartan, nateglinide, pitavastatin, pravastatin, rosuvastatin, telmisartan, torasemide, and valsartan) showed temperature-dependent uptake in human hepatocytes. In transporter-transfected cells, OATP1B1- and OATP1B3-mediated uptake was observed in all compounds except for telmisartan. RAF-based net CLuptake was mainly accounted for by OATP1B1 (72.3-99.7%) and fell within the 3-fold of PSinf,act observed in human hepatocytes in 11 out of 13 compounds (excluding telmisartan). This study demonstrated that the RAF approach provides a quantitative index of OATP1B1- and OATP1B3-mediated PSinf,act in human hepatocytes, which will facilitate the optimization of the pharmacokinetic properties of OATP1B substrates at nonclinical stages of drug development.

Comparison of the Predictability of Human Hepatic Clearance for Organic Anion Transporting Polypeptide Substrate Drugs Between Different In Vitro-In Vivo Extrapolation Approaches.

Prediction of human pharmacokinetic profiles of drug candidates is an essential step toward first-in-human studies. However, it remains difficult to quantitatively predict hepatic clearance, particularly when hepatic uptake is mediated by transporter(s). Using 15 organic anion transporting polypeptide (OATP) substrate drugs, we tested 3 in vitro-in vivo extrapolation (IVIVE) approaches to predict overall hepatic intrinsic clearance in vivo (CLint,all,vivo). IVIVE approaches involved metabolic intrinsic clearance in human liver microsomes (CLint,met) with or without hepatocyte-to-buffer maximum unbound concentration ratio (Kp,uu,max) correction and uptake intrinsic clearance at 37°C (PSinf,37°C) in human hepatocyte suspensions. Kp,uu,max and PSinf,37°C values were determined in 2 hepatocyte batches, and all tested compounds showed temperature-dependent uptake, consistent with the fact of transporter-mediated uptake. CLint,met substantially underestimated CLint,all,vivo. By multiplying CLint,met by Kp,uu,max values, the prediction performance was much improved; however, in vitro-in vivo correlation was poor. Among the IVIVE approaches, PSinf,37°C showed the most robust correlation with CLint,all,vivo, and one of the hepatocyte batches could predict CLint,all,vivo with a minimal empirical scaling factor. These results suggested IVIVE with hepatic uptake clearance determined in hepatocyte suspensions as one of the most practical approaches for predicting CLint,all,vivo of OATP substrate drugs.

Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays.

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of various drugs. Because OATP1B1 is a site of drug-drug interactions (DDIs), evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug development. For establishing a highly sensitive, high-throughput fluorescence-based OATP1B1 inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter-expressing human embryonic kidney 293 cells. We identified 2',7'-dichlorofluorescein (DCF), 4',5'-dibromofluorescein (DBF), and Oregon green (OG) as good OATP1B1 substrates with Km values of 5.29, 4.16, and 54.1 µM and Vmax values of 87.9, 48.1, and 187 pmol/min/mg protein, respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG, and DBF were identified as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates. Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The Ki values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good agreement with those obtained using [(3)H]estradiol-17ß-glucuronide (E2G) as a substrate, whereas [(3)H]estrone-3-sulfate and [(3)H]sulfobromophthalein yielded higher Ki values for all inhibitors than DCF. Mutually competitive inhibition observed between DCF and E2G suggested that they share the same binding site on OATP1B1. Therefore, DCF as well as E2G can be used as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variations.

Investigation of the impact of substrate selection on in vitro organic anion transporting polypeptide 1B1 inhibition profiles for the prediction of drug-drug interactions.

The risk assessment of organic anion transporting polypeptide (OATP) 1B1-mediated drug-drug interactions (DDIs) is an indispensable part of drug development. We previously reported that in vitro inhibitory potencies of several inhibitors on OATP1B1 depend on the substrates when prototypical substrates, estradiol-17ß-glucuronide (E2G), estrone-3-sulfate, and sulfobromophthalein were used as test substrates. The purpose of this study was to comprehensively investigate this substrate-dependent inhibition of OATP1B1 using clinically relevant OATP1B1 inhibitors and substrate drugs. Effects of cyclosporine A (CsA), rifampin, and gemfibrozil on OATP1B1-mediated uptake of 12 substrate drugs were examined in OATP1B1-expressing human embryonic kidney 293 cells. The Ki values (µM) for CsA varied from 0.0771 to 0.486 (6.3-fold), for rifampin from 0.358 to 1.23 (3.4-fold), and for gemfibrozil from 9.65 to 252 (26-fold). Except for the inhibition of torasemide uptake by CsA and that of nateglinide uptake by gemfibrozil, the Ki values were within 2.8-fold of those obtained using E2G as a substrate. Preincubation potentiated the inhibitory effect of CsA on OATP1B1 with similar magnitude regardless of the substrates. R values calculated based on a static model showed some variation depending on the Ki values determined with various substrates, and such variability could have an impact on the DDI predictions particularly for a weak-to-moderate inhibitor (gemfibrozil). OATP1B1 substrate drugs except for torasemide and nateglinide, or E2G as a surrogate, is recommended as an in vitro probe in the inhibition experiments, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent Ki variation.

Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17ß-glucuronide, estrone-3-sulfate, and sulfobromophthalein.

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of many drugs, and the evaluation of OATP1B1-mediated drug-drug interactions (DDIs) is emphasized in the latest DDI (draft) guidance documents from U.S. and E.U. regulatory agencies. It has been suggested that some OATP1B1 inhibitors show a discrepancy in their inhibitory potential, depending on the substrates used in the cell-based assay. In this study, inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of the prototypical substrates [³H]estradiol-17ß-glucuronide (E2G), [³H]estrone-3-sulfate (E1S), and [³H]sulfobromophthalein (BSP) were studied in OATP1B1-transfected cells. Inhibitory potencies of tested compounds varied depending on the substrates. Ritonavir, gemfibrozil, and erythromycin caused remarkable substrate-dependent inhibition with up to 117-, 14-, and 13-fold difference in their IC50 values, respectively. Also, the clinically relevant OATP inhibitors rifampin and cyclosporin A exhibited up to 12- and 6-fold variation in their IC50 values, respectively. Regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when [³H]E2G was used as a substrate. Mutual inhibition studies of OATP1B1 indicated that E2G and E1S competitively inhibited each other, whereas BSP noncompetitively inhibited E2G uptake. In addition, BSP inhibited E1S in a competitive manner, but E1S caused an atypical kinetics on BSP uptake. This study showed substrate-dependent inhibition of OATP1B1 and demonstrated that E2G was the most sensitive in vitro OATP1B1 probe substrate among three substrates tested. This will give us an insight into the assessment of clinically relevant OATP1B1-mediated DDI in vitro with minimum potential of false-negative prediction.