The need for human breast cancer resistance protein substrate and inhibition evaluation in drug discovery and development: why, when, and how?

Although the multiplicity in transport proteins assessed during drug development is continuously increasing, the clinical relevance of the breast cancer resistance protein (BCRP) is still under debate. Here, our aim is to rationalize the need to consider BCRP substrate and inhibitor interactions and to define optimum selection and acceptance criteria between cell-based and vesicle-based assays in vitro. Information on the preclinical and clinical pharmacokinetics (PK), drug-drug interactions, and pharmacogenomics data was collated for 13 marketed drugs whose PK is reportedly associated with BCRP interaction. Clinical examples where BCRP impacts drug PK and efficacy appear to be rare and confounded by interactions with other transporters. Thirty-seven compounds were selected to be tested as BCRP substrates in a cell-based assay using MDCKII cells (Madin-Darby canine kidney cells) and 18 in membrane vesicles. Depending on the physicochemical compound properties, we observed both in vitro systems to give false-negative readouts. In addition, the inhibition potential of 19 compounds against BCRP was assessed in vesicles and in MDCKII cells, where we observed significant system and substrate-dependent IC50 values. Therefore, neither of the two test systems is superior to the other. Instead, one system may offer advantages under certain situations (e.g., low permeability) and thus should be selected based on the physicochemical compound properties. Finally, given the clinical relevance of BCRP, we propose that its evaluation should remain issue-driven: for low permeable, low bioavailable drugs, in particular when other more common processes do not allow a mechanistic understanding of any unexpected absorption or brain disposition, and for drugs with a low therapeutic window.

Calibration of in vitro multidrug resistance protein 1 substrate and inhibition assays as a basis to support the prediction of clinically relevant interactions in vivo.

The multidrug resistance protein 1 (MDR1) is known to limit brain penetration of drugs and play a key role in drug-drug interactions (DDIs). Theoretical cut-offs from regulatory guidelines are used to extrapolate MDR1 interactions from in vitro to in vivo. However, these cut-offs do not account for interlaboratory variability. Our aim was to calibrate our experimental system to allow better in vivo predictions. We selected 166 central nervous system (CNS) and non-CNS drugs to calibrate the MDR1 transport screening assay using Lewis lung cancer porcine kidney 1 epithelial cells overexpressing MDR1 (L-MDR1). A threshold efflux ratio (ER) of 2 was established as one parameter to assess brain penetration in lead optimization. The inhibitory potential of 57 molecules was evaluated using IC50 values based on the digoxin ER-IC50(ER)-or apparent permeability-IC50(Papp)-in L-MDR1 cells. Published clinical data for 68 DDIs involving digoxin as the victim drug were collected. DDI risk assessments were based on intestinal concentrations ([I2]) as well as unbound [I1u] and total plasma [I1T] concentrations. A receiver operating characteristic analysis identified an [I2]/IC50(ER) of 6.5 as the best predictor of a potential interaction with digoxin in patients. The model was further evaluated with a test set of 11 digoxin DDIs and 16 nondigoxin DDIs, resulting in only one false negative for each test set, no false positives among the digoxin DDIs, and two among the nondigoxin DDIs. Future refinements might include using cerebrospinal fluid to unbound plasma concentration ratios rather than therapeutic class, better estimation of [I2], and dynamic modeling of MDR1-mediated DDIs.

Role of the intestinal peptide transporter PEPT1 in oseltamivir absorption: in vitro and in vivo studies.

It was reported that oseltamivir (Tamiflu) absorption was mediated by human peptide transporter (hPEPT) 1. Understanding the exact mechanism(s) of absorption is important in the context of drug-drug and diet-drug interactions. Hence, we investigated the mechanism governing the intestinal absorption of oseltamivir and its active metabolite (oseltamivir carboxylate) in wild-type [Chinese hamster ovary (CHO)-K1] and hPEPT1-transfected cells (CHO-PEPT1), in pharmacokinetic studies in juvenile and adult rats, and in healthy volunteers. In vitro cell culture studies showed that the intracellular accumulation of oseltamivir and its carboxylate into CHO-PEPT1 and CHO-K1 was always similar under a variety of experimental conditions, demonstrating that these compounds are not substrates of hPEPT1. Furthermore, neither oseltamivir nor its active metabolite was capable of inhibiting Gly-Sar uptake in CHO-PEPT1 cells. In vivo pharmacokinetic studies in juvenile and adult rats showed that the disposition of oseltamivir and oseltamivir carboxylate, after oral administration of oseltamivir, was sensitive to the feed status but insensitive to the presence of milk and Gly-Sar. Moreover, oseltamivir and oseltamivir carboxylate exhibited significantly higher exposure in rats under fasted conditions than under fed conditions. In humans, oral dosing after a high-fat meal resulted in a statistically significant but moderate lower exposure than after an overnight fasting. This change has no clinical implications. Taken together, the results do not implicate either rat Pept1 or hPEPT1 in the oral absorption of oseltamivir.

Design, data analysis, and simulation of in vitro drug transport kinetic experiments using a mechanistic in vitro model.

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, V(max) and K(m), as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (P(dif)). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for V(max) and K(m) were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5-6-fold higher permeability at 37 degrees C compared with that at 4 degrees C), for fexofenadine a 16-fold higher passive permeability was seen at 37 degrees C. Therefore, P(dif) was better predicted if it was evaluated under the same experimental conditions as V(max) and K(m), i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37 degrees C, instead of a parallel control evaluation at 4 degrees C.

Characterization of the transmembrane transport and absolute bioavailability of the HCV protease inhibitor danoprevir.

BACKGROUND AND OBJECTIVES: Understanding transmembrane transport provides a more complete understanding of the pharmacokinetics of a drug and mechanistic explanations for drug-drug interactions. Here, the transmembrane transport of danoprevir (hepatitis C virus protease inhibitor) and the effects of ritonavir and ciclosporin on transmembrane transport of danoprevir were evaluated and clinical pharmacokinetic studies of danoprevir co-administered with/without ritonavir and ciclosporin were conducted. METHODS: Transcellular transport of danoprevir was evaluated in Lewis lung cancer porcine kidney, Madin-Darby canine kidney, or Chinese hamster ovary cells transfected with human transport proteins, and in human hepatocytes. The pharmacokinetics of intravenous and oral danoprevir administered with/without ritonavir, and the impact of ciclosporin on danoprevir pharmacokinetics were evaluated in randomized, open-label, crossover studies in healthy subjects. RESULTS: Danoprevir transport in vitro involved organic anion transporting polypeptide (OATP) 1B1, OATP1B3, P-glycoprotein, and multidrug resistance protein-2, but not breast cancer resistance protein. Ritonavir and ciclosporin inhibited transport of danoprevir by human hepatocytes. The pharmacokinetics of intravenous danoprevir 6 mg were not altered by oral ritonavir 100 mg. In contrast, exposure to oral danoprevir 100 mg increased two- to threefold when co-administered with ritonavir. Absolute bioavailability of danoprevir 100 mg was low (1.15%), but increased more than threefold (3.86%) when co-administered with ritonavir. Oral ciclosporin 100 mg increased exposure to intravenous danoprevir 2 mg and oral ritonavir 100 mg. CONCLUSION: Collectively, these studies provide insight into the transmembrane transport and pharmacokinetics of danoprevir and the mechanisms that underlie a recently reported, three-way drug-drug interaction involving danoprevir, ritonavir, and ciclosporin.

Substrate-dependent drug-drug interactions between gemfibrozil, fluvastatin and other organic anion-transporting peptide (OATP) substrates on OATP1B1, OATP2B1, and OATP1B3.

Hepatic uptake carriers of the organic anion-transporting peptide (OATP) family of solute carriers are more and more recognized as being involved in hepatic elimination of many drugs and potentially associated drug-drug interactions. The gemfibrozil-statin interaction was studied at the level of active hepatic uptake as a model for such drug-drug interactions. Active, temperature-dependent uptake of fluvastatin into primary human hepatocytes was shown. Multiple transporters are involved in this uptake as Chinese hamster ovary or HEK293 cells expressing either OATP1B1 (K(m) = 1.4-3.5 microM), OATP2B1 (K(m) = 0.7-0.8 microM), or OATP1B3 showed significant fluvastatin uptake relative to control cells. For OATP1B1 the inhibition by gemfibrozil was substrate-dependent as the transport of fluvastatin (IC(50) of 63 microM), pravastatin, simvastatin, and taurocholate was inhibited by gemfibrozil, whereas the transport of estrone-3-sulfate and troglitazone sulfate (both used at 3 microM) was not affected. The OATP1B1- but not OATP2B1-mediated transport of estrone-3-sulfate displayed biphasic saturation kinetics, with two distinct affinity components for estrone-3-sulfate (0.23 and 45 microM). Only the high-affinity component was inhibited by gemfibrozil. Recombinant OATP1B1-, OATP2B1-, and OATP1B3-mediated fluvastatin transport was inhibited to 97, 70, and 62% by gemfibrozil (200 microM), respectively, whereas only a small inhibitory effect by gemfibrozil (200 microM) on fluvastatin uptake into primary human hepatocytes was observed (27% inhibition). The results indicate that the in vitro engineered systems can not always predict the behavior in more complex systems such as freshly isolated primary hepatocytes. Therefore, selection of substrate, substrate concentration, and in vitro transport system are critical for the conduct of in vitro interaction studies involving individual liver OATP carriers.