Correlation Analysis of Potential Breast Cancer Resistance Protein Probes in Different Monolayer Systems.

Breast cancer resistance protein (BCRP) is a point of interest in drug-drug interaction safety testing. Therefore, a consensus probe that can be applied as victim in multiple experimental settings is of great benefit. Identification of candidates has been driven by the amount and quality of available clinical data, and as a result, drugs such as sulfasalazine and rosuvastatin have been suggested. In this article, the in vitro performance of 5 possible alternatives was evaluated: atorvastatin, chlorothiazide, dantrolene, topotecan, and teriflunomide, and benchmarked against sulfasalazine and rosuvastatin in reference in vitro assays for BCRP drug-drug interaction testing. Based on the results, teriflunomide is proposed as an alternate in vitro BCRP probe.

Human OATP1B1 (SLCO1B1) transports sulfated bile acids and bile salts with particular efficiency.

Human OATP1B1 is highly expressed at the basolateral membrane of the hepatocyte. It plays an important role in the sodium-independent transport of bile acids and bile salts and contributes to the systemic clearance of many drugs. In this study, the interaction of at least one representative of all major chemical classes of bile acids and bile salts, which include the bile acid chenodeoxycholate (CDC), monovalent (amidated) bile salts glycochenodeoxycholate (GCDC), taurochenodeoxycholate (TCDC) and taurocholate (TC), a sulfated bile acid 3-sulfo-chenodeoxycholate (3S-CDC) and a divalent (amidated and sulfated) bile salt 3-sulfo-glycolithocholate (3S-GLC) were tested with OATP1B1 overexpressed in HEK293 cells. All bile acid derivatives except for CDC showed an efficient transport by OATP1B1. 3S-GLC gave the lowest KM (0.708 ±â€¯0.125 µM) and 3S-CDC showed the highest Vmax value (158 ±â€¯87.3 pmol/mg protein/min). The ranking of Clint values (3S-GLC > 3S-CDC > TCDC > GCDC > TC) also showed a preference for sulfated derivatives. In summary, human OATP1B1 transports sulfate esters of bile acids and bile salts more efficiently than monovalent bile salts.

Kinetic characterization of bile salt transport by human NTCP (SLC10A1).

The transport of bile acids facilitated by NTCP is an important factor in establishing bile flow. In this study, we examine the kinetics associated with human NTCP-dependent transport of two quantitatively important bile acids comprising the human bile acid pool, chenodeoxycholic acid and glycine-chenodeoxycholate, and secondary bile salt, 3-sulfo-glycolithocholate of potential toxicological significance. The study employed human NTCP overexpressing Chinese Hamster Ovary cells and results compared with taurocholate, a prototypical bile salt commonly used in transporter studies. GCDC and 3S-GLC but not CDCA were transported by NTCP. The efficient uptake of GCDC, TCA and 3S-GLC by NTCP enabled the determination of kinetics. GCDC displayed a lower KM (0.569±0.318µM) than TCA (6.44±3.83µM) and 3S-GLC (3.78±1.17µM). The apparent CLint value for GCDC was 20-fold greater (153±53µl/mg protein/min) than the apparent CLint for TCA (6.92±4.72µl/mg protein/min) and apparent CLint for 3S-GLC (8.05±1.33µl/mg protein/min). These kinetic results provide important complementary data on the substrate selectivity and specificity of NTCP to transport bile acids. NTCP transports GCDC with greater efficiency than TCA and has the same efficacy for 3S-GLC and TCA.

Species specificity profiling of rat and human organic cation/carnitine transporter Slc22a5/SLC22A5 (Octn2/OCTN2).

The purpose of this study was to characterize the uptake of carnitine, the physiological substrate, and the uptake of 3-(2,2,2-trimethylhydrazinium)propionate, a consensus substrate by rat Octn2 and human OCTN2 transporters as well as to characterize drug-mediated inhibition of l-carnitine uptake by the rat and human orthologs overexpressed in CHO-K1 cells. l-carnitine and 3-(2,2,2-trimethylhydrazinium)propionate were found to be a lower affinity substrate for rat Octn2 (KM = 32.66 ± 5.11 µM and 23.62 ± 4.99 µM respectively) than for human OCTN2 (KM = 3.08 ± 0.74 µM and 7.98 ± 0.63 µM). The intrinsic clearance (CLint) value for carnitine was higher for the human than for the rat transporter (22.82 ± 5.57 ml/min*mg vs 4.008 ± 0.675 ml/min*mg). For 3-(2,2,2-trimethylhydrazinium)propionate, in contrast, the CLint value for rat Octn2 was higher than for human OCTN2 (323.9 ± 72.8 ml/min*mg vs 65.11 ± 5.33 ml/min*mg). Furthermore, many pharmacologically important drugs were shown to affect l-carnitine transport by Octn2/OCTN2. The correlation between the IC50 datasets for the rat and human transporter resulted in an r value of 0.47 (p > 0.05). However, the greatest difference was less than seven-fold and 13 of 15 compounds yielded a difference less than 3-fold. Thus, the transporters from these two species showed an overlapping but somewhat different substrate and inhibitor specificity.

[Role of drug transporters in the central nervous system]. / Gyógyszertranszporterek szerepe a központi idegrendszerben.

Although the presence of blood-brain barrier in the mammalian organisms was discovered in the early 1900s, its precise structure and the drug transporter proteins localized in the blood-brain barrier were identified only in the last decades. Beside the ATP-binding cassette transporter proteins responsible for the protection of the brain, the Solute Carrier transporters play also an important role in the function of the central nervous system by its feeding, energy supply and cleaning function during the metabolism. This review provides an overview on the main types of transporters located in the brain, on their localization in different cell types and the main techniques for their investigation. In the second part of this article various neurodegenerative disorders and the pathology-related transporter proteins are presented. In the light of recent experimental results new therapeutic strategies may come into the focus of research for the treatment of disorders currently without effective therapy.

Structure and function of BCRP, a broad specificity transporter of xenobiotics and endobiotics.

The discovery and characterization of breast cancer resistance protein (BCRP) as an efflux transporter conferring multidrug resistance has set off a remarkable trajectory in the understanding of its role in physiology and disease. While the relevance in drug resistance and general pharmacokinetic properties quickly became apparent, the lack of a characteristic phenotype in genetically impaired animals and humans cast doubt on the physiological importance of this ATP-binding cassette family member, similarly to fellow multidrug transporters, despite well-known endogenous substrates. Later, high-performance genetic analyses and fine resolution tissue expression data forayed into unexpected territories concerning BCRP relevance, and ultimately, the rise of quantitative proteomics allows putting observed interactions into absolute frameworks for modeling and insight into interindividual and species differences. This overview summarizes existing knowledge on the BCRP transporter on molecular, tissue and system level, both in physiology and disease, and describes a selection of experimental procedures that are the most widely applied for the identification and characterization of substrate and inhibitor-type interactions.

A P-gp vesicular transport inhibition assay - optimization and validation for drug-drug interaction testing.

Accurate determination of potential drug-drug interaction mediated by efflux transporters (tDDI) is crucial to assess the risk of pharmacokinetic interaction and toxicity of drugs. Passive permeability and uptake transporter mediated transport are important covariates of cell-based inhibition assays that need to be taken into consideration when performing kinetic analysis of data. Vesicular uptake inhibition has been considered by regulatory agencies as a viable alternative for testing tDDI potential of low passive permeability drugs in particular. Membranes prepared from a P-gp overexpressing human cell line has superior transport properties over membranes prepared from Sf9 cells and cholesterol enriched Sf9 membranes. P-gp expressed in this membrane effluxes N-methyl-quinidine (NMQ) with high affinity (K(m) is 3.65 µM) and a high rate (V(max) is 656 pmol/mg protein/min). Digoxin, vinblastine and paclitaxel, established P-gp substrates inhibited transport of NMQ with estimated K(i) values of 250, 0.1 and 0.6 µM, respectively. A panel of 11 drugs that have been listed by regulatory agencies as reference inhibitors were used to validate the assay to predict clinical inhibition potential. All the drugs that have been implicated in P-gp mediated DDI were found to be inhibitors in a relevant concentration range.

Chlorothiazide is a substrate for the human uptake transporters OAT1 and OAT3.

The thiazide diuretic chlorothiazide is poorly metabolized, and is predominantly excreted via the kidneys. We have previously shown that chlorothiazide is transported by ATP-binding cassette transporter G2, suggesting a potential role for this transporter in apical efflux of chlorothiazide in the kidney. However, because of the poor passive permeability of the drug, it is likely that uptake transporters on the basolateral membrane are also involved to facilitate vectorial transport in the renal proximal tubule. Two suggested candidate transporters for this role are the human organic anion transporters, OAT1 and OAT3. By using mammalian cells stably expressing these transporters, we have demonstrated OAT1- and OAT3-dependent uptake of chlorothiazide with Michaelis constant values of 14.5 and 37.6 µM, respectively. Furthermore, we have found that probenecid, furosemide, and diclofenac inhibit chlorothiazide transport by OAT1 and OAT3, of which the probenecide-mediated inhibition may be of clinical importance. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1683-1687, 2013.

Synthesis and ABCG2 inhibitory activity of novel fumitremorgin C analogs--specificity and structure activity correlations.

The Ko family of fumitremorgin C analogs are potent and selective ABCG2 inhibitors. However, the most potent Ko compounds carry an ester linkage in their side-chain that makes them chemically and metabolically less stable. We have synthesized 16 tricyclic and 28 tetracyclic novel analogs devoid of ester linkages and tested them for ABCG2 inhibition potency and specificity. Unlike in the tricyclic analog group, potent ABCG2 inhibitory compounds were found among the tetracyclic analogs. The most potent compounds carried the 3S,6S,12aS configuration. We observed a marked stereospecificity as compounds with the 3S,6S,12aS configuration were at least 18-fold more potent inhibitors than their diastereoisomeric pairs with a 3S,6R,12aS configuration. This stereospecificity was not observed in ABCB1 and ABCC1 inhibition. Therefore, a single chiral center confers specificity for ABCG2 over ABCB1 and ABCC1. This is quite unexpected considering the large multivalent drug binding site these transporters harbor.

ABCG2 modulates chlorothiazide permeability--in vitro-characterization of its interactions.

We are showing that chlorothiazide, a diuretic, is an ABCG2 substrate. It is a Biopharmaceutics Classification System/Biopharmaceutics Drug Distribution and Classification System (BCS/BDDCS) Class IV drug with low bioavailability. Therefore, we tested if chlorothiazide interacts with major apically located intestinal efflux transporters. Our data show that chlorothiazide is transported by ABCG2 with a Km value of 334.6 µM and does not interact with ABCB1 or ABCC2. The chlorothiazide-ABCG2 interaction results in a vectorial transport in MDCKII-BCRP and Caco-2 cells with efflux ratios of 36 and 8.1 respectively. Inhibition of ABCG2 in Caco-2 cells reduced the efflux ratio to 1.4, suggesting that ABCG2 plays a role in limiting chlorothiazide bioavailability in humans.

Efflux transporters in the blood-brain interfaces--in vitro and in vivo methods and correlations.

INTRODUCTION: Sufficient brain exposure is crucial to the success of CNS drugs. The twofold greater attrition rate in clinical development of CNS drugs over the respective attrition rate of non-CNS drugs is due to lack of efficacy. It is generally thought that poor brain exposure is at least partly responsible for this, as the concentration-time profile at the brain target site is critical for efficacy. Efflux transporters in the blood-brain interfaces play a crucial role in modulation of permeability of drugs across these interfaces. Validation of preclinical tools to correctly predict brain exposure in humans is essential. AREAS COVERED: This review summarizes in vitro and in vivo tools to detect and characterize interactions of drugs with efflux transporters relevant to blood-brain interfaces. Furthermore, the article discusses the strengths and weaknesses of these methods and the limitations of their application, in addition to covering in vitro - in vivo correlations. EXPERT OPINION: A more detailed validation of in vitro efflux transporter assays employing primary brain endothelial cultures is needed. This should go along with mapping uptake transporters expressed in the blood-brain interfaces. With the availability of specific inhibitors, utilization of in vivo methods such as brain microdialysis is increasing. Once transporter-humanized mice are available, we may witness a further increase in applications of in vivo methods.

Quinidine as an ABCB1 probe for testing drug interactions at the blood-brain barrier: an in vitro in vivo correlation study.

This study provides evidence that quinidine can be used as a probe substrate for ABCB1 in multiple experimental systems both in vitro and in vivo relevant to the blood-brain barrier (BBB). The combination of quinidine and PSC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on ABCB1. Effects of quinidine and substrate-inhibitor interactions were tested in a membrane assay and in monolayer assays. The authors compared quinidine and digoxin as ABCB1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in ATPase and monolayer efflux assays employing MDCKII-MDR1 and the rat brain microcapillary endothelial cell system. Brain exposure to quinidine was tested in dual-/triple-probe microdialysis experiments in rats by assessing levels of quinidine in blood and brain. Comparing quinidine levels in dialysate samples from valspodar-treated and control animals, it is evident that systemic/local administration of the inhibitor diminishes the pumping function of ABCB1 at the BBB, resulting in an increased brain penetration of quinidine. In sum, quinidine is a good probe to study ABCB1 function at the BBB. Moreover, quinidine/PSC-833 is an ABCB1-specific substrate/inhibitor combination applicable to many assay systems both in vitro and in vivo.

ATP-binding cassette B1 transports seliciclib (R-roscovitine), a cyclin-dependent kinase inhibitor.

Seliciclib, a cyclin-dependent kinase inhibitor, is a promising candidate to treat a variety of cancers. Pharmacokinetic studies have shown high oral bioavailability but limited brain exposure to the drug. This study shows that seliciclib is a high-affinity substrate of ATP-binding cassette B1 (ABCB1) because it activates the ATPase activity of the transporter with an EC(50) of 4.2 µM and shows vectorial transport in MDCKII-MDR1 cells, yielding an efflux ratio of 8. This interaction may be behind the drug's limited penetration of the blood-brain barrier. ABCB1 overexpression, on the other hand, does not confer resistance to the drug in the models tested. These findings should be considered when treatment strategies using seliciclib are designed.