Emese Kis

Effect of Membrane Cholesterol on BSEP/Bsep Activity: Species Specificity Studies for Substrates and Inhibitors

The efflux transporter responsible for the canalicular elimination of bile salts from the hepatocytes is the bile salt export pump (BSEP, ABCB11). Absence or inhibition of this transporter leads to bile salt retention in the hepatocyte and in turn can lead to cholestatic liver disease. We expressed the BSEP/Bsep protein from three species (human, rat, and mouse) in a baculovirus-infected Sf9 system. Vesicles prepared from these cells were used to evaluate bile salt transport of four conjugated bile salts. Because the Sf9 system contains less membrane cholesterol than the liver canalicular membrane, the effect of added cholesterol on the kinetics of BSEP/Bsep-mediated bile salt transport was also investigated. Cholesterol treatment increased the Vmax values in all the species, with the most pronounced effect observed in the rat transporter. In contrast, Km values, with the exception of glycochenodeoxycholate, remained largely unchanged. The species-specific bile salt transport inhibition potential of three compounds known to cause clinical cholestasis was investigated in vesicles containing BSEP/Bsep. Troglitazone and glibenclamide inhibited the BSEP/Bsep-mediated transport of different bile salts with similar affinities, whereas the potential of cyclosporine A to inhibit bile salt transport showed species- and bile salt-specific variations. In conclusion, the cholesterol-loaded Sf9 vesicles overexpressing BSEP/Bsep seem to be a useful system for the identification of potential cholestatic compounds and can also be used for the investigation of species specificity. We observed greater differences in IC50 values for inhibitors than in Km values for substrates between species.

ABCG2 (breast cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool to detect drug-transporter interactions.

The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

BSEP inhibition – In vitro screens to assess cholestatic potential of drugs

Bile salt export pump (BSEP, ABC11) is a membrane protein that is localized in the cholesterol-rich canalicular membrane of hepatocytes. Its function is to eliminate unconjugated and conjugated bile acids/salts from hepatocyte into the bile. In humans there is no compensatory mechanism for the loss of this transporter. Mutations of BSEP result in a genetic disease, called progressive familial intrahepatic cholestasis type 2 (PFIC2), that is characterized with decreased biliary bile salt secretion, leading to decreased bile flow and accumulation of bile salts inside the hepatocyte, inflicting damage. BSEP inhibitor drugs produce similar bile salt retention that may lead to severe cholestasis and liver damage. Drug-induced liver injury is a relevant clinical issue, in severe cases ending in liver transplantation. Therefore, measurement of BSEP inhibition by candidate drugs has high importance in drug discovery and development. Although several methods are suitable to detect BSEP-drug interactions, due to interspecies differences in bile acid composition, differences in hepatobiliary transporter modulation, they have limitations. This review summarizes appropriate in vitro methods that could be able to predict BSEP-drug candidate interactions in humans before the start of clinical phases.

Characterization of 5(6)-Carboxy-2,′7′-Dichlorofluorescein Transport by MRP2 and Utilization of this Substrate as a Fluorescent Surrogate for LTC4

MRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug—endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties. (Journal of Biomolecular Screening 2008:295-301)

Multidrug Resistance Protein 2-Mediated Estradiol-17β-D-glucuronide Transport Potentiation : In Vitro-in Vivo Correlation and Species Specificity

Multidrug resistance protein 2 (MRP2) is a multispecific organic anion transporter expressed at important pharmacological barriers, including the canalicular membrane of hepatocytes. At this location it is involved in the elimination of both endogenous and exogenous waste products, mostly as conjugates, to the bile. Estradiol-17β-d-glucuronide (E217βG), a widely studied endogenous substrate of MRP2, was shown earlier to recognize two binding sites of the transporter in vesicular transport assays. MRP2 modulators (substrates and nonsubstrates) potentiate the transport of E217βG by MRP2. We correlated data obtained from studies of different complexities and investigated the species-specific differences between rat and human MRP2-mediated transport. We used vesicular transport assays, sandwich-cultured primary hepatocytes, and in vivo biliary efflux in rats. Our results demonstrate that the rat Mrp2 transporter, unlike the human MRP2, transports E217βG according to Michaelis-Menten type kinetics. Nevertheless, in the presence of modulator drugs E217βG transport mediated by the rat transporter also shows cooperative kinetics as potentiation of E217βG transport was observed in the vesicular transport assay. We also demonstrated that the potentiation exists both in rat and in human hepatocytes and in vivo in rats.

Ivermectin Interacts With Human ABCG2

Ivermectin is an antiparasitic drug frequently administered to humans. It has a limited brain exposure that is attributed to the efflux activity of ABCB1/Abcb1. ABCG2/Abcg2 is also a major transporter present in most pharmacologically important barriers. However, interaction of ivermectin with Abcg2 shows species specificity and in many studies was confounded by the masking effect of ABCB1/Abcb1. In this study using cellular and membrane assays we show that ivermectin displays a high-affinity interaction with human ABCG2 with IC(50) values in the 1-1.5  µM range. This interaction may have implications in human ABCG2-mediated drug-drug interactions of ivermectin.

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.

Mouse Bsep ATPase Assay: A Nonradioactive Tool for Assessment of the Cholestatic Potential of Drugs

The mouse ortholog of the human bile salt export pump (BSEP) transporter was expressed in a baculovirus-infected insect cell (Sf9) system to study the effect of membrane cholesterol content on the transporter function. The transport activity of cholesterol-loaded mouse Bsep-HAM-Sf9 vesicles was determined in a vesicular transport assay with taurochenodeoxycholate (TCDC), a known BSEP substrate. Mouse Bsep transports TCDC at a high rate that can be sensitively detected in the ATPase assay. Cholesterol upload of the Sf9 membrane potentiates both TCDC transport and TCDC-stimulated ATPase activities. Inhibitory effect of BSEP interactors on probe substrate transport was tested in both vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC50 values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate. This upgraded form of the mouse Bsep-HAM ATPase assay is a user friendly, sensitive, nonradioactive method for early high-throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay. (Journal of Biomolecular Screening 2009:10-15)

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.

Potentiation of MRP2/Mrp2‐Mediated Estradiol‐17β‐Glucuronide Transport by Drugs – A Concise Review

Introduction. – MRP2/Mrp2 (ABCC2, cMOAT) is a member of the ABC transporter family, a group of proteins using the energy of ATP to transport molecules across cell membranes. MRP2/Mrp2 is expressed in various tissues, including the liver, the kidneys, and the intestine. The transporter is localized at the apical membrane of polarized epithelial cells in these tissues. Probably, its most important role is in the biliary elimination of various endogenous and exogenous anions. These anions are structurally diverse; they can be both conjugates and unconjugated anions. In 2003, two groups simultaneously found that estradiol-17b-glucuronide (E217bG) transport mediated by human MRP2 has positive cooperativity [1] [2]. These laboratories observed a rather sigmoid curve when investigating the concentration dependence of E217bG transport. This phenomenon is due to the existence of at least two binding sites of MRP2. The Hill number of the linearized saturation curves was > 1.5, confirming the presence of multiple binding sites. These sites can interact with each other, e.g., if a modulator compound binds to one of the sites, it is able to stimulate the transport of E217bG or other substrates. E217bG is a cholestatic endogenous estradiol metabolite, causing reversible, dosedependent cholestasis [3]. The mechanism of E217bG-induced cholestasis is multifactorial: it induces endocytic internalization of BSEP and MRP2 proteins [4] [5] and increases paracellular permeability [6]. In addition, E217bG inhibits the transport of bile salts by Bsep via trans-inhibition [7]. Transport by MRP2/Mrp2, thus, contributes to the cholestatic activity of E217bG, making this transport physiologically and pharmacologically even more important. In the present minireview, we give an overview of recent studies focusing on the interactions of MRP2/Mrp2 with E217bG. The review is organized according to the experimental approaches used. In vitro – in vivo correlations (IVIVC) and speciesspecificity aspects will also be covered.