Elsevier

Biochemical Pharmacology

Volume 70, Issue 6, 15 September 2005, Pages 949-958
Biochemical Pharmacology

Expression of the drug transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver

https://doi.org/10.1016/j.bcp.2005.06.018Get rights and content

Abstract

ATP binding cassette (ABC)-transporters like P-glycoprotein (multidrug resistance (MDR)1/ABCB1), the multidrug resistance associated proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2), and the breast cancer resistance protein (BCRP/ABCG2) have a large impact on the pharmacokinetics of numerous drugs and may also modulate the effectiveness of drug therapy. Prediction of a patient's susceptibility to xenobiotics and individualization of drug therapy would become possible, if a simple test were available for an easy screening of transporter expression. This study quantified the mRNA expression of the four ABC-transporters and of the pregnane X receptor (PXR), a key regulator in drug metabolism and efflux, in peripheral blood mononuclear cells (PBMCs), and corresponding liver or small intestine samples of humans by real-time reverse transcription-polymerase chain reaction (RT-PCR). The results obtained prove the absence of a correlation between the expression of four major ABC-transporters in PBMCs and in the intestine or liver. For all transporters (except MRP1/ABCC1 in the intestine), mRNA amount of the ABC-transporters was positively correlated with PXR expression in PBMCs and intestine. In conclusion, the study suggests that basal expression levels of the transporters are directly influenced by PXR expression in liver and PBMCs and demonstrates that PBMCs do not qualify as surrogate tissue for the expression of the four ABC-transporters in small intestine and liver. However, the transporter status in PBMCs remains important for drugs, whose primary site of therapeutic action is the lymphocyte and which are known substrates of the transporters.

Introduction

ATP binding cassette (ABC)-transporters represent a large family of transmembrane proteins that bind ATP and use the energy of ATP hydrolysis to transport various compounds across cell membranes [1]. ABC drug transporters in the intestine, liver, and kidney have a large impact on the pharmacokinetics of numerous drugs. In particular, they may not only restrict absorption in the gut and thus reduce bioavailability but also substantially determine drug elimination into bile, urine, and even feces. Moreover, because ABC-transporters actively restrict drug distribution to the site of action (e.g. at the blood–brain barrier), they may also modulate the effectiveness of drug therapy.

Activity of ABC-transporters is highly variable and may be altered within hours by inhibitors and within days by inducing agents. Also, genetic influences [2] or endogenous factors like sex-hormones [3] or cholesterol [4] might influence transporter activity. Moreover, for multidrug resistance (MDR)1/ABCB1 and multidrug resistance associated protein (MRP)2/ABCC2, it is well known that the orphan receptor pregnane X receptor (PXR), which is a key regulator in drug metabolism and efflux, induces MDR1/ABCB1 and MRP2/ABCC2 when activated by PXR ligands like rifampin [5], [6]. In contrast, for MRP1/ABCC1, there are only few studies on this topic, which are furthermore controversial [7], [8], and for breast cancer resistance protein (BCRP)/ABCG2, no data exist so far.

Just because their activity is variable and also because the individual extent of drug–drug interactions at this level is almost unpredictable, a surrogate marker for the activity of the most important drug transporting systems would be very helpful.

Peripheral blood mononuclear cells (PBMCs) express many of the ABC-transporters important for drug transport which might restrict drug access to this important site of action [1], [9], [10], [11], [12]. Moreover, PBMCs are readily available, a prerequisite for a suitable surrogate marker. However, PBMCs only qualify as surrogate, if their transporter expression correlates with that of other tissues, especially with the intestine, whose transporter equipment appears to have the largest impact on the pharmacokinetics of numerous drugs.

We therefore investigated in humans by means of RT-PCR, whether the mRNA expression of MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, and BCRP/ABCG2 in PBMCs correlates with that in intestine and liver. For P-glycoprotein (P-gp), we also studied the influence of the G2677T and the C3435T polymorphism in the MDR1/ABCB1 gene on the expression and we evaluated the association between expression and function of P-gp as measured by rhodamine123 efflux from PBMCs. Finally, we quantified the mRNA expression of PXR in the different tissues and examined a possible correlation between PXR and transporter expression.

Section snippets

Materials

Medium (RPMI), medium supplements, PBS, and Hanks balanced salt solution (HBSS) were purchased from Invitrogen (Karlsruhe, Germany), fetal calf serum (FCS) from Biochrom AG (Berlin, Germany), and rhodamine123 from Calbiochem (Bad Soden, Germany). Vacutainer®CPT™ were purchased from Becton Dickinson (Heidelberg, Germany). K/EDTA monovettes® were from Sarstedt (Nümbrecht, Germany). DMSO was from AppliChem (Darmstadt, Germany). Ficoll-Paque™Plus was purchased from Amersham Biosciences (Freiburg,

Quantification of mRNA expression by real-time RT-PCR

For the mRNA expression of the target genes (MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR) and the housekeeping gene β2mg, a reliable and reproducible relative quantification method based on LightCycler™ technology was established and validated. The amplification curves, melting point analysis, and standard curves are exemplified for MDR1/ABCB1 and β2mg in Fig. 1. The specificity of the PCR products was verified by agarose gel electrophoresis and for the newly designed primers also

Discussion

The ABC-transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, and BCRP/ABCG2 may substantially impact the pharmacokinetic properties of many drugs and endogenous substrates, may confer the development of drug resistance, and may be a critical site of drug–drug interactions. Indeed, their activity is highly variable and changes in the in vivo activity of ABC-transporters have not altered drug elimination alone but also substantially modified drug distribution [17]. Hence, optimum doses of

Acknowledgments

We would like to thank Jutta Kocher, Corina Mueller, Stephanie Rosenzweig, and Alexandra Sauer for their excellent technical assistance, Heike Lindenmaier for her help with the study protocol, as well as Nina Wettschurek for giving us access to the flow cytometer. LY335979 was generously provided by Eli Lilly Company.

This work was supported by grant 01EC9902 from the German Ministry for Education and Research (Bundesministerium für Bildung und Forschung, BMBF) and the Friedrich-Fischer Nachlass

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