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
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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