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.

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.

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Recently we provided evidence for the involvement of a probenecid-inhibitable anion exchanger in cortisol release from primary cultures of bovine adrenocortical cells. In the present study, we further characterized this exchange transporter. Adrenocorticotropic hormone stimulated ³H-p-aminohippurate (³H-PAH) uptake into as well as cortisol release from the cells about two- and tenfold, respectively. Probenecid inhibited both ³H-PAH uptake and cortisol release by about 55 and 63%. Preincubation of the cells with 1 mM PAH trans-stimulated ³H-PAH uptake by 30%, whereas cortisol release was inhibited by 30%. ³H-PAH uptake was cis-inhibited by 1 mM glutarate or by 1 mM cortisol in the medium, while cortisol release was trans-stimulated by glutarate. PAH in the incubation medium showed saturable cis-inhibition of ³H-PAH uptake. The release of cyclic adenosine monophosphate, a substrate of the renal PAH exchanger, was also inhibited by probenecid and trans-stimulated by glutarate. In summary, the trans-stimulation and cis-inhibition experiments support the concept of an anion exchanger involved in cortisol and cyclic adenosine monophosphate release from and PAH uptake into adrenocortical cells.

ABSTRACT: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 M 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.

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.

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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.