ABSTRACT:The role of breast cancer resistance protein (Bcrp) and the combined activities of Bcrp and P-glycoprotein (P-gp, Mdr1a/1b) in limiting the brain penetration of drugs at the blood-brain barrier (BBB) were investigated using wild-type FVB, Mdr1a/1b(؊/؊), (؊/؊), Bcrp(؊/؊), and Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice. Four drugs, flavopiridol, imatinib mesylate (Gleevec), PF-407288, and prazosin, with different transport specificity for BCRP/Bcrp and MDR1/ Mdr1a were selected, and the drug levels in plasma, cerebrospinal fluid, and brain of mice were determined. Flavopiridol and prazosin were identified as substrates for both mouse Bcrp and Mdr1a with greater transport associated with Bcrp. The brain/plasma (B/P) ratios at 0.5 and 2 h in Mdr1a/1b(؊/؊), (؊/؊) and Bcrp(؊/؊) mice were 1-to 2-fold for both compounds, whereas the ratios in Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice were more than 5-fold of those observed in FVB mice. For imatinib, a better substrate of P-gp than Bcrp, the B/P ratios in Bcrp(؊/؊) were comparable to those in FVB mice, whereas the B/P ratios in Mdr1a/1b(؊/؊), (؊/؊) and Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice were more than 4-and 28-fold of those in FVB mice at both time points, respectively. Finally, the Bcrp-specific substrate PF-407288 exhibited comparable B/P ratios in Mdr1a/1b(؊/؊), (؊/؊) and Bcrp(؊/؊) mice and slightly but significantly increased B/P ratios in Mdr1a/ 1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice compared with those in FVB mice. The B/P ratios of compounds in Mdr1a/1b(؊/؊), (؊/؊)Bcrp(؊/؊) mice compared with those in Mdr1a/1b(؊/؊), (؊/؊) mice clearly demonstrate that Bcrp impairs the brain penetration of its substrates. Moreover, P-gp and Bcrp at BBB function synergistically to limit the brain penetration of shared substrates.It is widely recognized that the tight junctions between adjacent brain endothelial cells forming the blood-brain barrier (BBB) restrict the entry of compounds by paracellular diffusion from the blood to the brain. Moreover, the transcellular diffusion of compounds through the brain endothelial cells can also be impeded by transmembrane efflux transporters, such as P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). These efflux transporters can eliminate xenobiotics from the brain against a concentration gradient, thereby limiting central nervous system (CNS) exposure to these compounds. Indeed, the prominent effect of P-gp at the BBB is well established, and P-gp is functionally important in limiting the brain penetration of its substrates (Schinkel et al., 1994;Chen et al., 2003;Scherrmann, 2005). Like P-gp, BCRP is another major member of the ATP-binding cassette family of drug transporters and is highly expressed in the BBB as well. BCRP has been found at the luminal side of human brain capillary endothelial cells (Cooray et al., 2002), and the mRNA level of mouse analog Bcrp was ϳ700 times greater in brain microvessels than in the cortex of the mice (Cisternino et al., 2004), implying that mouse Bcrp may play a key role at the BBB. B...

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Over the last few years the importance of the intracellular C-terminus in the signaling of G-protein coupled receptors (GPCR

Review of these applications has demonstrated their utility in providing accurate results in a time-efficient manner, leading to acceleration of metabolite identification initiatives while highlighting the continued need for biotransformation expertise in the interpretation of more complex metabolic reactions.