The human brain endothelial capillary cell line hCMEC/D3 has been developed recently as a model for the human blood‐brain barrier. In this study a further characterization of this model was performed with special emphasis on permeability properties and active drug transport. Para‐ or transcellular permeabilities (Pe) of inulin (0.74 × 10−3 cm/min), sucrose (1.60 × 10−3 cm/min), lucifer yellow (1.33 × 10−3 cm/min), morphine (5.36 × 10−3 cm/min), propranolol (4.49 × 10−3 cm/min) and midazolam (5.13 × 10−3 cm/min) were measured. By addition of human serum the passive permeability of sucrose could be reduced significantly by up to 39%. Furthermore, the expression of a variety of drug transporters (ABCB1, ABCG2, ABCC1–5) as well as the human transferrin receptor was demonstrated on the mRNA level. ABCB1, ABCG2 and transferrin receptor proteins were detected and functional activity of ABCB1, ABCG2 and the ABCC family was quantified in efflux experiments. Furthermore, ABCB1‐mediated bidirectional transport of rhodamine 123 was studied. The transport rate from the apical to the basolateral compartment was significantly lower than that in the inverse direction, indicating directed p‐glycoprotein transport. The results of this study demonstrate the usefulness of the hCMEC/D3 cell line as an in vitro model to study drug transport at the level of the human blood‐brain barrier.

Sunitinib is an orally active, multitargeted tyrosine kinase inhibitor which has been used for the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. We aimed to investigate the in vivo roles of the ATP-binding cassette drug efflux transporters ABCB1 and ABCG2 in plasma pharmacokinetics and brain accumulation of oral sunitinib, and the feasibility of improving sunitinib kinetics using oral coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. We used in vitro transport assays and Abcb1a/1b 2/2 , Abcg2 2/2 and Abcb1a/1b/Abcg2 2/2 mice to study the roles of ABCB1 and ABCG2 in sunitinib disposition. In vitro, sunitinib was a good substrate of murine (mu)ABCG2 and a moderate substrate of human (hu)ABCB1 and huABCG2. In vivo, the systemic exposure of sunitinib after oral dosing (10 mg kg 21 ) was unchanged when muABCB1 and/or muABCG2 were absent. Brain accumulation of sunitinib was markedly (23-fold) increased in Abcb1a/b/Abcg2 2/2 mice, but only slightly (2.3-fold) in Abcb1a/b 2/2 mice, and not in Abcg2 2/2 mice. Importantly, a clinically realistic coadministration of oral elacridar and oral sunitinib to wild-type mice resulted in markedly increased sunitinib brain accumulation, equaling levels in Abcb1a/1b/Abcg2 2/2 mice. This indicates complete inhibition of the blood-brain barrier (BBB) transporters. High-dose intravenous sunitinib could saturate BBB muABCG2, but not muABCB1A, illustrating a dose-dependent relative impact of the BBB transporters. Brain accumulation of sunitinib is effectively restricted by both muABCB1 and muABCG2 activity. Complete inhibition of both transporters, leading to markedly increased brain accumulation of sunitinib, is feasible and safe with a clinically realistic oral elacridar/sunitinib coadministration.

Brain capillary endothelial cells form the blood-brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1beta, IL-6 and TNF-alpha. The strongest BCRP suppression at the protein level was observed after IL-1beta treatment. IL-1beta, IL-6 and TNF-alpha also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-alpha treatment. TNF-alpha also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.

Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 are drug transporters mediating the active hepatic uptake of their substrates. Because they exhibit overlapping substrate specificities, the contribution of each isoform to the net hepatic uptake needs to be considered when predicting drug-drug interactions. The relative contribution of OATP1B1-and OATP1B3-mediated uptake of statins into hepatocytes was estimated based on either relative transporter protein expression data or relative activity data. Therefore, kinetics of eight statins and OATP1B1-and OATP1B3-specific reference substrates was determined in OATP1B1-and OATP1B3-expressing human embryonic kidney 293 cells and in human cryopreserved hepatocytes. Absolute OATP1B1 and OATP1B3 protein abundance was determined by liquid chromatography-tandem mass spectrometry in all expression systems. Transporter activity data generated in recombinant cell lines were extrapolated to hepatocyte values using relative transporter expression factors (REF) or relative activity factors (RAF). Our results showed a pronounced OATP1B1 and comparatively low OATP1B3 protein expression in the investigated hepatocyte lot. Based on REF scaling, we demonstrated that the active hepatic uptake clearances of reference substrates, atorvastatin, pravastatin, rosuvastatin, and simvastatin were well predicted within twofold error, demonstrating that OATP1B1 and OATP1B3 were major contributors. For other statins, the net hepatic uptake clearance was underpredicted, suggesting the involvement of other hepatic uptake transporters. Summarized, we showed that REF-and RAF-based predictions were highly similar, indicating a direct transporter expressionactivity relationship. Moreover, we demonstrated that the REF-scaling method provided a powerful tool to quantitatively assess the transporterspecific contributions to the net uptake clearance of statins in hepatocytes.

The results show a marked effect of orally administered GLP-1 and PYY3-36 on appetite by showing enhanced fullness at meal onset and reduced energy intake. This trial was registered at clinicaltrials.gov as NCT00822705.

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P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) combination knockout mice display disproportionately increased brain penetration of shared substrates, including topotecan and several tyrosine kinase inhibitors, compared to mice deficient for only one transporter. To better study the interplay of both transporters also in vitro, we generated a transduced polarized MDCKII cell line stably coexpressing substantial levels of human ABCB1 and ABCG2 (MDCKII-ABCB1/ABCG2). Next, we measured concentration-dependent transepithelial transport of topotecan, sorafenib and sunitinib. By blocking either one or both of the transporters simultaneously, using specific inhibitors, we aimed to mimic the ABCB1-ABCG2 interplay at the blood-brain barrier in wild-type, single or combination knockout mice. ABCB1 and ABCG2 contributed to similar extents to topotecan transport, which was only partly saturable. For sorafenib transport, ABCG2 was the major determinant at low concentrations. However, saturation of ABCG2-mediated transport occurred at higher sorafenib concentrations, where ABCB1 was still fully active. Furthermore, sunitinib was transported equally by ABCB1 and ABCG2 at low concentrations, but ABCG2-mediated transport became saturated at lower concentrations than ABCB1-mediated transport. The relative impact of these transporters can thus be affected by the applied drug concentrations. A comparison of the in vitro observed (inverse) transport ratios and cellular accumulation of the drugs at low concentrations with in vivo brain penetration data from corresponding Abcb1a/1b⁻/⁻, Abcg2⁻/⁻ and Abcb1a/1b;Abcg2⁻/⁻ mouse strains revealed very similar qualitative patterns for each of the tested drugs. MDCKII-ABCB1/ABCG2 cells thus present a useful in vitro model to study the interplay of ABCB1 and ABCG2.

ABSTRACT:The second-generation tyrosine kinase inhibitor and anticancer drug axitinib is a potent, orally active inhibitor of the vascular endothelial growth factor receptors 1, 2, and 3. Axitinib has clinical activity against solid tumors such as metastatic renal cell carcinoma and advanced pancreatic cancer. We studied axitinib transport using Madin-Darby canine kidney II cells overexpressing human ABCB1 or ABCG2 or murine Abcg2. Axitinib was a good substrate of ABCB1 and Abcg2, whereas transport activity by ABCG2 was moderate. These transporters may therefore contribute to axitinib resistance in tumor cells. Upon oral administration of axitinib, Abcg2(؊/؊) and Abcb1a/1b;Abcg2(؊/؊) mice displayed 1.7-and 1.8-fold increased axitinib areas under the plasma concentration-time curve from 0 to 4 compared with those of wild-type mice. Plasma concentrations in Abcb1a/1b(؊/؊) mice were not significantly increased. In contrast, relative brain accumulation of axitinib in Abcb1a/1b(؊/؊) and Abcb1a/1b;Abcg2(؊/؊) mice was, respectively, 6.8-and 13.9-fold higher than that in wild-type mice at 1 h and 4.9-and 20.7-fold at 4 h after axitinib administration. In Abcg2(؊/؊) mice, we found no significant differences in brain accumulation compared with those in wild-type mice. Thus, Abcb1 strongly restricts axitinib brain accumulation and completely compensates for the loss of Abcg2 at the blood-brain barrier, whereas Abcg2 can only partially take over Abcb1-mediated axitinib efflux. Hence, Abcg2 has a stronger impact on axitinib oral plasma pharmacokinetics, whereas Abcb1 is the more important transporter at the blood-brain barrier. These findings illustrate that in vitro transport data for ABCB1 and ABCG2 cannot always be simply extrapolated to the prediction of the relative impact of these transporters on oral availability versus brain penetration. IntroductionThe ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) affect the disposition of a variety of endogenous and exogenous compounds, including many anticancer drugs. Both transporters are expressed at the apical membranes of enterocytes, hepatocytes, and renal tubular epithelial cells, where they potentially limit gastrointestinal absorption or mediate direct intestinal, hepatic, or renal excretion of their substrates. Moreover, ABCB1-and ABCG2-mediated efflux activity in brain endothelial capillary cells of the blood-brain barrier (BBB) is crucial for the protection of the central nervous system from harmful compounds (Schinkel and Jonker, 2003;Vlaming et al., 2009). In addition, ABC transporters are expressed in many tumor types, mediating multidrug resistance against anticancer drugs (Borst and Oude Elferink, 2002).Only recently, the combined role of ABCB1 and ABCG2 at the BBB in limiting brain accumulation of shared substrates has been studied in detail using Abcb1a/1b;Abcg2(Ϫ/Ϫ) combination knockout mice. It was found that brain penetration of topotecan and several tyrosine kinase inhibitors (TKIs) includ...