Correlation between Membrane Protein Expression Levels and Transcellular Transport Activity for Breast Cancer Resistance Protein

Abstract

Emerging evidence indicates an important role for the breast cancer resistance protein (BCRP) in limiting brain penetration of substrate drugs. While in vitro transwell assays can provide an indication of BCRP substrate potential, the predictability of these assays in relation to in vivo brain penetration is still under debate. The present study examined the correlation of BCRP membrane protein expression level and transcellular transport activity across Madin-Darby canine kidney (MDCK) II monolayers. We expressed human BCRP or murine BCRP1 in MDCKII wild-type cells using BacMam2 virus transduction. The selective P-glycoprotein (P-gp) inhibitor LY335979 (1 M) was included in the transport medium to measure BCRP-mediated transcellular transport for P-gp and BCRP cosubstrates. The BCRP levels in membrane extracts from MDCKII-BCRP or MDCKII-Bcrp1 cells were quantified by liquid chromatography-tandem mass spectrometry. The results are summarized as follows: 1) the membrane protein expression levels correlate with the corrected efflux ratios of substrates for human BCRP and murine BCRP1 within the efflux ratios investigated; 2) we demonstrate good concordance in rank order between the BCRP and BCRP1-mediated efflux ratios for 12 drugs; and 3) we propose an approach to contextualize in vitro BCRP transport data of discovery compounds by comparing them to the in vitro and in vivo transport data of the reference drug dantrolene and taking into account interbatch variation in BCRP expression. This approach correctly predicted compromised brain penetration for 25 discovery compounds in rodents, which were BCRP substrates but not P-gp or weak P-gp substrates. These results suggest that BCRP-expressing MDCKII cells are useful in predicting the in vivo role of BCRP in brain penetration.

Drug Distribution into Peripheral Nerve

“…e Passive permeability was measured in MDCKII-BCRP cells in the presence of 0.2 mM of Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino [19,29:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester] and 1 mM of LY335979 [(R)-4-[(1aR,6R,10bS)-1,2-difluoro-1,1a,6,10btetrahydrodibenzo-(a,e)cyclopropa(c)cycloheptan-6-yl]-a-[(5-quinoloyloxy)methyl]-1-piperazine ethanol trihydrochloride] at pH 7.4 (Liu et al, 2017).…”

“…For instance, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) do not have an appreciable impact on drug distribution into sciatic nerves, as indicated by the comparable sciatic nerve to plasma concentration ratios between wild-type and transgenic knockout rats for several P-gp and/or BCRP substrates (Huang et al, 2015). This is distinct from the CNS where P-gp and BCRP are two key gatekeepers preventing drug distribution into the brain (Schinkel et al, 1996;Liu et al, 2017). Interestingly, drug passage into the cell body-rich dorsal root ganglion (DRG), which is part of the peripheral nerve, appears unlimited for small and large molecule tracers, such as fluorescein (Abram et al, 2006), albumin (Olsson, 1971), horseradish peroxidase (Jacobs et al, 1976), and antibody IgG (Seitz et al, 1985).…”

“…From these results, the tissue-to-blood unbound concentration ratios (K p,uu ) for 11 small-molecule drugs were calculated for comparison between peripheral nerve and CNS tissues in rats. Imatinib is a substrate of both P-gp and BCRP (Kodaira et al, 2010;Liu et al, 2017). The influence of N-[4-[2-(6,7-dimethoxy-3,4dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide (GF120918), a P-gp and BCRP inhibitor (Matsson et al, 2009), on the penetration of imatinib into the peripheral nerve and CNS tissues in rats was also examined.…”

Muscle to Brain Partitioning as Measure of Transporter-Mediated Efflux at the Rat Blood–Brain Barrier and Its Implementation into Compound Optimization in Drug Discovery

“…For compound screening in the early drug discovery phase, several in vitro methods have been established in the pharmaceutical industry. P-gp and BCRP-transfected Madin-Darby canine kidney (MDCK) cells were shown to be valuable tools [12,13]. In such assays, the bidirectional transport of a drug across a cell monolayer grown on a Transwell ® insert is measured.…”

In-vitro and in situ assessment of the efflux of five antidepressants by breast cancer resistance protein

“…A significant difference among various experiment batches was found since P app values were decreased by approximately 80% in the presence of Ko143 in MDCKII‐WT cells (WT and BCRP without Ko143 as one batch and with Ko143 as another batch). Although the TEER values in all wells were greater than 200 Ω·cm 2 , the activity of cells may vary between experimental lots, thereby causing significant differences for similar treatments . Second, the intracellular accumulation assay showed that treatment with 1 and 5 μ m STL led to significantly greater intracellular STL levels in WT cells than in BCRP cells, and Ko143 significantly increased the intracellular accumulation of STL in BCRP cells at 1, 5 and 10 μ m concentrations (Figure ).…”