Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [3H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells ( n ¼ 3), whereas a 2.4-fold lower (p < 0.0 01) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.0 6± 0.22 fmol/mg protein, n ¼ 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97± 2.51 and 3.3 2± 0.6 6, n ¼ 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.
Author(s): Matthew Harwood, Sibylle Neuhoff, Amin Rostami-Hodjegan, Geoffrey Warhurst