Stephanie van Hoppe

146 Chapter 6 in the knockout mice methotrexate plasma levels were increased 5-fold, whereas liver levels were decreased 24-fold, and small intestinal levels 20-fold. Each of the three humanized transgenes partially reversed all of the three measured parameters, albeit not to the levels seen in wild-type mice. Plasma levels of methotrexate were reduced ~2-fold, and liver levels increased by 4- to 9-fold. Small intestinal levels were 2- to 4-fold increased. Qualitatively very similar results were obtained at the 2 mg/kg methotrexate dose. In general, transgenic OATP1B3 and OATP1A2 caused a ~2-fold more effective reversal of liver and small intestinal concentrations than OATP1B1, whereas the plasma reversal effects were similar between the three transgenes. The partial reversal by the humanized OATP1B1 and OATP1B3 proteins compared to wild-type parameters could relate to species differences in substrate preference between mouse and human Oatp/ OATP proteins, but also to differences in effective expression level. Also, in human liver OATP1B1 and OATP1B3 would presumably act additively towards methotrexate, and thus may cause larger pharmacokinetic effects than suggested by the effects seen in the single transgenic strains. Regardless, the data clearly show that human OATP1B1 and OATP1B3 can have major effects on the plasma level, hepatic uptake clearance, and subsequent hepatobiliary/intestinal excretion of methotrexate. Moreover, as OATP1B3 is also expressed in various gastrointestinal, hepatocellular, breast, and lung cancers, it may further affect susceptibility of these cancers to OATP1B3 substrate drugs (Abe et al., 2001; Cui et al., 2003; Monks et al., 2007; Muto et al., 2007). The data obtained for human OATP1A2 suggest that this protein could substantially affect methotrexate uptake in vivo in other relevant tissues and barriers, such as the blood-brain barrier, kidney tubules, and tumor cells that express OATP1A2 (Gao et al., 2000; Lee et al., 2005). 2 . 3 . 3 . Impa c t o f r i famp i c i n and te lmi s a r t an coadmi n i s t ra t i on on OAT P-me d i a te d me t ho t re xa te d i s po s i t i on The humanized mouse strains were subsequently used to further investigate OATP- dependent drug-drug interactions, starting with the inhibitory effect of rifampicin on in vivo methotrexate transport by human OATP1B1 and OATP1B3. Using various human OATP-overexpressing HEK293 cells, Durmus et al. (2015) showed that rifampicin inhibited methotrexate uptake in vitro , with IC 50 levels ranging from 0.3 to 0.9 µM. In the mouse models, rifampicin (20 mg/kg, i.v.) substantially inhibited both mouse and human OATP-mediated hepatic uptake and plasma disposition of methotrexate (10 mg/kg, i.v.) at clinically achievable concentrations for each of the drugs. Liver-to-plasma ratios of methotrexate were decreased 6- to 8-fold by inhibiting the mouse Oatp1a/1b proteins, ~4-fold by inhibiting human OATP1B1 and 11- to 18-fold by inhibiting human OATP1B3. This also led to increased plasma levels of methotrexate by 4- to 5-fold in mouse Oatp1a/1b background and up to 2-fold in humanized OATP1B1 or OATP1B3