Stephanie van Hoppe

140 Chapter 6 kg) over 6 days resulted in more extensive weight loss in the Oatp1a/1b(-/-) mice, and a more pronounced reduction in the white blood cell count than in the wild-type mice measured at the end of the experiment (day 7). Assessed at this same day 7, there was some toxic damage observed in the bone marrow, thymus, and small intestine of wild- type mice, but this was much more pronounced in the Oatp1a/1b(-/-) mice (Iusuf et al., 2014). These data were in line with a much higher trough plasma concentration of SN-38 in the Oatp1a/1b(-/-) mice measured 24 hours after the last irinotecan administration, whereas the irinotecan concentrations at this time point were not significantly different from wild-type mice. It seems very likely that the higher exposure to SN-38 in the Oatp1a/1b(-/-) mice was the main cause of the increased toxicity. The most obvious explanation for the higher SN-38 exposure was strongly reduced hepatic clearance of SN-38, and likely also of its precursor irinotecan, by removal of the mouse Oatp1a/1b proteins. 2 . 2 . 1 . Up re gu l a t i on o f p l a sma ca r box y l e s te ra s e i n Oa t p1a / 1b ( - / - ) mi ce Further analyses revealed, however, that the higher SN-38 exposure was not caused only by a decreased clearance of SN-38 directly due to a deficiency of Oatp1a/1b transporters. It turned out that, tested ex vivo, plasma of Oatp1a/1b(-/-) mice contained a highly increased level of irinotecan hydrolase activity, which caused a far more rapid formation of SN-38 from spiked irinotecan than observed in wild-type plasma. This difference was most likely the result of the highly increased expression of a number of carboxylesterase (Ces) genes in the liver of Oatp1a/1b(-/-) mice, namely Ces1b, Ces1c, Ces1d and Ces1e. Ces1c, which is relatively abundant, displayed an 80-fold increase in RNA levels (Iusuf et al., 2014). Interestingly, the mouse Ces1c enzyme is known to be mainly secreted from liver into plasma, due to the absence of a C-terminal endoplasmic reticulum retention signal in its amino acid sequence (Holmes et al., 2010). Application of the carboxylesterase inhibitor bis(4-nitrophenyl) phosphate (BNPP) supported that increased plasma carboxylesterase activity was responsible for the hydrolysis of irinotecan in Oatp1a/1b(-/-) plasma. Moreover, various other tested Ces1, Ces2, and Ces3 family genes, and genes for other candidate plasma esterase enzymes like butyrylcholinesterase, Aadac, and Pon1-3, were not upregulated in the Oatp1a/1b(-/-) liver. Altogether, Ces1c upregulation is thus the most likely cause of the increased hydrolysis of irinotecan in Oatp1a/1b(-/-) mice. That the increase in Ces1c expression was a direct consequence of the functional Oatp1a/1b deficiency was strongly supported by analysis of Ces gene expression in OATP1B1- and OATP1B3-humanized transgenic derivatives of the Oatp1a/1b(-/-) mice: the highly increased liver expression of Ces1c, but also of Ces1b, Ces1d and Ces1e, were markedly reduced, for Ces1c and Ces1d to even below the level of expression seen