Stephanie van Hoppe

34 Chapter 2 ;Abcg2 -/- mice compared to WT mice (Table I, Fig. 3C, Fig. 4C). However, this was not the case for the two single knockout strains. This suggests that in each case the remaining efflux transporter still present at the BBB could virtually completely take over the role of the knocked out transporter. This disproportionate increase in brain accumulation of afatinib observed in the Abcg2;Abcb1a/1b -/- mice compared to the single knockout strains was similar to that found for other TKIs ( e.g. axitinib, vemurafenib, lapatinib, gefitinib, erlotinib, sunitinib, dasatinib and imatinib [21, 30, 31, 33-37]), and can be explained by relatively straightforward pharmacokinetic models [35, 38]. These indicate that, if the two transporters each have a high contribution of efflux transport relative to the background efflux at the BBB in the absence of both transporters, the effect of single transporter ablation on brain accumulation will be far less than the effect of combined ablation. An interesting implication of these models is that, next to Abcg2 and Abcb1a/1b, there can be no other efflux transporters in the BBB that are remotely as efficient in keeping afatinib out of the brain, at least under the conditions we applied. The liver is pharmacologically a highly important organ; therefore we have also analyzed the accumulation of afatinib in the liver in vivo . We show that when the ABCB1 and ABCG2 efflux transporters are knocked out, there is a significant increase in the accumulation of afatinib in the liver (Fig. 3, 4). However, these changes appear to be driven mainly by the alterations in plasma concentrations between the strains, as suggested by the liver-to-plasma ratios (Fig. 4E; note that the liver-to-plasma ratio in the WT mice at 24 h could not be established because of undetectable plasma levels of afatinib, Fig. 3E). In previous studies we have found that for some anticancer drugs (everolimus, cabazitaxel) the upregulation of plasma carboxylesterase Ces1c in the ABC transporter knockout strains canaffect thepharmacokinetics of thesedrugs [39, 40].This canoccur by strong binding of some drugs to this protein, resulting in pronounced retention of drug in plasma. If this were the case for afatinib, one would expect a substantially decreased liver-to-plasma ratio in the knockout strains, as previously observed for everolimus and cabazitaxel. As there is no indication whatsoever of such a decrease (Fig. 4E), it appears that afatinib is not substantially affected by such a potentially confounding process. Clinical trials of afatinib up till now have shown positive results in tumor regression in NSCLC patients with EGFR-mutation positive tumors. Progression-free survival in these patients is enhanced in patients without, as well as with brain metastases [41-43]. A phase III trial in patients with second-line recurrent and/or metastatic head and neck squamous cell carcinoma comparing efficacy of afatinib versus methotrexate has shown favorable results for treatment with afatinib, with a higher progression-free survival rate [44]. Thus, although afatinib has been approved for treatment of NSCLC [5, 17], it may be beneficial for other tumor types containing the relevant EGFR-mutations as well.