Nilotinib efflux and resistance development: the effects of combination and concomitant therapies on the transport and efficacy of Nilotinib.

Abstract

Chronic myeloid leukaemia (CML) is characterised by the presence of Bcr-Abl tyrosine kinase. Tyrosine kinase inhibitors (TKIs), such as imatinib, and more recently nilotinib and dasatinib, act by specifically binding to the Bcr-Abl kinase domain. The advent of TKIs resulted in significantly improved treatment outcomes for the majority of patients with CML. However, the focus is now customised treatment regimes employing drug combinations to reduce resistance development and maximise treatment outcomes. The present study investigated the interaction of nilotinib with efflux transporters and 1) assessed how concomitant administration of additional drugs may enhance the effects of nilotinib in patients and 2) how altered expression or inhibition of these transporters affected nilotinib transport and function. Secondly, in vitro cell line models of nilotinib resistance were generated in order to replicate modes of nilotinib resistance in vivo. The reported relationship between nilotinib and efflux transporters ABCB1 and ABCG2 is conflicting and nilotinib has previously been reported to inhibit the function of OCT-1. Thus, in order to resolve conjecture, a novel approach was employed to determine the effect of ABCB1/ABCG2 inhibition on nilotinib-mediated Bcr-Abl kinase inhibition. Results demonstrated ABCB1-mediated nilotinib transport was concentration dependent: transport of nilotinib occurred at low concentrations whereas inhibition of both ABCB1 and ABCG2 occurred at high nilotinib concentrations. Additionally, data demonstrated nilotinib had no inhibitory effect on the functional activity of OCT-1 but may reduce intracellular imatinib concentrations by impairing passive influx. Bcr-Abl dependent modes of resistance relating to kinase domain mutations and Bcr-Abl overexpression are well documented. The mechanisms underlying Lyn-mediated resistance however, require further investigation and Bcr-Abl-independent resistance is even more poorly understood. Accordingly, in vitro cell line models of nilotinib resistance were developed. ABCB1 overexpression was consistently demonstrated as the initiator of nilotinib resistance in all cell lines, however, both Bcr-Abl dependent and Bcr-Abl independent resistance mechanisms were subsequently observed. These results suggest determination of ABCB1 expression levels at diagnosis and 3 months post-therapy, for example, may predict resistance in patients. Furthermore, this is the first reported nilotinib resistant, genuine Bcr-Abl independent cell line model and may provide insight into unexplained TKI resistance observed in patients. Additionally, both nilotinib resistant cell lines demonstrated ABCC6 overexpression suggesting this transporter may play a role in nilotinib resistance in vitro. Further investigation in patient mononuclear cells confirmed nilotinib as a likely substrate of ABCC6. This is the first report of ABCC6 involvement in nilotinib transport and concomitant administration of ABCC6 inhibitors may present an attractive option to enhance TKI efficacy and prevent resistance. Findings detailed in this thesis may assist in developing new therapeutic strategies using TKIs in combination with other medications in order to enhance the intracellular concentrations of TKI. Additionally, further insight into the modes of resistance to nilotinib, as well as the kinetics of resistance emergence, may assist in identifying patients at risk of developing resistance to TKIs. Finally, ABCB1/ABCC6 mRNA expression levels in de novo CML patients at diagnosis may present a novel technique for predicting response to nilotinib at 12 months.