Objective Tyrosine kinase inhibitors (TKI) are effective in treating malignant disorders and were suggested to have also an impact on non-malignant diseases such as rheumatoid arthritis (RA). To exert their effect, hydrophilic TKI have to be actively accumulated in target cells by membrane transporters, a process which is known to govern drug efficiency. This study aims to evaluate the importance of transport processes for TKI delivery in inflammatory diseases and their pathology-induced regulation at the example of the RA-treatment with the c-Abl and Src kinase inhibitor Saracatinib (AZD0530). Since Saracatinib is an organic cation and fibroblasts are major players in RA-development, we focused on its interaction with transporters for organic cations (OCTs) in synovial fibroblasts (SFs).
Methods Saracatinib transport was investigated in OCT-transfected HEK293 cells, in SF from from RA patients (RASF) and from hTNFtg mice (hTNFtg SF) using a HPLC method to quantify intracellular Saracatinib levels. The Saracatinib-transporter was identified in RASF by inhibition with specific competitors and by its siRNA-downregulation. Moreover, the impact of the transport process on the target c-Abl was determined by western blot analysis. Saracatinib transport under disease-relevant conditions like stimulation with TNFa was also assessed. Expression of OCTs in SF after TNFa stimulation was analysed using qRT-PCR.
Results Transport inhibition experiments in RASF and hTNFtg SF identified OCTN1 (organic cation transporter, novel, type 1) as mediator of the uptake process. OCTN1 transporter function governs effectiveness of saracatinib on tyrosine kinase c-Abl in RASF. The stimulation with TNFa enhanced Saracatinib uptake in RASF by increasing OCTN1 expression. Similarily, Saracatinib uptake was significantly increased in hTNFtg SF compared to wt SF.
Conclusion The uptake of Saracatinib in SF is mediated by OCTN1, whose activity is up-regulated by RA associated pro-inflammatory cytokines, such as TNFa. For these reasons, Saracatinib seems to be a suitable TKI for treatment of RA. Furthermore, our results show that investigating transporter mediated drug processing is important for developing intracellularly acting drugs used in inflammatory diseases.