Questions:

Tyrosine kinase inhibitors (TKI) are effective in treating malignant disorders and were suggested to also have an impact on non-malignant diseases such as rheumatoid arthritis (RA). To exert their effect hydrophilic TKI are 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 this process for TKI delivery in inflammatory diseases and its pathology induced regulation at the example of the treatment of RA with the SRC kinase inhibitor Saracatinib. Since Saracatinib is an organic cation and fibroblasts are major players in RA-development, we focused on its interaction with transporters for organic cations in human RA synovial fibroblasts (hRASF).

Methods:

Saracatinib transport was investigated in transporter-transfected HEK293 cells and hRASF by HPLC detection of Saracatinib accumulation. The anti-proliferative effect of Saracatinib on PDGF stimulated hRASF was quantified by cell counting. Saracatinib transport under disease-relevant conditions like an altered pH milieu, following stimulation with TNF-a and in the presence of specific transporter inhibitors was quantified by HPLC. Gene expression analysis for organic cation transporters (OCTs) with and without TNF-a was performed using quantitative RT-PCR.

Results:

Saracatinib demonstrated concentration dependent inhibition of hRASF cell proliferation at sub-micromolar levels. In transfection experiments, hOCTN1 (organic cation transporter, novel, type 1) showed the highest apparent affinity (IC50 = 72 nM) for Saracatinib among the OCTs. Experiments quantifying the Saracatinib uptake in the presence of specific inhibitors identified hOCTN1 to mediate the uptake in hRASF. This uptake was significantly reduced by an acidic extracellular pH as present in the inflamed joints of patients with rheumatoid arthritis. The stimulation with TNF-a enhanced Saracatinib uptake in hRASF by increasing hOCTN1-mRNA expression.

Conclusion:

We conclude that Saracatinib uptake is mediated by hOCTN1 and is regulated by the pH alterations and cytokine level (TNF-a) as present in RA (supported by IMF of the Medical Faculty Münster, BE121009).