Background/Purpose: 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 (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 human RA synovial fibroblasts (hRASF).

Methods: Saracatinib transport was investigated in OCT-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-α and in the presence of specific transporter inhibitors was quantified by HPLC. Gene expression analysis for OCTs with and without TNF-α 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 as mediator of 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-α enhanced Saracatinib uptake in hRASF by increasing hOCTN1-mRNA expression.

Conclusion: The uptake of Saracatinib in hRASF via hOCTN1 is regulated by the pH and RA associated pro-inflammatory cytokines. pH alterations and cytokine levels (TNF-α) as present in RA enhance the effective Saracatinib concentration in hRASF. Our results suggest that investigating transporter mediated drug processing is important for developing intracellularly acting drugs used in inflammatory diseases (supported by IMF of the Medical Faculty Münster, BE121009).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transporters-as-drug-gateway-into-the-cell-for-specific-targeting-of-tyrosine-kinase-signaling-pathway-in-rheumatoid-arthritis/