Transport Mechanisms and Their Pathology-Induced Regulation Govern Tyrosine Kinase Inhibitor Delivery in Rheumatoid Arthritis

BackgroundTyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions.
Methodology/Principal FindingsThe transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein.
Conclusion/SignificanceThe regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.

背景 酪氨酸激酶抑制剂（Tyrosine kinase inhibitors, TKIs）是治疗恶性疾病的有效手段，近期研究表明其对非恶性疾病亦具有潜在影响。然而在类风湿关节炎（rheumatoid arthritis, RA）等部分炎性疾病中，其体内效应似乎较为温和。由于多数TKIs可通过细胞膜转运蛋白被细胞主动摄取，本研究旨在评估此类转运蛋白在甲磺酸伊马替尼（Imatinib mesylates）在类风湿关节炎滑膜成纤维细胞中的蓄积过程中所发挥的作用，以及炎性条件下该类转运蛋白的调控机制。
方法与主要结果 本研究在转染了转运蛋白的HEK293细胞以及人类风湿关节炎滑膜成纤维细胞（human RA synovial fibroblasts, hRASF）中，探究了伊马替尼的转运与蓄积过程。通过实时定量聚合酶链反应（qRT-PCR）对转运蛋白的表达水平进行定量分析。转染实验结果显示，在所有已知的伊马替尼转运蛋白中，hMATE1对伊马替尼表现出最高的表观亲和力。通过添加特异性转运蛋白抑制剂以及利用小干扰RNA（siRNA）敲低hMATE1的表达后检测伊马替尼摄取量的实验，证实hMATE1确实介导了hRASF内伊马替尼的转运过程。通过细胞计数法量化了伊马替尼对血小板衍生生长因子（PDGF）刺激后的hRASF的抗增殖效应，该效应与hMATE1的摄取活性呈直接正相关。通过蛋白质印迹（Western blot）与免疫荧光技术检测了hMATE1的表达情况。此外，本研究还通过高效液相色谱法（HPLC）探究了疾病相关条件下（如细胞外pH改变以及不同细胞因子刺激后）伊马替尼的转运情况。结果发现，酸性细胞外pH以及TNFα、IL-1β、IL-6这三种细胞因子均可显著降低伊马替尼的摄取量，同时这三种因子均可下调hMATE1的mRNA与蛋白表达水平。
结论与意义 本研究表明，hMATE1介导的伊马替尼摄取调控及其对hRASF增殖的影响，或可解释伊马替尼在类风湿关节炎体内效应较为温和的原因。此外，本研究结果提示，针对正常与病理状态下转运蛋白介导的药物加工过程开展研究，对于开发用于炎性疾病的细胞内作用药物具有重要意义。