Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis

TY - JOUR

T1 - Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis

AU - Schmidt-Lauber, Christian

AU - Harrach, Saliha

AU - Pap, Thomas

AU - Fischer, Meike

AU - Victor, Marion

AU - Heitzmann, Marianne

AU - Hansen, Uwe

AU - Fobker, Manfred

AU - Brand, Stefan-Martin

AU - Sindic, Aleksandra

AU - Pavenstädt, Hermann

AU - Edemir, Bayram

AU - Schlatter, Eberhard

AU - Bertrand, Jessica

AU - Ciarimboli, Giuliano

PY - 2012

Y1 - 2012

N2 - BACKGROUND: Tyrosine 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 FINDINGS: The 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/SIGNIFICANCE: The 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.

AB - BACKGROUND: Tyrosine 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 FINDINGS: The 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/SIGNIFICANCE: The 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.

KW - Arthritis, Rheumatoid/enzymology

KW - Benzamides/pharmacology

KW - Cell Line

KW - Cell Proliferation/drug effects

KW - Chromatography, High Pressure Liquid

KW - Cytokines/pharmacology

KW - Humans

KW - Hydrogen-Ion Concentration

KW - Imatinib Mesylate

KW - Interleukin-1beta/pharmacology

KW - Interleukin-6/pharmacology

KW - Organic Cation Transport Proteins/genetics

KW - Piperazines/pharmacology

KW - Protein Kinase Inhibitors

KW - Protein-Tyrosine Kinases/antagonists & inhibitors

KW - Pyrimidines/pharmacology

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Tumor Necrosis Factor-alpha/pharmacology

U2 - 10.1371/journal.pone.0052247

DO - 10.1371/journal.pone.0052247

M3 - SCORING: Journal article

C2 - 23284953

VL - 7

SP - e52247

JO - PLOS ONE

JF - PLOS ONE

SN - 1932-6203

IS - 12

ER -