Abstract 4425: Characterisation of cellular transport and accumulation of six clinically approved tyrosine kinase inhibitors (TKIs) in colon cancer cells.
Authors: Honeywell Richard J. , Hitzerd Sarina , Kathmann Ietje , Giovannetti Elisa , Verheul Henk , Peters Frits 
Published: August 2014

TKIs are a loosely characterised family of small molecules which inhibit the ATP driven phosphorylation of signalling proteins that normally activate transduction cascades. Aberrations in signal cascades have been linked to the development of tumours and their survival pathways; TKIs are targeted at these pathways. However, promising preclinical studies on structurally different TKIs often did not translate into clinical success due to cellular resistance, cascade mutations and poor bioavailability. TKIs tend to be alkali in nature with a correspondingly high pKa (5-8) while absorption properties have been linked to both ABC and Organic Cation Transporters (OCT). Previously we demonstrated in a gut epithelium model that absorption of TKIs is relatively poor, while a correspondingly high negative flow was observed. In this study we aimed to characterize tumour cell uptake and accumulation of TKIs using specific transporter inhibitors. We used a sensitive and specific LC-MS-MS method to characterize the role of the transport inhibitors Ko143 (BCRP inhibitor), amantadine (Am;hOCT1&2), cimetidine (Ci;hOCT1&2&3), desipramine (Ds;hOCT1&2&3), β-estradiol (Be; hOCT1&3) and verapamil (Ve; hOCT1&P-gp).

Uptake of TKIs at 4°C and 37°C in CaCo2 and WiDr colon cancer cells showed large differences; uptake of Gefitinib (G - 10μM), Erlotinib (E - 10 μM) & Dasatinib (D - 1μM) was partially active since after 2hr uptake at 4°C was lower compared to 37°C. Uptake of Sunitinib (Su - 10μM), Sorafenib (So - 10 μM) and Crizotinib (Cr - 10 μM) was predominantly passive since values were comparable at 4°C and 37°C. Absolute accumulation over 24hr (37°C) was very different with E and D reaching 20-150 ng/mg protein, while G was 20 fold higher. Su, So and Cr accumulation were even higher reaching 2 to 12 μg/mg protein. This pattern matched accumulation of some TKIs observed in the clinical setting. Accumulation for Su and Cr showed a similar pattern suggesting that Cr accumulated in lysosomes similar to Su. Bafilomycin, a lysosomal inhibitor, decreased total accumulation of Su 14.1-fold, and that of Cr 2.4-fold indicating lysosomal accumulation. The hOCT inhibitor Ds consistently (25-55%) decreased accumulation of G, So Su and Cr; Ci, Be and Am had little effect on cellular accumulation for all TKIs tested. Ve, De and Ci increased accumulation of E, and Ve that of D, suggesting inhibition of efflux via P-gp, MRP or BCRP. Ko143 increased accumulation for G, E, So and Su in WiDr but only for Su in CaCo2 cells indicating a differential role for BCRP depending on TKI and tumour type. In conclusion, several transporters appear to be involved to differing degrees for each molecule, indicating TKIs pharmacokinetics should be investigated on an individual basis.

Citation Format: Richard J. Honeywell, Sarina Hitzerd, Ietje Kathmann, Elisa Giovannetti, Henk Verheul, Frits Peters. Characterisation of cellular transport and accumulation of six clinically approved tyrosine kinase inhibitors (TKIs) in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4425. doi:10.1158/1538-7445.AM2013-4425