Can “specific” OCT1 inhibitors be used to determine OCT1 transporter activity toward imatinib?

To the editor:

The human organic cation transporter (OCT1) has been implicated in the uptake of imatinib into chronic myeloid leukemia (CML) cells.^1-4  Moreover, OCT1 expression or activity is thought to be a critical determinant of imatinib response and is considered to predict the long-term outcome of chronic-phase CML.^5-7  Assessment of OCT1 activity was derived from the intracellular uptake and retention (IUR) of imatinib with and without inhibitors such as prazosin or amantadine.^1,3  These functional OCT1 activity assays, as originally devised by Thomas et al and later on also used by White and colleagues, solely rely on transporter inhibition.^1,3  For the correct interpretation of these measurements, it is essential that this inhibitor-based assay truly reflect OCT1 transporter activity. This is a clinically relevant issue in view of the ongoing debate on how to identify patients with chronic-phase CML whose disease is likely to respond poorly to imatinib. White and Hughes argue that functional OCT1 activity is the best classifier for stratification, whereas others consider the BCR-ABL transcript levels to be the superior prognostic marker.⁸ 

In a recent paper in Blood, Giannoudis et al provided evidence that OCT1 M420del modifies clinical outcome in patients with CML treated with imatinib.⁹  In vitro studies using OCT1-transfected KCL22 cells showed that M420del is associated with impaired IUR of imatinib, suggesting that patients carrying this M420del allele might do better if treated with a tyrosine kinase inhibitor that does not rely on OCT1 transporter activity. Functional OCT1 activity was determined by subtracting the uptake in the presence of amantadine, a specific OCT1 inhibitor according to the authors, from the uptake observed in unmanipulated cells. Examination of the data presented in their Figure 2 suggest that the IUR of imatinib in the absence of 500 µM of amantadine was increased by ∼50% in OCT1-transfected KCL22 cells compared with mock-transfected cells.⁹  It was assumed that this difference in imatinib uptake could be attributed to OCT1 activity. Remarkably, although mock-transfected KCL22 cells did not express detectable OCT1 mRNA and protein (Figure 4, Giannoudis et al⁹ ), a substantial IUR of imatinib was observed, which could be partially inhibited by amantadine. According to the authors (Figure 4, Giannoudis et al⁹ ), this decrease in IUR of imatinib is attributable to OCT1 activity. On the basis of these observations and our experimental data (see below), we argue that these inhibitor-based assays, designed to quantify functional OCT1 transporter activity, are in fact measuring additional, as yet unidentified, processes involved in imatinib IUR. Therefore, we have serious concerns about the use of “specific” OCT1 inhibitors to demonstrate OCT1-mediated uptake of imatinib. Using HEK293 (human embryonal kidney) cells, we show that overexpression of OCT1 resulted in a limited (∼25%) increase in imatinib IUR (Figure 1A-C). These results coincide with other imatinib uptake studies using OCT1-transfected leukemic cells or oocytes.^2,10  Furthermore, we show a dose-dependent decrease of imatinib uptake by amantadine in HEK293/OCT1 cells, but importantly, amantadine also decreased the imatinib uptake in HEK293/Neo control cells having an almost 4-log lower OCT1 expression (Figure 1D-E). Similar, even more profound results were obtained with prazosin (Figure 1F-G). Analogous inhibitory effects were also seen in the leukemic K562 cell line (Figure 1H-I). Importantly, both amantadine and prazosin decreased the IUR of imatinib independent of OCT1 expression levels.

In conclusion, we demonstrated that these inhibitors are not exclusively specific for OCT1 and, therefore, we would like to emphasize to be very cautious in the interpretation of inhibitor-based OCT1 data. In fact, our data indicate that prazosin and amantadine may also interfere with other imatinib uptake processes evidently distinct from that of OCT1-mediated IUR.