Low OCT-1 Activity Research Articles

Enhancing the Functional Activity of the OCT-1 Influx Pump May Overcome the Negative Impact of Low OCT-1 Activity in Imatinib Treated CML Patients

The human organic cation transporter-1 (hOCT-1) is the major active influx protein responsible for the transport of imatinib into blood cells 1,2. The functional activity of the OCT-1 protein is defined as the intracellular uptake and retention (IUR) of 14-C labelled imatinib into patient pre therapy mononuclear blood cells over a two hour period, which is inhibited by OCT-1 inhibitors such as prazosin or procainamide. The level of OCT-1 activity is a key determinant of the interpatient variation observed in intrinsic sensitivity to imatinib induced kinase inhibition (IC50imatinib3). We have previously demonstrated that a significantly greater proportion of de-novo CML patients with high functional activity of OCT-1, achieve a major molecular response (MMR: 3 log reduction in BCR-ABL mRNA from standardised baseline) when treated with imatinib, than patients with low OCT-1 Activity 4. We have also identified a link between dose and OCT-1 Activity, demonstrating that the negative impact of low OCT-1 Activity could be overcome to a variable extent by imatinib dose increase. However, not all patients can dose increase, largely because of tolerability issues. While the transport of second-generation ABL-kinase inhibitors (nilotinib and dasatinib5) is not OCT-1 mediated, the long term effect of these drugs is not yet known. Hence, we sought to identify strategies to increase OCT-1-mediated imatinib uptake. We queried the drug gene expression signatures in version 1 of the Connectivity Map (CMAP; Lamb J, Nat. Rev. Cancer 7; 54–60, 2007: http://www.broad.mit.edu/cmap) with 3 transporters including OCT-1. This identified the Rho kinase inhibitor fasudil and COX-2 inhibitor / celecoxib analogue LM1685 as potential up-regulators of OCT-1 mRNA. The impact of these drugs on OCT-1 mRNA expression and IC50imatinib (fasudil alone to date) has been analysed in two bcr-abl positive cell lines (K562 and KU812). The effect of these two candidate OCT-1 enhancers on OCT-1-mediated imatinib uptake was also assessed in 10 newly diagnosed chronic phase CML patients, previously demonstrated to have low OCT-1 Activity (4 with no demonstrable OCT-1 Activity), using the IUR assay.Table 1: Assessing the effects of fasudil and LM1685 on the intracellular transport of imatinib. These data demonstrate a statistically significant increase in OCT-1 Activity with LM1685, and show a strong trend towards significance with fasudil. Importantly, we show that patients with no demonstrable OCT-1 Activity (0ng/200,000 cells) have detectable Activity in the presence of both fasudil (Range 1.5 to 2ng/200,000 cells) and LM1685 (Range 1.5 to 4.5 ng/200,000 cells). We have previously demonstrated that patients with no detectable OCT-1 Activity universally fail to achieve imatinib therapeutic response milestones (imatinib failure), whereas 54% of patients with low, but detectable OCT-1 Activity achieve these milestones4. The ability to enhance the functional activity of the OCT-1 protein may therefore be of significant clinical relevance in this group. In addition we demonstrate an increase in imatinib IUR which, along with the increase in OCT-1 Activity, is likely associated with increased OCT-1 mRNA levels. Significantly, in the two CML cell lines tested we show a marked reduction in the IC50imatinib indicating that the observed increase in IUR and OCT-1 Activity translates to an increase in the kinase inhibitory activity of imatinib. Preliminary analysis in one patient analysed to date also indicates a reduction in IC50 from 0.48 to 0.35μM in the presence of fasudil. In the clinical scenario the use of such OCT-1 enhancers may improve the response of some imatinib treated patients to both standard and increased dose imatinib. Importantly, these findings validate the use of resources such as C-MAP to identify candidate drugs that may mediate desired changes in the levels of key proteins resulting in improved response to therapy.Fasudil (10μM) %increase from controlLM1685 (1μM) % increase from controlIUR of imatinibOCT-1 ActivityIC50imatinibIUR of imatinibOCT-1 ActivityK56276% (n=5)163% (n=5)51% reduction (n=3)41% (n=2)122% (n=2)KU81210.7% (n=2)75% (n=2)15% reduction (n=2)NANACML patients n=108%89%9%114%p value>0.050.08>0.050.03

Dasatinib Cellular Uptake and Efflux in Chronic Myeloid Leukemia Cells: Therapeutic Implications

The organic cation transporter OCT-1 mediates active transport of imatinib. We recently showed that low OCT-1 activity is a major contributor to suboptimal response in chronic myeloid leukemia (CML) patients treated with imatinib. The relevance of OCT-1 activity and efflux pumps in determining intracellular uptake and retention (IUR) of dasatinib was assessed. The effect of OCT inhibitors on [14C]dasatinib and [14C]imatinib IUR was compared using peripheral blood mononuclear cells from newly diagnosed CML patients. The role of efflux transporters was studied using ABCB1- and ABCG2-overexpressing cell lines and relevant inhibitors. Unlike imatinib, there was no significant difference in the dasatinib IUR at 37 degrees C and 4 degrees C (P = 0.8), and OCT-1 inhibitors including prazosin did not reduce dasatinib IUR significantly. In CML mononuclear cells, prazosin inhibitable IUR was significantly higher for imatinib than dasatinib (6.38 versus 1.48 ng/200,000 cells; P = 0.002; n = 11). Patients with high OCT-1 activity based on their imatinib uptake had IC50(dasatinib) values equivalent to patients with low OCT-1 activity. Dasatinib IUR was significantly lower in ABCB1-overexpressing cell lines compared with parental cell lines (P < 0.05). PSC833 (ABCB1 inhibitor) significantly increased the dasatinib IUR (P < 0.05) and reduced IC50(dasatinib) (from 100 to 8 nmol/L) in K562-DOX cell line. The ABCG2 inhibitor Ko143 significantly increased dasatinib IUR in ABCG2-overexpressing cell lines and reduced IC(50)(dasatinib). Unlike imatinib, dasatinib cellular uptake is not significantly affected by OCT-1 activity, so that expression and function of OCT-1 is unlikely to affect response to dasatinib. Dasatinib is a substrate of both efflux proteins, ABCB1 and ABCG2.

Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: higher doses of imatinib may overcome the negative impact of low OCT-1 activity

AbstractInterpatient variability in intracellular uptake and retention (IUR) of imatinib may be due to variable function of the OCT-1 influx pump. OCT-1 activity was measured in pretherapy blood from chronic myeloid leukemia (CML) patients by calculating the difference in IUR of [14C]-imatinib with and without OCT-1 inhibition. Of patients with higher than median (high) OCT-1 activity, 85% achieved major molecular response (MMR) by 24 months, versus 45% with no more than a median (low) OCT-1 activity. Assessing patients receiving 600 mg imatinib per day and those averaging fewer than 600 mg over 12 months of therapy revealed patients with high OCT-1 activity achieved excellent molecular response regardless of dose, whereas response of patients with low OCT-1 activity was highly dose dependent. Of patients with low OCT-1 activity who received fewer than 600 mg, 45% failed to achieve a 2-log reduction by 12 months, and 82% failed to achieve a MMR by 18 months, compared with 8% and 17% in the cohort with high OCT-1 activity and dose less than 600 mg/day (P = .017 and P = .022). OCT-1 activity is an important determinant of molecular response to imatinib, with predictive value closely linked to dose. This pretherapy assay identifies patients at greatest risk of suboptimal response where dose intensity is critical, and those likely to respond equally well to standard dose imatinib.

In Contrast to Imatinib, OCT-1 Mediated Influx Has Minimal Impact on Cellular Uptake of Dasatinib in CML Patients at Diagnosis.

Molecular response to imatinib in newly diagnosed CML patients correlates with intrinsic sensitivity to imatinib (IC50imatinib) which is related to imatinib intracellular uptake and retention (IUR). Imatinib cellular uptake is OCT-1 dependent and low OCT-1 activity in newly diagnosed CML patients is a major contributor to suboptimal response to imatinib. The cellular transport pathways for dasatinib, a more potent BCR-ABL kinase inhibitor, are yet to be defined. We have assessed dasatinib cellular transport pathways using mononuclear cells (MNCs) from newly diagnosed CML patients and cell lines. Dasatinib IUR was determined with 14C-labelled dasatinib (Bristol-Myers Squibb) at 4°C and 37°C, with/without influx/efflux inhibitors. There was no significant difference in dasatinib IUR at 37°C and 4°C in KU812 cell line (27 ng vs. 26 ng) and MNCs of CML patients (17 ng vs. 17 ng, n=10), indicating cellular uptake was not temperature dependent and most likely passive. Prazosin and progesterone (OCT-1 and OCT-3 inhibitors) did not significantly reduce IUR and did not reduce inter-patient variation in dasatinib IUR in contrast to imatinib IUR (P=0.45 and P=0.93 respectively, Fig. 1). Procainamide (OCT-1 and OCT-2 inhibitor), nicotinamide (OCT-2) and corticosterone (OCT-3 inhibitor) reduced dasatinib IUR but this did not reach significance (P=0.42, 0.74, 0.79 respectively). These data suggest that dasatinib uptake is not OCT dependent. Using ABCB1 overexpressing cell lines, we found that dasatinib IUR is lower in K562-Dox (P<0.001) and VBL-100 (P=0.05) compared to parental cell lines (K562 and CCRF-CEM respectively). PSC-833 (ABCB1 inhibitor, Novartis) significantly increased dasatinib IUR (P<0.0001), reduced IC50dasatinib in K562-Dox cell line (100 nM vs. 16 nM) and increased IUR in VBL-100 (P=0.01) suggesting ABCB1 actively efflux dasatinib. Median IC50dasatinib in 18 newly diagnosed CML patients was 2.20 nM (± 0.8), which is significantly below median plasma concentration achieved (100–200 nM). Interpatient variability in IC50dasatinib and IUR is therefore unlikely to be clinically significant for newly diagnosed CML patients receiving dasatinib. In conclusion1.dasatinib cellular uptake is predominantly passive and may be OCT independent.2.dasatinib is effluxed actively by ABCB1 in cell lines over-expressing these proteins.3.Inter-patient variation in dasatinib intracellular uptake and intrinsic sensitivity may not be clinically significant in newly diagnosed CML patients receiving dasatinib, in contrast to recipients of imatinib. [Display omitted]

Molecular Response to Imatinib Is Dependent on Dose in CML Patients with Low OCT-1 Influx Activity. Patients with High Activity May Respond Equally Well to Standard or Increased Dose Imatinib.

We have shown that IC50 is predictive of molecular response in newly diagnosed CML patients treated with 600mg of imatinib (TIDEL). OCT-1 is an influx transporter for imatinib and key determinant of interpatient variation in the IC50 assay. Using 14C imatinib we have performed an intracellular uptake and retention (IUR) assay and RQ-PCR for OCT-1 mRNA expression on blood cells from 42 patients at diagnosis, enrolled to TIDEL. Using the OCT-1 inhibitor Prazosin, we determined OCT-1 in-vitro activity to be the difference in IUR between assays performed with and without Prazosin. The median OCT-1 activity was 6.1ng (range of -3.6 to 27ng). Grouping patients about the median, into low and high OCT-1activity, we found a significant difference between the two groups in molecular response.Log Reduction BCR-ABL based on OCT-1 ActivityMonths6M12M18M24MLow OCT-1 Activity n=191.92.42.62.5High OCT-1 Activity n=232.072.93.63.8p value0.050.050.0030.007Low OCT-1 Activity<600mg n=101.41.71.72.4600mg n=92.72.62.93.6p value0.010.0230.0460.042High OCT-1 Activity<600mg n=112.42.93.33.7600mg n=122.42.83.33.9p value0.630.690.820.66Dividing groups into patients receiving 600mg imatinib and those receiving <600mg over the first 12 months of therapy revealed patients with high OCT-1 activity generally achieved excellent molecular response regardless of dose, whereas the response of patients with low OCT-1 activity was highly dose dependent. There was a significant difference in molecular response between the low and high OCT-1 activity groups on <600 mg (p=0.004 at 24 months) but no difference in the 600mg cohort, further indicating dose sensitivity of low OCT-1 activity patients. The level of OCT-1 mRNA (n=30) as determined by RQ-PCR correlated with the in-vitro OCT-1 activity (r=0.381 p=0.04). However, in this smaller cohort, OCT-1 mRNA level was not predictive of molecular response (low OCT-1 3.5 log(n=15); high OCT-1 3.9 log(n=16) p=0.169). The IC50 is predictive of molecular response to 12 months (low IC50 log reduction 3.0 (n=20) high IC50 2.5 (n=15) p=0.02), but not in this smaller cohort to 24 months (p=0.15). TIDEL mandated dose increase to 800mg where tolerable. We assessed the log reduction from 12 to 24 months based on dose.Log reduction from 12 to 24 months.Dose increaseYesNop valueLow OCT-1 activity0.33 n=120 n=70.002High OCT-1 activity0.59 n=140.46 n=90.637p value0.7030.034There was no statistical difference between the low and high OCT-1 activity groups when dose was escalated, however with constant dose, patients with low OCT-1 activity performed less well (Table 2). We conclude that OCT-1 influx activity is an important determinant of molecular response to imatinib, but its predictive value is closely linked to the dose received. This functional assay of OCT-1 activity performed at diagnosis may identify patients most likely to benefit from higher doses of imatinib or second generation ABL kinase inhibitors, and a cohort who are likely to respond well to standard dose imatinib.