Organic Cation Transporter 1 mRNA Research Articles

Dysregulation of Human Hepatic Drug Transporters by Proinflammatory Cytokines.

Proinflammatory cytokines, elevated during inflammation caused by infection and/or autoimmune disorders, result in reduced clearance of drugs eliminated primarily by cytochrome P450 enzymes (CYPs). However, the effect of cytokines on hepatic drug transporter expression or activity has not been well-studied. Here, using plated human hepatocytes (PHHs; n = 3 lots), we investigated the effect of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), on the mRNA expression and activity of hepatic drug transporters. PHHs were incubated for 72 hours at their pathophysiologically relevant plasma concentrations, both individually (0.01, 0.1, 1, 10 ng/ml) or as a cocktail (i.e., when each was combined at 0.1 or 1 ng/ml). Following cytokine cocktail exposure (1 ng/ml), significant downregulation of mRNA expression of organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, sodium/taurocholate cotransporting polypeptide (NTCP), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1, multidrug resistance proteins (MRP) 2, 3, and 4 was observed. While the mRNA expression of organic anion transporter (OAT) 2 and organic cation transporter (OCT) 1 was downregulated in two lots, it was upregulated in one lot. In agreement (mostly), the 1 ng/ml cytokine cocktail reduced OATP1B1/3, OATP2B1, OAT2, OCT1, and NTCP activity by 75%, 44%, 82%, 47%, and 80%, respectively. Interestingly, upregulation of OAT2 and OCT1 mRNA in one donor did not translate into the same directional change in activity. Although significant interlot variability was observed, in general, the above effects, using individual cytokines, could be attributed to IL-1β, TNF-α, and IFN-γ. SIGNIFICANCE STATEMENT: To date, this is the first comprehensive study to investigate the effect of four major proinflammatory cytokines, both individually and as a cocktail, on the mRNA expression and activity of human hepatic drug transporters. The data obtained can be used in the future to predict transporter-mediated drug clearance changes during inflammation through physiologically based pharmacokinetic modeling and simulation.

Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10μM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8μM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

Significance of OCT1 Expression in Acute Myeloid Leukemia.

Organic cation transporter 1 (OCT1) is one of the membrane proteins in the large solute carrier (SLC) family. It participates in the transport of organic cations, i.e. nutrients, neurotransmitters, metabolites or drugs in an electrogenic manner and translocate various cationic cytostatics. Knowledge concerning the expression of drug transporters in tumor cells may help to develop cytotoxic agents that are targeted to specific tumors. OCT1 expression and its relationship to the proliferation of cancer cells, development of metastases and resistance to chemotherapy has been observed in solid tumors. There is no data concerning the significance of OCT1 expression in the clinical course and treatment results in acute myeloid leukemia (AML). The objective of the study was firstly to evaluate OCT1 mRNA expression in patients with newly diagnosed de novo AML, and secondly to compare the obtained results to the healthy control group as well as analyze them according to leukemia subtypes, CD34 expression, cytogenetic and molecular factors and treatment results. 101 patients with AML, excluding the subtype classified as M3 by French-American-British (FAB) criteria, were analyzed. The control group consisted of 26 healthy individuals. The evaluated material was bone marrow (BM). Real-time quantitative polymerase chain reaction (RQ-PCR) was used in the study as a method of evaluating OCT1 mRNA expression. The study showed a statistically significant lower expression of OCT1 mRNA in patients with AML in comparison to the control group. The level of OCT1 mRNA expression was lowest for CD34+ leukemia. No significant correlation between OCT1 mRNA expression and cytogenetic and molecular factors was observed. A significant influence of OCT1 mRNA expression on the clinical outcome of the disease was observed: patients with lower expression had higher chances of achieving complete remission (CR) and longer overall survival (OS).

Abstract 257: Evaluation of organic cation transporter 1 (OCT1, SLC22A1) as transporter for sorafenib

Abstract The multikinase inhibitor sorafenib is approved for the treatment of advanced clear-cell renal and hepatocellular carcinomas, yet, patients’ survival is prolonged for only several months (Wilhelm et al., Mol Cancer Ther 2008). The understanding of this poor efficacy remains incomplete. One reason for treatment failure may be the high interindividual variability in pharmacokinetics of the orally-given sorafenib. Membrane transporters may contribute to pharmacokinetics of sorafenib and its metabolites as recently reported for the hepatic solute carrier (SLC) organic anion transporter family member 1B1 (OATP1B1) and OATP1B3 (Zimmerman et al, Clin Cancer Res 2013). In contrast, the role of organic cation transporter 1 (OCT1), which is also a major hepatic uptake transporter (Nies et al, Handb Exp Pharmacol 2011), for cellular uptake of sorafenib is controversial (Hu et al, Clin Cancer Res 2009; Swift et al, Drug Metab Dispos 2013; Herraez et al, Hepatology 2013). We therefore sought to comprehensively elucidate the impact of OCT1 on cellular sorafenib uptake by using a combination of different in vitro cell culture models and in vivo approaches involving experiments with Oct1 knockout (-/-) mice. Firstly, we analyzed cellular accumulation of radiolabeled sorafenib by the mammalian cell line HEK293 stably expressing OCT1. Despite overexpression of a functional OCT1 transporter, cellular uptake of neither sorafenib nor its active metabolite sorafenib N-oxide was increased. Next, we studied sorafenib and sorafenib N-oxide pharmacokinetics in mice with a homozygous genetic deletion of the Oct1 transporter. The deficiency of Oct1 had no influence on plasma and hepatic sorafenib and sorafenib N-oxide concentrations. Finally, we investigated sorafenib transport into the human hepatocellular carcinoma cell lines HepG2 and HuH7. Although neither OCT1 mRNA nor protein was detected, a considerable cellular accumulation of sorafenib was observed in both cell lines. Expression profiling of a panel of 55 relevant SLC drug uptake transporters identified several potential novel candidates mediating sorafenib accumulation. We conclude from these findings that cellular uptake of sorafenib and sorafenib N-oxide is independent of OCT1 and that other, as yet unidentified, transport mechanisms are responsible for cellular sorafenib uptake. Supported by the Robert Bosch Foundation, Stuttgart, Germany and the ICEPHA Grant Tübingen-Stuttgart, Germany. Citation Format: Claudia Neul, Sharyn D. Baker, Alex Sparreboom, Elke Schaeffeler, Stefan Laufer, Matthias Schwab, Anne T. Nies. Evaluation of organic cation transporter 1 (OCT1, SLC22A1) as transporter for sorafenib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 257.

The pregnane X receptor down-regulates organic cation transporter 1 (SLC22A1) in human hepatocytes by competing for ("squelching") SRC-1 coactivator.

The organic cation transporter 1 (OCT1) transports cationic drugs into hepatocytes. The high hepatic expression of OCT1 is controlled by the HNF4α and USF transcription factors. Pregnane X receptor (PXR) mediates induction of the principal xenobiotic metabolizing enzymes and transporters in the liver. Here, we have assessed the down-regulation of OCT1 expression by PXR activation. We used primary human hepatocytes and related cell lines to measure OCT1 expression and activity, by assaying MPP(+) accumulation. Western blotting, qRT-PCR, the OCT1 promoter gene reporter constructs and chromatin immunoprecipitation assays were also used. OCT1 mRNA in human hepatocytes was down-regulated along with reduced [(3) H]MPP(+) accumulation in differentiated HepaRG cells after treatment with rifampicin. Rifampicin and hyperforin as well as the constitutively active PXR mutant T248D suppressed activity of the 1.8kb OCT1 promoter construct in gene reporter assays. Silencing of both PXR and HNF4α in HepaRG cells blocked the PXR ligand-mediated down-regulation of OCT1 expression. The mutation of HNF4α and USF1 (E-box) responsive elements reversed the PXR-mediated inhibition in gene reporter assays. Chromatin immunoprecipitation assays indicated that PXR activation sequestrates the SRC-1 coactivator from the HNF4α response element and E-box of the OCT1 promoter. Consistent with these findings, exogenous overexpression of the SRC-1, but not the PGC1α coactivator, relieved the PXR-mediated repression of OCT1 transactivation. PXR ligands reduced the HNF4α-mediated and USF-mediated transactivation of OCT1 gene expression by competing for SRC-1 and decreased delivery of a model OCT1 substrate into hepatocytes.

Organic Cation Transporter 1 (OCT1) mRNA expression in hepatocellular carcinoma as a biomarker for sorafenib treatment.

BackgroundThe polyspecific organ cation transporter 1 (OCT1) is one of the most important active influx pumps for drugs like the kinase inhibitor sorafenib. The aim of this retrospective study was the definition of the role of intratumoral OCT1 mRNA expression in hepatocellular carcinoma (HCC) as a biomarker in systemic treatment with sorafenib.MethodsOCT1 mRNA expression levels were determined in biopsies from 60 primary human HCC by real time PCR. The data was retrospectively correlated with clinical parameters.ResultsIntratumoral OCT1 mRNA expression is a significant positive prognostic factor for patients treated with sorafenib according to Cox regression analysis (HR 0.653, 95 %-CI 0.430-0.992; p = 0.046). Under treatment with sorafenib, a survival benefit could be shown using the lower quartile of intratumoral OCT1 expression as a cut-off. Macrovascular invasion (MVI) was slightly more frequent in patients with low OCT1 mRNA expression (p = 0.037). Treatment-induced AFP response was not associated with intratumoral OCT1 mRNA expression levels (p = 0.633).ConclusionsThis study indicates a promising role for intratumoral OCT1 mRNA expression as a prognostic biomarker in therapeutic algorithms in HCC. Further prospective studies are warranted on this topic.

The Synergistic Effects of Pioglitazone on the Glucose-Lowering Action of Metformin in Relation to OCT1 and Gluts m-RNA Expression in Healthy Volunteer

Objective: Organic Cation Transporter 1 (OCT1) plays a key role in the disposition of metformin in hepatocytes; a recent non-clinical study reported that the peroxisome-proliferator activated receptor γ agonist pioglitazone increased the expression of Slc22a1 (Oct1) by 3.1-fold as well as its transporting function. We therefore evaluated the effect of pioglitazone on the glucose-lowering effect of metformin in 15 human participants using the Oral Glucose Tolerance Test (OGTT) and measuring the mRNA levels of OCT1, as well as those of Glucose Transporter 1 (GLUT 1) and GLUT4, which are also important in glucose utilization, in peripheral blood cells. Research design and methods: The glucose-lowering effects of metformin were evaluated by the OGTT before and after metformin treatment on Days 1 and 2 of the study and again on Days 18 and 19 after a 14-day course of pioglitazone 30 mg/day. Differences in maximum glucose levels (ΔGmax) and the areas under the glucose-time curve during the first 60 min after glucose ingestion (ΔAUCgluc60) and the entire 180-min test (ΔAUCgluc) caused by metformin treatment were determined before and after pioglitazone administration. Results: Pioglitazone increased ΔGmax by 50.0% (P=0.021), ΔAUCgluc60 by 88.1% (P=0.020), and ΔAUCgluc by 266%. Pioglitazone did not increase OCT1 and GLUT1 mRNA levels in peripheral blood cells. Conclusion: OCT1 induction plays a limited role in the synergistic effect of pioglitazone on the glucose-lowering activity of metformin. However, this synergistic effect lasted 3 days after pioglitazone treatment ended, which warrants further clinical investigation.

Cellular Uptake of Imatinib into Leukemic Cells Is Independent of Human Organic Cation Transporter 1 (OCT1)

In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). As data are conflicting as to whether OCT1 transports imatinib and may serve as a clinical biomarker, we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake. Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from patients with imatinib-naïve CML as well as in cell lines. Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations. These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1, and therefore OCT1 is apparently not a valid biomarker for imatinib resistance.

Inter-individual Variability in OCT1 Expression and Its Relationship with OCT1 Genotype in Liver Samples from a Korean Population

To clarify inter-individual variation in the expression of organic cation transporter 1 (OCT1), the levels of OCT1 mRNA and protein from 65 human liver samples were examined by real-time PCR and Western blot analysis and were associated with OCT1 genotypes. The expression levels of OCT1 mRNA and protein in 65 liver samples of Korean origin were not normally distributed and varied by 23.6- and 15.9-fold, respectively. OCT1 mRNA expression was correlated with OCT1 protein expression with a correlation coefficient of 0.641 (p < 0.0001). However, non-genetic factors, such as age, gender, and cholestasis, were not significantly associated with OCT1 expression. When quantitative expression levels were compared in relation to OCT1 promoter SNPs, there was no significant difference in OCT1 expression levels among the -1795 GG, GA, and AA genotypes. Moreover, expression levels of OCT1 were not changed in relation to the -1756 genotypes. Inter-individual variation in OCT1 mRNA and protein expression levels in the liver did not correlate with OCT1 genotypes or non-genetic factors, such as age, gender, and cholestasis. These results suggest that genetic and non-genetic factors may not be a significant contributing factor of variations in OCT1 expression from liver samples of Korean origin.

Lamotrigine is a substrate for OCT1 in brain endothelial cells

The mechanisms that underpin the passage of lamotrigine at the blood–brain barrier to its site of action in the brain is poorly understood. Lamotrigine has been postulated to be delivered to its site of action in the brain favourably despite its physicochemical properties. The aim of this study was to investigate the transport of lamotrigine in an in-vitro model of the BBB. In this study, lamotrigine was found to have a distribution coefficient of 0 at pH 7.4 indicating that it was not highly lipophilic. Human brain endothelial cells (hCMEC/D3) were used to probe the interaction of lamotrigine with drug transporters. The uptake of lamotrigine into hCMEC/D3 cells was found to be an active process (Km=62±14μM; Vmax=385±30pmol/min/million cells). Furthermore, use of a panel of transporter inhibitors indicated that this active uptake was mediated by organic cation transporter 1 (OCT1). OCT1 mRNA and protein were shown to be expressed in hCMEC/D3 cells. KCL22 cells overexpressing OCT1 were then used to validate these findings. Lamotrigine was confirmed to be a substrate and inhibitor in OCT1-transfected KCL22 cells. A putative pharmacokinetic drug–drug interaction (DDI) between quetiapine and lamotrigine was recently reported in patients and we show here that quetiapine is a potent inhibitor of the OCT1-mediated transport of lamotrigine. This is the first time that a specific influx transporter has been shown to transport lamotrigine. The clinical implications of these findings with respect to the efficacy of lamotrigine and its potential for DDI require further investigation.

Rifampin Enhances the Glucose-Lowering Effect of Metformin and Increases OCT1 mRNA Levels in Healthy Participants

We evaluated the effect of the pregnane X receptor (PXR) agonist rifampin on metformin pharmacokinetics, organic cation transporter 1 (OCT1) and OCT2 mRNA levels, and glucose levels, using the oral glucose tolerance test (OGTT) in 16 healthy subjects. The glucose-lowering effects of metformin were evaluated by OGTT before and after metformin treatment on days 1 and 2 and again on days 13 and 14 after a 10-day course of rifampin. Rifampin increased the difference in maximum glucose levels (ΔG(max)) by 41.9% (P = 0.024) and the area under the concentration-time curve (AUC) during the first 60min after glucose ingestion (ΔAUC(gluc60)) by 54.5% (P = 0.020). Renal clearance (CL(R)) of metformin was increased by 16% (P = 0.008), but the systemic exposure was only slightly increased (13%, P = 0.049), possibly because of increased absorption. Rifampin increased OCT1 mRNA levels 4.1-fold in peripheral blood cells (P = 0.001); however, OCT2 mRNA was not detected. Our results suggest that rifampin increases OCT1 expression and hepatic uptake of metformin, leading to enhanced glucose-lowering action.

Abstract 3538: An atomic absorption spectroscopy assay (AASA) as a surrogate method to measure imatinib (IM) uptake via the organic cation transporter 1 (OCT1)

Abstract Background. In chronic myeloid leukaemia (CML) 15 to 30% of patients (pts) treated with the ABL specific tyrosine kinase inhibitor IM may develop resistance. In these pts clonal evolution, amplification of the BCR-ABL gene or kinase domain mutations have been identified as underlying mechanisms. Additionally, pharmacokinetic alterations leading to reduced intracellular imatinib levels may also exist. IM is transported into the cell via the OCT1. The activity of this transporter could play a pivotal role for the intracellular IM concentration. Until now, uptake efficacy of IM is primarily measured by using radioactive labelled IM. Due to the limitations of these methods, we tried to establish alternative methods to measure the activity of OCT1 using cellular OCT1 dependent oxaliplatin (OX) uptake as a surrogate. Materials &amp; Methods. To quantitatively analyse OX uptake in human cell lines an AASA was established. Conversely, radioactive IM uptake was measured by scintillation analysis. We compared the uptake of OX and IM in different human cell lines, including JURKAT, Molt-4, K-562, KU-812, Lama-87, KCL-22 and OCI-AML-2. Ten different transport inhibitors were used to block IM or OX uptake, respectively. Furthermore, we investigated the expression levels of different influx (OCT1, OCT2, OCT3) and efflux transporters (ABCB1, ABCC1, ABCG2) in those cell lines. Results. IM and OX both were taken up linearly into cells within a range from 0,4 to 2 µM (IM) or 1 to 10 µM (OX). 150 mM verapamil and 100 µM prazosin could effectively inhibit cellular uptake of both drugs (21%±8% and 27%±8%). However, a differential uptake pattern between OX and IM was observed in relation to the different cell lines. In contrast, we could show that 15 µM citalopram inhibited the transport of 5 µM OX in a similar type as the uptake of 1-2 µM IM into all cell lines tested yet (1,14±0,15*). In this regard citalopram may be most useful for further studies on OCT1 activity. Using real-time PCR, an adequate expression of OCT1 mRNA could be detected in all cell lines tested, albeit the levels differed significantly from 150 to 27.000 fold less than in the internal control (ß-actin). However, the mRNA expression level of OCT1 does not seem to be a predictor for IM or OX uptake. The expression pattern of the efflux transporters showed no significant distribution, too. Conclusions. Here, we demonstrate the feasibility and accuracy of AASA testing of oxaliplatin as a surrogate marker for IM uptake via OCT1. As this methodology is secure, fast and sensitive, this approach may substitute conventional testing of OCT1 activity by radio labelled IM. On a clinical level, this procedure could be helpful as a clinical tool to test IM pharmacokinetics in CML patients. However, patient sample derived data will be needed to further evaluate this approach. * 1 would stand for 100% equality of uptake of imatinib or oxaliplatin Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3538.

Chronic Myeloid Leukemia CD34+ cells have reduced uptake of imatinib due to low OCT-1 Activity

Active influx of imatinib in chronic myeloid leukemia (CML) cells is mediated by the organic cation transporter 1 (OCT-1). Functional activity of OCT-1 (OCT-1 Activity) in mononuclear cells is an excellent predictor of molecular response over the first 24 months of imatinib therapy for chronic phase patients. CML progenitor cells are less sensitive to imatinib-induced apoptosis and are likely contributors to disease persistence. We investigated whether alterations in the expression and function of OCT-1 have a role in imatinib resistance in progenitors. We found the intracellular uptake and retention (IUR) of imatinib, OCT-1 Activity and OCT-1 mRNA expression are all significantly lower in CML CD34+ cells compared with mature CD34- cells (P<0.001). However, no differences in IUR or OCT-1 Activity were observed between these subsets in healthy donors. In contrast to OCT-1, ABCB1 and ABCG2 seemed to have no functional role in the transport of imatinib in CML CD34+ cells. Consistent with the observation that nilotinib uptake is not OCT-1 dependent, the IUR of nilotinib did not differ between CML CD34+ and CD34- cells. These results indicate that low imatinib accumulation in primitive CML cells, mediated through reduced OCT-1 Activity may be a critical determinant of long-term disease persistence.

Reduced Activity of the OCT-1 Protein in Primitive CML Cells: A Likely Determinant of Stem Cell Resistance in Imatinib Treated CML Patients

Despite cytogenetic and molecular remissions, residual chronic myeloid leukemia (CML) cells persist in the primitive CD34+ compartment in the majority of imatinib treated patients. It has been demonstrated that CD34+ CML cells have a reduced sensitivity to imatinib induced apoptosis. Factors which may contribute to this reduced sensitivity are reduced dependence on BCR-ABL, an increase in BCR-ABL transcripts, increased expression of efflux proteins or decreased expression of drug influx proteins. Our previous studies show that a patient's intrinsic sensitivity to imatinib-induced kinase inhibition (IC50) is related to the intracellular uptake and retention (IUR) of imatinib in peripheral blood mononuclear cells. The organic cation transporter 1 (OCT-1) is the major active influx transporter for imatinib in these cells, and the functional activity of this protein (determined using functional inhibition of OCT-1, in the IUR assay) directly correlates with molecular response to imatinib. In the present study we investigated the role that OCT-1 plays in stem cell resistance to imatinib.Primitive CD34+ and mature CD34− cells were isolated from CML patients and normal individuals using magnetic cell sorting. CML CD34+ cells had a significantly lower IURimatinib than that of CML CD34− cells (Table 1). The addition of the OCT-1 inhibitor, prazosin (100μM), eliminated this difference in IUR (Table 1), indicating that variation in IURimatinib is due to variation in the functional activity of the OCT-1 protein. In addition, the OCT-1 Activity (Table 1) and OCT-1 mRNA expression (expressed as % of BCR: mean CD34+=0.25; CD34−=4.9, p=0.040, n=10) was significantly lower in CML CD34+ cells compared with CML CD34− cells. These differences in IURimatinib and OCT-1 activity between CD34+ and CD34− cells were not observed in normal individuals (Table 1), suggesting this phenomenon is specific to leukemic cells. Furthermore, we isolated the more primitive compartment, CD34+38− cells and less primitive CD34+38+ cells in 4 CML patients. The CD34+38− cells demonstrated a 13% reduction in IURimatinib and a 41% reduction in OCT-1 activity compared with CD34+38+ cells. These data suggest a reduced IUR mediated by low OCT-1 function and/or expression may play a role in the resistance of CML stem cells to imatinib.Table 1: The IUR of 2μM imatinib expressed as ng/200,000 cells (standard deviation)nImatinib IURP value+ PrazosinP valueOCT-1 ActivityP valueCML CD34+1416.05 (4.53)0.00213.02 (3.34)0.5053.25 (2.32)<0.001CML CD34−1427.28 (12.92)14.76 (5.25)14.01 (12.08)Normal CD34+1111.13 (2.66)0.21210.43 (3.80)0.7432.03 (2.09)0.693Normal CD34−1113.92 (5.32)10.93 (3.30)3.52 (5.24)Increased expression of efflux transporters of imatinib (i.e. ABCB1 and ABCG2) has been suggested as an important mechanism for drug resistance. The effect of an ABCB1 inhibitor (PSC833) and ABCG2 inhibitor (Ko143) was assessed in CD34+ cells from 3 CML patients, using the IUR assay. Neither of these drugs had any effect on the IURimatinib in CML CD34+ cells. Additionally the mRNA expression of ABCB1 did not differ between CML CD34+ and CD34− cells (expressed as a % of BCR: mean CD34+=33.7; CD34−=33.77, p=0.064, n=10). These data suggest that alterations in imatinib influx (via OCT-1) are more critical for development of stem cell resistance rather than differences in efflux.We have previously demonstrated that, unlike imatinib, the OCT-1 protein is not involved in nilotinib transport, as the addition of OCT-1 inhibitors does not alter IURnilotinib in patients. Assessing the IUR of nilotinib in CD34+ and CD34− CML cells reveals no significant difference between the two populations (Table 2). Additionally, the IURnilotinib is significantly higher than IURimatinib in CML CD34+ cells (Table 2). In summary, the reduced OCT-1 mediated uptake of imatinib in more primitive, CD34+ CML cells may result in inadequate kinase inhibition and contribute to stem cell resistance in CML. Since nilotinib uptake into CML CD34+ cells is not impaired in the same manner as imatinib, more substantial depletion of the primitive CML cells may be achieved.Table 2: The IUR of 2μM imatinib and nilotinib in the same 11 CML patients. Expressed as ng/200,000 cells (standard deviation)nImatinib IURP valueNilotinib IURP valueP value between imatinib & nilotinib IURCML CD34+1117.80 (5.73)0.00626.35 (7.54)0.2300.007CML CD34−1130.28 (12.33)22.05 (8.68)0.076