Imatinib Uptake Research Articles

Release of intracellular calcium primes chronic myeloid leukaemia cells for tyrosine kinase inhibitor-induced apoptosis

Imatinib is a substrate for hOCT1 (SLC22A1) and inhibitors of this influx transporter, such as amantadine and prazosin, have previously been shown to decrease cellular imatinib uptake. However, here we report that in longer term experiments, both drugs paradoxically increase the cytotoxicity of all three currently licensed tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib. This effect is due to release of intracellular calcium from the endoplasmic reticulum (ER), with changes in mitochondrial calcium and alterations in mitochondrial membrane permeability, resulting in caspase-mediated apoptosis. The effect is confined to BCR-ABL-positive cells, and is greater in primary cells than in cell lines. Furthermore, in primary cells at original diagnosis, the effect is only seen in samples from patients destined to become complete cytogenetic responders to imatinib. These results indicate that calcium release from the ER, here induced by amantadine or prazosin, may prime BCR-ABL-positive cells to TKI-induced apoptosis. Amantadine/prazosin primed TKI cytotoxicity in vitro may be a useful test for the level of ER-releasable calcium, and may be of prognostic value.

Metronidazole leads to enhanced uptake of imatinib in brain, liver and kidney without affecting its plasma pharmacokinetics in mice

The pharmacokinetic interaction between metronidazole, an antibiotic-antiparasitic drug used to treat anaerobic bacterial and protozoal infections, and imatinib, a CYP3A4, P-glycoprotein substrate kinase inhibitor anticancer drug, was evaluated. Male imprinting control region mice were given 50 mg/kg imatinib PO (control group) or 50 mg/kg imatinib PO, 15 min after 40 mg/kg PO metronidazole (study group). Imatinib plasma, brain, kidney and liver concentrations were measured by HPLC and non-compartmental pharmacokinetic parameters estimated. Metronidazole coadministration resulted in a double-peak imatinib disposition profile. The maximum concentration (C(max)) decreased by 38%, the area under the curve (AUC(0-∞)) decreased by 14% and the time to C(max) (T(max)) was earlier (50%) in plasma. Apparent volume of distribution (V(SS)/F) and oral clearance (Cl/F) increased by 21% and 17%, respectively. Imatinib tissue penetration was higher after metronidazole coadministration, with 1.7 and 2.1-fold AUC(0-∞) increases in liver and kidney, respectively. Metronidazole increased imatinib's tissue-to-plasma AUC(0-∞) ratio in liver from 2.29 to 4.53 and in kidney from 3.04 to 7.57, suggesting higher uptake efficiency. Brain C(max) was 3.9-fold higher than control and AUC(0-t last) was 2.3-fold greater than plasma (3.5% in control group). No tissue-plasma concentration correlation was found. Metronidazole slightly decreased imatinib systemic exposure but enhanced liver, kidney and brain penetration, probably due to metronidazole-mediated inhibition of P-glycoprotein and other efflux transporters. The high brain exposure opens possibilities for treatment of glioma and glioblastoma. Renal and hepatic functions may need to be monitored due to potential renal and hepatic toxicity.

Pharmacokinetic Impact of SLCO1A2 Polymorphisms on Imatinib Disposition in Patients With Chronic Myeloid Leukemia

The purpose of this study was to explore the role of the organic anion-transporting polypeptide (OATP) 1A2, which is encoded by SLCO1A2, in the cellular uptake of the Bcr-Abl tyrosine kinase inhibitor imatinib, and the relationship between SLCO1A2 polymorphisms and the pharmacokinetics of imatinib in patients with chronic myeloid leukemia (CML). Imatinib uptake was significantly enhanced in OATP1A2-transfected human embryonic kidney (HEK) 293 cells (P = 0.002). Naringin, an OATP1A2 inhibitor, decreased the transport of imatinib in OATP1A2-transfected HEK293 cells, the human intestinal cell line Caco-2, and K562 CML cells. Linkage disequilibrium was found between the SLCO1A2 -1105G>A and -1032G>A genotypes in 34 CML patients and 100 healthy subjects. Imatinib clearance in CML patients was influenced by the SLCO1A2 -1105G>A/-1032G>A genotype (P = 0.075) and the SLCO1A2 -361GG genotype (P = 0.005). These findings suggest that imatinib is transported into cells by OATP1A2, and that SLCO1A2 polymorphisms significantly affect imatinib pharmacokinetics.

Drug Transporters and Imatinib Treatment: Implications for Clinical Practice

Imatinib mesylate is approved for the treatment of chronic myeloid leukemia (CML) and advanced gastrointestinal stromal tumors (GIST). Unfortunately, in the course of treatment, disease progression occurs in the majority of patients with GIST. Lowered plasma trough levels of imatinib over time potentially cause disease progression, a phenomenon known as "acquired pharmacokinetic drug resistance." This outcome may be the result of an altered expression pattern or activity of drug transporters. To date, the role of both efflux transporters (ATP-binding cassette transporters, such as ABCB1 and ABCG2) and uptake transporters [solute carriers such as organic cation transporter 1 (OCT1) and organic anion transporting polypeptide 1A2 (OATP1A2)] in imatinib pharmacokinetics and pharmacodynamics has been studied. In vitro experiments show a significant role of ABCB1 and ABCG2 in cellular uptake and retention of imatinib, although pharmacokinetic and pharmacogenetic data are still scarce and contradictory. ABCB1 and ABCC1 expression was shown in GIST, whereas ABCB1, ABCG2, and OCT1 were found in mononuclear cells in CML patients. Several studies have reported a clinical relevance of tumor expression or activity of OCT1 in CML patients. Further (clinical) studies are required to quantify drug transporter expression over time in organs involved in imatinib metabolism, as well as in tumor tissue. In addition, more pharmacogenetic studies will be needed to validate associations.

Specific Drug Transporter Genotypes Are Significantly Associated with Increased Rates of Major and Complete Molecular Responses In Newly Diagnosed Chronic Myeloid Leukemia Patients Treated with Imatinib – A TOPS Correlative Substudy

AbstractAbstract 670The availability of multiple options for chronic myeloid leukemia (CML) treatment is not paralleled by the availability of biological predictors of outcome allowing to identify patients (pts) who are more likely to benefit from dasatinib or nilotinib rather than imatinib (IM). Pharmacogenetics has proven a potential source of biomarkers given the known influence of polymorphisms in key genes encoding drug transporters and metabolizing enzymes on drug delivery – hence effectiveness. In CML, only two studies had so far explored this field, but both were conducted in heterogeneous populations including pts at different stages of disease, not all receiving IM first-line. We thus aimed to investigate a panel of 20 single nucleotide polymorphisms (SNPs) in ABCB1, ABCG2, SLC22A1, OATP1A2, OCTN1, CYP3A4 and CYP3A5 genes that can be hypothesized to influence IM transport and metabolism in 189 newly diagnosed CML pts enrolled in the TOPS phase III trial (Cortes et al, J Clin Oncol 2010). Pts selection was exclusively based on availability of written informed consent and sufficient amount of archived material. Median age was 46 years; male to female ratio was 103 to 86; 156 (83%) pts were Caucasian and 23 (12%) were Asian; low, intermediate and high Sokal risk pts were 84 (44.4%), 65 (34.4%) and 40 (21.2%), respectively. Baseline demographic/clinical features did not differ significantly from those of the overall population. Treatment outcomes (complete cytogenetic response [CCyR]; major molecular response [MMR] and complete molecular response [CMR]) were compared according to i) each candidate genotype ii) summary measures based on combinations of SNPs in the same gene and iii) summary measures based on combinations of SNPs in functionally related genes (uptake; efflux). CC genotype in OCTN1 had a favorable impact on the achievement of MMR at 12 months (MMR@12m; P = 0.03). With respect to the summary measures, combination of SNPs in the SLC22A1 gene was significantly correlated with MMR@12m (P = 0.03). When considering summary measures of uptake and efflux, the former was found to be associated with both MMR@12m and CMR@12m (P = 0.003 and P = 0.01, respectively). A separate analysis limited to Caucasian pts (n=156) yielded similar results (Table 1). In addition, the analysis in the Caucasian subgroup evidenced a significant association between the CC genotype in ABCB1 rs60023214 and MMR@12m (P = 0.005) (Table 1). Cumulative incidence plots based on the Kaplan-Meier method were also analyzed in the overall population and in Caucasians, with comparable results. Representative plots are shown in Figure 1. There was evidence for difference among MMR cumulative incidence curves for 2 single SNPs and 2 score measures. Presence of the major allele in OCTN1 (CC) and of the minor allele in CYP3A4 rs2740574 (GG) were associated with increased MMR rate (P = 0.028 and P = 0.042, respectively, in the overall population and P = 0.027 and P = 0.038, respectively, in Caucasians). Similarly, an increase in the number of favorable alleles in the SLC22A1 gene was associated with increased MMR rate (P = 0.030 and P = 0.043 in the overall population and in Caucasians, respectively). In addition, the combination of favorable alleles in the genes involved in IM uptake was associated with increased rates of both MMR and CMR (P = 0.004 and P = 0.015, respectively, in the overall population and P = 0.005 and P = 0.009, respectively, in Caucasians). Our results suggest that SNP genotyping might be helpful in selecting pts who are more likely to benefit from first-line use of more potent inhibitors. Further assessment of the SNPs here identified in larger series of pts is warranted.Table 1Summary of significant associationsOverall population (n=189)Caucasians (n=156)Gene, rsOutcomePHazard ratio[95% CI]PHazard ratio[95% CI]OCTN1 - rs1050152MMR@12m0.030.78 (0.63, 0.98)0.030.77 (0.60, 0.97)ABCB1 (MDR1) - rs60023214CMR@12m0.060.71 (0.49, 1.02)0.0050.56 (0.37, 0.84)SLC22A1 (hOCT1) - SNP combinationMMR@12m0.031.24 (1.02, 1.50)0.041.24 (1.00, 1.54)Uptake - SNP combinationMMR@12m0.0031.21 (1.06, 1.37)0.0051.24 (1.06, 1.44)Uptake - SNP combinationCMR@12m0.011.30 (1.05, 1.61)0.011.37 (1.08, 1.74) [Display omitted] Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures:Hughes:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria. White:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Saglio:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Hatfield:Novartis: Employment. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Early Switching From Imatinib to Nilotinib In CML Patients Failing to Achieve Early Molecular Targets May Not Be An Effective Approach In Patients with Very Low OCT-1 Activity: A TIDEL II Sub-Study

AbstractAbstract 356We have previously demonstrated that CML patients with very low OCT-1 activity (Quartile 1 (Q1)<4 ng/200,000 cells) have significantly poorer outcomes with respect to response, mutation development, progression, and survival than patients with higher OCT-1 activity(OA). We have also demonstrated that the influx pump OCT-1 is associated with the active uptake of imatinib, but not nilotinib (NIL), into target CML cells. Hence, if the prognostic significance of OA primarily reflects IM transport we predicted that patients with very low OA who had poor initial responses to imatinib, should respond well when switched to NIL. In the Tidel II study CML-CP patients are treated with 600mg/day imatinib (IM) upfront and are dose escalated to 400mg BID or switched to NIL 400mg BID for either intolerance (Grade III/IV or persistent Grade II non-haematological toxicity) or failure to meet pre defined molecular targets. These targets are early markers of sub-optimal response, but not IM failure: failure to achieve <10% BCR-ABL IS by 3 months, <1% by 6 months and <0.1% by 12months. Hence this study is designed to enable early efficacious intervention in the case of poor response, to prevent frank therapeutic failure. In this study we examine the cohort of patients who have switched to NIL and compare their response on NIL to that achieved on IM. To date 63 patients on this trial have greater than 12 month molecular follow up, and OA quantified. 26/63 patients have OA<4ng/200,000 cells. As demonstrated in Table 1, and in keeping with previous findings, patients with low OA have significantly poorer rates of MMR by 12 months, and higher rates of study discontinuation when compared to patients with ≥4.0ng/200,000 (high OA). Importantly the study design of Tidel II has enabled the more careful assessment of therapeutic intervention. Of note 23% of patients with low OA had a dose increase and 42% switched to nilotinib because of failure to meet pre-stated milestones. This compares with only 8% of remaining patients being dose increased and 14% switching to NIL(p=0.007).Table 1:Showing the response of patients within the two OCT-1 Activity Groups.MMR by 12 moMutationsOff StudyIncreased dose 400mg BID IMMMR on 400mg BID IMSwitched to NILMMR on NIL<4.0ng/200,000 cells n=2637%19%19%23%16.6%42%10%≥4.0ng/200,000 cells n=3781%0%2.7%8.1%100%13.5%100%p=<0.0010.0060.0370.1410.050.0070.007With a median follow up after switching to NIL of 12 months (Low OA R=6-21 high OA R=9-12 p=0.887) all patients with high OA, not previously in MMR (n=4), achieved MMR within 6 months. In contrast 1/10 low OA patients not previously in MMR achieved MMR on NIL. Interestingly, not all patients with low OA were switched to NIL because of sub-optimal IM response. Four of this cohort were intolerant to 600mg IM (one of these 4 achieved MMR), while all patients in the high OA cohort were switched because of intolerance. This implies that a low OA is not overcome with early switch to NIL, despite NIL transport being unrelated to OCT-1. This may be due to other intrinsic factors, possibly related to leukaemia biology and majority cell type for which the OA assay may be a surrogate measure. Alternatively, the poor response to imatinib in low OA patients has facilitated the activation of other resistance mechanisms, that in turn lead to a poor response after switching implying that patients with low OA may require upfront treatment with a TKI that is OCT-1 independent. Importantly only 1 of the patients on NIL had a mutation prior to switching, with another patient transforming to blast crisis with a T315I mutation while on NIL. Thus the presence of kinase domain mutations does not explain these findings. Conclusions:Low OA is associated with poor response to IM. Here we show that the MMR rate for patients with low OA was not substantially improved by the TIDEL II treatment strategy. This is despite the protocol allowing for early dose escalation or switch for suboptimal response. This preliminary data suggests that patients with low OA may have more generalized insensitivity to kinase inhibitor therapy which is not simply related to drug transport. These are important findings in the setting of multiple TKIs being available, and suggest that in patients with very low OA a regimen of selective TKI intensification may not be the optimal approach. Disclosures:White:Novartis: Honoraria, Research Funding; BMS: Research Funding. Slader:Novartis Pharmaceuticals: Employment, Equity Ownership. Osborn:Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding. Mills:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria.

The poor response to imatinib observed in CML patients with low OCT-1 activity is not attributable to lower uptake of imatinib into their CD34+ cells

AbstractThe functional activity of the organic cation transporter 1 (OCT-1) protein in chronic myeloid leukemia (CML) mononuclear cells (MNCs) is highly predictive of molecular response in imatinib treated patients. Here we investigate whether the MNC OCT-1 activity (OA) provides a surrogate indicator of effective targeting of the more immature CD34+ cells. While confirming our previous findings that high MNC OA is significantly associated with the achievement of major molecular response (MMR; P = .017), the present studies found no relationship between high CD34+ OA and the achievement of MMR. Furthermore, no correlation was found between the MNC OA and the CD34+ OA in matched CML samples. These results suggest that the predictive value of the MNC OA may primarily reflect the effective targeting and subsequent reduction of mature CML cells. Therefore kinase inhibition in these mature cells, and not the CD34+ cells, may be the key determinant of response in CML.

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.

OCT-1 Activity in CML CD34+ Cells Is Not Predictive of Molecular Response to Imatinib Treatment in CP-CML Patients, Despite the Strong Predictive Value of MNC OCT-1 Activity.

Abstract 2189Poster Board II-166 Introduction.The organic cation transporter 1 (OCT-1) is the major active influx transporter for imatinib in PB mononuclear cells (MNC) from CML patients. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 Activity, OA) in MNC from chronic phase (CP) CML patients is highly variable (mean: 7.2 ng/200,000 cells, standard deviation: 6.4). We also demonstrated that the MNC OA is significantly related to a patient's sensitivity to imatinib-induced kinase inhibition, and their subsequent molecular response. In addition, we have recently demonstrated that OA is predictive of event free and transformation free survival to 5 years (White ASH submitted 2009). This is of a greater importance for those patients on lower doses of imatinib (below 600mg). For these patients who have a low MNC OA, 82% failed to achieve a major molecular response (MMR) by 18 months of imatinib treatment, as opposed to 17% for patients with a high MNC OA (p=0.022). Recently, we have shown that the OA in CML CD34+ cells is significantly lower than that found in mature CML cells (mean CD34+: 3.9, CD34-: 11.6 ng/200,000 cells, p<0.001, n=36), however interpatient variation in CD34+ OA still remains (standard deviation: 2.65). Given the importance of low MNC OA as a prognostic indicator of poor molecular response, we sought to ascertain whether a patient's MNC OA was related to the OA in their CD34+ cells, and as such could be used as a surrogate indicator for efficient targeting of the primitive population. To do this, we firstly looked for a correlation between the OA in a patient's MNC and their CD34+ cells. Secondly, we assessed the predictive value of CD34+ OA for MMR in imatinib treated patients. Methods.MNC and CD34+ cells were isolated from imatinib naïve, newly diagnosed CP-CML patients using density gradient centrifugation and magnetic cell sorting, respectively. OA was determined using [14C]-labeled imatinib and the OCT-1 inhibitor prazosin. OA is calculated as the difference between the uptake and retention of imatinib in the absence and presence of prazosin. Statistical analyses were performed using the Mann-Whitney Rank Sum or Student's t-test, correlations were performed using the Spearman-Rank Order. Results.In 34 CML patients no correlation was observed between the OA in their MNC and the OA in their CD34+ cells (R=0.09, p=0.577). Furthermore, when these patients were divided as having high or low OA in their MNC (about a previously described median of 7.2 ng/200,000 cells), no difference was seen between the OA in their corresponding CD34+ cells (Table 1).Table 1The mean OCT-1 Activity (OA) in a patient's CD34+ cells expressed as ng/200,000 cells (Standard Deviation)nCD34+ OAP ValueLow MNC OA254.02 (2.78)0.854High MNC OA93.83 (2.53)Finally, in 19 patients where 12 month response data was available, patients were grouped according to the achievement or not of MMR by 12 months. MNC OA was found to be significantly associated with the achievement of MMR (Table 2). This suggests that patients with a high OA in their MNC achieve better molecular responses, which is in agreement with our previous findings. However, assessment of CD34+ OA failed to demonstrate a relationship between OA and the achievement of MMR (Table 2). Therefore, a low OA in patient's CD34+ cells (unlike MNC) does not appear to be associated with poor molecular responses.Table 2The mean OCT-1 Activity (OA) in patients MNC and CD34+ cells expressed as ng/200,000 cells (Standard Deviation)nMNC OAP ValueCD34+ OAP ValueNo MMR73.13 (2.90)0.0313.65 (2.29)0.979Yes MMR128.38 (5.44)3.63 (1.81) Conclusion.No relationship was identified between the OA measured in a patient's MNC and that in their CD34+ cells. Furthermore, CD34+ OA is not predictive of a patient's molecular response to imatinib treatment. This is in contrast to MNC OA, which is predictive of event free and transformation free survival to 5 years of imatinib treatment. This data suggests that the predictive value of the MNC OA may primarily reflect the effective targeting and subsequent eradication of mature CML cells. And that kinase inhibition in these mature cells (as opposed to CD34+ progenitor cells) may be the key determinant of MMR achievement in CP-CML.Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures:Zannettino:Novartis Pharmaceuticals: Research Funding. White:Novartis Pharmaceuticals: Honoraria, Research Funding. Hughes:Novartis Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau.

Imatinib and Nilotinib Intracytoplasmic Determination Levels.

Abstract 2750Poster Board II-726 IntroductionCellular uptake of imatinib is known to be mediated by Oct-1 but efflux by MDR1. In the cell, metabolism, particularly by CYP3A4, produces metabolites that could be carried by other transporters such as MRP2. Nilotinib seems to suffer less from the influence from such transporters. The aim of this study was to determine the impact of cellular exchange on the intracellular concentrations of imatinib, nilotinib or both in vitro but also among patients in order to improve the therapeutic drug monitoring of these medicines. MethodsIn vitro studies used the chronic myeloid leukemia cell line K562 overexpressing MDR1 (K562mdr) and wild-type K562 cells (K562wt). To investigate cellular uptake and the intra/extracellular concentration relationship, these were incubated with imatinib or nilotinib for 2h in the presence or absence of transporter inhibitors (verapamil, PSC833). Cells were precipitated by acetonitrile (with internal standards) and analysis was performed using HPLC-tandem mass spectrometry (Waters Acquity-TQD). The intracellular concentration of peripheral blood mononuclear cells (PBMC) of patients treated by these drugs was also investigated. ResultsIn K562wt, the presence of verapamil, an inhibitor of both Oct-1 and MDR1, reduced imatinib intracellular concentration compared to the steady-state untreated K652wt, but the presence of PSC833, a specific inhibitor of MDR1, had little effect on the level of imatinib. K562mdr, that had lower steady-state imatinib cellular concentration than K562wt, recovered higher levels with verapamil but PSC833 allowed the greatest concentrations. Experiments investigating nilotinib found no difference between cell lines with or without inhibitors. Finally, the presence of both imatinib and nilotinib seemed to modify the steady-state concentration found individually.Intracellular imatinib concentration was correlated with that found in the culture medium (extracellular), but the gradient was different for each conditions for both K562wt and K562mdr, with or without inhibitors. At every concentration tested, we found the same differences in levels than in the kinetic study. Saturation was observed with imatinib, especially for untreated K562wt indicating probable involvement of active transport.Investigation of nilotinib under the same conditions did not show any difference in the gradient of each conditions and the relationship was more linear.The raw intracellular concentrations of PBMC obtained from patients (n=30) were very variable, especially for the imatinib. Correction by plasma concentration indicates individual patient's capacity for intracellular incorporation and a trend was found with response to the treatment. ConclusionContrary to imatinib, nilotinib did not seem to be under influence of MDR1, nor of OCT1. The intracellular concentration appears as a good way to investigate pharmacokinetic resistance and could be an additional help for the management of the treatment, especially for imatinib. Disclosures:Mahon:Amgen: Honoraria; Novartis Pharma: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria.

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

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

Analysis of Genetic Variants and Expression Levels of Human Organic Cation Transporter 1 (HOCT1) and Genetic Variants in MDR1 in CML: Weak Associations Were Detected but a Major Role in Clinical Response to Imatinib Resistance Is Unlikely

It has previously been shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human Organic Cation Transporter 1 (hOCT1; SLC22A1). In more recent work on clinical samples it was further shown that low hOCT1 expression of this influx transporter may be an important mechanism of imatinib resistance. To further evaluate this issue we have retrospectively quantified pretreatment hOCT1 mRNA expression in 92 CML patients (pts) that responded with major molecular remission within the first year of treatment and compared these results to 19 pts with primary resistance to imatinib. We found that all 19 resistant pts had low hOCT1 expression (median: 2.032 (expressed as %hOCT1/ABL); range 0.18–4.24). Although the median hOCT1 expression at diagnosis in the responders was higher (median 8.417) the range was very heterogeneous (0.45–188.2) with only 30% of all responders having a significantly higher expression than the resistant pts. As in vitro studies have shown that genetic variants of the SLC22A1 gene that codes for hOCT1 can have a negative effect on the transport of some substrates we hypothesized that not only certain hOTC1 expression levels but also different genetic variants within the SLC22A1 gene may be associated with different efficiencies of imatinib uptake. Using high resoluting melting and subsequent sequencing we have genotyped exons 1, 2, 5, 6, 7, 9, 10, and 11 in 109 responders as well as in 55 resistant pts, thus each 326 alleles were evaluated. We detected 12 different exonic polymorphisms. Two of these, a G38D and a Y404C were so far undescribed variants. Both nonsynonymous variants were detected in heterozygeous forms, the G38D in one responder and the Y404C variant in one resistant pt. All other variants were detected in frequencies similar to those that have already been described (R61C: 0.07, L160F: 0.76, P341L: 0.01, G401S: <0.01, M408V: 0.60, delM420: 0.19, G465R: 0.05, V519I: <0.01). In addition the silent variants S51S and V501V were detected with frequencies of 0.26% and 0.01% respectively. All variants in heterozygous as well as in homozygous form were distributed equally between responders and resistant patients. Thus we did not find any correlation between SLC22A1 genotype and imatinib response. In addition there was also no correlation of any of these polymorphisms to the high expressers. We found that those polymorphisms that have been described to severely affect hOTC1 functions in vitro were very rare (P341L and G401S with <0.01% each) or even never detected (P283L and R287L) in our cohort. Thus, although in vitro studies have shown that hOCT1 polymorphisms may severely affect function with respect to substrat specificity and transport efficiency of imatinib they do not seem to play a major role in response of CML patients to imatinib. In addition, we analyzed the three most frequent polymorphisms in exons 12, 21, and 26 in the multidrug resistence gene (MDR1) that codes for an efflux transporter implicated in imatinib efflux. In total 84 responders and 38 resistant patients were analyzed. We found that the exon12 nt1236t allele is more frequently observerd in resistant patients (p=0.045) whereas there was only a week association for the exon21 nt 2677t allele (p=0.121) and the exon26 nt3435t allele (p=0.139) to resistance. In conclusion,it seems to be unlikely that genetic variants of hOCT1 play a major role in imatinib resistanceif at all, also the hOCT1 expression levels account for the response of only a few cases.It remains unclear whether hOCT1 plays a role in influx of imatinib or whether its function may be overwritten by other influx transporters like the very homologous and functionally redundant hOCT3 just in the vicinity of hOCT1. 3) The role of efflux transporters in imatinb resistance may be more important, however we detected only a weak association to certain polymorphisms in MDR1 to resistance in our cohort.

Effective dasatinib uptake may occur without human organic cation transporter 1 (hOCT1): implications for the treatment of imatinib-resistant chronic myeloid leukemia

AbstractWe have previously shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human organic cation transporter 1 (hOCT1; SLC22A1), and that low hOCT1 expression is an important determinant of clinical outcome to imatinib treatment. We hypothesized that dasatinib might be transported differently than imatinib, possibly accounting for its favorable effects in imatinib-resistant patients. 14C-dasatinib uptake was greater in KCL22-transfected cells with pcDNA3-hOCT1 plasmid (high hOCT1-expressing cells) than in control cells (P = .02). However, hOCT inhibitors did not decrease dasatinib uptake into either control or primary cells, in contrast to their block on imatinib uptake. Dasa-tinib decreased the level of phosphorylated CrkL to 49.9% in control and 40.3% in high hOCT1-expressing cells. Dasa-tinib efflux was investigated in confluent ABCB1-transfected MDCKII cell monolayers. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating that they were transported by ABCB1, which was confirmed using the ABCB1 inhibitor PSC833 (P = .001 and P < .001, respectively). Compared with imatinib, dasatinib achieved superior intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1. Efflux of dasatinib and imatinib appear similar via ABCB1. Dasatinib may therefore offer an advantage over imatinib in patients with low hOCT1 expression.

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.

Unlike Imatinib, Dasatinib Uptake into Chronic Myeloid Leukaemia Cells Is Independent of hOCT1 Expression.

In chronic myeloid leukaemia (CML), resistance to imatinib is an important clinical issue. We have previously shown that imatinib uptake into CML cells and cell lines is dependent on the uptake transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). Furthermore, in clinical samples, low hOCT1 expression is an important mechanism of imatinib resistance (Wang L et al, Clinical Pharmacology and Therapeutics; epub June 13th 2007). Dasatinib is a second generation novel tyrosine kinase inhibitor that is effective in many imatinib-resistant patients. Its mechanism of transport into and out of CML cells has not been previously investigated. We hypothesised that dasatinib might be transported differently to imatinib, which might account for its favourable effects in imatinib-resistant patients. The CML cell line KCL22 was selected for transfection work as it has a particularly low basal level of hOCT1 expression. KCL22 cells were transfected with pcDNA3-hOCT1 plasmid (kind gift of D Gründemann, Germany) and stable lines were selected with high hOCT1 expression. The uptake of radiolabelled dasatinib (kind gift from Bristol Myers Squibb) was greater in high-hOCT1 expressing cells than in mock transfected cells (p=0.0149, n=3). However, prazosin and amantadine, both inhibitors of hOCT transport, did not decrease dasatinib uptake into mock transfected KCL22 cells, in sharp contrast to the block on imatinib uptake seen with both these inhibitors. Dasatinib decreased the level of phosphorylated CRKL (surrogate marker for BCR-ABL) by 49.9% in mock transfected KCL22 cells and 40.3% in high-hOCT1 expressing cells. The distribution coefficient (logD) of dasatinib between 1-octanol and HEPES medium was 2.05, compared with 0.81 for imatinib, demonstrating that dasatinib is more lipophilic. The efflux of dasatinib was investigated in confluent monolayers of Madin-Darby canine kidney (MDCKII) cells on a semipermeable membrane. These cells stably express ABCB1 (MDR1) on their apical but not their basal aspect. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating ABCB1 transporter-mediated efflux of both drugs (p=0.001, p<0.0001, respectively). Addition of the ABCB1 inhibitor PSC833 blocked transport of both drugs (p=0.0013, p<0.0001 respectively). Taken together, the data are consistent with the view that dasatinib, unlike imatinib, may achieve adequate intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1 function. However, in cells with high hOCT1 expression, dasatinib uptake may be augmented. Efflux of dasatinib and imatinib appear similar and via ABCB1. Dasatinib may be useful therapeutically in patients with imatinib resistance related to low hOCT1 expression.

Should the Initial Starting Dose of Imatinib for Treatment of Some Patients with Chronic Myelocytic Leukemia (CML) Be Lower Than 400 mg Daily?.

A 62-year-old female was referred to me, on January 23, 2004, because of leukocyosis. She was obese with the weight of 141.5 Kg. Her WBC was 37.6 x 10e9/L with neutrophillia. The findings on examination of her blood smear were strongly suggestive of CML. Her blood was sent for BCR-ABL detection and bone marrow aspirate was sent for cytogenetic studies. No therapy was given while awaiting the result of these tests. On March 12, 2004, Hgb was 144 G/L, platelet count 318 x 10e9/L, WBC 66.5 x10e9/L. WBC differential showed 57% neutrophils, 11% bands, 4% metamyelocytes, 8% myelocytes, 2% promyelocytes.... BCR-ABL fusion gene product had been detected in her peripheral blood; and cytogenetic studies had demonstrated Philadelphia (Ph) chromosome in all of the cells examined. The diagnosis of Ph + CML in chronic phase with low Sokal score was made. Despite her morbid obesity, she was started on standard recommended dose of imatinib 400 mg daily. After 6 weeks of therapy her WBC was 9.5 x 10e9/L with normal WBC differential and she was in complete hematological remission. When she was tested after 7 months of therapy, there was a three log decrease in BCR-ABL fusion gene. BCR-ABL fusion gene was undetectable in the next study that was done at 16 months of therapy. Since then, she has remained in complete molecular remission, with no detectable BCR-ABL fusion gene (last test 3 years after starting therapy), on imatinib 200 mg daily. The only repeat cytogenetic study of the bone marrow carried out 2 years after starting therapy showed normal karyotype and no Ph + chromosome. Discussion: There is a marked difference in active transport of imatinib in and out of the cells and sensitivity of Ph + CML cells of different patients to imatinib. In the initial trials the dose of 400 mg daily was selected because, in most of the patients, irrespective of their weight, this dose was associated with adequate uptake of imatinib which could inhibit cellularr tyrosin kinase activity. Furthermore, in the majority of the cases, the side effects of this dose of imatinib were tolerable. However, this drug is very expensive and because it has to be taken for many years, it creates a major financial burden for the patient or for the health care system. The question then arises whether it is justified to give unnecessary higher doses of imatinib that are needed, to many patients in order to produce higher rate of remission in patients with CML. In the case cited above, considering her weight, she was given a dose of imatinib which would be the equivalent of 200 mg daily for average weight patients. Nevertheless, she responded promptly to such a relatively low dose of imatinib without experiencing any side effects attributed to imatinib. Carella and Lerma (Ann Hematol 2007: June 19, Epub ahead of print) recently reported the outcome of four CML patients who were imatinib-intolerant but who had undetectable residual disease. The imatinib dose in these patients was reduced to 200 mg daily. All four patients have remained in complete remission with undetectable residual disease from 4 to 37 months. Conclusion: Until an in vitro test of imatinib sensitivity of Ph + cells becomes available, it seems reasonable that an average weight patient with CML in chronic phase and low Sokal score be started on imatinib 200 mg daily initially. Only if such patient fails to show a complete hematological response within three months, should the dose of imatinib be increased to 400 mg daily.

Influence of breast cancer resistance protein (Abcg2) and p‐glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec®) across the mouse blood–brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.