ABCB1 Overexpressing Research Articles

Abstract 5447: Investigating the relationship between pharmacogenetic variation in ABCB1 and ABCC2 tagSNPs and actinomycin D pharmacokinetics in children

Abstract Actinomycin D (Act D) has been used successfully to treat adult and childhood cancers for over 30 years. It continues to play a key role in the treatment of Wilms tumour patients, with 5-year survival rates >80% currently observed. While treatment is generally well tolerated, veno-occlusive disease (VOD) is a potentially life-threatening side effect observed in 1.7-13.5% of patients. Dose-intensity of Act D treatment has been highlighted as a possible risk-factor for VOD in paediatric patient populations. ATP binding cassette (ABC) transporters are expressed throughout the body to protect against exogenous compounds. In the liver and kidney, ABC transporters facilitate the removal of compounds via biliary or renal excretion and have previously been shown to play a major role in the transport of anti-cancer agents such as doxorubicin and irinotecan. Common single nucleotide polymorphisms (SNPs) for these transporters have been shown to alter clinical exposure to various anti-cancer agents. We have therefore carried out translational and clinical studies focusing on Act D pharmacokinetics in Wilms tumour patients and pharmacogenetic variation in key ABC transporters. Initial experiments were carried out to determine the potential importance of ABC transporter expression on the in vitro activity and transport of Act D, using MDCKII-WT alongside ABCB1, ABCG2 and ABCC2 over-expressing cell lines. Growth inhibition (GI) and intracellular accumulation experiments were carried out over a range of Act D concentrations (0-10µM). Pharmacokinetic analysis of samples obtained from patients receiving Act D was carried out using a validated LC/MS method and blood samples were obtained from all patients for pharmacogenetic analysis. Finally, the International HapMap database was used to determine tagSNPs for ABCB1 and ABCC2 with minor allele frequencies of greater than 0.1 in a Caucasian population, allowing the link between ABCB1 and ABCC2 haplotypes and Act D exposure to be investigated. GI experiments indicated a 40-fold decrease in Act D sensitivity in cells over-expressing ABCB1 (GI50: 0.85µM) and a 3-fold decrease in cells over-expressing ABCC2 (GI50: 0.06µM) compared to the control cell line (GI50: 0.02µM). Higher intracellular levels of Act D (normalized per mg protein) were observed in the WT parental cell line (650nM) as compared to those over-expressing ABCB1 (215nM), following a 6h incubation with 2µM Act D. This effect was abrogated by use of the ABCB1 inhibitor verapamil. Pharmacokinetic analysis of samples obtained from 56 patients indicated a large inter-patient variability in Act D exposure (AUC0-24h range: 1.6 – 11.4 µg/ml.min). Ongoing investigations into pharmacogenetic variation in ABCB1 and ABCC2 tagSNPs in these patients provide novel preliminary data relating to links between ABCB1 and ABCC2 haplotypes and Act D exposure in children with cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5447. doi:10.1158/1538-7445.AM2011-5447

Proton Pump Inhibitors Augment Nilotinib and Dasatinib Mediated Bcr-Abl Kinase Inhibition

Abstract Abstract 3991 Proton pump inhibitors (PPIs) are widely used for the treatment of gastro-oesophageal reflux and peptic ulcer disease. PPIs may not only alter absorption and metabolism of tyrosine kinase inhibitors (TKIs) but could also alter intracellular uptake and retention (IUR) into leukemic cells by interacting with ABCB1 and ABCG2 pump. There is limited literature assessing the interaction of PPI with TKI's at the cellular level. Here we compare the interaction of PPI with dasatinib, imatinib and nilotinib. Mononuclear cells (MNC) of CML-CP patients, K562 and K562-Dox (ABCB1 overexpressing), K562-ABCG2 cells were cultured with 14C-labelled dasatinib, imatinib and nilotinib with or without PPI for 2h and IUR were assessed. The effect of combining PPI with TKI's on Bcr-Abl kinase activity was assessed by measuring p-Crkl (surrogate marker of Bcr-Abl kinase activity). High concentrations of pantoprazole (1 and 2 mM) significantly increased dasatinib IUR in K562-Dox cells (p=0.004 and p=0.0006 respectively) but not in K562 cells (p=0.7 and p=0.1). Similarly, 1 mM (p=0.01) and 2 mM (p=0.007) esomeprazole significantly increased dasatinib IUR in K562-Dox cells but not in K562 cells. These data suggest that high concentration of PPI inhibit ABCB1 mediated dasatinib efflux. This was further supported by significant reduction in IC50dasatinib in K562-Dox cells by 1 mM pantoprazole (112±37 nM to 28±6; p=0.02; n=4). Similarly, 1 mM esomeprazole reduced IC50dasatinib (112±37 nM to 12±3 nM; n=2). This demonstrates that PPI mediated increase in IUR translates to increased kinase inhibition and lower IC50dasatinib. At lower concentrations (100 to 400 μM), neither pantoprazole nor esomeprazole significantly changed the dasatinib IUR or the IC50dasatinib in K562-Dox cells. In K562-ABCG2 cell line 100, 200, 400, 500, 1000 μM pantoprazole reduced IC50dasatinib from 27 nM to 15, 7, 11, 9.5 and 6.5 nM respectively. Similarly, 100, 200, 400, 500 and 1000 μM esomeprazole reduced IC50dasatinib from 21 nM to 15, 11, 8.5, 4.5 and 3.5 nM respectively. These data suggest that PPI enhances dasatinib mediated Bcr-Abl kinase inhibition in ABCG2-overexpressing cells. Although PPI did not change dasatinib IUR significantly in primary CML-MNC (n=10, Table I), it reduced IC50dasatinib (n=4, Table II). Similarly, pantoprazole and esomeprazole (5 to 400 μM) significantly increased nilotinib IUR (n=10, Table I) and significantly reduced IC50nilotinib in CML-MNC (n=3, Table II). Pantoprazole also increased the nilotinib IUR in K562 and K562-Dox cells, and reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 230 nM) cells. Similarly esomeprazole reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 150 nM) cells. The effect of PPI on IC50nilotinib was dose dependent. Pantoprazole and esomeprazole reduced imatinib IUR in K562, K562-Dox and CML-MNC. Pantoprazole increased IC50imatinib in K562 (3.8 to 4 μM) and K562-Dox (6.5 to 8 μM) cells. Similarly, 200 μM of pantoprazole and esomeprazole significantly reduced IM IUR in CML-MNC. However, the effect of pantoprazole on IC50imatinib in CML-MNC was variable and modest (Table II). Our data provide evidence that PPI might interfere with TKI activity. PPI's can enhance the dasatinib and nilotinib mediated Bcr-Abl kinase inhibition in primary CML cells. Table I: Effect of PPI on dasatinib, imatinib and nilotinib intracellular uptake and retention (IUR) in CML-MNC (n=10) TKI at 1μM TKI at 2μM Pantoprazole 0 μM 100μM 200μM 400μM 0uM 100μM 200μM 400μM Imatinib 13.3 10.9 9.9 8.8 23.7 20.4 18.9 16.3 p-value 0.06 0.01 0.004 0.2 0.09 0.01 Nilotinib 13.0 16.4 16.6 17.4 30.5 56.9 66.2 74.7 p-value 0.04 0.02 0.03 0.004 0.001 <0.001 Dasatinib 10.3 11.1 9.4 7.9 18.9 18.3 17.7 18.9 p-value 0.6 0.1 0.08 0.4 0.4 0.2 Table II: Effect of PPI on IC50 of nilotinib, dasatinib and imatinib in CML-MNC Nilotinib Nilotinib + 200 μM pantoprazole (% change) Nilotinib + 200 μM esomeprazole (% change) CML1 62.5 nM 30 nM (−52) 55 nM (−12) CML2 80 nM 32 nM (−60) 17 nM (−78) CML3 75 nM 68 nM (−9.4) 27 nM (−64) Dasatinib Dasatinib + 200 μM pantoprazole (% change) Dasatinib + 200 μM esomeprazole (% change) CML1 1.8 nM 0.7 nM (−61) 1.2 nM (−33) CML2 3 nM 2.6 nM (−13) – CML3 7 nM 2.5 nM (−64) – CML4 2.25 nM 1.7 nM (−25) 1.5 nM (−33) Imatinib Imatinib + 200 μM pantoprazole (% change) Imatinib + 200 μM esomeprazole (% change) CML1 1.05 μM 0.325 μM (−69) 0.8 μM (−23) CML2 1.8 μM 1.35 μM (−25) CML3 0.83 μM 1.15 μM (+38) 0.65 μM (−21) Disclosures: Hughes: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Research Funding.

Sensitization of ABCB1 overexpressing cells to chemotherapeutic agents by FG020326 via binding to ABCB1 and inhibiting its function

The effectiveness of chemotherapeutic treatment is usually limited by the overexpression of adenosine triphosphate binding cassette (ABC) transporters, which mediate multidrug resistance (MDR) by acting as efflux pumps to remove chemotherapeutic agents from MDR cancer cells. Thus, the inhibition of ABC transporters may represent a promising strategy to reverse MDR. This study was to characterize the actions of FG020326, a newly synthesized triaryl-substituted imidazole derivative, to reverse MDR in vitro and in vivo. FG020326 significantly potentiated the cytotoxicity of paclitaxel, doxorubicin, and vincristine in the ABCB1 (P-glycoprotein, P-gp) overexpressing cells KBv200 and MCF-7/adr, but not in the ABCB1 negative parental cell lines KB and MCF-7. However, FG020326 did not alter the cytotoxicity of the aforementioned drugs in ABCC1 (MRP1), ABCC4 (MRP4), ABCG2 (BCRP) and LRP overexpressing cell lines, KB-CV60, NIH3T3/MRP4-2, S1-M1-80 and SW1573/2R120, respectively. FG020326, following p.o. administration, was present in concentrations sufficient for reversal of MDR in mice. The co-administration of FG020326 with paclitaxel or vincristine significantly enhanced the antitumor activity of these drugs without significantly increasing toxicity in the mice bearing the KBv200 cell xenografts. In addition, FG020326, at concentrations that reversed MDR, did not significantly affect the activity of CYP3A4 or alter the pharmacokinetic profile of paclitaxel after co-administration with paclitaxel. FG020326 produced a significant concentration-dependent displacement of [3H]azidopine and inhibition of efflux of drug from cells. Furthermore, FG020326 was co-localized with ABCB1 in cell membranes. Hence, FG020326 is characterized as a third generation MDR modulator that holds great promise for the treatment of cancer patients with ABCB1-mediated MDR.

Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2

Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we evaluated the possible interaction of sunitinib with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP) in vitro. Our results showed that sunitinib completely reverse drug resistance mediated by ABCG2 at a non-toxic concentration of 2.5muM and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1 overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1 overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine 123 and methotrexate by ABCG2 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [(3)H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Overall, we conclude that sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic.

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

Abstract 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 conclusion dasatinib cellular uptake is predominantly passive and may be OCT independent. dasatinib is effluxed actively by ABCB1 in cell lines over-expressing these proteins. 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. Figure 1: Prazosin significantly reduced imatinib (IM) IUR and eliminated interpatient variability, however it did not reduce dasatinib IUR and did not eliminate interpatient variability. Figure 1:. Prazosin significantly reduced imatinib (IM) IUR and eliminated interpatient variability, however it did not reduce dasatinib IUR and did not eliminate interpatient variability.