Abstract
Single-nucleotide polymorphisms (SNPs) in the gene coding for the efflux-transport protein ABCB1 (P-glycoprotein) are commonly inherited as haplotypes. ABCB1 SNPs and haplotypes have been suggested to influence the pharmacokinetics and therapeutic outcome of the tyrosine kinase inhibitor (TKI) imatinib, used for treatment of chronic myeloid leukemia (CML). However, no consensus has yet been reached with respect to the significance of variant ABCB1 in CML treatment. Functional studies of variant ABCB1 transport of imatinib as well as other TKIs might aid the interpretation of results from in vivo association studies, but are currently lacking. The aim of this study was to investigate the consequences of ABCB1 variant haplotypes for transport and efficacy of TKIs (imatinib, its major metabolite N-desmethyl imatinib [CGP74588], dasatinib, nilotinib, and bosutinib) in CML cells. Variant haplotypes – including the 61A>G, 1199G>A, 1236C>T, 1795G>A, 2677G>T/A, and 3435T>C SNPs – were constructed in ABCB1 complementary DNA and transduced to K562 cells using retroviral gene transfer. The ability of variant cells to express ABCB1 protein and protect against TKI cytotoxicity was investigated. It was found that dasatinib and the imatinib metabolite CGP74588 are effectively transported by ABCB1, while imatinib, nilotinib, and bosutinib are comparatively weaker ABCB1 substrates. None of the investigated haplotypes altered the protective effect of ABCB1 expression against TKI cytotoxicity. These findings imply that the ABCB1 haplotypes investigated here are not likely to influence TKI pharmacokinetics or therapeutic efficacy in vivo.
Supplementary materials
Method for quantification of intracellular TKI accumulation
K562, K562/ve, and K562/ABCB1 TTT were incubated with TKIs, followed by quantification of intracellular drug concentrations using a method modified from a previous report.Citation1 Two methods were developed: one for quantification of imatinib, CGP74588, and bosutinib where dasatinib (800 ng/mL) served as the internal standard, and another for dasatinib and nilotinib, using imatinib (80 ng/mL) as the internal standard.
Cells were seeded 400,000/mL in 5 mL of growth medium and incubated for 120 minutes (imatinib, CGP74588, or bosutinib) or 180 minutes (dasatinib or nilotinib), depending on the time to attain influx–efflux equilibrium. TKIs were used in concentrations in the same range as mean IC50 concentrations found in parental K562 cells using the MTT assay (imatinib 0.5 μM, CGP74588 2.0 μM, dasatinib 1.5 nM, nilotinib 20 nM, bosutinib 2.0 μM). Cells were separated from the medium by centrifugation (4,000 g, 5 minutes at 22°C) on 1.5 mL silicone oil. Cell pellets were disrupted by adding 200 μL of internal standard in 4% formic acid (FA) in water (v/v), with the exception of pellets incubated with dasatinib or nilotinib, which were disrupted using 100 μL of 4% FA. Lysates were centrifuged at 10,000 g for 10 minutes at 4°C, and supernatants were collected and diluted 1:10 in water before analysis, with the exception of extracts from dasatinib and nilotinib incubations, which were analyzed as concentrates.
Samples were analyzed on a chromatographic system (Acquity UPLC System; Waters, Milford, MA, USA) coupled to the tandem-quadrupole mass spectrometer Xevo TQ MS (Waters). Five microliters of samples were separated on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) column (Waters), using a gradient mobile phase of 0.1% FA (v/v) in water (A) and 0.1% FA (v/v) in acetonitrile (B). A gradient was delivered at 0.6 mL/minute − 0.0–0.4 minutes, 80% A; 0.4–3.0 minutes, linear gradient to 20% A; 3.0–3.5 minutes, 20% A – followed by reequilibration with 80% A to 4.0 minutes. Multiple-reaction monitoring was applied and TKIs were monitored at transitions m/z 494 > 394 for imatinib, 480 > 394 for CGP74588, 488 > 232 and 488 > 401 for dasatinib, 530 > 289 for nilotinib, and 530 > 141 for bosutinib.
Calibrators were prepared in blank lysates and extracted in accordance with the same procedure as for the samples. The calibration curve ranges were 10–3,000 ng/mL for imatinib, CGP7488 and bosutinib; 1–500 ng/mL for dasatinib; and 25–500 ng/mL for nilotinib. Quality-control samples were prepared in two concentrations in blank lysates for each calibration curve: imatinib, CGP74588, and bosutinib were analyzed at 100 ng/mL and 2,500 ng/mL; dasatinib at 8 ng/mL and 200 ng/mL; and nilotinib at 30 ng/mL and 200 ng/mL.
The calibration curves were used for calculation of the TKI concentration in samples and normalized to the internal standard. All compounds had assay imprecision < 10%, with accuracy ranging from 85% to 113% at the investigated quality-control concentrations (n = 5).
Reference
- MlejnekPNovakODolezelPA non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite in Bcr-Abl positive cellsTalanta2011831466147121238737
Acknowledgments
The authors would like to thank the Swedish Research Council, the Swedish Cancer Society, the Medical Research Council of Southeast Sweden and the Linköping University Cancer Research Network for funding this study. We are also grateful to Novartis Pharma AG for providing imatinib and CGP74588.
Disclosure
The authors report no conflicts of interest in this work.