Influence of CYP3A enzymes and ABC transporters on the activity of tyrosine kinase inhibitors in chronic myeloid leukemia

...................................................................................................................... 5 POPULÄRVETENSKAPLIG SAMMANFATTNING ............................................ 7 INTRODUCTION........................................................................................................... 9 Chronic myeloid leukemia ............................................................................................ 9 CML treatment ............................................................................................................. 11 The tyrosine kinase inhibitor revolution ............................................................... 11 Monitoring of response ........................................................................................... 12 CML treatment today ............................................................................................... 13 Mechanisms of imatinib resistance ......................................................................... 15 Imatinib pharmacokinetics.......................................................................................... 16 Metabolism ................................................................................................................ 17 Cellular transport ...................................................................................................... 19 Single nucleotide polymorphisms in ABCB1 and ABCG2 ................................. 21 Predicting the response to CML therapy .................................................................. 23 AIMS OF THE THESIS ............................................................................................... 25 METHODS ..................................................................................................................... 27 In vitro studies of ABC transporters (Papers I and II) ............................................. 27 K562 cells as a model system .................................................................................. 27 Selection and construction of variant ABCB1 and ABCG2 ............................... 28 Retroviral gene transfer ........................................................................................... 31 Analyzing EYFP, ABCB1, and ABCG2 with flow cytometry ............................ 33 Cell survival assay ..................................................................................................... 34 Quantification of TKIs in cell lysates .................................................................... 35 In vivo studies on CML patients .................................................................................. 36 Pilot study design (Paper III) .................................................................................. 37 Follow-up study design (Paper IV) ........................................................................ 37 CYP3A phenotyping ................................................................................................ 39 Quantification of imatinib and CGP74588 in patient plasma ............................ 41 RESULTS AND DISCUSSION ................................................................................... 43 In vitro studies of ABCB1 and ABCG2 (Papers I and II) ........................................ 43 TKIs as substrates of ABCB1 and ABCG2 .......................................................... 43 ABCB1 haplotypes and their influence on TKI transport .................................. 46 ABCG2 SNPs and their influence on TKI transport .......................................... 49 In vivo studies of CYP3A activity (Papers III and IV) ............................................. 53 CYP3A significance for imatinib outcome in the pilot study ............................. 53 Influence of CYP3A on imatinib pharmacokinetics and outcome .................... 54 CONCLUSIONS ............................................................................................................ 59 FUTURE ASPECTS....................................................................................................... 61 ACKNOWLEDGEMENTS ......................................................................................... 63 REFERENCES ............................................................................................................... 67 APPENDIX ..................................................................................................................... 79