There are several reports in the literature describing the importance of imatinib uptake into the target cells by an organic cation transporter 1 (OCT1). It has also been proposed that the OCT1 activity determination could provide a valuable tool for the prediction of treatment success in patients with CML. With that purpose we developed a method for imatinib uptake determination that could be performed also in hematological laboratory, at least to the last analytical step. We incubated the mononuclear cells (MNC) and granulocytes (Gran) of a healthy volunteer with imatinib in the presence or absence of prazosin before and after Ficoll cell sorting. The cells were lysed with liquid nitrogen and extracted with organic solvents. The intracellular concentration of imatinib was determined by LC-MS/MS method after cell extract concentration. The results of imatinib uptake correlated well with the data published in literature. By means of this test, we also intend to determine the correlation of OCT1 activity with treatment success in the population of Slovenian CML patients.
COBISS.SI-ID: 3414641
Imatinib, dasatinib and nilotinib are three tyrosine kinase inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML). However, achieving maximum benefit with these drugs may require optimal dosing and adherence to therapy. In those cases, therapeutic drug monitoring (TDM) can be a useful tool in managing patients with CML. The paper presents simple and high throughput method for simultaneous determination of all three TKIs in dried blood spot (DBS) samples from CML patients. DBS samples were prepared by applying 10 μL of spiked whole blood onto an Agilent DBS cards. Whole blood spot was punched out of the card, transferred to a well in a 96-well Captiva ND Lipids filter plate. After the addition of isotopically labelled internal standard, the drug was extracted with 0.1% formic acid in methanol. The collected extract (1 μL) was injected onto a Phenomenex Kinetex 50 mm 2.1 mm C18 column and eluted with acetonitrile gradient into a triple quadrupole ESI-MS/MS Agilent 6460 operated in positive mode. The total run time was only 2.6 min. The method was validated in terms of linearity, selectivity, specificity, accuracy, precision, absolute and relative matrix effect and stability. The effect of haematocrit (Hct) on the accurate concentration determination was also examined. The method was linear in the range of 50-5000 micro g/L for imatinib and nilotinib and in the range of 2.5- 250 micro g/L for dasatinib, with correlation coefficient values higher than 0.997. Lower limits of quantification (LLOQ) were 50 micro g/L for imatinib and and 2.5 microg/L for dasatinib. The method proved to be accurate (% bias ( 13.20 and precise (CV ( 10.3%) on intra- as well as on inter-day basis. Sample matrix (% ME = 94.5-106.7) and different Hct values had no significant effect on the accuracy of measured concentrations. Samples proved to be stable whilst stored on DBS cards at room temperature or in the refrigerator; however,at 40 °C the stability of dasatinib was compromised. The method presented was successfully applied to clinical samples.
COBISS.SI-ID: 3292273
The acquired mutations in the BCR-ABL1 kinase domain (KD) may contribute to resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) patients. The aim of our work was to perform the BCRABL1 mutation analysis in a cohort of CML patients receiving TKIs, who did not achieve a major molecular response (MMR) by 18 months or more, or who lost MMR. The T315I mutation was detected in one out of nine CML patients by direct sequencing and ASO-PCR. It is a highly-resistant mutation to imatinib, nilotinib and dasatinib. The silent mutation p.Glu499Glu was detected in another CML patient by direct sequencing. It seems that the BCR-ABl1 mutations are rare in patients who do not achieve a MMR by 18 months or more or who have lost MMR. The T315I mutation detected in one patient in our cohort of CML patients indicates that the BCR-ABL1 mutation analysis could be recommended in these cases.