Page 169 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

1.0%)3 would determine the total number of patients required in the study. These patients would then arise from an expected minimum population of 1,100 treated patients. To account for a proportion of patients not evaluable for the study, the target accrual was set at 1,250 patients. Given the very low allele frequency of the c.1679T>G variant, it was considered not feasible to power this study for this particular variant. The estimated frequency of c.1236G>A is 3% and of DPYD*2A 1%, which means that the calculated sample size would be adequate for those individual variants, or when analyzing all four variants together (estimated frequency of 5%).
Pharmacokinetic analyses
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Supplement
For pharmacokinetic analyses, peripheral blood was collected on the first day of treatment. Blood was collected in lithium heparin tubes at nine different time points up to eight hours after capecitabine intake (pre-dose, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 hours after capecitabine intake). Samples were centrifuged immediately after the blood was drawn and plasma was stored at -80°C until analysis.
Capecitabine and the metabolites 5’-deoxy-5-fluorocytidine (5’DFCR), 5’-deoxy-5- fluorourdine (5’DFUR), 5-fluorouracil (5-FU), and fluoro-β-alanine (FBAL) were quantified in plasma samples using a validated ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) method. Lower limit of quantifications were 25 ng/ml for capecitabine, 10 ng/ml for 5’DFCR, 5’DFUR and 5-FU, and 50 ng/ml for FBAL. Stable isotopes were used as internal standard for all analytes. To a sample volume of 300 μl of plasma, 900 μl of methanol-acetonitrile (50:50 v/v) was added to precipitate the plasma proteins. Samples were vortex-mixed for 10 seconds, shaken for 10 minutes at 1,250 rpm and centrifuged at 14,000 rpm for 10 minutes. The clear supernatants were dried under a stream of nitrogen at 40°C and reconstituted in 100 μl of 0.1% formic acid in water. An Acquity UPLC® HSS T3 column (150 x 2.1 mm ID, 1.8 μlm particles) was used for chromatographic separation, at a flow rate of 300 μl/min and a gradient of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The following gradient was applied: 100% A from 0─2.5 minutes, an increase from 0% to 90% B from 2.5─7.5 minutes, and 100% A from 7.5─9 minutes. For detection an API5500 triple quadruple mass spectrometer (Sciex) equipped with a turbo ionspray interphase was used, using optimized mass transitions m/z 360.0243.9 for capecitabine, 244.9128.8 for 5’DFUR, 128.942.1 for 5-FU, and 105.985.9 for FBAL.
Pharmacokinetic parameters were calculated using non-compartmental analysis and the calculated area under the plasma concentration-time curve (AUC) and half-life (t1/2) were compared with pharmacokinetic data described in literature,4 measured at the same laboratory as the current study.
Data sharing statement
Data collected in the study, including individual participant data, will not be made available to others, except to researchers involved in the study. However, upon request, data sharing for additional research is possible and will be supported. Requests will be judged on scientific and clinical rationale and may need to be reviewed by an authorized institutional review
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