ISR Failure for 5-Fluorouracil Analysis from Patients Dosed with CapecitabineJanine McKnight1, Chris Schmidt1, Kevin Lam1, Kristy Strooisma1, Sara Andersen1, Brian Dean2 and Xiao Ding21Covance Laboratories Inc., Madison, WI; 2Genentech, A Member of the Roche Group, South San Francisco, CA

Presented at CFABS-WRIB 2014

Introduction▶ Here we describe the investigation of an Incurred Sample Reanalysis (ISR)

failure for the measurement of 5-fluorouracil (5-FU) in human subjects dosed with capecitabine.

▶ A subgroup analysis of 5-FU ISR results from patients dosed with either 5-FU or capecitabine suggested the measurement of 5-FU following capecitabine administration is complicated by the conversion of upstream metabolites (5-fluoro-5’-deoxycytidine, 5’-DFCR and 5-fluoro-5’-deoxyuridine, 5’-DFUR) to 5-FU following sample collection.

▶ Experimental evidence indicates that 5’-DFUR converting to 5-FU was the primary cause for ISR failure.

▶ This investigation led to the development of a specialized collection procedure to prevent the conversion of 5’-DFUR to 5-FU.

BackgroundCapecitabine (Xeloda, Roche) is an orally-administered chemotherapy agent for treating breast and colorectal cancers. Capecitabine is a prodrug which is enzymatically converted to the active drug 5-fluorouracil (5-FU) within the tumor. 5-Fluorouracil (Adrucil, Teva Generics) is administered by infusion and inhibits action of the enzyme, thymidylate synthase (required for DNA replication) which causes cancerous cells to undergo cell death. It is used to treat breast, colorectal, stomach, pancreatic and skin cancers. A clinical study involved the measurement of 5-FU from patients who were dosed orally with capecitabine and from patients dosed by infusion with 5-FU. ISR analysis for 5-fluorouracil on samples from patients that were dosed orally with capecitabine failed this evaluation while 5-FU IV dosed patients in the same study had acceptable ISR results, shown at right.

Materials and MethodsOutline of Method▶ Blood collected in a custom tube containing THU/EDTA▶ Blood tube to be on wet ice at all times until spun down in a refrigerated

centrifuge to plasma (should be centrifuged immediately after collection)▶ 20 mM citric acid to be spiked into plasma tubes while on wet ice▶ Plasma aliquots to be stored at -70°C immediately▶ 5-Fluorouracil and the internal standard (ISTD), 15N2-5-fluorouracil, are

extracted from human plasma by supported liquid extraction (SLE)▶ Samples should be extracted on wet ice▶ Separation and detection using LC-MS/MS with negative ion electrospray

Extraction Method1) Aliquot 100 μL plasma 2) Add 25 μL internal standard solution3) Add 100 μL 10% acetic acid in water4) Vortex-mix, load sample to SLE 200+ plate (Isolute)5) Elute with 1000 μL ethyl acetate6) Dry eluent under nitrogen 7) Reconstitute in 100 μL 5% methanol in water

LC-MS/MS Method▶ LC system: LC-20AD, Shimadzu▶ HPLC column: Waters Atlantis dC18, 150 x 2.1 mm, 5 μm particle size▶ Mobile phases: 10 mM ammonium formate, methanol▶ Flow Rate: 0.400 mL/min▶ Typical injection volume: 5 μL▶ Mass spectrometer: API 5000 (AB Sciex)▶ Detection: ESI negative mode▶ 5-Fluorouracil 129 → 42▶ 15N2-5-Fluorouracil 131 → 43▶ Data processing: Analyst (AB Sciex), Watson LIMS (Thermo)

Metabolism of Capecitabine

Results5-FU ISR Results for Clinical Study

Investigation Part 1: Which metabolite is converting to 5-FU?▶ Blank plasma was spiked individually with capecitabine, 5’-DFCR and

5’-DFUR at potential Cmax concentrations. ▶ These individually fortified test pools were left at room temperature for

2 hours and then analyzed to determine 5-FU concentrations.

▶ Remaining individual test pools were stored at -70°C for at least 24 hours, thawed and then analyzed to determine 5-FU concentrations.

Chromatograms of Calibration Standard at LLOQ and Blank Sample

Chromatograms of Capecitabine, 5’-DFCR and 5’-DFUR Prepared Fresh and after 1 Freeze/Thaw Cycle at -70°C

Investigation Part 2: How to stop the conversion▶ To test if an inhibitor was suitable to prevent the conversion, plasma was spiked with 5’-DFUR

at the expected approximate Cmax.

▶ Following fortification with 5’-DFUR, the following inhibitors were tested: tetrahydrouridine (THU) with formic acid, acetic acid, and citric acid; 5-nitrouracil and 7-deazaxanthine (7DX).

▶ The test samples treated with the inhibitors were subjected to the following conditions prior to measurement of the 5-FU concentrations: 1) freshly prepared; 2) 6 and 24 hours on wet ice; 3) 6 and 24 hours at room temperature; and 4) freeze/thaw cycles on wet ice and room temperature.

ReferencesMontange, D., Berard, M., Demarchi, M., Muret, P., Piedoux, S., Kantelip, J. P. & Royer, B. (2010). An APCI LC-MS/MS method for routine determination of capecitabine and its metabolites in human plasma. Journal of Mass Spectrometry, 45, 670-677.Vainchtein, L.D., Rosing, H., Schellens, J.H.M. & Beijnen, J.H. (2010). A new, validated HPLC-MS/MS method for the simultaneous determination of the anti-cancer agent capecitabine and its metabolites: 5’-deoxy-5-fluorocytidine, 5’-deoxy-5-fluorouridine, 5-fluorouracil and 5-flurordihydrouracil, in human plasma. Biomedical Chromatography, 24, 374-386.Besnard, T., Renee, N., Etienne-Grimaldi, M.C., Francois, E. & Milano, G. (2008). Optimized blood sampling with cytidine deaminase inhibitor for improved analysis of capecitabine metabolites. Journal of Chromatography B, 870, 117-120.Mayer, R.J. (2007). Should Capecitabine Replace Infusional Fluorouracil and Leucovorin When Combined With Oxaliplatin in Metastatic Colorectal Cancer? Journal of Clinical Oncology, 25(27), 4165-4167.

5-FU LLOQ 5.00 ng/mL

Blank matrix

Capecitabine-QC-5000-fresh

Capecitabine-QC-5000-1/F/T

5’-DFCR-QC-10000-fresh

5’-DFCR-QC-10000-1 F/T

5’-DFUR-QC-15000-fresh

5’-DFUR-QC-15000-1 F/T

Impact of Inhibitors on Conversion of 5’-DFUR to 5-FU in Plasma

Impact of Acids on Conversion of 5’-DFUR to 5-FU in Plasma

Conclusion▶ After testing capecitabine, 5’-DFCR and 5’-DFUR, it was determined that 5’-DFUR was

continuing to convert to 5-FU even after samples had been stored at -70°C.

▶ Ultimately, it was determined that blood would be collected in a THU/K2EDTA tube, with harvested plasma spiked with citric acid prior to samples being frozen. The entire collection procedure would be performed on wet ice.

▶ A bioanalytical method was then developed and validated for the determination of 5-FU (curve range of 1-1,000 ng/mL) in human plasma from patients dosed with capecitabine utilizing the above appropriate sample collection procedure.

▶ This new approach has resulted in a clinical study with acceptable ISR results for 5-FU from patients dosed with capecitabine.

5-FU ISR Results for New Clinical Study for Patients Dosed with Capecitabine

AcknowledgementsThe authors gratefully acknowledge Fengxia Li, Yifan Shi, Harumi Shimizu, and Abigail Trenchak for their expertise.

Inhibitor Test condition Fold increase in 5-FU

concentration

No inhibitor 1 F/T cycle 1.8 fold 24 h on wet ice 6.0 fold

24 h at RT 41.5 fold

0.3 mM 5-Nitrouracil 1 F/T cycle 1.0 (No significant increase)24 h on wet ice 1.0 (No significant increase)

24 h at RT 1.3 fold

0.3 mM 7DX 1 F/T cycle 1.2 fold 24 h on wet ice 3.4 fold

24 h at RT 14.3 fold

5-Nitrouracil is better inhibitor than 7DX

Acid Test condition Fold increase in 5-FU

concentration

0.1% formic acid 5 F/T cycles 2.3 fold 0.1 mg/mL THU 6 h on wet ice 2.5 fold

24 h on wet ice 6.9 fold

0.1% acetic acid 5 F/T cycles 2.1 fold 0.1 mg/mL THU 6 h on wet ice 2.6 fold

24 h on wet ice 6.4 fold

20 mM Citric acid 5 F/T cycles 0.9 fold (No significant

increase)

0.1 mg/mL THU 6 h on wet ice 1.1 fold (No significant

increase) 24 h on wet ice 1.4 fold

Citric acid + THU has better inhibition than formic acid + THU or acetic acid + THU Citric acid + THU is as effective as 5-Nitrouracil at tested condition

TreatmentOriginal Conc.

(ng/mL)Reassay

Conc. (ng/mL) % Difference

Cap 246 245 -0.4Cap 3.43 3.72 8.1Cap 205 202 -1.5Cap 4.99 5.39 7.7Cap 64.5 64.2 -0.5Cap 4.07 4.19 2.9Cap 175 176 0.6Cap 11.0 11.1 0.9Cap 191 187 -2.1Cap 5.05 4.97 -1.6Cap 104 105 1.0Cap 3.07 2.68 -13.6