Cytotoxic activity of gemcitabine and correlation with expression profile of drug-related genes in human lymphoid cells.

Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA polymerization with promising activity in hematologic malignancies. Gemcitabine enters the cell mostly via the human equilibrative nucleoside transporter-1 (hENT1), while drug metabolism occurs by phosphorylation by deoxycytidine kinase (dCK), 5'-nucleotidase (cN-II) and cytidine deaminase (CDA) are the main inactivating enzymes. The aim of this study was to investigate the role of these determinants in gemcitabine cytotoxicity and analyze their expression in lymphoid cells. Cytotoxicity was assessed by MTT, and modulated by simultaneous addition of 2'-deoxycytidine (dCK natural substrate), tetrahydrouridine (CDA competitive inhibitor) and diethylpyrocarbonate (cN-II non-competitive inhibitor), while the expression of hENT1, dCK, cN-II, CDA and RR in WIL2-S, Jurkat and CCRF-CEM cells as well as in lymphoid cells from 25 chronic lymphocytic B-leukemia (B-CLL) patients was studied with quantitative-PCR. Cell cycle modulation and induction of apoptosis were analyzed by cytofluorimetry and bisbenzimide staining. Gemcitabine was highly cytotoxic, increased the cells in S-phase and significantly enhanced apoptosis. The crucial role of metabolism in gemcitabine activity was confirmed by the significant modulation of cytotoxicity by inhibitors of dCK, CDA and cN-II. Furthermore, PCR demonstrated a correlation between gemcitabine sensitivity and expression of its determinants, and that their values were within those observed in patients. These data indicate that gemcitabine is cytotoxic against lymphoid cells, affecting cell cycle and apoptosis. Furthermore, chemosensitivity may be predicted on the basis of gene expression profile of critical determinants involved in gemcitabine mechanism of action, suggesting the use of pharmacogenetic profiling for treatment optimization.

A pharmacokinetic-based test to prevent severe 5-fluorouracil toxicity.

BACKGROUND AND OBJECTIVES: Dihydropyrimidine dehydrogenase (DPD) plays a key role in the catabolism of 5-fluorouracil (5-FU) to 5-fluoro-5,6-dihydrouracil (5-FDHU), and as such, an impairment of DPD has been recognized as an important factor for altered 5-FU and 5-FDHU pharmacokinetics, predisposing patients to the development of severe 5-FU-associated toxicity. Our objectives were to avoid severe 5-FU toxicities in patients with greatly impaired 5-FU and 5-FDHU pharmacokinetics after the administration of a reduced test dose of 5-FU and to investigate possible 5-FU or 5-FDHU pharmacokinetic parameters of the test dose related to the most common drug toxicities that affect patients after the first cycle of 5-FU chemotherapy. METHODS: Pharmacokinetics of 5-FU/5-FDHU and DPD activity in peripheral blood mononuclear cells (PBMCs) were examined in 188 gastrointestinal cancer patients given a test dose of 5-FU, 250 mg/m2, 2 weeks before starting the planned 5-FU treatment of 370 mg/m2 plus l-folinic acid, 100 mg/m2, for 5 days every 4 weeks. Drug levels were examined by HPLC, and toxicities were graded according to World Health Organization criteria. RESULTS: The 5-FU test dose was well tolerated in all patients. Of 188 patients, 3 (1.6%) had marked alterations of 5-FU/5-FDHU pharmacokinetics (ie, 5-FU half-life [t(1/2 beta)] >5 hours, 5-FU total body clearance [CL(TB)] <1 L x h(-1) x m(-2), and 5-FDHU time to reach maximum plasma concentration [t max] > or = 45 minutes); they were excluded from 5-FU treatments and treated with irinotecan, which was well tolerated. The plasma disposition of 5-FU in the remaining 185 patients revealed an area under the curve (AUC) of 3.73 +/- 2.18 h x microg/mL (mean +/- SD), maximum plasma concentration (Cmax) of 16.78 +/- 8.61 microg/mL, and t(1/2 beta) of 0.16 +/- 0.15 hour, whereas the CL(TB) was 65.67 +/- 31.86 L x h(-1) x m(-2). The 5-FDHU plasma profile showed a Cmax value of 3.64 +/- 1.94 microg/mL, whereas the t max value was 26.63 +/- 10.06 minutes, with an AUC value of 3.71 +/- 1.90 h x microg/mL. The PBMC DPD activity was 202.15 +/- 141.14 pmol 5-FDHU x min(-1) x mg(-1) protein (95% confidence interval, 165-239.3 pmol 5-FDHU x min(-1) x mg(-1) protein). A significant correlation between 5-FU AUC and 5-FDHU AUC was found (r = 0.5492, P < .0001), whereas a weaker correlation between PBMC DPD activity and both 5-FDHU AUC (r = 0.328, P = .0121) and 5-FDHU Cmax (r = 0.369, P = .0044) was found. Interestingly, no relationships between PBMC DPD activity and common toxicities were found, whereas 5-FDHU t max values greater than 30 minutes were associated with the risk of moderate to severe neutropenia and diarrhea (P = .0323 and P = .0138, respectively; chi-square test). CONCLUSIONS: This study suggests a successful approach for preventing severe or life-threatening toxicities in gastrointestinal cancer patients who are candidates for standard 5-FU treatment by analyzing the 5-FU and 5-FDHU pharmacokinetic parameters after the administration of a reduced 5-FU test dose.