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Cellular Elimination of 2',2'-Difluorodeoxycytidine 5'-Triphosphate: A Mechanism of Self-Potentiation¹

Department of Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [V. H., Y. X., S. C., A. S., W. P.], and Lilly Research Laboratories, Indianapolis, Indiana 46285 [L. W. H., G. B. G.]

2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (<50 µM), dFdCTP elimination was linear (t = 3.3 h), but it became biphasic at intracellular dFdCTP levels > 100 µM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 µM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.

¹ Supported in part by Grant CH-130 from the American Cancer Society and Grant CA28596 from the National Cancer Institute, Department of Health and Human Services.

¹ Present address: Department of Internal Medicine. Hematology/Oncology, Klinikum Großihadcrn, University of Munich, Munich. Germany.

³ To whom requests for reprints should be addressed.

Received 8/21/91. Accepted 11/13/91.

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