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Pharmacodynamics of Immediate and Delayed Effects of Paclitaxel: Role of Slow Apoptosis and Intracellular Drug Retention¹

College of Pharmacy [J. L-S. A., D. L., Y. G., X. G., A. L. J., J. J., N. J. M., S. H. J., H-J. K., C-T. C., M. G. W.] and Comprehensive Cancer Center [J. L-S. A., Y. G., M. G. W.], The Ohio State University, Columbus, Ohio 43210

The kinetics of the time-dependent antitumor effects of paclitaxel are not fully understood; some literature reports indicate a higher activity by prolonging treatment durations, whereas other reports indicate no enhancement under in vitro conditions. The present study was designed to address this controversy and to determine the mechanism of the higher cytotoxicity associated with longer treatment durations. Six human epithelial cancer cell lines (bladder RT4, breast MCF7, pharynx FaDu, ovarian SKOV3, and prostate PC3 and DU145) were used. To determine whether the higher activity observed for the longer treatment durations is due to a delayed exhibition of drug effects and/or a reflection of cumulative effects that required a continuous drug exposure, cells were treated with paclitaxel for 3–96 h and then either: (a) immediately processed for drug effect measurement; or (b) washed, incubated in drug-free medium, and processed for drug effect measurement at 96 h. The overall drug effect (i.e., combination of cytostatic and apoptotic effects) was determined by the sulforhodamine B assay, which measures the cellular protein. In addition, to determine whether apoptosis occurs with a time delay, apoptosis was measured in cells that were collected immediately after drug treatment for various durations or in cells that were treated with drugs for 3 h but collected at later time points. Apoptosis was determined using agarose gel electrophoresis and by measuring the cytoplasmic DNA-histone complex using ELISA. The contribution of the intracellularly retained drug to the delayed drug effect was studied by characterizing the kinetics of cellular drug uptake and efflux and by examining the effect of removal of the intracellularly retained drug. All six cell lines showed similar results, as follows: (a) paclitaxel produced cytotoxicity that was exhibited immediately after treatment (immediate effect) and after treatment was terminated (delayed effect); (b) the immediate and delayed effects showed different pharmacodynamics. The immediate effect increased with treatment duration and drug concentration. For the delayed effect, all treatments produced the same maximum effect at 96 h, although treatments for 12 h showed higher IC₅₀s than longer treatments, whereas treatments for 24 h showed indistinguishable IC₅₀s; (c) treatment for as brief as 3 h was sufficient to induce apoptosis, which occurred with a lag time of about 24 h, although longer treatments produced a greater extent of apoptosis; (d) The intracellular and extracellular concentrations reached an equilibrium at 5 h, which rules out slow and/or insufficient uptake as the cause of the lower effects at shorter treatment times (i.e., <24 h); (e) upon removal of drug-containing medium, the amount of drug retained intracellularly was about 10% of the applied dose and was reduced to 0.5% after three successive washes, separated by 3-h equilibration periods; and (f) the delayed effect of the 3-h treatment was largely due to the drug retained intracellularly, whereas the delayed effect of the 24 h treatment was independent of the drug retained intracellularly. In conclusion, in human epithelial cancer cells, paclitaxel-induced cytotoxicity occurred after termination of drug treatment, which was partly due to the slow manifestation of apoptosis and partly due to the significant amount of drug retained intracellularly. Based on these findings and recognizing that some previous studies measured the immediate effect whereas the other studies measured the delayed effect, we propose that the conflicting data in the literature regarding the effect of treatment duration on paclitaxel activity under in vitro conditions are in part due to the different pharmacodynamics of the immediate and delayed drug effects. Furthermore, differences in the delayed effects for treatments of <24 h and the minimal differences for treatments of 24 h indicate that the delayed effect is maximally elicited by 24-h drug exposure.

¹ Supported in part by R37 CA49816 and R01 CA63363 from the National Cancer Institute, NIH. A. L. J. was supported in part by Grant T32CA09338 from the National Cancer Institute. D. L. was supported in part by the Pharmacia-Upjohn Fellowship. N. J. M. was supported in part by the American Foundation of Pharmaceutical Education Fellowship.

² To whom requests for reprints should be addressed, at College of Pharmacy and Medicine, Ohio State University, 508 Vernal G. Riff Building, 500 West 12th Avenue, Columbus, OH 43210. Phone: (614) 292-4244; Fax: (614) 688-3223.

Received 10/17/97. Accepted 3/17/98.

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