In vitro pharmacodynamic assay for cancer drug development: application to crisnatol, a new DNA intercalator

Abstract

A microtiter pharmacodynamic assay is described that evaluates antitumor activity in vitro within a matrix of extracellular drug concentrations (C) and exposure times (T). The results were analyzed according to the pharmacodynamic principle: Cn x T = k, where n is the concentration coefficient and k is the drug exposure constant. This assay was used to characterize the antitumor activity of crisnatol (BW A770U), a member of the new arylmethylaminopropanediol class of DNA intercalators, in MCF-7 human breast cancer cells. The assay showed that drug action was a function of k, the extracellular drug exposure. Crisnatol had no effect at k less than 30 (n less than or equal to 1); was growth inhibitory at k = 30-1000 (n = 1), cytostatic at 1500, and cytotoxic at k greater than 2000 microMn-h (n = 2). These effects were directly related to increasing cellular retention of crisnatol. The threshold for growth inhibition was 0.02 fmol/cell, while cytoreduction required over 1 fmol/cell. The assay also yielded concentration-time curves of the form C = (k/T)1/n at specific surviving fractions, which were useful in selecting exposure conditions for further studies and emphasized the impact of exposure time on crisnatol activity. The hyperbolic nature of these curves suggested a unique parameter for comparing antitumor agents: the minimum C x T. This parameter represents the minimum exposure conditions required for a specified level of antitumor activity and accounts for differences in concentration coefficients among agents. The pharmacodynamic assay for crisnatol illustrates the importance of both concentration and exposure time in drug action and suggests a pharmacodynamic basis for comparing antitumor agents that conform to the Cn x T = k principle. Such agents include doxorubicin, 5-fluorouracil, cisplatin, etoposide, and tamoxifen. Analysis of these agents in the MCF-7 model shows that the minimum C x T parameter gives a relative cytotoxicity profile distinct from that found with the standard IC90 end point. This disparity was also seen in another, less differentiated breast cancer cell line (MDA-MB-231), and in normal human skin fibroblasts. Regardless of the end point, the in vitro cytotoxicity of crisnatol compares favorably with that of some clinically useful antitumor agents.