The most effective radioprotective drug now available is S-2(3-aminopropylamino)-ethyl phosphorothioic acid (WR-2721, RSP), a drug currently undergoing clinical trials as an agent which radioprotects and chemoprotects normal but not tumor tissue. Studies of the mechanism of action have been hindered by the lack of methods for measurement of RSP, its hydrolysis product (WR-1065, RSH), corresponding disulfide forms (RSS), other metabolites, and also by the failure to achieve good protection in cultured cell systems where key factors such as temperature, oxygen tension, and drug level can be accurately known and controlled. Using recently developed methods based upon fluorescent labeling and HPLC analysis for determination of RSP, RSH, and RSS we have found that alkaline phosphatase added to culture media containing RSP rapidly converts it to RSH and RSS, and promotes intracellular accumulation of RSH, with accompanying radioprotection, in fully oxygenated V-79 cells. We propose to exploit and expand this advance in the technology in order to: (1) establish the mechanisms of transport of RSP and its metabolites, (2) elaborate the mechanisms of toxicity associated with the drug, and (3) identify the drug forms and the mechanisms involved in radioprotection. The rates at which RSP, RSH, and specific RSS forms are taken up and exported by cells will be determined. The forms of the drug responsible for cell toxicity will be examined. The mechanism of radioprotection of V-79 cells will be investigated by determining the relationship between radioprotection and intracellular drug level under both oxic and anoxic conditions, and determining whether protection involves initial damage or biochemical repair processes. Equilibrium binding of specific drug forms to calf thymus DNA will be used as an initial probe to assess the importance of DNA-drug interactions. Significant findings with V-79 cells will be tested in other normal cell lines to assess species and tissue variation, and in selected tumor cell lines to ascertain whether the in vivo difference between normal vs tumor cells has a biochemical basis that can be measured in vitro. The results of these studies should greatly clarify our understanding of how WR-2721 functions, will identify key factors to be monitored in clinical studies, and may indicate features to be considered in improved drug design.
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