In vitro metabolism of the phosphorothioate radioprotectors WR-2721 and WR-3689.

The phosphorothioate drugs S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) and S-(2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689) are effective in protecting certain normal tissues in vivo against radiation damage (Yuhas et al. , 1980; Menard et al., 1984; Spence et al. , 1986). However, studies with cultured cells demonstrate little or no protection when cells are treated with these phosphorothioate drugs (Purdie, 1979). Enzymatic action on WR-2721 is known to increase its radioprotective efficacy in vitro (Mori et al., 1983); incubation with purified alkaline phosphatase (AP) improves in vitro radioprotection in tissue culture (Calabro-Jones et al., 1985). We have investigated the kinetics of this metabolism in vitro, using radiolabeled drugs and thin layer chromatographic separation of the metabolites. These studies of in vitro metabolism have been correlated with the uptake and radioprotective efficacy in tumor cell lines in culture. Our conditions for studying metabolism involved incubation of radioactively labeled drug in various tissue culture media with Escherichia coli AP. [3H]-WR-3689 (450 mg/ml; 1.8 mM) or [35S]-WR-2721 (450 mg/ml; 1.75 mM) was incubated in Hanks' balanced salt solution (BSS) or Waymouth's MB752/1 (without added serum) and with 1.0 IU/ml AP. These incubations were carried out at 37°C, and the incubation vessel was bubbled with either air or argon to maintain a controlled atmosphere. At selected time points, samples of the incubate were removed. The enzyme was quenched by the addition of acid and frozen in liquid nitrogen. Samples were subsequently spotted on Whatman C 18 reverse phase TLC plates and developed in methanol/ ,0.5 M NaCI (1:1). The silica surface of the plates was then cut into 1 cm sections, scraped into scintillation vials, mixed with Insta-gel, and counted in a liquid scintillation spectrometer. Data are expressed as the percent of total radioactivity applied to the plate that was recovered at an Rf corresponding to standards of phosphorothioate and its associated thiol or disulfides (symmetrical or mixed). Figure 1 shows the results obtained from an incubation of WR-3689 wth AP in Hanks' BSS. With 1.0 IU/ml AP and an initial drug concentration of 1.8 raM, the phosphorothioate was rapidly metabolized with a half-life in solution of about 10 min. WR-3689-thiol appearance coincided with the disappearance of the parent drug. The thiol was only slowly oxidized in BSS, as only a small increase in WR-3689-disulfide was observed. This result was observed whether the incubation was carried out under an atmosphere of air or argon, indicating reasonable stability of the WR-3689-thiol to oxidation. However, the ratio of products was greatly influenced by the nature of the cell culture medium. Although WR-3689 disappearance was the same in Waymouth's MB752/1 cell culture medium, only a small amount of thiol accumulated in this medium and a steady rise in WR-3689-disulfide was observed. Figure 2 shows the results of an experiment in which metabolism was begun in Hanks' BSS, but at 30 min the reaction mixture was diluted with an equal volume of Waymouth's medium. A rapid decrease in WR-3689-thiol and a concomitant increase in WR-3689-disulfide(s) were observed. Similar results have been observed with WR-2721, viz. rapid hydrolysis and medium-dependent recovery of metabolic products.