WR-2721 is an FDA-approved agent which protects normal cells from toxic effects of alkylating agents. It is a prodrug that is dephosphorylated by alkaline phosphatase to the active free thiol form, WR-1065. We administered WR-2721 (825 mg/m2 IV over 15 min) to 7 patients immediately prior to and 3 hrs after ifosfamide and cyclophosphamide (POG #9457). Drug levels in whole blood (WB), plasma (P), and blood cells (BC) were measured following each infusion. For each time point, 1 ml of blood was added to a purple-top Vacutainer tube containing 30 mM monobromobimane. Samples were mixed and shipped on ice. The biman derivatives were prepared, separated by HPLC, and detected by fluorescence as previously described (Fahey, R.C. and Newton, G.L., Int. J. Rad. Onc. Biol. Phys. 1985;11:1193-7). There were no significant differences in WR-2721 and its metabolites between the ifosfamide and cyclophosphamide courses. The mean±SD(n) level of WR-2721 in WB at the end of infusion was 106±132(11) μM (range: 0-391), declining with a half-life of 4.2±1.9(6) min (range: 1.8-6.6). The mean±SD(n) level of WR-1065 at the end of infusion in WB was 66±32(21) μM (range: 12-125), in P 89±31(10) μM (range: 17-22), and in BC 84±36(10) μM (range: 40-146). The WR-1065 levels declined biexponentially; the mean ±SD(n) alpha half-lives and beta half-lives were 16.8±7.0(22) min (range: 4.1-29.2) and 67±14(3) min (range: 54-82) in WB, 13.9±6.6(10) min (range: 4.8-24.5) and 91±35(2) min in P. and 16.2±7.2(12) min (range: 5.9-29.2) and≈44 min in BC, respectively. A several-fold increase in the cystein:cystine ratio was observed in P and BC, lasting for at least 4 hrs. These results demonstrate that WR-2721 treatment can substantially modulate the extracellular and intracellular redox states and provide prolonged cytoprotection.