In vitro microassay for biological activity of cyclophosphamide metabolites.

A rapid tissue culture assay for detecting the activated metabolites of cyclophosphamide in serum is described. Serum obtained shortly after drug administration was added to microtest wells containing a known number of previously planted target cells. The presence and amount of drug was directly correlated with the degree of inhibition of target cell replication. Either transformed or untransformed cells could be used as the target cells. No inhibition of cell growth was observed if cyclophosphamide was added directly to target cells without prior in vivo activation. The appearance and persistence of activated drug in hamster serum was followed by using in vitro microassay. Cyclophosphamide administered intraperitoneally was metabolized to its active form very rapidly, reaching peak activity in 15 min. After 2 h, activity was no longer detectable. Drug given subcutaneously was activated less rapidly and persisted for a longer period of time. The kinetics of drug activation, as determined by tissue culture assay, appeared to be similar to that obtained by chemical assay for other species.