TUBULIN POLYMERIZATION BY PACLITAXEL (TAXOL) PHOSPHATE PRODRUGS AFTERMETABOLIC-ACTIVATION WITH ALKALINE-PHOSPHATASE

Paclitaxel (taxol) phosphate derivatives BMY46366, BMY46489, BMS180661 and BMS180820 were used to determine the ability of alkaline phosphat ase to convert these water-soluble potential prodrugs to tubulin-polym erizing metabolites (i.e., paclitaxel). Compounds were treated up to 1 80 min with an in vitro metabolic activation system composed of 10% bo vine alkaline phosphatase in 0.2 M tris, pH 7.4, or in 0.2 M glycine, pH 8.8, plus 0.05 M MgCl2. Samples were tested (either by direct addit ion or after methylene chloride extraction/dimethylsulfoxide resuspens ion) in spectrophotometric tubulin polymerization assays utilizing bov ine-derived microtubule protein. Pretreatment of 2'- and 7-phosphonoxy phenylpropionate prodrugs BMS180661 and BMS180820 with alkaline phosph atase for 30 to 120 min yielded relative initial slopes of about 20 to 100% at test concentrations equimolar to paclitaxel. High-performance liquid chromatography/mass spectrometry of BMS180661 treated with alk aline phosphatase confirmed the production of paclitaxel from the prod rug. In contrast, 2'- and 7-phosphate analogs BMY46366 and BMY46489 tr eated with alkaline phosphatase were not active in tubulin assays. Non e of the paclitaxel phosphate prodrugs polymerized tubulin in the abse nce of metabolic activation. The differences in tubulin polymerization with metabolic activation may be related both to accessibility of the phosphate group to the enzyme and to anionic charge effects. These re sults demonstrate that certain paclitaxel phosphate prodrugs can be me tabolized by alkaline phosphatase to yield effective tubulin polymeriz ation.