In vitro studies on stability and phosphatase activity of the anticancer agent LB-100 and its active hydrolysis product endothall

LB-100, a small molecule PP2A inhibitor currently in phase 1 and 2 dose-finding and efficacy trials, is an amide that is stable at pH ≥ 10.5, but can hydrolyze at lower pH values to endothall and N-methylpiperazine. Endothall has been detected in plasma of patients after i.v. LB-100 administration, but in vitro LB-100 hydrolysis has not been studied in detail. LC-MS/MS assays of various LB-100 solutions showed that LB-100 rapidly hydrolyzes in aqueous solutions at room temperature (RT) at pH 5.6–6.5 to endothall (t_1/2 = 2.1–3.3 h). Although hydrolysis is much slower in PP2A assay medium HEPES pH 7.5 at RT (t_1/2 ∼ 20 h), sufficiently high concentrations of the potent phosphatase inhibitor endothall (IC₅₀ = 95 nM) are formed during a 2-hour incubation at RT to inhibit PP2A activity. At 37 °C LB-100 hydrolysis is much faster, with t_1/2 values of 3.2 h and 4.9 h in cell culture medium pH 6.8 and pH 7.4, respectively. LB-100 stock in DMSO contains low endothall concentrations, from 0.2 % of LB-100 when dissolved at RT, to 3 % of LB-100 when DMSO is heated at 65 °C. Endothall added via stock solutions and/or formed during incubations, is thus always present in PP2A assays of LB-100. Data from in vitro PP2A assays using DMSO stocks made at RT show that LB-100 is a weak PP2A inhibitor with an apparent IC₅₀ of 12.2 μM, and is 20-fold more potent (IC₅₀ = 0.59 μM), when DMSO stocks are heated to 65 °C and endothall concentrations in assay medium are higher. Endothall concentrations present in LB-100 assays can account for the observed PP2A inhibition, indicating that inhibitory activity measured in LB-100 assays is mainly due to endothall. These data suggest that LB-100 is a prodrug that acts as a PP2A inhibitor through hydrolysis to endothall, which is ultimately responsible for PP2A inhibition and LB-100′s pharmacological activity.