Pharmacokinetic and Pharmacodynamic Studies of Lestaurtinib (CEP-701) and PKC-412: Cytotoxicity Is Often Dependent on Non-FLT3-Mediated Effects.

We undertook a study to compare two small molecule FLT3 inhibitors, CEP-701(lestaurtinib) and PKC-412. Both compounds have been shown to have activity as monotherapy in FLT3 mutant acute myeloid leukemia (AML), and both are being evaluated in combination with chemotherapy. Using in vitro phosphorylation and proliferation assays with leukemia cell lines and primary AML samples, we compared PKC-412 and one of its metabolites, CGP-52421, with CEP-701. We also studied the FLT3-inhibitory activity of plasma from clinical trial patients receiving these drugs using an ex-vivo bioassay. While both CEP-701 and PKC-412 could effectively inhibit FLT3 in vitro, CEP-701 was much more cytotoxic to primary samples at comparable levels of FLT3 inhibition. We conclude that inhibition of FLT3 by itself is often insufficient to kill AML blasts, even those harboring FLT3 activating mutations. The cytotoxic effect of CEP-701 in primary samples correlates with STAT5 inhibition, suggesting that CEP-701 inhibits other cellular targets upstream of STAT5 to induce cytotoxicity. PKC412 appears to be more selective for FLT3 than CEP-701, and is therefore less effective at inducing cytotoxicity in primary AML samples in vitro. However, the metabolite CGP-52421 is much less selective than its parent compound PKC-412 as determined using an IL-3 rescue assay, and CGP-52421 is more cytotoxic than PKC-412 against primary blast samples at comparable levels of FLT3 inhibition. In vitro, CGP-52421 is 24-fold less potent than PKC-412 at inhibiting FLT3 autophosphorylation, but in plasma, because it is less protein-bound, CGP-52421 is only 7-fold less potent than PKC-412. In patients receiving oral PKC-412, steady state levels of CGP-52421 are 10- to 20-fold higher than PKC-412 levels. After testing plasma samples from PKC-412 trial patients, we concluded that the metabolite CGP-52421 probably constitutes a substantial part of the anti-leukemia activity observed in some patients receiving oral PKC-412. More importantly, these studies demonstrate that (1) AML blasts harboring FLT3 mutations vary considerably in their dependence on FLT3 signaling for survival, (2) concurrent inhibition of other targets is usually necessary for cytotoxicity, and finally, (3) that our ex-vivo plasma assay represents an ideal surrogate test for monitoring the efficacy of FLT3 inhibitors.