Enzastaurine (Enza) is a novel protein kinase C-β (PKCβ) inhibitor with anti-angiogenic and anti-signaling activity. Pemetrexed (PMX) inhibits thymidylate synthase (TS) and is active against non-small cell lung cancer (NSCLC). In combination anti-angiogenic and enhanced cytotoxic effects can be expected, which have been studied mechanistically and in a clinical Phase I study.

The combination Enza-PMX was evaluated in SW1573 and A549 NSCLC cells using the multiple drug effect analysis, in which a Combination Index (CI) of <0.9 indicates synergism. Western blotting, ELISA and FACS were used to study protein phosphorylation, cell cycle effects and cell kill at IC50 concentrations. TS catalytic activity was measured radioactively. In a phase 1 study PMX (500 mg/m2) was given at day 1 and 22; Enza at day 4 and thereafter daily (500 mg). Heparinized blood samples from 15 patients (day 1, 4 and 22) were used to measure pharmacokinetics, VEGF and a marker for TS inhibition (deoxyuridine; UdR).

Simultaneous Enza-PMX was highly synergistic at a fixed (SW1573, CI=0.5; A549, CI=0.3) and variable ratio (fixed IC25 of Enza, CIs <0.1). PMX induced an S-phase arrest at 24 hr followed by an G2M arrest at 72 hr (increase of 15-20 %); Enza arrested cells in the G1 phase (increase of 10%), but Enza-PMX in G2M (increase of 25%). Enza-PMX increased cell kill at 72 hr (SW1573: >25%, versus <10% for each drug; A549: additive). These effects were preceded by decreased signalling: Enza, but not PMX reduced phosphorylation of Akt and GSK3β (downstream of PKCβ), but in Enza-PMX reduction was enhanced. Enza inhibited CDK2 phosphorylation, leading to downregulation of the S-phase regulator E2F-1, while both cdc25 and its phosphorylation were either decreased or completely abrogated by Enza-PMX, explaining the arrest at the G1/S and G2/M checkpoints, resp. E2F-1 down-regulation was associated with a decrease in TS expression, while Enza prevented PMX induced TS overexpression. Enza itself also inhibited TS and enhanced PMX mediated TS inhibition in intact cells (-89 and -80% in A549 and SW1573 cells, resp).

In patients PMX treatment increased plasma VEGF levels 4-fold after 2 hr and 6-fold after 4 to 6 hr. VEGF normalized after 4 days. At 22 days Enza-PMX treatment led to a 7-fold VEGF increase after 2, 4 and 6 hr. PMX treatment at day 1 induced a mean 1.5-fold UdR accumulation after 2-4 hr normalizing after 6 hr. Enza-PMX treatment at day 22 induced UdR 1.5-fold after 2 to 4 hr, but 2-fold at 6 hr and 2.5-fold after 4 days, indicating an increased TS inhibition by Enza-PMX in patients.

In conclusion: Enza-PMX is a very synergistic, cytotoxic combination, mediated by inhibition of signalling processes required for proliferation and by abrogation of cell cycle checkpoints enabling enhanced TS inhibition and increased apoptosis. Evaluation of biomarkers revealed an increased VEGF accumulation and an increased TS inhibition.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA