Pivanex, a histone deacetylase inhibitor, is synergistic with Velcade in inhibiting growth of human non-small cell lung cancer cells

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Pivanex® (pivaloyloxymethyl butyrate) is a histone deacetylase inhibitor with anti-neoplastic activity inhibiting tumor cell growth and inducing tumor cell differentiation and apoptosis. Pivanex has shown single-agent activity in non-small cell lung cancer (NSCLC) patients. To evaluate the potential for combining Pivanex with Velcade® (bortezomib) in advanced NSCLC, Pivanex was evaluated alone and in combination with Velcade for growth inhibitory activity in the human NSCLC cell lines A549 and NCI-H460. Single drug treatments with either Pivanex or Velcade were initially performed using two different schedules. In the pretreatment schedule, cells were initially treated for 72 hours with the respective drug. After washing, the cells were incubated in media without drug for another 72 hours. In the post-treatment schedule, cells were incubated for 72 hours in media without drug, and then followed by 72 hour treatment with either Pivanex or Velcade. At the end of the study cell viability was assessed by the MTS assay. Each drug concentration experiment was performed in eight independent wells. IC₅₀ values were calculated using GraphPad™. Single-agent Pivanex IC₅₀ values in A549 and NCI-H460 cells were 28.7 μM and 28.2 μM for the pretreatment schedule, and 198.2 and 176.1 μM for the post-treatment schedule, respectively. Single-agent Velcade treatment resulted in IC₅₀ values of 1.99 and 0.67 μM, for the pre-treatment schedule and 1.64 and 0.63 μM for the post-treatment schedule, for A549 and NCI-H460 cells, respectively. Combination experiments were performed using the 72 hours pre- and post-treatment schedules with dose ranges for Pivanex and Velcade selected based on the single agent experiments. In the post-treatment protocol the dose response curve for Velcade was significantly shifted to the left by 1-2 log units in the presence of Pivanex. In the A549 line, the IC₅₀ value for Velcade alone was 2.64 μM compared to 0.44 μM in the presence of increasing concentrations of Pivanex. In the NCI-H460 line, the IC₅₀ value for Velcade alone was 2.55 μM, compared to 0.22 μM in the presence of increasing concentrations of Pivanex. Isobologram analysis of cells pre-treated with Velcade followed by Pivanex demonstrated significant synergistic effects on cell viability across a range of doses in both A549 and NCI-H460 lines (Combination index for both cell lines ranged from 0.1-0.5). These results suggest that combination treatment of Velcade, a proteasome inhibitor, followed by Pivanex, a histone deacetylase inhibitor, may have superior anticancer efficacy to treatment with either agent alone. Further studies are planned to evaluate this possibility in the clinic.