Pivanex inhibits histone deacetylase activity in non small cell lung cancer patients

Proc Amer Assoc Cancer Res, Volume 45, 2004 4016 Pivanex™ (pivaloyloxymethyl butyrate) is a histone deacetylase (HDAC) inhibitor that distributes rapidly into tissues and induces cell differentiation, leading to apoptosis in a wide range of tumor cells examined. Pivanex is very well tolerated, and has demonstrated phase II single-agent activity in non-small cell lung cancer (NSCLC) patients with refractory or relapsed disease. It is currently in a phase IIb randomized, controlled study in combination with docetaxel (Taxotere™) in second line treatment of NSCLC. Using the NCI-H23 human NSCLC cell line as a model for Pivanex’s activity, we examined HDAC inhibition at various time points following a single in vitro addition of Pivanex. Hyperacetylation of histones 3 and 4 were quantified by Western blot analysis, and HDAC activity was determined with a fluorescent-substrate based enzyme activity assay (Biomol, Inc.). HDAC activity was then evaluated in NSCLC patient peripheral blood mononuclear cells (PBMCs) before and after treatment with Pivanex. Pivanex was administered by 6-hr infusions on 3 consecutive days at a dose of 2.5 g/m2. Blood samples were collected just before starting Pivanex treatment (day 1) and the day after a course of Pivanex infusion (day 4). PBMCs were isolated with sodium citrate Vacutainer-CPT tubes (Becton Dickinson) and frozen until analysis. PBMC pellets were lysed in osmotic lysing buffer containing 1% protease and phosphatase inhibitor cocktails (Sigma) with or without 0.3% SDS, respectively, for Western blot and HDAC activity assays. The total protein concentration was determined with BCA reagent (Pierce) and equal amounts of total protein were analyzed. Results: For NCI-H23 NSCLC cells, a single treatment with Pivanex resulted in the hyperacetylation of histone 3 at 1, 3, 7, 24 and 48 hours. HDAC enzyme activity was found to be inhibited at all time points examined: 1, 3, 5, 7, 24, 48, and 72 hours; with maximum inhibition to 25% at 72 hours. Pivanex-treatment in the clinic also resulted in the hyperacetylation of histones 3 and 4 in patient-derived PBMCs, one day after initial infusion. In addition, HDAC activity from patient PBMCs on day 4 was also strongly inhibited after Pivanex treatment. Conclusion: Pivanex treatment in the clinic results in the inhibition of HDAC enzyme activity leading to the hyperacetylation of histones as previously demonstrated in in vitro experiments. PBMCs from patients undergoing Pivanex therapy can thus be monitored for the activity of this potent HDAC inhibitor.