Abstract 759: Quantitation of the HDAC inhibitor belinostat (PXD-101) and metabolites in human plasma by a novel LC-MS/MS assay.
Authors: Kiesel Brian F. , Parise Robert A. , Tjornelund Jette , Christensen Mette K. , Loza Einars , Chu Edward , Kummar Shivaani , Beumer Jan H. 
Published: August 2012

Introduction: Histone deacetylases (HDAC) are frequently deregulated in human cancers and their inhibition allows re-expression of silenced genes. Belinostat is an HDAC inhibitor with in vitro and in vivo activity in multiple malignancies, currently in phase II trials. To date, the pharmacokinetics and metabolism of belinostat have not been adequately characterized. To support an organ dysfunction study and clinical development of belinostat, we developed and validated an LC-MS/MS assay for the sensitive, accurate, and precise quantitation of belinostat and its metabolites belinostat-glucuronide, methylated-belinostat, belinostat amide, 3-ASBA, and belinostat acid in human plasma. Methods: The assay used 50 µL of plasma, [13C6]-belinostat and [D5]-3-ASBA as internal standards and acetonitrile (0.1% TFA) for protein precipitation. A UPLC C18 column was used with a gradient elution from 90:10 to 10:90 water-acetonitrile (0.1% formic acid) over the course of 4 min followed by re-equilibration for 3 min (7 min total run time). Flow rate was 0.5 mL/min. An ABI 4000 tandem MS/MS with ESI positive and negative ionization in MRM mode was used to detect the analytes. Bioanalytical method validation was performed based on FDA guidelines. The applicability of the assay was demonstrated by quantitating belinostat and its metabolites in plasma of a human patient sampled over the course of 24 h. Results: Belinostat eluted at 2.9 min, while all metabolites eluted between 2.7 and 3.6 min. The assay was linear and accurate between 30 and 5000 ng/mL for all analytes; 3 triplicate standard curves assayed on 3 separate days displayed a CV &lt;19% at each of 6 different calibration points (30, 100, 300, 1000, 3000, 5000 ng/mL) for belinostat and CV &lt;29% for the metabolites. The accuracy of the assay was between 101% and 106% at 4 different QC levels (30, 100, 2500, and 4000 ng/mL) for belinostat and between 96% and 117% for metabolites, while the precision was &lt;17% at each concentration for belinostat and &lt;26% for metabolites. Recovery was &gt;69% for belinostat and &gt;64% for metabolites. Belinostat and all metabolites could be quantitated in human plasma after IV administration of belinostat over 30 min. Belinostat glucuronide appeared to be the major metabolite with up to 10-fold exposure relative to belinostat. Conclusion: We have developed a sensitive, accurate and precise LC-MS/MS assay that allows quantitation of belinostat and its 5 metabolites in human plasma. UGT1A1 is responsible for glucuronidation, which appears to be a major metabolic route. This assay will be a valuable tool to assess the pharmacokinetics and metabolism of belinostat in humans, and it is being used to support clinical studies employing belinostat in multiple clinical trials. Supported by Grants U01-CA099168 and N01-CM62209, P30-CA47904 from the NCI, and Spectrum Pharmaceuticals Inc and Topotarget A/S

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 759. doi:1538-7445.AM2012-759