title: Development and validation of a UPLC-MS/MS method for the novel folate-targeted small molecule drug conjugate EC1456 and its metabolites in tumor homogenates from mice.
abstract: EC1456 is a novel folate-targeted small molecule drug conjugate of tubulysin B hydrazide being developed as an anticancer agent for patients with advanced solid tumors expressing the folate receptor. To try and correlate circulating systemic levels of EC1456 and its metabolites to tumor concentrations and potentially develop a PK/PD model, a sensitive bioanalytical method was developed and validated for the quantitation of the analytes in KB tumor homogenates. The method involved homogenizing tumors with buffer containing N-maleoyl-β-alanine, mannitol and acetic acid, precipitation of the homogenate with acetone followed by heating at 55°C for 1h to convert tubulysin B hydrazide to its corresponding hydrazone. The extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method demonstrated good inter-day (3 runs, n=18) accuracy (-2.3% to 7.3%) and precision (1.7% to 10.3%) for all three analytes. Stability was established for three freeze-thaw cycles, 4h on the bench-top on ice, 20h in the autosampler at 8°C and for at least 46days frozen at -70°C. This method was successfully used to determine concentration of EC1456 and its metabolites tubulysin B hydrazide and tubulysin B in tumor homogenates in preliminary experiments with KB tumor bearing mice dosed intravenously with EC1456.
authors: Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA. Electronic address: srao749@gmail.com.
authors: Satish I
authors: SI
authors: Rao
authors: Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA.
authors: Michael
authors: M
authors: Pugh
authors: Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA.
authors: Melissa
authors: M
authors: Nelson
authors: Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA.
authors: Joseph A
authors: JA
authors: Reddy
authors: Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA.
authors: Patrick J
authors: PJ
authors: Klein
authors: Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA.
authors: Christopher P
authors: CP
authors: Leamon
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005492:Folic Acid / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009369:Neoplasms / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000378:metabolism*; D010875:Pipecolic Acids / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D009842:Oligopeptides; D004364:Pharmaceutical Preparations; D010875:Pipecolic Acids; D054852:Small Molecule Libraries; C546650:tubulysin B; D005492:Folic Acid
keywords: EC1456; LC–MS/MS; Mouse; Tumor homogenate; Validation