Abstract 2981: Carboxylesterase 2 hydrolyzes a gemcitabine prodrug (LY2334737) and provides a patient tailoring biomarker
Authors: Pratt Susan E. , Shepard Robert L. , Durland-Busbice Sara , Heinz-Taheny Kathleen , Dantzig Anne H. 
Published: June 2012

Abstract
LY2334737 (LY) is a prodrug analog of gemcitabine with an amide linked valproate. LY is orally bioavailable, non-toxic, and cleaved in the liver to release gemcitabine systemically. The studies presented identify carboxylesterase 2 (CES2) as the enzyme responsible for the hydrolysis of LY, and further outlines how tumor expression of CES2 would enhance LY efficacy.
Employing HPLC and enzyme inhibitors, CES2, and not CES1 or CES3, was identified as the liver enzyme responsible for cleaving LY to gemcitabine. Carboxylesterases rapidly hydrolyze ester bonds; however the hydrolysis of the prodrug amide bond occurs at a much slower rate. This observation supports the findings that only a fraction of administered LY is converted to gemcitabine, the two agents circulating at a ratio of 10:1 respectively in humans.
In view of the persistent levels of LY in the blood, it was thought that CES2 expression in tumors might provide an additional site of prodrug activation. A number of cancer cell lines, including the NCI60 panel, were screened in vitro for sensitivity to LY, which was defined as the ratio of the prodrug EC50 versus gemcitabine EC50 being less than 15. Cells which met this criterion were all found to express CES2. The ability of CES2 to confer cellular sensitivity to LY was confirmed through inhibition of cytotoxicity by 10uM loperamide, a CES2-specific inhibitory dose, and CES2 knockdown with shRNA in a sensitive cell line. Transfection of CES2 into the LY insensitive cell line HCT116 resulted in increased responsiveness to LY that directly correlated to the level of CES2 expression.
In vivo studies comparing LY efficacy with xenografts of mock- and CES2-transfected HCT116 cell lines also demonstrated that CES2 expressing tumors are more sensitive to LY treatment than non-expressors. As such, employing CES2 as a tailoring biomarker would identify those tumors that would have enhanced sensitivity through their ability to intracellularly cleave circulating LY and result in increased levels of gemcitabine directly at the tumor site. Immunohistochemical characterization of human tumor types revealed that head and neck, colorectal, and squamous NSCLC tumors have a high incidence of CES2 expression.
Gemcitabine administered IV is first or second line therapy in many cancers. LY, as an oral agent, is ideally suited to be dosed, and deliver gemcitabine, metronomically. The advantages of a metronomic dosing strategy include the ability to deliver sustained dosing of chemotherapeutic agents with lower toxicity and potential antiangiogenic activity. Additionally, tailoring treatment to CES2 expressing tumors could enhance efficacy through both a systemic and a local generation of gemcitabine.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2981. doi:10.1158/1538-7445.AM2011-2981