CZ48, the 20-O-propionate ester of Camptothecin (CPT), is a non-toxic prodrug of CPT first described by Cao et al. (1998). The propionate side-chain is enzymatically cleaved in target tissues. This gives rise to CPT, an inhibitor of Topoisomerase I, which then blocks cell division causing selective tumor toxicity. We have previously established the preferential activation of CZ48 in tumor tissues compared to normal tissues and an ultra-sensitive assay to detect it (2009). The present studies explore the specifics of that activation in vitro using both cells in culture and surgically removed xenografted tumors from nude mice. DOY lung adenocarcinoma was grown subcutaneously in nude mice and then surgically excised, homogenized and serially centrifuged to produce specific subcellular fractions. The bulk of esterase activity was found to be concentrated in the cytosol, while the microsomal fraction showed minimal activity. All other fractions tested were below the limit of detection. Microsomes showed only a fraction of the esterase activity of the cytosol (99.78 ± 14.89 ng/ml versus 487.48 ± 16.69 ng/ml CPT produced overnight), but even that little activity was eradicated in the presence of an NADPH redux regeneration system (10.0 ± 7.26 ng/ml). This inhibition was also observed in the cytosolic fractions. Deconstructing the redux system led to the conclusion that NADPH was inhibiting the activation of CZ48: when incubating cytosol with CZ48 in the presence of 26.1 mM NADPH, 66mM MgCl2, or 66mM Glucose-6-phosphate, only NADPH exhibited any effect on the esterase activity of CZ48, resulting in only 106.00 ± 15.49 ng/ml CPT produced overnight. These data reinforce the notion that CZ48 is metabolized to CPT by a non-microsomal, non CYP450 enzymatic process in tumor tissues.

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 3232. doi:1538-7445.AM2012-3232