Characterization Of The Activation Of The Non-Toxic Produg Cz48: Ultra-Sensitive, Simultaneous Detection With Cpt And Tumor-Specific In Vitro Activation

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO 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. To detect trace amounts of CZ48 and its active metabolite CPT in tumor and normal tissue, a novel, high throughput LC/tandem MS chromatographic method was developed and validated. Ultra high pressure liquid chromatography was employed to bring run times under five minutes and MRM transitions were used to selectively and sensitively detect both CPT and CZ48 on the MS/MS. Lower limits of detection were determined to be under 10pg and 30pg for CPT and CZ48 respectively while upper limits of detection exceeded physiologically relevant levels for both compounds. Using this method, we investigated the activation of CZ48 in human tumors and normal mouse and human tissues. Tumors were grown as xenografts in nude mice and then surgically excised along with normal mouse tissues. Cells were disrupted by mechanical homogenization and resulting suspensions gently centrifuged to remove debris. Total protein concentrations were normalized to 20 mg/ml (using BSA standards) and incubated overnight with 1000ng/ml CZ48. After extraction, CPT and CZ48 were analyzed with the chromatographic method described previously. Additionally, homogenates were serially centrifuged to yield various subcellular fractions. Each of these was incubated with CZ48 as described above in an effort to localize the esterase activity responsible for the activation of CZ48. Overall, tumor tissues show a much greater ability to hydrolyze CZ48 to CPT -thus unlocking its anticancer potential - than normal tissues do. This property of some tumor tissues may be the reason for the extreme selective toxicity exhibited by CZ48. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1707.