Abstract 1682: Assessment of pharmacokinetics and pharmacodynamics correlation of a novel fluorescent anticancer prodrug

Abstract Prodrug therapies provide high promise in improving current cancer treatments. A novel anticancer prodrug 6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (CNOB) and a bacterial nitroreductase (ChrR6) that can efficiently activate reductive prodrugs have recently been discovered by our group. This CNOB/ChrR6 regimen is a superior anticancer treatment compared to the prodrugs currently in clinical use (Mitomycin C) or in clinical trials. Both CNOB and its activated compound 9-amino-6-chloro-5H-benzo[a]phenoxazine-5-one (MCHB) are fluorescent with distinctive emission wavelengths at 550 nm and 625 nm, respectively. This feature provides a unique opportunity to evaluate prodrug activation and pharmacokinetics (PK). Moreover, it likely constitutes a reliable tool to predict pharmacodymamic (PD) outcomes based on PK evaluations. Our objectives are to appraise the correlation between the PK and PD parameters, especially based on the quantitative fluorescence measurements, which will provide an innovative approach for drug development and therapeutic monitoring. Murine mammary cancer (4T1) cells expressing luciferase gene (4T1-luc) and 4T1-luc transfected with humanized ChrR6 (4T1-luc/hChrR6) were utilized. In vitro cell viability from CNOB (15 μM) treatment was determined by both the MTS and SRB assays. Fluorescence of CNOB and MCHB in cell culture medium was measured using a Tecan plate reader. Both types of cells were implanted in the mammary fat pad of nude mice to generate tumor xenografts. CNOB was administered intravenously (3.3 mg/kg), and fluorescence images were taken at various times (1, 2, 4, 6, 12 and 24 h) via IVIS® Spectrum, followed by sacrifice for tissue drug analysis using high performance liquid chromatography (HPLC). Correlation and statistical analysis were performed via GraphPad Prism®. In vitro results demonstrate significant (p < 0.01) time-dependent responses both in MCHB fluorescence generation and cell viability reduction. MCHB fluorescence (PK) is highly correlated (ρ = 0.96) with the reduction of cell viability (PD) only in cells expressing HChrR6. We found a direct correlation between tumor MCHB levels determined via fluorescence imaging and HPLC analysis from in vivo studies. The imaging results show significantly higher (p < 0.05) MCHB fluorescence levels in 4T1-luc/hChrR6 tumors as compared to 4T1-luc tumors, with calculated MCHB area under the concentration curve (AUC) of 582.0 ± 246.2 and 50.7 ± 23.7 μg·h/L (n = 5), respectively. We are currently assessing other PD parameters, such as cell apoptosis and/or tumor regression, as well as more in depth PK analysis in order to establish the PK-PD correlation in vivo. In conclusion, the unique fluorescent prodrug described here provides a reliable marker for PK determination of the therapeutic compound (MCHB) in tumors, which can be further utilized for the prediction of cancer treatment outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1682.