Abstract 3504: Debio 0507 primarily forms diaminocyclohexane-d(GpG) and -d(ApG) DNA adducts in HCT116 cells

Abstract

The major DNA adducts formed by Debio 0507 in HCT116 human colon carcinoma cells were determined by a combination of inductively coupled mass spectrometry (ICP-MS) and ultraperformance liquid chromatography mass spectrometry (UPLC-MS/MS). Debio 0507 is a micellular copolymer of trans-RR-diaminocyclohexane-platinum(II) (DACH-Pt) with polyethylene-glycol (PEG)-polyglutamate (pGlu). Debio 0507 is long lasting compared to oxaliplatin (platinum is detectable in plasma and tumor for up to 14 days) and has shown anti-tumor activity and low toxicity in several tumor models. Debio 0507 has been shown to accumulate in tumors, but the intracellular activation mechanism and type of DNA adducts formed were not previously known. We have previously reported an UPLC-MS/MS method for the identification and quantification of the intrastrand GG DNA adducts formed by cisplatin (Baskerville-Abraham et al, Chem. Res. Toxicol., 22: 905-912, 2009). The limit of quantification of this assay was 3 fmol or 3.7 adducts per 108 nucleotides. This assay was modified slightly to identify the major DNA adducts formed by Debio 0507 and to compare them to the major DNA adducts formed by oxaliplatin. HCT116 cells were incubated with equitoxic doses of Debio 0507 (0.014 ug Pt/ml) and oxaliplatin (0.0006 ug Pt/ml) for 3 days. DNA was isolated from the cells and digested as described previously for cisplatin-treated DNA. ICP-MS was used to quantify Pt adducts in the DNA digests. The Pt adducts were 7.4/104 deoxynucleosides for Debio 0507-treated cells and 5.5/104 deoxynucleosides for oxaliplatin-treated cells following the DNA digestion step. DACH-Pt-d(GpG) and DACH-Pt-d(ApG) adducts were then separated from deoxynucleosides by HPLC. Following HPLC purification, the total Pt DNA adducts recovered were 63 pmoles (29% recovery) for Debio 0507 and 58 pmoles (38% recovery) for oxalipltin by ICP-MS, with approximately equal amounts of DACH-Pt-d(GpG) and DACH-Pt-d(ApG) adducts recovered at this stage (The DACH-Pt-d(GpG) peak overlapped with the deoxythymidine peak, which diminished the recovery of DACH-Pt-d(GpG)). Finally, UPLC-MS/MS in the positive ion mode was used to confirm the identity of the DACH-Pt-d(GpG) (904.2 m/z→459 m/z and 904.2 m/z→610 m/z) and DACH-Pt-d(ApG) (888.2 m/z→459 m/z and 888.2 m/z→594 m/z). These data show that the major DNA adducts formed by Debio 0507 are the DACH-Pt-GG and DACH-Pt-AG adducts and at equitoxic doses Debio 0507 and oxaliplatin form similar levels of DACH-Pt-GG and DACH-Pt-AG adducts which makes it unlikely that Debio 0507 forms significant amounts of other DNA adducts of equal or greater cytotoxicity. (Supported by Research Contract Debio 0507-069 with Debiopharm SA and P30-ES10126) 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 3504.