Abstract 4552: Defining the metabolic pathways of 13-cis retinoic acid.

Abstract

Abstract Introduction: Isotretinoin (13-cRA) induces differentiation of human neuroblastoma cells. Althoughhigh-dose, pulse 13-cis-RA treatment significantly improves overall survival of high-risk neuroblastoma patients, most recently together with ch14.18 antibody + cytokines, many children develop recurrent disease during or after 13-cis-RA treatment. Pharmacokinetic data from clinical trials showed a large variation in the levels of 13-cRA or a metabolite 4-oxo-13-cis-retinoic acid (4-oxo-13-cRA) with the metabolite being present up to 7-fold higher than 13-cRA in the plasma at steady-state (Br J Cancer 96:424-31, 2007). We sought to define the enzymes involved in metabolism of 13-cis-RA using liver microsomes. Methods: Phase I metabolism was assessed using pooled human liver (HLM) and intestinal (HIM) microsomes as well as supersomes expressing the recombinant human CYP isoenzymes (2B6, 2C9*1, 2C9*3, 3A5, 3A4, 2C19 and 2C8) for phase I metabolism. We tested inhibitors of CYP enzymes: fluconazole (50μM) (CYP2C9) and ketoconazole (50μM) (CYP3A4). For UGT (phase II) metabolism, we utilized UGT1A1, UGT1A3, UGT1A7, UGT1A9 and UGT1A8 isoenzymes. The generation of metabolites was analyzed by high performance liquid chromatography (HPLC). Results: We identified 4-oxo-13-cRA, monohydroxy-13-cRA, monohydroxy-4-oxo-13-cRA, and 13-cRA glucuronide and 4-oxo-13-cRA-glucuronide as metabolites in the plasma of a 13-cRA treated patient, of which 4-oxo-13-cRA was by far the most abundant. The enzyme kinetic studies revealed that the formation of 4-oxo-13-cRA by HLM (velocity: v=1.67) was much greater compared with HIM (v=0.08) at 100 μM 13-cRA. The formation of 4-oxo-13-cRA from 13-cRA by CYP3A4 isoenzyme was the highest, with CYP3A4 generating more than twice the 4-oxo-13ciRA compared with other isoenzymes tested at a given range of concentrations. In HLM, the CYP3A4 inhibitor ketoconazole (50μM) caused an 83% ± 0.0009 reduction in the formation of 4-oxo-13-cRA relative to no inhibitor controls, supporting that CYP3A4 is a key enzyme in the generation of 4-oxo-13-cRA. Other CYP isoenzymes (2B6, 2C9*1, 2C9*3, 3A5, 2C19 and 2C8) generated minimal amounts of 4-oxo-13-cRA. Glucuronidation of 13-cRA was >5 fold higher by UGT1A3 as compared to other UGT isoenzymes, while 4-oxo-13-cRA was glucuronized at similar rates by multiple isoenzymes of UGT (UGT1A1, UGT1A3, UGT1A7, UGT1A9, UGT1A8). Conclusions: CYP3A4 and UGT1A3 are the major enzymes metabolizing 13-cRA while the major metabolite, 4-oxo-13-cRA is a substrate for a broader spectrum of UGT enzymes. Future studies on the association between enzymatic polymorphisms and pharmacokinetics of 13-cRA may inform strategies to optimize dosing of 13-cRA in high-risk neuroblastoma patients. Citation Format: Poonam Sonawane, Hardeep Singh, C Patrick Reynolds, Min H. Kang. Defining the metabolic pathways of 13-cis retinoic acid. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4552. doi:10.1158/1538-7445.AM2013-4552