Impaired dacarbazine activation and 7-ethoxyresorufin deethylation in vitro by polymorphic variants of CYP1A1 and CYP1A2: implications for cancer therapy Benjamin C. Lewis a,b , Porntipa Korprasertthaworn c and John O. Miners b Objectives To extend our understanding of how interindividual variability mediates the efficacy of cancer treatment. Materials and methods The kinetics of dacarbazine (DTIC) N-demethylation by the most frequent polymorphic variants of CYP1A1 (T461N, I462V) and CYP1A2 (F186L, D348N, I386F, R431W, R456H) were characterized, along with kinetic parameters for the O-deethylation of the prototypic CYP1A substrate 7-ethoxyresorufin, using recombinant protein expression and high-performance liquid chromatographic techniques. Results A reduction of ∼ 30% in the catalytic efficiencies (measured as in-vitro intrinsic clearance, CL int ) was observed for DTIC N-demethylation by the two CYP1A1 variants relative to wild type. Although a modest increase in the CL int value for DTIC N-demethylation was observed for the CYP1A2 D348N variant relative to the wild type, the CL int for the F186L variant was reduced and the I386F, R431W, and R456H variants all showed loss of catalytic function. Conclusion Comparison of the kinetic data for DTIC N-demethylation and 7-ethoxyresorufin O-deethylation indicated that alterations in the kinetic parameters (K m , V max , CL int ) observed with each of the CYP1A1 and CYP1A2 polymorphic variants were substrate dependent. These data indicate that cancer patients treated with DTIC who possess any of the CYP1A1-T461N and I462V variants or the CYP1A2-F186L, D348N, I386F, R431W, and R456H variants are likely to have decreased prodrug activation, and hence may respond less favorably to DTIC treatment compared with individuals with wild-type CYP1A alleles. Pharmacogenetics and Genomics 26:453–461 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. Pharmacogenetics and Genomics 2016, 26:453–461 Keywords: CYP1A1, CYP1A2, cytochrome P450, dacarbazine, enzyme kinetics, ethoxyresorufin, genetic polymorphism, prodrug activation a Department of Clinical Pharmacology, b Flinders Centre for Innovation in Cancer, School of Medicine, Flinders University, Adelaide, South Australia, Australia and c Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand Correspondence to Benjamin C. Lewis, BSc, PhD, MRACI CCHEM, Department of Clinical Pharmacology, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia Tel: + 61 8 82044031; fax: + 61 8 82045114; e-mail: ben.lewis@flinders.edu.au Received 18 April 2016 Accepted 21 June 2016 Introduction The activation of prodrugs is generally dependent on the intrinsic ability of the liver and/or other tissues to metabo- lize the compound. As such, activation is highly sensitive to changes in enzyme activity. Interindividual variability in CYP-mediated metabolism, resulting from genetic poly- morphism and other factors [1], renders each patient unique with respect to prodrug activation, as well as drug dosage and kinetics. Thus, altered metabolic activation of antic- ancer agents in patients carrying variant CYP alleles may have significant therapeutic consequences. Chemotherapy remains an important option for the treatment of metastatic melanoma. In Australia and the USA, the prodrug dacarbazine (DTIC; Fig. 1) is typically used in a curative regimen with other agents for the majority of patients [2]. DTIC produces a modest anti- tumor response in patients with melanoma (∼19%) [3]. Treatment of stage-I melanoma has a reasonable prog- nosis with the use of adjuvant interferon-α-2b therapy, although 5-year survival rates in patients with stage-IV disease remain poor at only 5–10%. Currently, the mechanism(s) responsible for melanoma chemoresistance to DTIC remain unknown. Fig. 1 N N N N N NH 2 CH 3 CH 3 CH 3 NH O O O O α α Dacarbazine 7-Ethoxyresorufin The chemical structures of dacarbazine [5-(3,3-dimethyltriazen-1-yl)- 1-imidazole-4-carboxamide] and 7-ethoxyresorufin (7-ethoxy-3H- phenoxazin-3-one). The site of dealkylation of each substrate by CYP1A1 and CYP1A2 is shown as ‘α’ . Original article 453 1744-6872 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/FPC.0000000000000236 Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.