The Use Of Deuterium Isotope Effects To Shift The Partitioning Of Cyclophosphamide And Ifosfamide Among Competing, P450-Dependent, Oxidative Pathways

Prodrugs cyclophosphamide and ifosfamide are each ‘activated’ through a P450 oxidation at the C-4 position of the oxazaphosphorine ring. This reaction triggers a series of spontaneous (non-enzymatic) steps resulting in the formation of the ultimate DNA crosslinking agents, phosphoramide mustard and isophosphoramide mustard. Competing with reaction at C-4 are P450-catalyzed oxidations at the alpha positions of the chloroethyl side-chains; reactions at the side chains result in the production of the neurotoxic chloroacetaldehyde. Predominance of one pathway over another is isozyme-dependent and of greatest impact in ifosfamide therapy where about 50% of this drug is lost to side-chain oxidation (relative to an average of 10% for cyclophosphamide) and dose-limiting neurotoxicity. In this project, deuterium isotope effects are being explored as a means to effect ‘metabolic switching’ in the oxidations of cyclophosphamide and ifosfamide; that is, favoring oxidation at the C-4 position by disfavoring oxidations at the side chains. Cyclophosphamide and ifosfamide were synthesized with deuterium at the alpha and alpha’ positions of the chloroethyl chains. After optimizing reaction times, drug and enzyme concentrations, cyclophosphamide and ifosfamide (unlabeled and labeled) were incubated (20 min) with three, relevant, cDNA-expressed supersomes (human CYP2B6, CYP3A4, CYP3A5). The concentration of each metabolite produced by C-4 and the two possible side-chain oxidations was measured quantitatively using LC-MS-MS techniques developed for this project. For unlabeled cyclophosphamide, the fraction of products derived from C-4 oxidation was 73% (3A4), 90% (3A5) and 100% (2B6). Use of deuterium labeled cyclophosphamide gave 89% (3A4), 94% (3A5) and 100% (2B6); the amount of product produced by 2B6 and labeled drug was more than twice that given by unlabeled drug (56 micromoles versus 23 micromoles, respectively). For unlabeled ifosfamide, the fraction of products given by C-4 oxidation was 63% (3A4), 55% (3A5) and 36% (2B6). Use of deuterium labeled ifosfamide gave 82% (3A4), 75% (3A5) and 88% (2B6). Aside from the initial oxidation, the metabolic steps most likely to be impacted by deuterium isotope effects are those of the alkylation sequence by the metabolites phosphoramide mustard and isophosphoramide mustard. To determine the effect, if any, phosphoramide mustard and isophosphoramide mustard are being synthesized with deuterium at the alpha and alpha’ positions of the chloroethyl chains and their alkylation kinetics, relative to unlabeled compounds are being determined using 31 P NMR spectroscopy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4766. doi:1538-7445.AM2012-4766