Abstract
Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO–induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.
Highlights
Even though xenobiotic metabolism during biotransformation serves mostly as biochemical detoxification process, resulting metabolites might cause adverse drug reactions and complications (Kirchmair et al 2015; Park et al 2011)
Human CPA metabolites produced by MroUPO Four unspecific peroxygenases secreted by different fungi were tested for their ability to selectively oxyfunctionalize cyclophosphamide (CPA)
The reaction mixtures were analyzed by HPLC-ELSD and the formed products determined by highresolution mass spectrometry (HRMS) using authentic standards of 4-ketocyclophosphamide (4-keto-CPA) and reduced perphosphamide (PPA)
Summary
Even though xenobiotic metabolism during biotransformation serves mostly as biochemical detoxification process, resulting metabolites might cause adverse drug reactions and complications (Kirchmair et al 2015; Park et al 2011). Depending on the metabolite structure and chemistry involved, a classical chemical synthesis of HDMs can be very complicated, time and resource consuming (Atzrodt et al 2012; Derdau et al 2010). The main pathway for metabolic clearance of pharmaceuticals is through oxidative mechanism predominantly catalyzed by liver cytochrome P450 monooxygenases (CYPs, EC 1.14.). They introduce in a highly selective manner oxygen into C-H-bonds of complex organic structures in order to convert lipophilic compounds into more hydrophilic and more excretable molecules (Guengerich 2008). Despite the versatility of those described methods the majority of these reactions are accompanied by low yields and selectivities and often lack in scalability (Genovino et al 2016; Zollner et al 2010)
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