In vitro metabolism of 4-MAA
To identify the CYPs responsible for the metabolism of 4-MAA, the principal metabolite of metamizole, we started with investigating the metabolism of 4-MAA in vitro using supersomes. To ensure the functionality of the supersomes used, we first studied the metabolism of the specific CYP substrates in the absence or presence of the corresponding inhibitors. The recombinant CYPs investigated were functional as evidenced by the metabolism of the specific substrates and the formation of the respective metabolites, and the reactions could be blocked or impaired by the addition of the respective inhibitors (suppl. Figure 1). The incubation of 4-MAA with the same supersomes revealed that CYP1A2, CYP2C19 and CYP2D6 formed 4-AA most efficiently (Figure 2). In comparison, CYP2B6, CYP2C9 and CYP2C8 had a measurable but minor 4-MAA demethylation activity, while CYP2E1 and CYP3A4 exhibited no detectable activity. Similar to the experiments with specific substrates, the formation of 4-AA could be prevented or slowed by the addition of a specific inhibitor. In addition to 4-MAA N-demethylation, the assessment of the formation of 4-FAA showed that the only CYP capable of producing 4-FAA was CYP1A2 (data not shown). To the best of our knowledge, no CYP has so far been identified that catalyzes the formation of 4-FAA from 4-MAA.