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.