Introduction

Metamizole (dipyrone) is an analgesic with antipyretic and spasmolytic properties, which is in use since almost 100 years. Despite many investigations concerning the analgesic effect of metamizole, the analgesic mechanism is currently not completely clear. In a well-designed study, Pierre et al. have shown that the two major metabolites of metamizole, 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA), inhibit COX-1 and COX-2 by interfering with the Fe3+ atom in the heme of the cyclooxygenases [1]. However, since the typical anti-inflammatory effect of the COX-inhibitors is not observed with metamizole in patients [2], additional, COX-independent mechanisms, may also be involved [3-7]. Similar to the analgesic activity, the spasmolytic effect of metamizole is clinically evident and experimentally established, but the mechanisms are not entirely established. Several possibilities have been proposed, among them opening of ATP sensitive potassium channels [8] and inhibition of G protein-coupled receptor (GPCR) mediated constriction of vascular smooth muscle cells [9].
Metamizole is a prodrug, which is converted to 4-MAA already pre-systemically in the intestinal tract and/or in the liver. 4-MAA has a high oral bioavailability (>80%) and is the principal metabolite in plasma [10, 11]. As shown in Figure 1, 4-MAA can be formylated to 4-formylaminoantipyrine (4-FAA) or demethylated to 4-aminoantipyrine (4-AA), which can be acetylated to 4-acetylaminoantipyrine (4-AAA). Less than 5% of orally administered metamizole is excreted in the urine as 4-MAA, the rest is excreted as 4-AAA, 4-FAA and 4-AA, as well as additional, quantitatively less important metabolites [11-13].
Although the four main metabolites of metamizole have been well-described, only the enzyme responsible for the formation of 4-AAA, a N-acetyltransferase [11, 14, 15] eventually recognized as N-acetyltransferase type 2 (NAT2) [16], has been unequivocally identified. In contrast, the enzymes performing the demethylation and formylation of 4-MAA are so far not known with certainty. Experiments with N,N-dimethyl-4-aminoantipyrine (4-DMAA), which carries two instead of one methyl group at the amino position of 4-aminoantipyrine, revealed that 4-DMAA can be converted to 4-AA by rat and rabbit liver microsomes, suggesting a cytochrome P450 (CYP)-mediated reaction [17-20]. La Du et al. showed that 4-MAA can be demethylated by isolated rabbit microsomes in a reaction using NADPH, Mg2+ and oxygen and producing formaldehyde, but this reaction accounted for less than 50% of 4-MAA degradation [20]. Twenty years after the publication of La Du et al., Noda et al. demonstrated that the oxidative conversion of 4-MAA to 4-FAA accounted for most of the microsomal activity that had not been identified by La Du et al. [21, 22]. In support of these findings, Geisslinger et al. verified that 4-MAA could be converted to 4-AA at a slow rate by human liver microsomes [23]. This reaction could be inhibited by ketoconazole, indicating the involvement of CYP3A4. In addition, patients with impaired liver cirrhosis have a prolonged half-life of 4-MAA, supporting the notion that 4-MAA is metabolized by the liver [15]. In a recent in vitro study, we could confirm that different hepatic CYPs are involved in the N-demethylation of 4-MAA but we also found demethylation activity by myeloperoxidase in neutrophil granulocytes, suggesting that a portion of 4-MAA might be extrahepatically metabolized to 4-AA [24].
Considering the uncertainties regarding N-demethylation of 4-MAA, the aim of the current study was to investigate the metabolism of 4-MAAin vitro using human recombinant CYP isoforms and in humans using established CYP inhibitors. The in vitro experiments were used to identify the most efficient CYPs regarding 4-MAA demethylation, whose contribution was subsequently investigated in vivo . The information in humans could also be used to estimate the clinical significance of potential interactions with the CYPs involved in the metabolism of 4-MAA.