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.