Discussion
The aim of the current study was to find out which CYPs are involved in
the metabolism of 4-MAA and whether inhibition of the CYPs involved
leads to clinically relevant drug interactions. The in vitroexperiments with supersomes revealed that CYP1A2 is the most important
enzyme for 4-MAA metabolism and that CYP2C19 and CYP2D6 contribute to
4-MAA N-demethylation. Our in vivo data confirmed these findings
and showed that the effects of ciprofloxacin and fluconazole regarding
inhibition of the formation of 4-AA and 4-FAA are additive.
Both our in vitro and in vivo experiments showed that
CYP1A2 is the dominant enzyme for the demethylation of 4-MAA to 4-AA but
also for the conversion of 4-MAA to 4-FAA. So far, it had been
demonstrated that both reactions are catalyzed by hepatic microsomes
[22-24] but the CYPs involved had not been clearly identified and
verified in a clinical study. The results of the current study are in
agreement with those in a recent study where we investigated the effect
of metamizole on the activity of different CYPs [30]. In this study,
we found that metamizole inhibits the conversion of caffeine to
paraxanthine, which is catalyzed by CYP1A2, most likely in a competitive
fashion. The results of the current study support this interpretation.
However, the findings in the current study disagree with the reports of
Geisslinger et al. [23] and of Bachmann et al. [24], which were
both performed with human microsomes as the enzyme source. Geisslinger
et al. showed that the demethylation of 4-MAA to 4-AA by human hepatic
microsomes could partially be inhibited by ketoconazole, suggesting a
major contribution of CYP3A4 [23]. Geisslinger et al. used a
ketoconazole concentration of 10 µM in their study, a concentration far
below the Ki of ketoconazole for CYP1A2 and 2C19
[31], excluding inhibition of CYP1A2 and CYPC19 as an explanation
for their findings. Bachmann et al. investigated the conversion of 4-MAA
to 4-AA by human liver microsomes using specific CYP inhibitors and
identified CYP2B6, 2C8, 2C9 and 3A4 as the most important contributors
to 4-MAA demethylation [24]. The discrepancies between the current
study and the studies of Geisslinger et al. and Bachmann et al. may be
due to the different enzyme sources used, human recombinant CYPs
expressed in supersomes versus human liver microsomes. The discrepancy
is relevant, indicating that results obtained in in vitro studies
should be confirmed in another in vitro system or, preferably, by
a clinical study. The fact that we obtained almost identical results for
the in vitro and in vivo investigations in the current
study supports the notion that CYP1A2 is the most important CYP for
4-MAA N-demethylation and conversion to 4-FAA.
The FDA defines strong, moderate and weak enzyme inhibitors as drugs
that increase the AUC of a sensitive substrate by ≥5-fold, ≥2 to
<5-fold, and ≥1.25 to <2-fold, respectively.
[32]. The FDA lists ciprofloxacin as a strong inhibitor of CYP1A2
and fluconazole as a strong inhibitor of CYP2C19 and a moderate
inhibitor of CYP2C9 and CYP3A4. In the current study, ciprofloxacin
increased the AUC of 4-MAA time-dependently by a factor of 1.31, 1.56
and 1.51 and fluconazole by a factor of 1.10, 1.32 and 1.17 at 6, 8 and
12 h, respectively (suppl. Table 1). According to the FDA, the
inhibition of 4-MAA metabolism by ciprofloxacin and fluconazole was
therefore weak. The reason for an only weak inhibition in the presence
of strong inhibitors could be the existence of alternative metabolic
pathways. Therefore, we also assessed the combined application of
ciprofloxacin and fluconazole, which increased the effect on the AUC of
4-MAA, resulting in AUC ratios of 1.54, 1.90 and 1.92 at 6, 8 and 12 h,
respectively. The combined application of ciprofloxacin and fluconazole
showed that the effects of ciprofloxacin and fluconazole on the AUCs of
the metamizole metabolites are additive, excluding a mutual compensation
between CYP1A2 and CYP2C19. However, Volz and Kellner have investigated
the pharmacokinetics of orally administered14C-labelled metamizole in healthy volunteers
[12]. They detected 7 metabolites in serum and could identify four
of them as 4-MAA, 4-AA, 4-FAA and 4-AAA. Forty-eight hours after
administration, 90% of the radioactivity had been excreted renally, but
the four metabolites accounted only for approximately 60% of the
excreted radioactivity. The study therefore indicated the existence of
additional metabolic pathways that may compensate for the inhibition of
CYP1A2 and CYP2C19.
Based on the results in the supersomes, compensation by CYP2D6 is an
obvious possibility, which we didn’t study in our clinical trial.
However, the activity of CYP2D6 in supersomes was not higher than for
CYP2C19, and, in contrast to CYP1A2 and CYP2C19, the CYP2D6 genotype
showed no correlation with the metabolic activity, rendering a major
contribution of CYP2D6 to the metabolism of 4-MAA unlikely.
An additional possibility is the contribution of myeloperoxidase in
granulocytes, as proposed in the report of Bachmann et al. [24]. The
results of the current study do not exclude this possibility but suggest
that the contribution of this pathway would most likely be less than the
contribution by the described hepatic metabolism. If the extrahepatic
metabolism were dominant, inhibition of the (in that case minor) hepatic
pathway would not be expected to increase the AUC of 4-MAA and to
decrease the formation of 4-AA. The inhibition of the (minor) hepatic
pathway should be compensated by the dominant extrahepatic pathway. In
the current study, pretreatment with ciprofloxacin/fluconazole increased
the AUC0-12h of 4-MAA by 92% and decreased the
AUC0-12h of 4-AA by 24%, rendering the existence of a
dominant extrahepatic pathway unlikely.
Even if the interaction with CYP1A2 and CYP2C19 inhibitors is
quantitatively small, this does not mean that this interaction is
clinically negligible. Important adverse reactions of metamizole are
hypotensive reactions, skin eruptions, myelotoxicity possibly leading to
agranulocytosis and hepatic injury [33]. While hepatic injuries and
skin toxicities are mainly dose-independent, immunological reactions
[33, 34], hypotensive reactions and myelotoxicity appear to be
dose-dependent. Regarding myelotoxicity, a recent genome-wide
association study failed to reveal an HLA association and suggested that
impaired antioxidative defense mechanisms in granulocytes or granulocyte
precursors could be a risk factor for neutropenia and agranulocytosis
[35]. In vitro studies using HL60 cells support toxicological
mechanisms associated with the formation of reactive metabolites, which
is dose-dependent [36, 37]. An increase in the plasma concentration
of 4-MAA by drug interactions and/or enzyme polymorphisms could
therefore enhance the risk for certain adverse reactions associated with
metamizole.
In conclusion, we provide evidence that CYP1A2 is the major CYP for the
conversion of 4-MAA to 4-AA and 4-FAA. CYP1A2 inhibition increases the
4-MAA exposure by a factor of approximately 1.5, which could be relevant
for dose-dependent adverse reactions.