European Journal of Pharmacology 378 1999 339–347
Regulation of cyclooxygenase activity by metamizol
, Raquel Garcıa-Nieto
, Federico Gago
, Susana Alemany
Medical Department, Boehringer Ingelheim, Spain
Received 18 March 1999; received in revised form 24 June 1999; accepted 29 June 1999
The ability of metamizol to inhibit cyclooxygenase-1 and cyclooxygenase-2 activities has been evaluated using different cyclooxy-
genase sources. Metamizol inhibited purified cyclooxygenase-1 and cyclooxygenase-2 with an IC
of about 150 mgrml. A similar IC
value for cyclooxygenase-2 was obtained in lipopolysaccharide-activated broken murine macrophages. Consistent with these findings,
derivative of metamizol, suggested a common binding mode to both isoforms. In intact cells, however, the inhibition profiles were
markedly different. The IC
platelets were 1730 " 150 mgrml and 486 " 56 mgrml, respectively. Inhibition of cyclooxygenase-2 activity in murine macrophages
values of 12 " 1.8 mgrml and 21 " 2.9 mgrml,
respectively. These data indicate that the IC
the cyclooxygenase inhibitory activity of nonsteroidal antiinflammatory drugs NSAIDs in intact cells. The data presented here also
indicate that cyclooxygenase-2 inhibition could play an important role in the pharmacological effects of metamizol. q 1999 Elsevier
Science B.V. All rights reserved.
Keywords: Cyclooxygenase; Nonsteroidal antiinflammatory drug NSAID ; Metamizol; Molecular modelling
The rate limiting step in the synthesis of prostaglandins
and thromboxanes is the conversion of arachidonate to
prostaglandin H , which is catalyzed by two isozymes
cyclooxygenase-1 and cyclooxygenase-2 for a review, see
Smith et al., 1998 and Vane et al., 1998 . The first
isozyme, cyclooxygenase-1, is a constitutive enzyme ex-
pressed in many tissues and also in platelets Funk et al.,
1991; Simmons et al., 1991 . Cyclooxygenase-1 has been
involved in ‘‘housekeeping’’ functions, such as coordinat-
ing the actions of circulating hormones, mucose gastric
protection and regulating vascular homeostasis Smith et
al., 1989 . In contrast, cyclooxygenase-2 is an inducible
Corresponding author. Tel.: q34-91-3975445; fax: q34-91-5854587;
R. de Gregorio and C. Campos have contributed equally to this work.
Cyclooxygenase-2 is induced in a variety of cell types by
diverse stimuli including cytokines, growth factors, mito-
gens and tumour promoters Kujubu et al., 1991; Lee et
Coffey et al., 1997 . In brain and spinal cord cyclooxy-
genase-2 is constitutively expressed
Cashman, 1996; Yaksh et al., 1998 .
by different genes Yokoyama and Tanabe, 1989; Kraemer
et al., 1992 but they exhibit a 62% homology at the
aminoacid level. The three-dimensional structures of both
enzymes have been solved by X-ray crystallography Picot
et al., 1994; Kurumbail et al., 1996 , and all the residues
that make up the cyclooxygenase active site have been
shown to be identical except for the amino acid at position
523, which is an isoleucine in cyclooxygenase-1 but a
valine in cyclooxygenase-2. This residue is found at the
bottom of a hydrophobic channel where nonsteroidal anti-
0014-2999r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 1 4 - 2 9 9 9 9 9 0 0 4 7 7 - X
inflammatory drugs NSAIDs bind. A mutant of cyclo-
shows inhibitor binding and selectivity profiles comparable
to those of wild-type cyclooxygenase-1
1996; Guo et al., 1996 .
in an atmosphere with oxygen, is spontaneously, nonenzy-
matically converted to 4-methylaminoantipyrine
1986; Levy et al., 1995 . Subsequently, the N-methyl side
formylaminoantipyrine, which is further converted to 4-
aminoantipyrine. Therefore, it is accepted that metamizol
in aqueous solution and in the presence of oxygen consists
of a group of several pyrazolone derivatives Brodgen,
1986; Levy, 1986; Levy et al., 1995 , of which 4-methyl-
aminoantipyrine is pharmacologically the most important
Brodgen, 1986; Levy, 1986; Levy et al., 1995; Vlahov et
al., 1990 . In the present paper hereafter, the term metami-
zol will encompass this whole set of pyrazolone deriva-
and relatively weak antiinflammatory properties.
It has been proposed that the antinociceptive action of
metamizol is at least partially centrally mediated Carlsson
1995 , even though an association of metamizol with
opioid receptors is unlikely Beirith et al., 1998; Taylor et
al., 1998 . On the other hand, a dissociation between the
inhibition of prostaglandins synthesis was reported prior to
the discovery of cyclooxygenase-2 Dembinska-Kiec et al.,
Ferreira, 1985 .
of cyclooxygenase is highlighted by the differences in
pharmacology between the two isozymes. The antiinflam-
matory capacity of the different NSAIDs is now proposed
to be associated with the capacity of inhibiting cyclooxy-
genase-2 activity. A central analgesic effect of NSAIDs
has also been proposed McCormak, 1994; Cashman, 1996;
Yaksh et al., 1998 , which is probably mediated by regula-
tion of cyclooxygenase-2 activity in the spinal cord Dirig
et al., 1997; Smith et al., 1998 . Regulation of cyclooxy-
genase-1 activity, on the other hand, is thought to be
responsible for the gastric and renal side effects of NSAIDs,
as well as for their antithrombotic activity for a review,
see Pairet and Engelhardt, 1996; Vane and Botting, 1998 .
Cyclooxygenase-2 is presently regarded as an important
target for the development of novel NSAIDs with im-
proved toxicological profiles.
In this paper we evaluate the capacity of metamizol to
inhibit cyclooxygenase-1 and cyclooxygenase-2 activities
using different cell systems, as well as purified cyclooxy-
Fig. 1. Binding mode of 4-methylaminoantipyrine in the active site of cyclooxygenase-2. Above Chemical formulae of metamizol and its active
cyclooxygenase-2, as proposed by programs GRID and AutoDock. In this orientation the interactions between the inhibitor and the enzyme are optimized.
Relevant residues are shown as ball-and-stick and have been labeled the numbering scheme follows the convention for the sheep cyclooxygenase-1
structure . This binding mode is identical to that found in the 4-methylaminoantipyrine-cyclooxygenase-1 complex and is presumed to be the same for the
active derivatives shown in A.
Fig. 1 continued .
lar model of the interaction between 4-methylaminoan-
tipyrine, the pharmacologically most important derivative
of metamizol, and the cyclooxygenase active sites of both
cyclooxygenase-1 and cyclooxygenase-2.
2. Material and methods
2.1. Docking of 4-methyl-amino-antipyrine into the cyclo-
oxygenase actiÕe sites of human cyclooxygenase-1 and
The experimentally determined three-dimensional struc-
tures of ovine cyclooxygenase-1 Picot et al., 1994 and
mouse cyclooxygenase-2 Kurumbail et al., 1996 , together
ble conformers of 4-methylaminoantipyrine were initially
substituent relative to the plane of the pyrazole ring, and
their geometries were optimized by using the semiempiri-
Atom-centered charges were then derived by fitting the
calculated molecular electrostatic potential to a mono-
Monte Carlo simulated annealing technique implemented
in AutoDock Morris et al., 1996 was used to generate
aminoantipyrine within the enzyme binding sites and to
evaluate the energy of each configuration. In addition, the
active sites of these enzymes were probed for regions of
steric and electrostatic complementarity with the functional
groups present in 4-methylaminoantipyrine i.e., aliphatic
ford, 1985 . The 4-methylaminoantipyrine favoured by
which automatically produces the ligand orientation that
best matches the calculated maps for the atom probes.
2.2. Measurement of purified cyclooxygenase actiÕities
Purified cyclooxygenase-1 and cyclooxygenase-2 were
obtained from Cayman Chemicals Ann Arbor, MI . En-
donic acid to prostaglandins, after separation by thin layer
Mitchell et al., 1994 . The reac-
tion was started by the addition of 10 units of the different
enzymes to a reaction mixture made up to a final volume
of 1 ml, containing 50 mM Tris buffer pH 8, glutathione 5
were incubated in a shaking water bath at 378C for 10 min,
after which the reaction was stopped by adding 30 ml of 1
mM HCl. One milliliter of saturated NaCl solution was
added to each sample followed by 1.5 ml of ethylacetate.
After mixing the samples by vortexing, they were cen-
trifuged at 575 = g for 10 min and the ethyl acetate layer
was transferred to a clean tube and dried under nitrogen
stream. Prostaglandins were separated by TLC. Each sam-
ple was redissolved in 30 ml of chloroformrmethanol, 2:1
vrv , and 20 ml were applied onto a glass-backed silica
approximately 90 min at room temperature in a solvent
consisting of the upper phase of ethyl acetatertrimethyl
tection of the
C-labelled prostaglandins an autoradiog-
fetal calf serum, 4 mM glutamine, 50 mgrml gentamicin
at 378C, were incubated for 30 min with different concen-
was added to a final concentration of 30 mM. Cells were
incubated for further 15 min at 378C. The medium was
was used to measure the formation of 6-keto-prostaglandin
F . Metamizol did not interfere with the measurement of
6-keto-prostaglandin F .
Fresh human platelets were isolated as described by
Font et al.
blood samples were centrifuged at 180 = g for 10 min at
208C and the supernatant containing the platelets was
diluted 1r1 vrv with phosphate saline buffer PBS .
boxane B in the incubation media after 5 min of stimula-
tion were measured with a thromboxane B
munoassay system Amersham . Metamizol did not inter-
fere with the measurement of thromboxane B .
2.4. Measurement of cyclooxygenase-2 actiÕity in intact
Murine monocyte–macrophage J774A.1 cells, in RPMI
1640 medium supplemented with 10% fetal calf serum,
gentamicin 50 mgrml
cellsrml, were treated with lipopolysaccharide at 1 mgrml
for 12 h to induce cyclooxygenase-2. Complete medium
was then changed and different concentrations of metami-
zol or indomethacin were added and the cells incubated for
30 min at 378C. The medium was then removed and
prostaglandin E levels were analyzed by prostaglandin E
enzyme immunoassay system Amersham . Metamizol did
Human peripheral leukocytes from Buffy-Coat were
separated using Histopaque 1099 Sigma as described by
Tavares and Bennett
and incubated for 2 h in
complete medium at 0.5 = 10
cellsrml. Subsequently, the
different concentrations of metamizol. The prostaglandin
released to the incubation media was measured by
radioimmunoassay as described above.
Lipopolysaccharide at 1 mgrml was added for 12 h to
confluent murine macrophages after which cells were
washed and homogenized with a glass teflon homogenizer
in 50 mM Tris buffer pH 7.4 containing 1 mM phenyl-
leupeptin. Broken cells were incubated at 378C in the
presence of different concentrations of metamizol or indo-
methacin for 30 min. About 30 mM of arachidonic acid
was then added, and the reaction continued for a further 15
min. The reaction was stopped by boiling, and after cen-
trifugation at 10,000 = g for 30 min, prostaglandin E was
measured in the supernatant as described above.
activity by metamizol. Cyclooxygenase enzymes were incubated with
saturating doses of arachidonic acid in the presence of different doses of
metamizol. The data are expressed as mean"S.D. from three different
experiments performed in duplicate. ^: Cyclooxygenase-1 activity. B:
Both GRID and AutoDock found the same binding
orientation for 4-methylaminoantipyrine, the main active
derivative of metamizol, irrespective of the isozyme con-
sidered. The molecule binds at the bottom of the substrate
channel placing the phenyl ring above the plane of Tyr
the 4-amino group in close proximity to the hydroxyl of
ring nitrogen facing the residue at
position 523 Fig. 1B . The smaller size of the side chain
allows the main substrate channel in cyclooxygenase-2 to
Fig. 3. Effects of metamizol on cyclooxygenase-1 activity measured in
formation after exposure to exogenous 30 mM arachidonic acid for 10
min. 100% of activity 1.6 ngrml"0.5 is given to the value without
measured as thromboxane B
after stimulation with calcium ionophore
for 5 min. The data are expressed as mean"S.D. from three different
Fig. 4. Effect of metamizol on cyclooxygenase-2 activity measured in
intact murine macrophages. Cells were treated with lipopolysaccharide
for 12 h and cyclooxygenase-2 activity was measured as prostaglandin E
formation. 100% is given to the value of 860"35 prostaglandinrml
S.D. from three different experiments performed in duplicate.
extend into a side pocket that is not accessible to inhibitors
in cyclooxygenase-1. 4-Methylaminoantipyrine does not
appear to exploit this adjacent binding pocket, the occu-
pancy of which is thought to be important for achieving
selectivity towards cyclooxygenase-2 Kurumbail et al.,
1996 , and a similar binding mode can be envisaged for
the other derivatives shown in Fig. 1A.
3.2. Inhibition of purified cyclooxygenase-1 and cyclooxy-
genase-2 by metamizol
Experiments to determine the activity of purified cyclo-
oxygenase-1 and cyclooxygenase-2 in the presence of
metamizol were carried out. Both enzymes were incubated
with saturating concentrations of arachidonic acid in the
presence of different concentrations of metamizol 60–2000
mgrml . Consistent with the common binding mode re-
ported above, we found that the inhibition curves of cyclo-
oxygenase-1 and cyclooxygenase-2 activity by metamizol
were very similar, the IC
of metamizol being about 150
mgrml for either cyclooxygenase-1 or cyclooxygenase-2
Fig. 2 .
To measure the capacity of metamizol to inhibit cyclo-
oxygenase-1 and cyclooxygenase-2 activities in a more
physiological context, we next decided to use different
intact cell systems as a source of cyclooxygenase-1 or
cyclooxygenase-2 activities. BAEC cells do not contain
cyclooxygenase-2 but a constitutive cyclooxygenase-1 ac-
tivity, which results in a release of prostaglandins to the
extracellular medium Mitchell et al., 1994 . High concen-
the incubation media after stimulation or not with 5 mgrml lipopoly-
genase-2 activity by metamizol measured as prostaglandin E formation.
100% is given to the value obtained in the absence of metamizol. The
formed in duplicate.
decrease of prostaglandins production. Metamizol, when
added to the incubation media of BAEC cells, showed an
for cyclooxygenase-1 activity of 1730 " 150 mgrml,
measured as the decrease in 6-keto-prostaglandin F
els Fig. 3A . The IC
of indomethacin in these condi-
tions was 8.5 " 0.5 mgrml data not shown .
We next decided to test the capacity of metamizol to
inhibit cyclooxygenase-1 activity in human platelets as a
model of freshly isolated intact human cells. Isolated
platelets were incubated with calcium ionophore A23187
at a concentration of 0.25 mM and cyclooxygenase-1
activity was measured as thromboxane production. As
shown in Fig. 3B, the IC
of metamizol for cyclooxy-
genase-1 was 486 " 56 mgrml.
Murine monocyte–macrophage J774A.1 cells have been
used as a model to determine cyclooxygenase-2 activity
detected in the incubation media, unless cells were stimu-
and as a result of inducing cyclooxygenase-2 expression,
the concentration of prostaglandin E released to the incu-
bation media in 30 min was 860 " 35 pgrml. Incubation
of metamizol for cyclooxygenase-2 in this cell system
Fig. 4 . Incubation with indo-
methacin also inhibited the release of prostaglandin E
with an IC
of 0.23 " 0.016 mgrml data not shown .
Similar experiments to determine the IC
for cyclooxygenase-2 in intact human primary cells were
lation of these cells with 3 mgrml lipopolysaccharide
increased the prostaglandin E
levels in the incubation
as a result of cyclooxygenase-2 induction. Incubation of
leukocytes with metamizol dramatically decreased the lev-
els of prostaglandin E
in the incubation media, with an
of 21 " 2.9 mgrml Fig. 5B .
Õalue of metamizol for
The 10-fold difference between the metamizol IC
values for purified cyclooxygenase-2 and in intact cells
mizol on cyclooxygenase-2 activity, using as a source of
enzyme lipopolysaccharide-activated broken murine mono-
cyte–macrophage cells. In these conditions, metamizol
inhibited prostaglandin E synthesis with a IC
of 145 "
18 mgrml Fig. 6 . When indomethacin was tested in
these conditions the IC
obtained was 0.60 " 0.009
Fig. 6. IC
of metamizol on cyclooxygenase-2 activity in broken lipo-
polysaccharide-activated murine macrophage preparation. Cell extracts
was measured after incubation with saturating doses of arachidonic acid
mean"from at least three different experiments performed in duplicate.
The discovery of an inducible isoform of cyclooxy-
genase allows a reinterpretation and refinement of cyclo-
oxygenase activity inhibition, as well as an explanation of
the therapeutic and side effects of NSAIDs. It is now
accepted that the antiinflammatory and analgesic actions of
NSAIDs are due to inhibition of cyclooxygenase-2 and the
unwanted side effects such as gastric and renal toxicity are
due to the inhibition of the constitutive enzyme cyclooxy-
genase-1. We have evaluated the ability of metamizol to
using different cyclooxygenase sources. We also propose a
molecular model of the interaction between these enzymes
and the main active derivative of metamizol based on
automated docking calculations.
The common binding mode of 4-methylaminoantipyrine
to cyclooxygenase-1 and cyclooxygenase-2 is in agreement
with the data obtained with the purified enzymes showing
metamizol to be equally potent in its inhibition of cyclo-
oxygenase-1 and cyclooxygenase-2 activities, with an IC
of about 150 mgrml. Metamizol also has an IC
150 mgrml when tested for cyclooxygenase-2 inhibition in
in inhibiting cyclooxygenase-2 activity when intact cells
are used as a source of cyclooxygenase-2. The IC
in intact lipopolysaccharide-activated murine monocyte–
macrophage cells is in accordance with previous data
demonstrating that the inhibition of the release of a noci-
ceptive factor from lipopolysaccharide-activated macro-
phages occurs at 3.5 " 0.35 mgrml metamizol Campos et
al., 1988 .
The low IC
cyclooxygenase-2 using intact cells as a source of cyclo-
described for metamizol
1984 can be due to cyclooxygenase-2 inhibition since
derivatives of metamizol are about 30 mgrml Levy et al.,
tegrity of the cell plasma membrane should facilitate the
access of metamizol to the endoplasmic reticulum and
nuclear envelope, where cyclooxygenase-2 is located
Morita et al., 1995 , cell disruption is in fact detrimental
purified enzymes have also been described for other
Carabaza et al., 1996 . The NSAID BF 389, for example,
value for cyclooxygenase-2
in intact cells when compared with the IC
the purified enzyme Mitchell et al., 1994 . The reasons for
these differences in the IC
values depending on assay
conditions remain to be established. It is conceivable that
homogenization in such a way that it loses affinity for
some inhibitors or, alternatively, that binding of some
inhibitors is facilitated by another cellular component pre-
sent only in intact cells.
In intact cells the IC
value of metamizol for cyclo-
oxygenase-1 was more than 20-fold higher than that ob-
genase-2 may be selectively inhibited by therapeutic con-
centrations of metamizol. In fact, its cyclooxygenase-2
selectivity profile is superior to that of indomethacin see
Section 3 or other classical NSAIDs Smith and DeWitt,
1994; Mitchell et al., 1994; Cashman, 1996 . The IC
value obtained for cyclooxygenase-1 in human platelets is
These data agree with the fact that the risk of gastrointes-
tinal bleeding associated with metamizol use is comparable
to that of paracetamol and clearly lower than that of
acetylsalicylic acid and other NSAIDs Laporte and Carne,
1987; Laporte et al., 1991 .
Different authors, prior to the discovery of the cyclo-
oxygenase-2 isozyme, had proposed that the ability of
metamizol to inhibit prostaglandin synthesis depends on
the biological system used, providing evidence that this
Dembinska-Kiec et al., 1976; Weithmann and Alpermann,
1985; Brodgen, 1986 . The specificity of metamizol to
inhibit prostaglandin synthesis in brain tissue can now be
reinterpreted in the light of the selectivity shown by
metamizol for cyclooxygenase-2 inhibition in intact cells.
This selectivity could also underlie the pharmacological
profile of this drug which is endowed with strong anal-
gesic and antipyretic effects together with weak antiinflam-
In conclusion, in this paper we present evidence that the
values obtained with purified enzymes or disrupted
cells cannot always be extrapolated to the cyclooxygenase
inhibitory capacity of the NSAIDs in intact cells and
probably ‘‘in vivo’’. The data presented here also indicate
that metamizol does not regulate cyclooxygenase-1 activity
in intact cells at therapeutic concentrations and that the
inhibition of cyclooxygenase-2 achieved at these concen-
trations could play an important role in the analgesic
effects of metamizol.
We thank the technical assistance of Joaquın Perez.
Rosa de Gregorio is supported by a ‘‘CSIC-sector empre-
AutoDock by Dr. Peter Goodford Molecular Discovery,
UK and Dr. Garrett Morris The Scripps Research Insti-
tute , respectively, is gratefully acknowledged. This work
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