Inhibition of cyclooxygenases by dipyrone

Article abstract of DOI:10.1038/sj.bjp.0707239

Background and Purpose: Dipyrone is a potent analgesic drug that has been demonstrated to inhibit cyclooxygenase (COX). In contrast to classical COX-inhibitors, such as aspirin-like drugs, dipyrone has no anti-inflammatory effect and a low gastrointestinal toxicity, indicating a different mode of action. Here, we aimed to investigate the effects of dipyrone on COX. Experimental approach: The four major metabolites of dipyrone, including the two pharmacologically active metabolites, 4-methyl-amino-antipyrine (MAA) and amino-antipyrine (AA), were used to characterise their binding to COX and haem as well as their effects on the biochemical properties of COX. Mass spectrometry, UV and visible photometry were used to study binding and prostaglandin production. Levels of anti-oxidant enzymes were assessed by Western blotting. Key results: The pharmacologically active metabolites of dipyrone, MAA and AA, did not inhibit COX activity in vitro like classical COX inhibitors, but instead redirected the prostaglandin synthesis, ruling out inhibition of COX through binding to its active site. We found that MAA and AA formed stable complexes with haem and reacted with hydrogen peroxide in presence of haem, ferrous ions (Fe²⁺) or COX. Moreover, MAA reduced Fe ³⁺ to Fe²⁺ and accordingly increased lipid peroxidation and the expression of anti-oxidant enzymes in cultured cells and in vivo. Conclusions and implications: Our data suggest that the pharmacologically active metabolites of dipyrone inhibit COX activity by sequestering radicals which initiate the catalytic activity of this enzyme or through the reduction of the oxidative states of the COX protein.

Full text of DOI:10.1038/sj.bjp.0707239

British Journal of Pharmacology (2007) 151, 494–503
2007 Nature Publishing Group All rights reserved 0007–1188/07 30.00
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$
www.brjpharmacol.org
RESEARCH PAPER
Inhibition of Cyclooxygenases by Dipyrone
1
1
1
2
1
1
SC Pierre , R Schmidt , C Brenneis , M Michaelis , G Geisslinger and K Scholich
1
Pharmazentrum frankfurt, ZAFES, Klinikum der Johann Wolfgang Goethe-Universit a¨ t Frankfurt, Frankfurt, Germany;
Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany
2
Background and Purpose: Dipyrone is a potent analgesic drug that has been demonstrated to inhibit cyclooxygenase (COX).
In contrast to classical COX-inhibitors, such as aspirin-like drugs, dipyrone has no anti-inflammatory effect and a low
gastrointestinal toxicity, indicating a different mode of action. Here, we aimed to investigate the effects of dipyrone on COX.
Experimental approach: The four major metabolites of dipyrone, including the two pharmacologically active metabolites,
4-methyl-amino-antipyrine (MAA) and amino-antipyrine (AA), were used to characterise their binding to COX and haem as
well as their effects on the biochemical properties of COX. Mass spectrometry, UV and visible photometry were used to study
binding and prostaglandin production. Levels of anti-oxidant enzymes were assessed by Western blotting.
Key results: The pharmacologically active metabolites of dipyrone, MAA and AA, did not inhibit COX activity in vitro like
classical COX inhibitors, but instead redirected the prostaglandin synthesis, ruling out inhibition of COX through binding to its
active site. We found that MAA and AA formed stable complexes with haem and reacted with hydrogen peroxide in presence
2
þ
3 þ
2 þ
to Fe and accordingly increased lipid peroxidation and
of haem, ferrous ions (Fe ) or COX. Moreover, MAA reduced Fe
the expression of anti-oxidant enzymes in cultured cells and in vivo.
Conclusions and implications: Our data suggest that the pharmacologically active metabolites of dipyrone inhibit COX
activity by sequestering radicals which initiate the catalytic activity of this enzyme or through the reduction of the oxidative
states of the COX protein.
British Journal of Pharmacology (2007) 151, 494–503; doi:10.1038/sj.bjp.0707239; published online 16 April 2007
Keywords: dipyrone; prostaglandin E2; cyclooxygenase; prostaglandin H
2
synthase; radical; haem
Abbreviations: AA, amino-antipyrine; AAA, 4-acetyl-amino-antipyrine; COX, cyclooxygenase; FAA, 4-formyl-amino-antipyrine;
MAA, 4-methyl-amino-antipyrine; NSAID, non-steroidal anti-inflammatory drug; PGE , prostaglandin E ; TXB
Thromboxan B ; TLC, thin layer chromatography
2
2
2
,
2
Introduction
Prostaglandin
E
2
(PGE
2
)
is an important mediator of
2
PGH by a two-step process both steps of which depend on
inflammation, pain and fever (Woolf and Costigan, 1999;
Vanegas and Schaible, 2001). It is released at the site of an
injury and, subsequently, in the dorsal horn of the spinal
cord where it is involved in the development of central
sensitization (Woolf and Costigan, 1999; Vanegas and
Schaible, 2001). Prostaglandins are formed from arachidonic
acid by the catalytic activity of the enzyme cyclooxygenase
haem as the prosthetic group: First, by reduction of a
hydroperoxide to its corresponding hydroxy compound,
the COX-peroxidase, oxidizes the haem from its resting state
(ferric haem) to a ferryloxo-protoporphyrin radical cation
analogous to compound I of classic peroxidases. By intra-
molecular electron transfer, this generates a tyrosyl radical in
the COX-cyclooxygenase site that is required for oxygena-
(
COX; also referred to as prostaglandin H
2
(PGH
2
) synthase;
tion of arachidonic acid to PGG
catalysed reaction, the peroxidase reaction, the hydroperoxy
group of PGG is reduced by an electron transfer from the
haem group, to the hydroxy compound PGH (Lambeir et al.,
2
. In the second COX-
EC 1.14.99.1) that exists in two isoforms, COX-1 and -2, and
several splice variants (Vanegas and Schaible 2001; Chan-
drasekharan et al., 2002). COX is a bifunctional enzyme that
possesses two enzyme activities, a cyclooxygenase as well as
a peroxidase activity. COX converts arachidonic acid into
2
2
1985; Tsai et al., 1998; Marnett et al., 1999; Marnett, 2000).
Dipyrone is a potent analgesic and antipyretic drug that
has been used clinically for more than 80 years. In aqueous
solution, dipyrone is immediately hydrolysed to 4-methyl-
amino-antipyrine (MAA; Figure 1a) which can then be
further metabolized to 4-amino-antipyrine (AA), 4-formyl-
amino-antipyrine (FAA) or 4-acetyl-amino-antipyrine (AAA).
Of these four major dipyrone metabolites, MAA has been
demonstrated to be the pharmacologically active compound,
Correspondence: Dr K Scholich, Pharmazentrum frankfurt, ZAFES, Klinikum
der Johann Wolfgang Goethe-Universit a¨ t Frankfurt, Theodor-Stern-Kai 7,
Frankfurt 60590, Germany.
E-mail: scholich@em.uni-frankfurt.de
Received 15 September 2006; revised 7 December 2006; accepted 19
February 2007; published online 16 April 2007

Inhibition of COX by dipyrone
SC Pierre et al
495
while AA has only a weak pharmacological effect and FAA
or AAA are pharmacologically inactive (Vlahov et al., 1990;
Levy et al., 1995). Over the last 20 years, several groups have
demonstrated the inhibition of COX by dipyrone in vitro
RPMI medium with 10% fetal calf serum, 1% penicillin–
streptomycin. After 24 h, the cells were treated for 16 h in
ꢁ
1
serum-free medium with 1 mg ml bacterial lipopolysacchar-
ide (LPS) in presence or absence of the dipyrone metabolites
(
Abbate et al., 1990; Campos et al., 1999; Chandrasekharan
et al., 2002) and in vivo (Eldor et al., 1984; Geisslinger et al.,
996; Levy et al., 1998; Ayoub et al., 2004). Based on these
at the indicated concentrations. The PGE
2
concentrations in
ELISA kit from
the medium were determined using the PGE
2
1
Assay Designs (Ann Arbor, MI, USA) according to the
instructions of the manufacturer.
findings, it has been proposed that the analgesic and
antipyretic effects of dipyrone depend, at least in part, on
COX inhibition and thus on decreased PGE
2
synthesis
Prostaglandin measurement by LC-MS/MS
(Carlsson et al., 1986; Gelgor et al., 1992; Tortorici and
Cell media (500 ml) were mixed with 500 ml 35 mM H
3 4
PO ,
Vanegas 1995; Warner and Mitchell 2002; Wickelgren 2002).
Hence, although the inhibitory effect of dipyrone on COX has
long been known, the mechanism by which MAA inhibits
COX activity remains unclear. Most COX inhibitors belong to
the class of non-steroidal anti-inflammatory drugs (NSAIDs),
which block the enzyme activity by competing with arachi-
donic acid for the cyclooxygenase active site (Marnett 2000;
Smith et al., 2000). Our data suggest that MAA inhibits COX
enzyme activity through an iron-dependant mechanism,
resulting in the sequestration of radicals that are necessary
to initiate the catalytic cycle of COX.
2
0 ml methanol and 20 ml internal standard solution. Pros-
taglandins were extracted with solid-phase extraction using
absolut Nexus cartridges (1 ml, Varian, Darmstadt, Ger-
many), which were washed with 2 ml of hexane:ethyl
acetate:isopropanol (35:60:5), dried for 20 s and conditioned
with 1 ml of methanol and 1 ml of water. The prepared cell
media was loaded onto the column and washed with 1 ml of
water and 1 ml of methanol/water (40:60). The cartridges
were then dried for 7 min and eluted with 1 ml of
hexane:ethyl acetate:isopropanol (30:65:5). The organic
phase was removed at a temperature of 451C under a gentle
stream of nitrogen. The residues were reconstituted with
5
0 ml of acetonitrile:water:formic acid (20:80:0.0025, pH 4.0),
Materials and methods
and injected into the LC-MS/MS system.
Sample analysis was performed by using liquid chromato-
graphy-electrospray ionization-tandem mass spectrometry
Animals
In all experiments, the ethics guidelines for investigations
in conscious animals were obeyed and the experimental
procedures were approved by the local Ethics Committee.
Male Sprague–Dawley rats weighing 250–300 g were pur-
chased from Charles River Wiga GmbH (Sulzfeld, Germany).
(LC-ESI-MS/MS). The LC-MS/MS system consisted of an API
4
000 triple-mass spectrometer (Applied Biosystems, Darm-
stadt, Germany) equipped with a Turbo-V-source operating
in negative ESI mode, an Agilent 1100 binary HPLC pump
and degasser (Agilent, B o¨ blingen, Germany). An inlet valve
was used to truncate non-relevant signals (10-port, VICI
Valco Instruments, Houston, TX, USA). For the chromato-
graphic separation, a Synergi Hydro-RP column and pre-
2
COX-1 assay using Thromboxane B measurements
Platelets were purified from blood from healthy volunteers
who had not taken acetylsalicylic acid or any other drug in
the previous 2 weeks. Blood was collected using S Monocuv-
ette – 9 NC syringes (Sarstedt, N u¨ mbrecht, Germany). After
an incubation of 20 min at room temperature, they were
centrifuged at 210 g for 20 min. The platelet-rich plasma was
mixed with 50% Hanks Balanced Salt Solution supplemented
with 25 mM HEPES (HHBSS) and 30% anticoagulant solution
column were used (150 ꢀ 2 mm ID, 4 mm particle size and
80 A˚ pore size from Phenomenex, Aschaffenburg, Germany).
ꢁ
1
A linear gradient was employed at a flow rate of 300 ml min
.
Mobile phase A was water:formic acid (100:0.0025, pH 4.0)
and mobile phase B was acetonitrile:formic acid (100:
0.0025). Total run time was 16 min and injection volume
of samples was 45 ml. The mass spectrometer was operated
in the negative ion mode with an electrospray voltage of
ꢁ3300 V at 4501C. Multiple reaction monitoring was used for
quantification. All quadrupoles were working at unit resolu-
tion. Quantitation was performed with Analyst Software
V1.4 (Applied Biosystems, Darmstadt, Germany) using the
internal standard method (isotope-dilution mass spectro-
metry). Ratios of analyte peak area and internal standard
peak area (y axis) were plotted against concentration (x axis)
and calibration curves for each prostaglandin were calcu-
(65 mM citric acid, 85 mM sodium citrate, 2% glucose) and
again centrifuged at 1000 g for 10 min. The pellet was washed
once more with HHBSS and 10% anticoagulant and then
6
resuspended in the same buffer. A total of 4 ꢀ 10 platelets
were preincubated with the dipyrone metabolites for 30 min
at 37 1C. Then calcium ionophore A23187 (Sigma, St Louis,
MO, USA) was added to a final concentration of 2 mM in a
total volume of 200 ml. The reaction was terminated after
1
0 min by addition of 100 ml methanol. After centrifugation,
ꢁ2
thromboxane B (TXB ), the stable metabolite of TXA , was
2
2
2
lated by least-squares regression with concentration
ꢁ
1
determined in the supernatant using the enzyme immu-
noassay from Assay Designs (Ann Arbor, MI, USA) according
to the instructions of the manufacturer.
weighting
.
Metabolic labelling
Cells were incubated with 1 mM radioactive arachidonic acid
14
COX-2 assay using PGE
2
measurements
[1– C] (Moravek Biochemicals, Brea, CA, USA) for 8 h. Then
the cells were washed with phosphate-buffered saline (PBS)
and stimulated as indicated for 15h. The medium (1ml) was
Murine RAW 264.7 (LGC Promochem, Wesel, Germany)
6
(
1 ꢀ 10 cells) were plated on 35 mm dishes and incubated in
British Journal of Pharmacology (2007) 151 494–503

Inhibition of COX by dipyrone
4
96
SC Pierre et al
extracted with one volume chloroform containing 1% formic
acid. After drying the extracts, eicosanoids were resuspended
in 50 ml chloroform and subjected to thin layer chromato-
graphy (TLC) as described previously (Brenneis et al., 2006).
Bathophenanthroline. Bathophenanthroline is a highly se-
2
þ
lective photometric reagent for Fe
determination. Bath-
ophenanthroline was dissolved in 128 mM NaCl to yield a
final concentration of 100 mM. The indicated dipyrone
metabolites and FeCl
of 100 and 500 mM, respectively. Reduction of Fe
induced a colorimetric change that was measured after
0 min at 540 nm in a multiwell reader spectrophotometer
SpectraFluor Plus, Tecan, Crailsheim, Germany).
3
were added to a final concentration
3
þ
2 þ
to Fe
Cell-free prostaglandin synthesis
Murine RAW macrophages were treated for 16 h in serum-
1
ꢁ
1
free medium with 1 mg ml
LPS. Cells were collected in
(
1
00 mM Tris-HCl, pH 7.5, sonicated and the final cell protein
ꢁ1
concentration adjusted at 1 mg ml with Tris buffer. In a
total volume of 200 ml, 100 mg protein was incubated with
the indicated drugs for 15 min. Alternatively, 20 units of
reconstituted COX-1 were used. The reaction was started
Potassium ferricyanide. The dipyrone metabolites were
added at the indicated concentrations to a solution of
1 mM FeCl
3
, 1 mM potassium ferricyanide. Ferricyanide ions
2
þ
react with the Fe
(KFe(III)Fe(II)(CN)
ions to form a dark blue compound
1
4
3 þ
2 þ
with the addition of arachidonic acid [1– C] (Moravek
Biochemicals, Brea, CA, USA) to a final concentration of
6
). Thus, reduction of Fe
to Fe
induced a colorimetric change that was determined at
690 nm in the multiwell reader spectrophotometer.
0
.25 mM. After 10 min incubation at room temperature, the
reaction was stopped by adding 1.4 ml chloroform:methanol
1:1) containing 1% formic acid. Cell debris was removed by
centrifugation with 13 000 g and 0.6 ml of chloroform, and
.32 ml of 0.88% formic acid were added to the supernatant.
The organic phase was dried under N , redissolved in 50 ml of
(
Lipid peroxidation
6
RAW 264.7 (3 ꢀ 10 cells per 35 mm dish) were incubated
0
2 þ
with 100 mM MAA or Fe
for 24 h in presence or absence of
2
1
00 mM deferoxamine before harvesting. Lipid peroxidation
chloroform and spotted on a Silica Gel 60 TLC plate. TLC
plates were run for 1 h benzene:dioxane:formic acid:acetic
acid (82:14:1:1) at 41C. After drying, the TLC plates were
analysed by a phosphoimager reader (Bio-imaging Analyzer
System BAS-1500, Fujifilm, D u¨ sseldorf, Germany).
was detected as described previously (Wong et al., 2001).
Expression studies in spinal cords
ꢁ
1
Male adult rats were given 1 g kg dipyrone intraperitone-
ally. After 4 or 24 h, they were killed and the spinal cord was
removed. Spinal cords were lysed in PBS by sonication. The
insoluble fraction was removed by centrifugation at 18 000 g
for 20 min. Aliquots of the supernatant were stored at ꢁ801C
until further use. Western blots for superoxide dismutase and
peroxiredoxin V were performed using 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. About 30 mg pro-
tein was loaded per lane and an anti-Hsp70 antibody was
used to control for equal loading.
MS–MS
The four dipyrone metabolites were incubated with or
without haem under the same conditions as described for
the spectrophotometric analysis. Detection was performed
using a PE Sciex API 3000 triple quadrupole mass spectro-
meter (Applied Biosystems, Langen, Germany) equipped
with a turbo ion spray interface. Nitrogen (high purity) was
supplied by a Whatman nitrogen generator (Parker Hannifin
GmbH, Kaarst, Germany).
Data analysis
Results are shown as means7s.e.m. Statistical analysis was
performed using Student’s t-test and P-values of less than
UV-visible absorbance spectra
0
.05 were considered to be significant.
The dipyrone metabolites (100 mM) were incubated for
1
0 min at room temperature with 10 mM H
haem. Where indicated, ferrous chloride (FeCl
chloride (FeCl ) was used instead of haem. COX-1 was
2
O
2
and 10 mM
2
) or ferric
Materials
3
Dipyrone, MAA, AA, FAA and AAA were a generous gift from
Aventis (Frankfurt, Germany). RPMI medium and fetal
bovine serum were purchased from Invitrogen (Karlsruhe,
Germany). Purified COX isoforms and prostaglandins used
as substrate or standards for TLC and LC-MS/MS experiments
were purchased from Cayman (Ann Arbor, MI, USA). All
other chemicals were purchased from Sigma (St. Louis, MO,
reconstituted with 10 mM haem prior to incubation with
MAA. To allow photometric analysis of the dipyrone
metabolites, after incubation of 100 mM MAA with 40 units
COX-1 (65 nM), the metabolites were separated from COX-1
by extraction with chloroform/1% formic acid. After eva-
poration of the chloroform, the metabolites were resus-
pended in water and analysed. The reactions were carried out
with identical results using either no reagents to stabilize the
pH or 25 mM Tris (pH 7.4). The spectra were recorded using
the Biowave S2100 diode array spectrophotometer (Walden
Precision Apparatus Ltd, Cambridge, UK).
1
4
USA) unless otherwise specified. Arachidonic acid [1– C]
ꢁ1
ꢁ1
(
53 mCi/mmol, 0.05 mCi ml , 287 mg ml ) was purchased
from Moravek Biochemicals (Brea, CA, USA).
Results
2
þ
measurements
Fe
The concentrations of Fe
The dipyrone metabolites MAA and AA inhibit COX-1 and -2
We determined the effect of the four main metabolites of
dipyrone (MAA, AA, FAA and AAA) on COX-2 activity by
2
þ
ions were measured photome-
trically using two different methods.
British Journal of Pharmacology (2007) 151 494–503

Inhibition of COX by dipyrone
SC Pierre et al
497
monitoring the release of PGE
2
from LPS-stimulated RAW
a
2
64.7 cells. To determine the effects of the metabolites on
COX-1 activity, TXB
2
synthesis in human platelets was
measured. MAA inhibited both COX isoforms, with COX-2
being more sensitive to inhibition by MAA (Figure 1b).
Similarly, AA inhibited COX-2 effectively at 100 mM but
not COX-1 (Figure 1c). In contrast, FAA and AAA did not
decrease COX-1 or COX-2 activity (Figure 1c). As plasma
concentrations in human subjects after a single dose of 1 g
dipyrone are about 100 mM for MAA and 5–8 mM for AA, FAA
and AAA (Damm 1989; Geisslinger et al., 1996), concentra-
tions over 100 mM were not tested.
R
Metabolite
N
CH3
O
N
H
CH3
CHO
AA
MAA
FAA
CH3
CH3CO AAA
R
N
140
b
1
1
20
00
PGE2
TXB2
MAA and AA react with PGG
environment
2
in a cell-free but not in a cellular
80
To investigate whether MAA and AA inhibit COX activity by
competing with arachidonic acid for the active site, we first
incubated lysates from LPS-stimulated RAW 264.7 cells with
60
4
0
0
0
2
1
4
[
C]arachidonic acid and determined the prostaglandin
synthesis by TLC. Surprisingly, while aspirin abolished
prostaglandin synthesis, MAA and AA did not block
prostaglandin synthesis, but instead redirected the prosta-
0
0.1
1
10
100
1000
MAA (µM)
c
160
glandin synthesis from PGE
2 2
and PGD towards PGF2a and
PGE2
140
20
other unidentified prostaglandins (Figure 2a). Also incuba-
TXB2
1
4
1
tion of purified COX-1 with [ C]arachidonic acid (Figure 2b)
caused a redirection of prostaglandin synthesis towards
PGF2a and other prostaglandins in presence of MAA.
100
80
2
We hypothesized that MAA may react with PGG or other
6
4
2
0
0
0
0
*
prostaglandins and determined, by mass spectroscopy, the
effect of MAA on several prostaglandins. Our results showed
*
*
that MAA concentration-dependently decreased PGG
centrations in vitro (Figure 2c). This reaction was specific for
MAA and AA, since FAA and AAA did not alter PGG
concentrations (Figure 2d–h). In contrast PGE , PGD , PGF2a
or PGH concentrations were not altered by MAA (Supple-
mentary data 1). Since PGG differs from the other tested
2
con-
**
MAA
AA
AAA
FAA
2
Figure 1 MAA inhibits PGE
2
and TXB synthesis. (a) Structures of
2
2
2
the four dipyrone metabolites. (b) LPS-stimulated RAW 264.7 were
treated with the indicated MAA concentrations for 16 h. Human
platelets were incubated with varying amounts of MAA for 30 min
before induction of the release of intracellular arachidonic acid with
A23187 for 10 min. PGE and TXB synthesis was determined as
2 2
described in the ‘Materials and methods’ section. The mean7s.e.m.
of at least three experiments are shown. The control values (100%)
2
2
prostaglandins by possessing a free hydroperoxide group and
MAA is known to be oxidized by radical donors (Iogannsen
et al., 1993), MAA may first react with this peroxide which
then could lead to a non-specific oxidation of the endoper-
ꢁ1
ꢁ1
correspond to 6 ng ml
for PGE2 and 70 ng ml for TXB . (c)
2
oxide and the generation of prostaglandins, such as PGF2a
Indeed nuclear magnetic resonance (NMR) spectrometry
indicated that MAA oxidized PGG rapidly to PGF2a, 5-trans
.
Conditions as in (b) except that all dipyrone metabolites were used
at 100 mM. The mean7s.e.m. of at least three experiments, each in
triplicate, are shown. Students t-test *Po0.01, **Po0.001 metabo-
lite vs control.
2
PGF2a and possibly further to 15-keto PGF2a and 13,14-
dihydro-15-keto PGF2a (data not shown). However this
redirection of the prostaglandin synthesis after MAA treat-
ment to PGF2a and its metabolites was not observed in
cultured LPS-stimulated RAW 264.7 cells (Figure 2i and j;
Supplementary data 2) or spinal cord neurons (Supplemen-
2
observed reaction between PGG and MAA does not take
place and an alternative mechanism must be responsible for
the inhibition of COX.
tary data 3), indicating that the reaction between PGG
MAA does not occur in a cellular environment.
2
and
Taken together, the data show that competition of MAA
with arachidonic acid for the catalytic site of COX, as known
to occur with traditional NSAIDs, is highly unlikely, since no
decrease of the prostaglandin synthesis was seen in the cell-
free settings. Instead in the cell-free assays arachidonic acid is
Dipyrone metabolites can form complexes with haem
Recently it was reported that at high concentrations,
paracetamol inhibits COX activity by reducing the higher
oxidative states of the protein that are part of the catalytic
cycle of COX (Aronoff et al., 2003; Lucas et al., 2005). Since
aminopyrine, a close structural homolog of dipyrone, is
2 2
converted into either PGG or PGH by COX, which then
2
þ
seems to be oxidized by MAA to a number of prostaglandins,
known to reduce peroxide in the presence of Fe
ions
such as PGF2a. However in a cellular environment, the
(Iogannsen et al., 1993), we hypothesized that MAA may
British Journal of Pharmacology (2007) 151 494–503

Inhibition of COX by dipyrone
4
98
SC Pierre et al
interact with the haem group of COX, as described for
paracetamol. To study whether a molecular interaction
between MAA and haem can be observed, we used mass
spectrometry to identify possible MAA–haem complexes. A
full spectrum scan in the negative ion mode of haem alone
yielded a signal with the expected m/z of 632.6 [M–H]-
(Figure 3a). Haem was not detectable using the positive ion
mode. After incubation of haem with MAA (100 mM), a new
a
bMAA
−
+
+
−
+
+
−
−
−
+
+
+
+
−
haem
MAA
AAA
AA FAA
Aspirin
PGHS-1
−
−
−
+
+
+
+
-
a.a.
a.a.
PGD2
PGE2
PGF2α
PGE
2
*
*
*
*
*
*
PGF2α
c
d
e
2
.4
.0
.6
.2
.8
.4
6
5
4
3
2
1
MAA
6
4
2
0
PGG
2
PGG
2
2
1
1
0
0
0
0
2
4
6
8
10
2
0
40
60
80 100 120
2
4
6
8
10
MAA (µM)
Time (min)
Time (min)
f
g
h
2
.0
.6
.2
.8
.4
4.0
3.2
2.4
1.6
0.8
0
3
.6
.8
.0
.2
.4
AA
AAA
FAA
1
2
1
2
0
1
0
0
0
2
4
6
8
10
2
4
6
8
c
10
0
1
2
3
c
4
5
6
7
8
9 10
Time (min)
Time (min)
Time (min)
LPS
MAA
−
−
+
−
−
+
+
+
j
i
m
c
m
c
m
m
12
9
6
3
PGE2
*
*
0
basal
LPS
LPS + MAA
MAA
British Journal of Pharmacology (2007) 151 494–503

Inhibition of COX by dipyrone
SC Pierre et al
499
þ
substance with an m/z of 833.6 [M þ H] was detected in the
metabolite–haem complexes are reaction intermediates
common to all four dipyrone metabolites.
positive ion mode (Figure 3b) that was not present in the
spectrum derived from haem or MAA alone. In the negative
ꢁ
ion mode, a matching signal at m/z 831.2 [M–H] appeared
MAA and AA react with peroxide in presence of Fe ions
which was considerably weaker (data not shown). The
precursor ion spectrum of the 833.6- and the 831.5-
substances matched the one of the haem alone and suggests
the formation of a new product that contains haem (Figure
Next we monitored the absorption spectra of the dipyrone
metabolites to study their interaction with haem and
hydrogen peroxide, as the UV- and visible spectra of haem
as well as dipyrone metabolites depend on their oxidation
state (Damm 1989; Iogannsen et al., 1993; Uetrecht et al.,
1995; Van Eps et al., 2003; Voegtle et al., 2003). Addition of
10 mM haem to the four metabolites slightly increased the
absorption of all four metabolites (Figure 4a and b), while the
haem spectrum itself was not altered by the metabolites
(data not shown). Most interestingly, a dramatic decrease in
the absorption spectrum for MAA and AA, but not FAA and
AAA, was observed between 230 and 270 nm in presence of
haem and hydrogen peroxide (Figure 4a and b). In contrast,
addition of peroxide had no effect on the absorption spectra
of the dipyrone metabolites (data not shown). A slight
3
a and b). The calculated mass of 832.6 equals the mass of a
haem–MAA adduct, assuming that MAA substitutes the iron
bound hydroxyl group with elimination of a molecule of
water. Accordingly, fragmentation of the newly formed
substance yielded a major fragment with m/z of 615.1 in
the negative ion mode that corresponds to the mass of haem
lacking a hydroxyl group (Figure 3c). However, the forma-
tion of this complex alone cannot be correlated with the
inhibitory effect of MAA on the COX activities, since haem
formed similar complexes with AA, FAA and AAA (Figure 3d,
Supplementary data 4). Instead it seems more likely that the
a
632.6
4
3
2
1
0
c
3
2
1
0
3
2
1
0
615.1
6
29 632 635 638
831.2
800
654.4
200
300
400
500
m/z,amu
600
700
600
650
700
750
800
850
m/z,amu
b
d
4
4
3
2
1
0
3
2
1
0
.5
.5
.5
758.7
8
33.6
6
74.6
3
6
16.3
2
1
0
861.7
8
30 833 836 839
616.4
.5
0
600
650
700
750
m/z,amu
800
850
6
00
650
700
750
800
850
m/z,amu
Figure 3 Dipyrone metabolites form stable complexes with haem. (a) Full scan mass spectrometric analysis (ESI-MS) in the negative ion mode
of haem alone. The inset shows a magnification of the indicated signals. (b) Full scan mass spectrometric analysis (ESI-MS) in the positive ion
mode of haem and MAA. The inset shows a magnification of the indicated signals. (c) Fragmentation of the suspected haem–MAA complex
with the m/z of 831.2 in the negative ion mode. (d) Full scan mass spectrometric analysis in the positive ion mode of haem with FAA. Arrows
indicate the position of the proposed haem–metabolite adduct.
Figure 2 MAA does not inhibit COX activity by competitive binding to the active site. (a) LPS-stimulated RAW 264.7 were lysed and
prostaglandin synthesis was measured in the presence of 100 mM MAA, AA, FAA, AAA or 1.25 mM aspirin, as described in the ‘Methods’ section.
Asterisks indicate the prostaglandins that were found only in MAA and AA treated samples. (b) Purified COX-1 (20 U) was incubated with
1
4
[
1– C]-arachidonic acid (a.a.) in the presence or absence of 100 mM MAA and 1 mM haem and the products analysed by TLC. (c) 1.3 mM PGG
was incubated with the indicated concentrations of MAA for 30 min and analysed by LC-MS/MS as described in the ‘Materials and methods’
section. (d–h) 13mM PGG alone (d) or incubated with 100 mM MAA (e), AA (f), AAA (g) or FAA (g) were incubated at room temperature for
0 min prior to analysis with LC-MS/MS. The arrows indicate the positions of the PGG -specific signals. (i) RAW 264.7 cells were metabolically
2
2
3
2
ꢁ1
labelled as described in the ‘Methods’ section. Cells were stimulated with 5 mg/ml LPS 100 mM MAA and the prostaglandins in the medium (m)
or in the cells (c) were analysed by TLC. (j) RAW 264.7 cells were incubated with LPS for 16 h in presence and absence of 100 mM MAA. PGF2a
levels were analysed with LC-MS/MS as described under the ‘Methods’ section. Students t-test *Po0.001 MAA vs LPS-treatment without MAA.
British Journal of Pharmacology (2007) 151 494–503

Inhibition of COX by dipyrone
5
00
SC Pierre et al
a
2.0
.6
.2
.8
.4
0
.4
1
0
2
50
350
450
1
-0.4
-0.8
0
MAA
MAA + haem
MAA + haem
0
+
H O2
2
0
2
00
300
400
500
600
700
800
Wavelength(nm)
b
0.4
0
c
0.4
0
2
50
350
450
250
350
450
-
0.4
-
-
0.4
0.8
AA
FAA
AAA
3+
Fe
haem
-0.8
Wavelength (nm)
Wavelength (nm)
350
e
0
d
0.4
2
50
450
-
0.04
0.08
0
0.4
0.8
2
50
350
450
-
-
-
COX-1
3+
-0.12
Fe
haem
Wavelength (nm)
Wavelength (nm)
Figure 4 MAA and AA react with hydrogen peroxide in presence of an iron source. (a) UV-visible absorption spectra of MAA in absence or
presence of haem or haem and peroxide. The inset shows absorption changes of the metabolite–haem complexes after addition of hydrogen
peroxide. The arrows indicate MAA specific changes. (b) As in (a) except that AA, AAA or FAA were used instead of MAA. (c–e) Changes in the
3
þ
2 þ
absorption spectrum of MAA after addition of 100 mM hydrogen peroxide in presence of 10 mM, Fe (c), Fe (d) or 65 nM reconstituted COX-
. (e) For better comparison, the absorption changes in presence of haem (dotted line) were included in (c and d).
1
decrease in the absorption spectrum of haem occurred after
hydrogen peroxide addition, independantly of the metabo-
lites, and is believed to reflect a change in the redox state of
the haem (Van Eps et al., 2003; Voegtle et al., 2003). To
elucidate the relevance of the presence of the protoporphyric
component of the haem, we repeated the experiments in
was necessary to remove the protein before the absorption
measurements, a quantitative comparison of the absorption
changes is not possible. Taken together, the data suggest that
MAA and AA are able to react with electron donors, such as
2
þ
3 þ
hydrogen peroxide in the presence of haem, Fe
or Fe
ions. In this way, MAA and AA can either prevent the start of
the catalytic cycle of COX or, alternatively, reduce the
already activated states of COX.
3
þ
2 þ
presence of Fe
or Fe
and hydrogen peroxide. The MAA
absorption spectrum was also altered by hydrogen peroxide
3
þ
2 þ
or Fe ,
in presence of Fe
although the observed
absorption changes after addition of hydrogen peroxide
were significantly smaller as compared to the absorption
changes seen in presence of haem (Figure 4c and d). In
3
þ
2 þ
to Fe ions
MAA and AA reduce Fe
Next, we studied the influence of the metabolites on the
3
þ
3 þ
presence of 10 mM Fe , the decrease in the absorption was
redox state of Fe , present in the resting state of haem in
2
þ
only 35% of the decrease seen with an equimolar amount of
COX. We determined the Fe
formation in presence of the
2
þ
haem (Figure 4c). Fe
was even less effective and decreased
four dipyrone metabolites using two different detection
methods, potassium ferricyanide and bathophenanthroline.
Interestingly, only the pharmacologically active metabolites
the absorption only by 25%, compared to haem (Figure 4d).
Most importantly, the same changes in the absorption
spectrum of MAA occurred in presence of COX-1 and
peroxide (Figure 4e). As we used significantly less COX
3
þ
2 þ
MAA and AA were able to reduce Fe
to Fe , while no
reducing effect was seen with FAA and AAA (Figure 5a and b).
Since MAA and AA interact with peroxide only in the
(65 nM) compared to haem (10 mM) and an extraction step
British Journal of Pharmacology (2007) 151 494–503

Inhibition of COX by dipyrone
SC Pierre et al
501
a
1.2
.0
.8
.6
.4
.2
FeCl3 + K4[Fe(CN)6]
b
0
.5
0.4
.3
.2
Bathophenanthroline
*
*
1
*
*
*
*
0
*
*
0
0
0
0
0
0.1
0
0
-
AAA
FAA
AA
MAA
Ascorbate
-
AAA
FAA AA
MAA
24h
c 2.5
d
*
2
.0
*
Dipyrone
Hsp70
0h
4h
1.5
Peroxiredoxin V
1.0
Hsp70
SOD
0.5
0
-
MAA
2 þ
Fe2+
DFO
DFO+
MAA
DFO +
Fe2+
3
þ
2 þ
formation after addition of dipyrone metabolites or ascorbate
Figure 5 MAA and AA reduce Fe
to FeCl
least three experiments each carried out in duplicate are shown. Student’s t-test **Po0.001 MAA or AA vs control. (c) Lipid oxidation in RAW
to Fe . (a and b) Determination of Fe
3
by potassium ferricyanide (a) or bathophenanthroline (b) as described in the ‘Materials and methods’ section. The mean7s.e.m. of at
2
þ
2
64.7 cells after incubation with 100 mM Fe
malondialdehyde (MDA). The mean7s.e.m. of at least four experiments is shown. Students t-test *Po0.05 MAA or Fe
Western blot analysis of the expression of superoxide dismutase and peroxiredoxin V in spinal cords of adult rats. The spinal cords were excised
or MAA in presence or absence of 100 mM deferoxamine (DFO) as determined by levels of
2
þ
vs control. (d)
ꢁ
1
4
or 24 h after i.p. application of 1 g kg dipyrone. Hsp 70 expression was used to control for even loading.
presence of Fe ions and this interaction depends on the
redox state of the iron, so far the data suggest that the
acetylation of serine-530 in the arachidonic acid cavity
(Marnett 2000; Smith et al., 2000). Our data clearly show that
MAA does not compete with arachidonic acid for the
catalytic site of COX. Instead it seems that MAA can oxidize
radical donors thus blocking the initiation of the catalytic
reaction and/or the further progress of the catalytic cycle by
reduction of the higher oxidative states of the enzyme
protein, in a mechanism similar to the one proposed for
paracetamol (Aronoff et al., 2003; Lucas et al., 2005). This
model would be in line with recent reports describing that
dipyrone and its metabolites are effective scavengers for
several ROS species (Costa et al., 2006a, b).
3
þ
oxidation of MAA and AA by Fe
is required for their
reaction with peroxide. Next, we wanted to determine if the
3
þ
occurs also in a cellular
reaction between MAA and Fe
environment. Since the free Fe pool in cells consists mainly
3
þ
of Fe (Lipinski et al., 2000; Imlay 2003; Huang et al., 2004),
an interaction of MAA with the free Fe would be expected to
2
þ
increase at least temporarily the Fe
concentration in cells
and may result in the increased generation of reactive
oxygen species (ROS) (Wang et al., 2002; Imlay 2003; Huang
et al., 2004). Indeed an increased lipid oxidation was
observed in MAA-treated RAW macrophages, which was
A possible inhibitory mechanism of MAA could start with
2
þ
3 þ
prevented by the Fe -scavenger deferoxamine (Figure 5c).
Furthermore, adult rats that received a single dose of
dipyrone showed, in the spinal cord, an increased expression
of superoxide dismutase and peroxiredoxin V, two enzymes
that are necessary to neutralize superoxide and other ROS
species. The expression levels of both enzymes were
increased in spinal cords of dipyrone-treated animals 4 and
the binding of MAA to the Fe
in the haem. The mass
determination of the MAA–haem complex by mass spectro-
metry suggests the formation of a bond between the 4-amino
group of the dipyrone metabolites and the Fe with release of
a water molecule. However, the complex formation by itself
does not cause inhibition of COX activity, since all four
metabolites form similar complexes with haem. In the next
3
þ
2
4 h after treatment (Figure 5d).
step, the Fe
is reduced and an MAA radical is formed. This
assumption is in accordance with a previous report which
3
þ
shows that AA forms radicals after direct oxidation by Fe
Discussion
(Iogannsen et al., 1993). Since these radicals are stabilized by
alkyl groups (Iogannsen et al., 1993), the stabilization of a
radical intermediate by the 4-methyl group of MAA would
explain why MAA is the only metabolite that is active
at pharmacologically relevant concentrations. The other
three tested metabolites form either less stable radical
Traditional NSAIDs inhibit COX enzyme activity by compet-
ing with arachidonic acid for binding to the cyclooxygenase
active site (Smith and Dewitt 1996; Marnett et al., 1999). One
exception is aspirin which inhibits PGG
2
synthesis by
British Journal of Pharmacology (2007) 151 494–503

Inhibition of COX by dipyrone
5
02
SC Pierre et al
intermediates, as in the case of AA, or even prevent
formation of the radicals due to the destabilizing effect of
a 4-acetyl (AAA) or a 4-formyl (FAA) group. The reaction of
MAA and AA with Fe seems necessary for the reaction with
hydrogen peroxide, since no effect of peroxide on the
absorption spectra of MAA and AA was observed in the
absence of haem or Fe ions Thus, MAA could block COX
activity by sequestering the peroxide and thereby suppres-
sing the generation of the tyrosyl radical in the COX-
cyclooxygenase site that is required for oxygenation of
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British Journal of Pharmacology (2007) 151 494–503