European Journal of Pharmacology 378 1999 339-347

Yüklə 158,49 Kb.

Pdf görüntüsü

tarix	21.04.2017
ölçüsü	158,49 Kb.
	#14874

European Journal of Pharmacology 378 1999 339–347

www.elsevier.nlrlocaterejphar

Regulation of cyclooxygenase activity by metamizol

Carmen Campos

c,1

, Rosa de Gregorio

, Raquel Garcıa-Nieto

, Federico Gago

,

´

Pablo Ortiz

, Susana Alemany

a,)

a

(

)

Instituto de InÕestigaciones Biomedicas CSIC , Departmento de Bioquımica de UAM, Arturo Duperier 4, E-28029 Madrid, Spain

b

Departamento de Farmacologıa, UniÕersidad de Alcala, E-28871 Alcala de Henares, Madrid, Spain

c

Medical Department, Boehringer Ingelheim, Spain

Received 18 March 1999; received in revised form 24 June 1999; accepted 29 June 1999

Abstract

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

50

value for cyclooxygenase-2 was obtained in lipopolysaccharide-activated broken murine macrophages. Consistent with these findings,

molecular models of the complexes between cyclooxygenase-1 or cyclooxygenase-2 with 4-methylaminoantipyrine, the major active

derivative of metamizol, suggested a common binding mode to both isoforms. In intact cells, however, the inhibition profiles were

.

markedly different. The IC

values of metamizol for cyclooxygenase-1 in intact bovine aortic endothelial cells BAEC cells and human

platelets were 1730 " 150 mgrml and 486 " 56 mgrml, respectively. Inhibition of cyclooxygenase-2 activity in murine macrophages

and primary human leukocytes activated by lipopolysaccharide yielded IC

values of 12 " 1.8 mgrml and 21 " 2.9 mgrml,

respectively. These data indicate that the IC

values obtained with purified enzymes or disrupted cells cannot always be extrapolated to

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

.

Keywords: Cyclooxygenase; Nonsteroidal antiinflammatory drug NSAID ; Metamizol; Molecular modelling

1. Introduction

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

Ž

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;

E-mail: salemany@biomed.iib.uam.es

R. de Gregorio and C. Campos have contributed equally to this work.

enzyme and is primarily associated with inflammation.

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

al., 1992; DeWitt and Meade, 1993; Ristimaki et al., 1994;

Coffey et al., 1997 . In brain and spinal cord cyclooxy-

genase-2 is constitutively expressed

McCormak, 1994;

Cashman, 1996; Yaksh et al., 1998 .

Cyclooxygenase-1 and cyclooxygenase-2 are encoded

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-

.

PII: S 0 0 1 4 - 2 9 9 9 9 9 0 0 4 7 7 - X

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

340

inflammatory drugs NSAIDs bind. A mutant of cyclo-

oxygenase-2 in which valine

523

is exchanged for isoleucine

shows inhibitor binding and selectivity profiles comparable

to those of wild-type cyclooxygenase-1

Gierse et al.,

1996; Guo et al., 1996 .

Metamizol is a prodrug which, at room temperature and

in an atmosphere with oxygen, is spontaneously, nonenzy-

matically converted to 4-methylaminoantipyrine

Levy,

1986; Levy et al., 1995 . Subsequently, the N-methyl side

chain of 4-methylaminoantipyrine is oxidized to yield 4-

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-

tives Fig. 1A , all of which have analgesic, antipyretic,

and relatively weak antiinflammatory properties.

It has been proposed that the antinociceptive action of

metamizol is at least partially centrally mediated Carlsson

et al., 1986; Gelgor et al., 1992; Tortorici and Vanegas,

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

antiinflammatory and analgesic effects of metamizol, and

inhibition of prostaglandins synthesis was reported prior to

the discovery of cyclooxygenase-2 Dembinska-Kiec et al.,

1976; Brune, 1983; Eldor et al., 1984; Lorenzetti and

Ferreira, 1985 .

The importance of the discovery of the inducible form

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

derivatives. Right page Molscript representation Kraulis, 1991 of 4-methylaminoantipyrine thicker bonds docked in the active site of human

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.

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

341

Fig. 1 continued .

genase-1 and cyclooxygenase-2. We also present a molecu-

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

cyclooxygenase-2

The experimentally determined three-dimensional struc-

.

tures of ovine cyclooxygenase-1 Picot et al., 1994 and

mouse cyclooxygenase-2 Kurumbail et al., 1996 , together

with our homology-based models of their human counter-

parts Garcıa-Nieto et al., 1999 , were studied. Two possi-

ble conformers of 4-methylaminoantipyrine were initially

considered differing in the orientation of the N-methyl

substituent relative to the plane of the pyrazole ring, and

their geometries were optimized by using the semiempiri-

cal molecular orbital AM1 method Dewar et al., 1985 .

Atom-centered charges were then derived by fitting the

calculated molecular electrostatic potential to a mono-

pole–monopole expression Besler et al., 1990 . Then, the

Monte Carlo simulated annealing technique implemented

.

in AutoDock Morris et al., 1996 was used to generate

different conformations and orientations of 4-methyl-

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

and aromatic carbons, carbonyl oxygen, and amino and

heterocyclic ring nitrogens using program GRID Good-

ford, 1985 . The 4-methylaminoantipyrine favoured by

AutoDock was then used as a rigid ligand for the GROUP

module of GRID version 16, Molecular Discovery, 1997 ,

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-

zyme activity was measured by conversion of

C arachi-

donic acid to prostaglandins, after separation by thin layer

. Ž

chromatography TLC

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

mM , epinephrine 5 mM , hematin 1 mM , 1 = 10 dpm

C arachidonic acid Amersham , 6.6 mM arachidonic

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

342

acid and different concentrations of metamizol. Samples

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

gel plate

Merck . The TLC plate was developed for

approximately 90 min at room temperature in a solvent

consisting of the upper phase of ethyl acetatertrimethyl

pentaneracetic acidrwater, 110:50:20:100 vrv . For de-

tection of the

C-labelled prostaglandins an autoradiog-

raphy in an Instant Imager was performed.

2.3. Measurement of cyclooxygenase-1 actiÕity in intact

cells

Bovine aortic endothelial cells BAEC , maintained in

Dulbecco’s Modified Eagle Medium DMEM with 10%

fetal calf serum, 4 mM glutamine, 50 mgrml gentamicin

at 378C, were incubated for 30 min with different concen-

trations of metamizol 125–4000 mgrml or with indo-

methacin 0.1–100 mgrml , after which arachidonic acid

was added to a final concentration of 30 mM. Cells were

incubated for further 15 min at 378C. The medium was

then removed, and a radioimmunoassay kit Amersham

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.

1992 . Acid–citrate–dextrose–anticoagulant

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 .

Cells were incubated with different concentrations of

metamizol 125–4000 mgrml and stimulated with cal-

cium ionophore A23187 0.25 mM . The levels of throm-

boxane B in the incubation media after 5 min of stimula-

tion were measured with a thromboxane B

enzyme im-

munoassay system Amersham . Metamizol did not inter-

fere with the measurement of thromboxane B .

2

2.4. Measurement of cyclooxygenase-2 actiÕity in intact

cells

Murine monocyte–macrophage J774A.1 cells, in RPMI

1640 medium supplemented with 10% fetal calf serum,

. Ž

gentamicin 50 mgrml

complete medium at 0.5 = 10

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

not interfere with the measurement of prostaglandin E .

Human peripheral leukocytes from Buffy-Coat were

separated using Histopaque 1099 Sigma as described by

Tavares and Bennett

1993

and incubated for 2 h in

complete medium at 0.5 = 10

cellsrml. Subsequently, the

cells were incubated for 12 h with or without lipopoly-

saccharide 3 mgrml , and in the presence or absence of

different concentrations of metamizol. The prostaglandin

released to the incubation media was measured by

radioimmunoassay as described above.

2.5.

Measurement

of

cyclooxygenase-2

actiÕity

in

macrophage cell extracts

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-

methyl sulfonyl fluoride, 50 mM pepstatin A, and 0.2 mM

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.

Fig. 2. Regulation of purified cyclooxygenase-1 and cyclooxygenase-2

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:

Cyclooxygenase-2 activity.

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

343

3. Results

3.1. Docking of 4-methylaminoantipyrine into the actiÕe

sites of human cyclooxygenase-1 and cyclooxygenase-2

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

385

,

the 4-amino group in close proximity to the hydroxyl of

Tyr

355

, and the N

ring nitrogen facing the residue at

position 523 Fig. 1B . The smaller size of the side chain

of valine relative to that of isoleucine at this position

allows the main substrate channel in cyclooxygenase-2 to

Fig. 3. Effects of metamizol on cyclooxygenase-1 activity measured in

intact cells: A on BAEC cells measured as 6-keto-prostaglandin F

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

metamizol incubation. The data are expressed as mean"S.D. from three

Ž .

different experiments performed in duplicate; B on human platelets

measured as thromboxane B

after stimulation with calcium ionophore

for 5 min. The data are expressed as mean"S.D. from three different

experiments performed in duplicate.

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

obtained in the absence of metamizol. The data are expressed as mean"

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 .

3.3. Regulation of cyclooxygenase-1 actiÕity in intact cells

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-

trations of metamizol in the incubation media resulted in a

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

344

Fig. 5. Induction of cyclooxygenase-2 activity in human freshly isolated

leukocytes and inhibition by metamizol. A Prostaglandin E

levels in

the incubation media after stimulation or not with 5 mgrml lipopoly-

saccharide for 12 h. The data are expressed as mean"S.D. from four

Ž .

different experiments performed in duplicate. B Inhibition of cyclooxy-

genase-2 activity by metamizol measured as prostaglandin E formation.

100% is given to the value obtained in the absence of metamizol. The

data are expressed as mean"S.D. from four different experiments per-

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

lev-

1a

Ž

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.

3.4. Regulation of cyclooxygenase-2 actiÕity by metamizol

in intact cells

Murine monocyte–macrophage J774A.1 cells have been

used as a model to determine cyclooxygenase-2 activity

Mitchell et al., 1994 . No prostaglandin E

levels were

detected in the incubation media, unless cells were stimu-

lated with lipopolysaccharide for 12 h. In these conditions,

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

with metamizol inhibited prostaglandin E

release. The

IC

of metamizol for cyclooxygenase-2 in this cell system

was 12 " 1.8 mgrml

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

of metamizol

for cyclooxygenase-2 in intact human primary cells were

carried out using freshly isolated human leukocytes. Stimu-

lation of these cells with 3 mgrml lipopolysaccharide

increased the prostaglandin E

levels in the incubation

Ž

.

media from 200 " 30 up to 4200 " 400 pgrml Fig. 5A ,

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 .

50

3.5. Determination of the IC

Õalue of metamizol for

50

cyclooxygenase-2 in cell extracts

The 10-fold difference between the metamizol IC

values for purified cyclooxygenase-2 and in intact cells

prompted us to determine the inhibitory capacity of meta-

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

Ž

.

mgrml data not shown .

Fig. 6. IC

of metamizol on cyclooxygenase-2 activity in broken lipo-

polysaccharide-activated murine macrophage preparation. Cell extracts

were prepared as described under Section 2. Prostaglandin E

formation

was measured after incubation with saturating doses of arachidonic acid

in the presence of different doses of metamizol. The data are expressed as

mean"from at least three different experiments performed in duplicate.

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

345

4. Discussion

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

inhibit cyclooxygenase-1 and cyclooxygenase-2 activities

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

of about

150 mgrml when tested for cyclooxygenase-2 inhibition in

macrophage cell extracts.

Metamizol is substantially more potent about 10-fold

in inhibiting cyclooxygenase-2 activity when intact cells

are used as a source of cyclooxygenase-2. The IC

about 12 " 1.8 mgrml of metamizol for cyclooxygenase-2

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

value 15–20 mgrml of metamizol for

cyclooxygenase-2 using intact cells as a source of cyclo-

oxygenase-2 leads us to conclude that the analgesic effects

described for metamizol

Brune, 1983; Matzner et al.,

1984 can be due to cyclooxygenase-2 inhibition since

after drug administration the blood levels of the active

derivatives of metamizol are about 30 mgrml Levy et al.,

1995 .

Our data also indicate that, although breaking the in-

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

for cyclooxygenase inhibition by metamizol. Differences

in IC

values obtained in intact cells, broken cells, or

purified enzymes have also been described for other

NSAIDs

Barnett et al., 1994; Mitchell et al., 1994;

Carabaza et al., 1996 . The NSAID BF 389, for example,

exhibits a 250-fold lower IC

value for cyclooxygenase-2

in intact cells when compared with the IC

obtained with

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

the conformation of the enzyme is altered upon membrane

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-

tained for cyclooxygenase-2, indicating that cyclooxy-

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

about 15-fold higher than the blood levels of metamizol

after the administration of the drug Levy et al., 1995 .

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

inhibition

was

particularly

potent

the

brain

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-

atory activity.

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.

Acknowledgements

We thank the technical assistance of Joaquın Perez.

´

Rosa de Gregorio is supported by a ‘‘CSIC-sector empre-

sarial’’ fellowship. Provision of programs GRID and

AutoDock by Dr. Peter Goodford Molecular Discovery,

UK and Dr. Garrett Morris The Scripps Research Insti-

tute , respectively, is gratefully acknowledged. This work

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

346

was supported by Plan Nacional, Comunidad de Madrid

and Boehringer Ingelheim-Europharma.

References

Barnett, J., Chow, J., Ives, D., Chiou, M., Mackenzie, R., Osen, E.,

Nguyen, B., 1994. Purification, characterization and selective inhibi-

tion of human prostaglandin GrH synthase 1 and 2 expressed in the

baculovirus system. Biochim. Biophys. Acta 1209, 130–139.

Beirith, A., Santos, A.R.S., Rodrigues, A.L.S., Creczynski-Pasa, T.B.,

Calixto, J.B., 1998. Spinal and supraspinal antinociceptive action of

dipyrone in formalin, capsaicin and glutamate tests. Study of the

mechanism of action. Eur. J. Pharmacol. 345, 233–245.

Besler, B.H., Merz, K.M. Jr., Kollman, P.A., 1990. Atomic charges

derived from semiempirical methods. J. Comp. Chem. 11, 431–439.

Brodgen, R.N., 1986. Pyrazolone derivatives. Drugs 32, 60–70, Suppl. 4.

Brune, K., 1983. Prostglandins and the mode of action of antipyretic

analgesic drugs. Am. J. Med. 14, 19–23.

Campos, D.I., Cunha, F.Q., Ferreira, S.H., 1988. A new mechanism of

action of dipyrone: blockade of the release of a nociceptive factor

from macrophages. Brasilian J. Med. Biol. Res. 21, 565–568.

Carabaza, A., Cabre, F., Rotllan, E., Gomez, M., Gutierrez, M., Garcia,

M.L., Mauleon, D., 1996. Stereoselective inhibition of inducible

cyclooxygenase by chiral nonsteroidal antiinflammatory drugs. J.

Clin. Pharmacol. 36, 505–512.

Carlsson, K.H., Helmreich, J., Jurna, I., 1986. Activation of inhibition

from the periaqueductal grey matter mediates central analgesic effect

of metamizol dipyrone . Pain 27, 373–390.

Cashman, J.N., 1996. The mechamisn of action of NSAIDs in analgesia.

Drugs 52, 13–23, Suppl. 5.

Coffey, R.J., Hawkey, C.J., Damstrup, L., Graves-Deal, R., Daniel, V.C.,

Dempsey, P.J., Chinery, R., Kirkland, S.C., DuBois, R.N., Jetton,

T.L., Morrow, J.D., 1997. Epidermal growth factor receptor activation

induces nuclear targeting of cyclooxygenase-2, basolateral release of

prostaglandins and mitogenesis in polarizing colon cancer cells. Proc.

Natl. Acad. Sci. U.S.A. 94, 657–662.

Dembinska-Kiec, A., Zmuda, A., Kupinska, J., 1976. Inhibition of prosta-

glandin synthetase by aspirin-like drugs in different microsomal

preparations. In: Samuesson, B., Pooletti, R.

Eds. , Adv. in

Prostaglandin and Thromboxane Research, Vol. 1. Raven Press, New

York.

Dewar, M.J.S., Zoebisch, E.G., Healy, E.F., Stewart, J.J.P., 1985. AM1: a

new general purpose quantum mechanical molecular model. J. Am.

Chem. Soc. 107, 3902–3909.

DeWitt, D.L., Meade, E.A., 1993. Serum and glucocorticoid regulation of

gene transcription and expression of the prostaglandin H synthase-1

and prostaglandin synthase-2 isozymes. Arch. Biochem. Biophys.

306, 94–102.

Dirig, D.M., Konin, G.P., Isakson, P.C., Yaksh, L.T., 1997. Effect of

spinal cyclooxygenase inhibitors in rat using the formalin test and in

vitro prostaglandin E release. Eur. J. Pharmacol. 331, 155–160.

Eldor, A., Zylber-Katz, E., Levy, M., 1984. The effects of oral adminis-

tration of dypirone on the capacity of blood platelets to synthesize

thromboxane A

in man. Eur. J. Clin. Pharmacol. 26, 171–176.

Font, J., Azula, F.J., Marino, A., Nieva, N., Trueba, M., Macarulla, J.M.,

1992. Intracellular Ca2

mobilization and not calcium influx pro-

motes phorbol ester-stimulated thromboxane A

synthesis in human

platelets. Prostaglandins 43, 383–395.

Funk, C.D., Funk, L.B., Kennedy, M.E., Pong, A.S., Fitzgerald, G.A.,

1991. Human plateletrerythroleukemia cell prostaglandin GrH syn-

thase cDNA cloning, expression, and gene chromosomal assignment.

FASEB J. 5, 2304–2312.

Garcıa-Nieto, R., Perez, C., Gago, F., 1999. Automated docking and

molecular dynamics simulations of nimesulide in the cyclooxygenase

active site of human prostaglandin-endoperoxide synthase-2 COX-2 .

J. Comp.-Aided Mol. Design, in press.

Gelgor, L., Cartmell, S., Mitchell, D., 1992. Intracerebroventricular mi-

cro-injections of non-steroidal anti-inflammatory drug abolish reperfu-

sion hyperalgesia in the rat’s tail. Pain 50, 323–329.

Gierse, J.K., Mcdonald, J.J., Hauser, S.D., Rangwala, S.H., Koboldt,

C.M., Seibert, K., 1996. A single amino acid difference between

cyclooxygenase-1 and -2 reverses the selectivity of cyclooxygenase-2

specific inhibitors. J. Biol. Chem. 271, 15810–15814.

Goodford, P.J.A., 1985. Computational procedure for determining ener-

getically favorable binding sites on biologically important macro-

molecules. J. Med. Chem. 28, 849–857.

Guo, Q., Wang, L.H., Ruan, K.H., Kulmacz, R.J., 1996. Role of Val

509

in time-dependent inhibition of human prostaglandin H synthase-2

cyclooxygenase activity by isoform-selective agents. J. Biol. Chem.

271, 19134–19139.

Kraemer, S.A., Meade, E.A., Dewitt, D.L., 1992. Prostaglandin endoper-

oxide synthase gene structure identification of the transcriptional start

site and 5

flanking regulatory sequences. Arch. Biochem. Biophys.

293, 391–400.

Kraulis, P.J., 1991. Molscript: a program to produce both detailed and

schematic plots of protein structures. J. Appl. Crystall. 24, 946–950.

Kujubu, D.A., Fletcher, B.S., Varnum, B.C., Lim, R.W., Herschman,

H.R., 1991. TIS10, a phorbolester tumor promoter-inducible mRNA

from Swiss 3T3 cells, encodes a novel prostaglandin synthasercyclo-

oxygenase homologue. J. Biol. Chem. 266, 12866–12872.

Kurumbail, R.G., Stevens, A.M., Gierse, J.K., McDonald, J.J., Stegeman,

R.A., Pak, J.Y., Gildehaus, D., Miyashiro, J.M., Penning, T.D.,

Seibert, K., Isakson, P.C., Stallings, W.C., 1996. Structural basis for

selective inhibition of cyclooxygenase-2 by anti-inflammatory agents.

Nature 384, 644–648.

Laporte, J.R., Carne, X., 1987. Blood dyscrasias and the relative safety of

non-narcotic analgesics. Lancet 4, 809–811.

Laporte, J.R., Carne, X., Vidal, X., Moreno, V., Juan, J., 1991. Upper

gastrointestinal bleeding in relation to previous use of analgesics and

non-steroidal anti-inflammatory drugs. Catalan countries study on

upper gastrointestinal bleeding. Lancet 337, 85–89.

Lee, S.H., Soyoola, E., Chanmugam, P., Hart, S., Sun, W., Zhong, H.,

Liou, S., Simmons, D., Hwang, D., 1992. Selective expression of

mitogen-inducible cyclooxygenase in macrophages stimulated with

lipopolysaccharide. J. Biol. Chem. 267, 25934–25938.

Levy, M., 1986. Pharmakokinetics of metamizol metabolites. Agents

Actions Suppl. 19, 199–204.

Levy, M., Zylber-Katz, E., Rosenkranz, B., 1995. Clinical pharmacoki-

netics of dipyrone and its metabolites. Clin. Pharmacokinet. 28,

216–234.

Lorenzetti, B.B., Ferreira, S.H., 1985. Mode of analgesic action of

dipyrone: direct antagonism of inflammatory hyperalgesia. Eur. J.

Pharmacol. 114, 375–381.

Matzner, Y., Drexler, R., Levy, M., 1984. Effect of dipyrone, acetylsali-

cylic acid and acetaminophen on human neutrophil chemotaxis. Eur.

J. Clin. Invest. 14, 440–443.

McCormak, K., 1994. Non-steroidal anti-inflammatory drugs and spinal

nociceptive processing. Pain 59, 9–43.

Mitchell, J.A., Akarasereenont, P., Thiemermann, C., Flower, R.J., Vane,

J.R., 1994. Selectivity of nonsteroidal antiinflammatory drugs as

inhibitors of constitutive and inducible cyclooxygenase. Proc. Natl.

Acad. Sci. U.S.A. 90, 11693–11697.

Morita, Y., Schindler, M., Regier, M.K., Otto, J.C., Hori, T., DeWitt,

D.L., Smith, W.L., 1995. Different intracellular locations for prosta-

glandin endoperoxidase H synthase-1 and -2. J. Biol. Chem. 270,

10902–10908.

Morris, G.M., Goodsell, D.S., Huey, R., Olson, A.J., 1996. AutoDock:

automated docking of flexible ligands to receptors, version 2.4. The

Scripps Research Institute, La Jolla, CA 92037-5025.

.

Pairet, M., Engelhardt, G., 1996. Distinct isoforms COX-1 and COX-2

of cyclooxygenase: possible physiological and therapeutic implica-

tions. Fundam. Clin. Pharmacol. 10, 1–15.

Picot, D., Loll, P.J., Garavito, R.M., 1994. The X-ray crystal structure of

(

)

C. Campos et al.r European Journal of Pharmacology 378 1999 339–347

347

the membrane protein prostaglandin H

synthase-1. Nature 367,

243–249.

Ristimaki, A., Garfinkel, S., Wessendrof, J., Maciag, T., Hla, T., 1994.

Induction of cyclooxygenase-2 by interleukin-1. J. Biol. Chem. 269,

11769–11775.

Simmons, D.L., Xie, W., Chipman, J.G., Evett, G.E., 1991. Multiple

cyclooxygenases: cloning of a mitogen-inducible form. In: Bailey,

J.M. Ed. , Prostaglandins, Leukotrienes, Lipoxins, and PAF. Plenum,

New York, 67–78.

Smith, W.L., DeWitt, D.L., 1994. Differential interactions of prosta-

glandin endoperoxide synthase with nonsteroidal anti-inflammatory

drugs. Curr. Opin. Invest. Drugs 3, 1–11.

Smith, W.L., Sonnenburg, W.K., Allen, M.L., Watanabe, T., Zhu, J.,

El-Harith, E.A., 1989. The biosynthesis and actions of prostaglandin

in the renal collecting tubule and thick ascending limb. In: Dunn,

.

M.J., Patrono, C., Cinotti, G.A. Eds. , Renal Eicosanoids. Plenum,

New York, 131–148.

Smith, C.J., Zhang, Y., Koboldt, C.M., Muhammad, J., Zweifel, B.S.,

Shaffer, A., Talley, J.J., Masferrer, J.L., Seibert, K., Isakson, P.C.,

1998. Prostaglandin biosynthesis and its compartmentation in vascular

smooth muscle and endothelial cells. Annu. Rev. Physiol. 48, 251–

262.

Tavares, I.A., Bennett, A., 1993. Acemetacin and indomethacin: differen-

tial inhibition of constitutive and inducible cyclo-oxygenases in hu-

man gastric mucosa and leucocytes. Int. J. Tiss. React. XV, 49–53.

Taylor, J., Mellstrom, B., Fernaud, I., Naranjo, J.R., 1998. Metamizol

potentiates morphine effects on visceral pain and evoked c-Fos im-

munoreactivity in spinal cord. Eur. J. Pharmacol. 351, 39–47.

Tortorici, V., Vanegas, H., 1995. Anti-nociception induced by systemic

or PAG-microinjected lysine-acetylsalicylate in rats. Effects on tail-

flick related activity of medullary off- and on-cells. Eur. J. Neurosci.

7, 1857–1865.

Vane, J.R., Botting, R.M., 1998. Mechanism of action of nonsteroidal

anti-inflammatory drugs. Am. J. Med. 104, 2–8.

Vane, J.R., Bakhle, Y.S., Botting, R.M., 1998. Cyclooxygenases 1 and 2.

Annu. Rev. Pharmacol. Toxicol. 38, 97–120.

Vlahov, V., Badian, M., Verho, M., Bacracheva, N., 1990. Pharmacoki-

netics of metamizol metabolites in healthy subjects after a single oral

dose of metamizol sodium. Eur. J. Clin. Pharmacol. 38, 61–65.

Weithmann, K.U., Alpermann, H.-G., 1985. Biochemical and pharmaco-

logical effects of dipyrone and its metabolites in a model system

related to arachidonic acid cascade. Arzneimittel-forschung 35, 947–

952.

Yaksh, T.L., Dirig, D.M., Malmberg, A.B., 1998. Mechanism of action of

nonsteroidal anti-inflammatory drugs. Cancer Invest. 16, 509–527.

Yokoyama, C., Tanabe, T., 1989. Cloning of human gene encoding

prostaglandin endoperoxide synthase and primary structure of the

enzyme. Biochem. Biophys. Res. Commun. 165, 888–894.

Yüklə 158,49 Kb.

Dostları ilə paylaş: