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Published as doi: 10.1096/fj.06-8061com.
 (The FASEB Journal. 2007;21:2343-2351.)
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Dipyrone elicits substantial inhibition of peripheral cyclooxygenases in humans: new insights into the pharmacology of an old analgesic

Burkhard Hinz1, Olga Cheremina, Jouri Bachmakov, Bertold Renner, Oliver Zolk, Martin F. Fromm and Kay Brune

Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University Erlangen-Nürnberg, Erlangen, Germany

1Correspondence: Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany. E-mail: hinz{at}pharmakologie.uni-erlangen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dipyrone (INN, metamizol) is a common analgesic used worldwide. Its widespread prescription or over-the-counter use in many countries (e.g., Brazil, Israel, Mexico, Russia, Spain) requires insight into its mode of action. This study therefore addressed the impact of its metabolites 4-methyl-amino-antipyrine (MAA) and 4-amino-antipyrine (AA) on peripheral cyclooxygenases (COX). Pharmacokinetics of metabolites and ex vivo COX inhibition were assessed in five volunteers receiving dipyrone at single oral doses of 500 or 1000 mg. Coagulation-induced thromboxane B2 formation and lipopolysaccharide-induced prostaglandin E2 synthesis were measured in vitro and ex vivo in human whole blood as indices of COX-1 and COX-2 activity. In vitro, metabolites elicited no substantial COX-1/COX-2 selectivity with MAA (IC50=2.55 µmol/L for COX-1; IC50=4.65 µmol/L for COX-2), being ~8.2- or 9-fold more potent than AA. After administration of dipyrone, MAA plasma concentrations remained above the IC50 values for each isoform for at least 8 h (500 mg) and 12 h (1000 mg) postdose. COX inhibition correlated with MAA plasma levels (ex vivo IC50 values of 1.03 µmol/L [COX-1] and 0.87 µmol/L [COX-2]). By contrast, plasma peak concentrations of AA after the 1000 mg dose were 2.8- and 6.5-fold below its IC50 values for COX-1 and COX-2, respectively. Maximal inhibitions of COX-1 and COX-2 were 94% and 87% (500 mg), 97% and 94% (1000 mg). Taken together, dipyrone elicits a substantial and virtually equipotent inhibition of COX isoforms via MAA. Given the profound COX-2 suppression by dipyrone, which was considerably above COX-2 inhibition by single analgesic doses of celecoxib and rofecoxib, a significant portion of its analgesic action may be ascribed to peripheral mechanisms. In view of the observed COX-1 suppression, physicochemical factors (lack of acidity) rather than differential COX-1 inhibition may be responsible for dipyrone’s favorable gastrointestinal tolerability compared with acidic COX inhibitors.—Hinz, B., Cheremina, O., Bachmakov, J., Renner, B., Zolk, O., Fromm, M. F., Brune, K. Dipyrone elicits substantial inhibition of peripheral cyclooxygenases in humans: new insights into the pharmacology of an old analgesic.


Key Words: dipyrone metabolites • human whole blood assay • pharmacokinetics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DIPYRONE (INN, METAMIZOL) is an antipyretic analgesic introduced into clinical practice in 1922. The water-soluble pyrazolinone derivative is available in oral, rectal, and injectable forms (for a review, see ref. 1 ). The drug is indicated for severe pain conditions, especially those associated with smooth muscle spasm or colics affecting the gastrointestinal, biliary, or urinary tracts. Moreover, dipyrone is useful for treatment of cancer pain and migraine as well as fever refractory to other treatments. Dipyrone (Fig. 1 ) is a prodrug. After oral administration, it is rapidly hydrolyzed in the gastric juice to its main metabolite 4-methyl-amino-antipyrine (MAA; Fig. 1 ) and absorbed in this form. MAA is converted to a variety of metabolites, including 4-formyl-amino-antipyrine (FAAP) and 4-amino-antipyrine (AA; Fig. 1 ), the latter being acetylated to 4-acetyl-amino-antipyrine (AAAP) by the polymorphic enzyme N-acetyl-transferase (2 , 3) .


Figure 1
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  		Figure 1. Structure of dipyrone and the investigated metabolites MAA and AA.

Although dipyrone is widely used as a prescription or over-the-counter analgesic in many countries (e.g., Brazil, Israel, Mexico, Russia, Spain), its mechanism of action is not entirely clear. Unlike the acidic nonsteroidal anti-inflammatory drugs (NSAIDs), dipyrone produces analgesic effects associated with a less potent anti-inflammatory action in different animal models (4 , 5) . Therefore is has been proposed that the antinociceptive effect of dipyrone is mediated at least in part by central mechanisms (6 7 8 9 10) . As a matter of fact, pyrazolinone derivatives as compounds with almost neutral pKa value and a low binding to plasma proteins are distributed homogeneously and quickly throughout the body due to their ability to penetrate the blood-brain barrier easily (11) . In line with this notion, a time-related decrease in cerebrospinal fluid thromboxane (Tx) B2 levels was noted in patients receiving dipyrone (12) . However, dipyrone has also been shown to interfere with the peripheral formation of eicosanoids, including TxB2 from human platelets (13 14 15 16) , prostaglandin (PG) E2 from human gastric mucosa (17) , and gastric juice (15) as well as prostacyclin from rat aorta (14) . In another study, MAA did not elicit an obvious difference in its inhibitory potency on PG formation in astrocytes and macrophages (18) , excluding a cell-specific modulation of eicosanoids by this compound. In accordance with these earlier findings, dipyrone was recently shown to interfere with the enzymatic activities of both PG- and Tx-synthesizing cyclooxygenase (COX) isoforms, COX-1 and COX-2, in preparations of the purified enzymes as well as in different peripheral cellular systems (19) .

However, there are many issues concerning inhibition of peripheral COX enzymes by dipyrone that remain unresolved. Accordingly, a detailed comparative analysis addressing selectivity, dose dependency, and duration of inhibition of peripheral COX enzymes in probands after oral administration of dipyrone is still missing. On the basis of 50% inhibitory concentration (IC50) values obtained from in vitro studies, it was recently claimed that dipyrone might elicit a selective COX-2 inhibition in vivo (19) —a potentially important issue that could explain why use of dipyrone, in contrast to aspirin and other NSAIDs, is not associated with an increased risk of gastrointestinal bleeding (20 , 21) , but that has to be proven in vivo. Moreover, despite the established inhibitory action of MAA on prostanoid production as well as the well-known inactivity of AAAP and FAAP (14) , the question as to whether AA contributes to inhibition of eicosanoid formation after dipyrone intake has not been answered (1) . For all these reasons the present study investigated the contribution of MAA and AA to the action of dipyrone in terms of prostanoid synthesis. To address this issue, we first performed an in vitro analysis of both compounds on the activity of the COX enzymes in human whole blood. The second and major objective of this study was to compare the kinetics of MAA and AA and the concomitant ex vivo inhibition of the COX enzymes in healthy male volunteers taking clinically recommended oral doses of either 500 mg or 1000 mg dipyrone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In vitro investigations
Materials
MAA and AA were kindly provided by Sanofi-Aventis (Frankfurt/Main, Germany). Aspirin and lipopolysaccharide (LPS) from Escherichia coli (serotype 026:B6) were obtained from Sigma (Deisenhofen, Germany). PGE2 and TxB2 enzyme immunoassay kits were from Cayman (Ann Arbor, MI, USA).

Effects of dipyrone metabolites on COX-1 and COX-2 activity in human whole blood
COX-1 assay
Blood was drawn from healthy volunteers who had not taken any NSAID for 2 wk prior to blood sampling. Aliquots of whole blood without anticoagulant were immediately transferred to glass tubes containing test agent (MAA or AA at 0.01, 0.1, 0.3, 1, 3, 10, 30, 100, or 300 µmol/L) or vehicle (phosphate-buffered saline containing DMSO). Final concentrations of DMSO in blood aliquots were 0.1% (v/v) [for concentrations of 0.01–100 µmol/L and its vehicle control] and 0.3% (v/v) [for 300 µmol/L and its vehicle control], respectively. Blood was allowed to clot for 1 h at 37°C (22) . Serum was separated by centrifugation and serum TxB2 levels were determined.

COX-2 assay
Aliquots of heparinized whole blood from healthy volunteers were incubated with LPS (10 µg/ml) plus test agent or vehicle for 24 h at 37°C (22) . The contribution of platelet COX-1 activity was suppressed by the addition of aspirin (10 µg/ml) at the start of the incubation. Plasma was separated by centrifugation and PGE2 levels were determined subsequently.

For both enzymatic assays, concentration response curves were fitted by a sigmoidal regression with variable slope, and 50% inhibitory concentration (IC50) values were derived by use of PRISM® Version 3.0 (GraphPad, San Diego, CA, USA).

Pharmacokinetics of dipyrone metabolites and ex vivo inhibition of COX activities after dipyrone treatment
Subjects and study design
Five male volunteers (all doctors of medicine), aged 39 to 65 years (mean age: 44.8 years) with a mean weight of 70.2 ± 3.0 kg (mean±SEM), took 500 or 1000 mg dipyrone (Novalgin® Filmtabletten, Sanofi-Aventis, Frankfurt/Main, Germany). Doses were chosen on the basis of clinical trials and recommendations of the manufacturer (23) . The doses were administered between 08:00 and 09:00 AM after an overnight fast. Subjects did not take any other medication (including aspirin or other NSAIDs) within 2 wk before and throughout the study. For metabolite analyses and COX activity assays, peripheral venous blood samples were taken from each volunteer immediately before and 0.25, 0.75, 1.5, 3, 5, 8, and 12 h after administration of dipyrone via an indwelling forearm vein catheter. For pharmacokinetic purposes, an additional blood sample was taken 24 h after dosing by venepuncture. To determine dipyrone metabolites, heparinized blood samples were centrifuged and plasma aliquots were frozen. Until further analysis, plasma samples were stored at –20°C for a maximum of 1 month.

Ex vivo inhibition of COX activities
COX-1 assay
Immediately after blood sampling, whole blood samples without anticoagulant were incubated for 1 h at 37°C, subsequently centrifuged, and serum TxB2 levels were determined.

COX-2 assay
Immediately after blood sampling, heparinized whole blood samples were incubated with 10 µg/ml LPS for 24 h at 37°C. The contribution of platelet COX-1 activity was suppressed by the addition of aspirin (10 µg/ml) at the start of the incubation. Plasma was separated by centrifugation and PGE2 levels were determined as an index of COX-2 activity.

Determination of MAA and AA in human plasma
Dipyrone metabolites were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Samples were prepared by adding 0.2 ml 1 mol/L sodium hydroxide and 0.1 ml internal standard stock solution (10 µg antipyrine/ml distilled water) to 0.5 ml plasma, followed by the addition of 4 ml chloroform. The tubes were capped, agitated in an overhead shaker for 5 min, and centrifuged at 4000 r.p.m. for 5 min. The organic layer was transferred into a glass tube and evaporated under a stream of nitrogen at room temperature. Before analysis, the residue was dissolved in 150 µl of mobile phase and 100 µl was used for HPLC analysis. The analytes were separated using a reversed-phase column (125/4 Nucleodur C18 Pyramid, 3 µm; Macherey-Nagel, Düren, Germany) and a C18 precolumn insert (3 µm). The mobile phase consisted of an 87:13 (v/v) mixture of 30 mM sodium acetate (pH=5.6) and acetonitrile. The flow rate was 1 ml/min. UV detection was set at 257 nm. AA and MAA were eluted at 9 min and 12 min, respectively. The retention time of antipyrine (internal standard) was 7.6 min. Quantification of peaks was achieved by the internal standard peak-area ratio method and linear regression. Linearity of the standard curves was determined over a concentration range of 50 to 20,000 ng/ml. In all cases, the regression coefficient was >0.997.

Pharmacodynamic analysis
The degree of COX-1 or COX-2 inhibition was calculated as the percentage change of plasma eicosanoid (COX-1: TxB2; COX-2: PGE2) measured at different time points postadministration relative to predose plasma eicosanoid levels. In the case of COX-2, for each value, basal PGE2 levels measured in the absence of LPS were substracted from PGE2 levels determined in LPS-treated blood aliquots. Maximal observed inhibition of the COX isoforms and times to reach it were obtained directly from the effect vs. time curves. The areas within the effect-time curves (AWECs) from time zero up to 12 h after drug administration were calculated using the linear trapezoidal rule. Larger AWECs correspond to greater levels of COX inhibition.

Pharmacokinetic analysis
Plasma concentration-time curves of dipyrone metabolites were evaluated by noncompartmental analysis using WinNonlin® Version 3.3 (Pharsight, Mountain View, CA, USA). Maximal plasma concentrations (Cmax) and times to Cmax (tmax) were obtained directly from the individual plasma concentration vs. time curves. The area under the plasma concentration-time curve up to the last quantifiable plasma concentration (AUCt) was determined according to the linear trapezoidal method.

Correlation between pharmacodynamics and pharmacokinetics
To assess the correlation between plasma concentrations of MAA and changes in COX-1 or COX-2 inhibition, plasma concentration-response curves were fitted using a sigmoidal regression with variable slope, and the ex vivo IC50 values for COX-1 or COX-2 inhibition were derived by using PRISM® Version 3.0 (GraphPad, San Diego, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vitro effects of MAA and AA on COX-1 and COX-2 activity in human whole blood
Using the in vitro approaches to determine COX inhibitory profiles described above, both MAA and AA were shown to elicit a concentration-dependent inhibition of either isoform (Table 1 ). However, none of the two metabolites exerted a substantial (>2-fold) selectivity toward one isoenzyme. Compared with AA, IC50 values obtained after incubation with MAA were ~8.2- and 9-fold lower.


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  		Table 1. Apparent in vitro potencies of MAA and AA as inhibitors of COX-1 and COX-2 activity in whole blood assaysa

Ex vivo inhibition of COX activity
Time courses of ex vivo LPS-induced PGE2 levels and coagulation-induced TxB2 levels in blood from dipyrone-treated volunteers are shown in Fig. 2 . Pharmacodynamic parameters are presented in Table 2 . Administration of both dipyrone doses resulted in a profound inhibition of either isoform (Table 2) . Statistical analysis revealed significantly lower maximal and mean COX-1 inhibitions in the 500 mg group compared with the data obtained after administration of dipyrone at 1000 mg (Table 2) . Moreover, the AWEC in the 500 mg group was significantly lower than the respective value in the 1000 mg group (Table 2) . There was also a tendency of an increase in the respective COX-2 values in the 1000 mg group (Table 2) . However, data were not statistically different. Likewise, there was no significant difference in COX-1 and COX-2 inhibition values when comparing data within one dosage group.


Figure 2
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  		Figure 2. Time-dependent ex vivo inhibition of COX-1 (A) and COX-2 activity (B) after oral administration of 500 mg or 1000 mg dipyrone to 5 volunteers. Values are means ± SEM.


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  		Table 2. Pharmacodynamic data of dipyrone after single-dose administration at 500 mg and 1000 mg, respectivelya

Pharmacokinetics of dipyrone metabolites
Time courses of plasma concentrations of MAA and AA after oral administration of dipyrone at 500 or 1000 mg are shown in Fig. 3 . Average pharmacokinetic parameters are summarized in Table 3 . Plasma peak concentrations of MAA were measured at 1.2 and 1.7 h, whereas maximal plasma concentrations of AA occurred in a delayed manner with tmax values of 3.1 and 5.2 h, respectively (Table 3) . Moreover, compared with those of MAA, the plasma levels of AA declined more slowly, with apparent terminal half-lives of 6.3 (500 mg dose) and 5.7 h (1000 mg dose), respectively (Table 3) . Comparing both doses, increases of AUC values of MAA and AA were ~5- and 3.2-fold, respectively.


Figure 3
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  		Figure 3. Plasma concentrations of MAA (A) and AA (B) after oral administration of 500 mg or 1000 mg dipyrone. Values are means ± SEM from n = 5 volunteers.


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  		Table 3. Pharmacokinetic data of MAA and AA after oral administration of dipyrone at 500 mg and 1000 mg, respectivelya

Plasma concentrations of AA were below the in vitro IC50 values for COX-1 and COX-2 inhibition at all time points studied (compare Table 1 and Fig. 3 ). On the other hand, plasma levels of MAA remained above the IC50 values for each isoenzyme for 8 h (500 mg dose) and 12 h (1000 mg dose) postadministration (Fig. 3) . MAA plasma concentrations close to or above in vitro IC50 values for inhibition of COX enzymes were measured as early as 15 min after administration (500 mg group: 3.12 µmol/L; 1000 mg group: 6.47 µmol/L).

Correlation between MAA plasma concentrations and ex vivo inhibition of COX isoforms
The relationship of MAA plasma concentrations to ex vivo inhibition of COX-1 and COX-2 was examined graphically and explored by estimating the MAA plasma concentration required to produce 50% inhibition of the respective COX isoform. The calculated ex vivo IC50 values were 1.03 µmol/L for COX-1 inhibition and 0.87 µmol/L for COX-2 inhibition (Fig. 4 ).


Figure 4
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  		Figure 4. Relationship between ex vivo inhibition of COX-1 (A) and COX-2 activity (B) and plasma concentrations of MAA. For analysis of ex vivo IC50 values, plasma drug concentrations and corresponding COX inhibitions determined after single dose administration of 500 mg and 1000 mg dipyrone were included. Values were derived from n = 5 volunteers.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanism of action of dipyrone has been elusive. Whereas the drug was first associated with a predominant suppression of PG synthesis in brain tissue (24) , a subsequent study contradicted a cell-specific modulation by showing comparable inhibitory effects of MAA on prostanoid release by astrocytes and macrophages (18) . Moreover, dipyrone has recently been claimed to selectively inhibit a splicing variant of COX-1 arising via retention of its intron 1, and referred to as COX-3 (25) . However, in this study performed on constructs of canine COX-3 and murine COX-1 and -2 enzymes with disparate activity levels, dipyrone displayed only a weak COX-3 inhibition (IC50=52 µmol/L). A further problem is that both induction and maintenance of hyperalgesia have been associated with centrally COX-2- rather than COX-1-derived PGs (26) . In addition to these concerns, the existence of a functional human COX-3 has been questioned, given that retention of intron 1 in human COX-3 leads to a shift in the reading frame, premature termination, and a truncated, COX-inactive protein (27 , 28) . Qin et al. (29) reported a low-level expression of three splice variants of COX-1 in human tissues, but were not able to show a significantly different potency of dipyrone in inhibiting human COX-1 vs. an intron 1-retained COX-1 splice variant. For all these reasons, attempts to explain the pharmacological action of nonacidic analgesics such as dipyrone and acetaminophen with inhibition of a central COX-3 have been rejected. Though other excellent papers have been published supporting central mechanisms of dipyrone’s antinociceptive effects (6 7 8 9 10) , its peripheral site of action has been largely neglected.

The present study is the first investigation to address the selectivity of dipyrone toward the peripheral COX isoenzymes in vivo and the drug metabolites involved in this response. Here, we show that the major metabolite of dipyrone, MAA, elicits a pronounced inhibition of both COX-1 and COX-2 in vitro and ex vivo in the blood of patients treated with clinically recommended dipyrone doses. In vitro, MAA inhibited the isoforms in a virtually equipotent manner. After oral administration of dipyrone, plasma peak concentrations of MAA were up to 23.2- and 12.7-fold higher (1000 mg dose) than the respective in vitro IC50 values for COX-1 and COX-2, respectively. Due to the rapid occurrence of relevant MAA plasma levels as early as 15 min postadministration, inhibition of either isoform showed a virtually instantaneous onset even in the 500 mg group. Plasma levels of MAA remained above the IC50 values for each isoenzyme for 8 h (500 mg dose) and 12 h (1000 mg dose) postadministration. Ex vivo inhibition of the COX enzymes correlated with the plasma concentration of MAA, with ex vivo-generated IC50 values being 2.5- and 5.3-fold lower than the respective in vitro values. In line with previous data on different COX-2 inhibitors (30) , we observed a substantial between-patient variability in the plasma concentrations of MAA and the corresponding degree of ex vivo COX-2 inhibition, which may also explain the above-mentioned, albeit rather small, difference in IC50 values. In line with this notion, significant differences between COX inhibition parameters (maximal and mean inhibition, AWEC values) of the two dipyrone doses were obtained for COX-1 but not for COX-2. The reasons for this are unclear, but several factors, including genetic variability in the target protein or intersubject variability in pharmacokinetics, appear feasible (31) .

In contrast to MAA, the maximum plasma concentration of AA after the 1000 mg dipyrone dose was 2.8- and 5.7-fold below the in vitro IC50 values for COX-1 and COX-2, respectively. Thus, a significant contribution of AA to inhibition of both enzymes by orally administered single doses of dipyrone appears unlikely. The question of why AA displays a lower COX inhibition than MAA remains unanswered and deserves further investigation. A docking mode for MAA in the active side of human COX-2 has been proposed (19) . MAA appears not to exploit the "side pocket" within the hydrophobic COX-2 channel, the occupancy of which is thought to be important for achieving selectivity toward COX-2 (19) .

The pharmacokinetic profile of MAA (i.e., short time to achieve maximal plasma concentration and short half-life) is in line with the clinically required rapid onset of effect and compatible with an up to four times a day dosage regimen. In support of a nonlinear pharmacokinetics (1) , we observed a >2-fold increase in AUC values of both metabolites after doubling the ingested dipyrone dose. This increase was more pronounced with MAA. Compared with MAA, maximal plasma levels of AA occurred later and declined in a slower and more variable manner. This variability may be explained by the fact that AA is acetylated to AAA by the polymorphic N-acetyl-transferase, with the reported half-lives of AA being ~3.8 h in rapid acetylators and 5.5 h in slow acetylators, respectively (1) .

Due to a rapid nonenzymatic hydrolysis in the gastric fluid to MAA, unchanged dipyrone cannot be detected in plasma after oral treatment (1 , 32 33 34 35) . Likewise, dipyrone undergoes substantial hydrolysis in vitro when tested at physiological temperature, concentration, and pH (36) . Despite this knowledge, many in vitro studies used dipyrone instead of MAA (19 , 25 , 29 , 37 , 38) , which certainly hampers a prediction of the actual in vivo activity of diyprone and confuses a comparison with clinical observations. Therefore, we did not attempt to analyze the impact of the unstable prodrug dipyrone on COX enzymes in the human whole blood assay that involves prolonged incubation periods in human blood, a medium in which intravenously administered dipyrone displays a half-life of ~14 min (23) .

Dipyrone has been accused of causing agranulocytosis. Although there appears to be a statistically significant link, the incidence is extremely rare (1 case per million treatment periods) (39 40 41) . Another evaluation of epidemiological studies showed that the estimated excess mortality per million users due to community-acquired agranulocytosis, aplastic anemia, anaphylaxis, and serious upper gastrointestinal complications is 0.25 for dipyrone, which is comparable to acetaminophen (0.2) but significantly lower than that for aspirin (1.85) or diclofenac (5.92) (42) . These differences are due to gastrointestinal complications that represent the main cause of NSAID-induced mortality. However, a persisting debate concerning dipyrone's risk of agranulocytosis hampered further clinical investigations with the drug. Accordingly, there are no clinical studies addressing dipyrone’s impact on chronic noncancer pain (43) . Likewise, animal studies suggesting a minor anti-inflammatory action of dipyrone compared with NSAIDs (4 , 5) have not been readdressed in humans.

In our hands, dipyrone elicited a substantial suppression of COX-2 activity, which was clearly above COX-2 inhibition by single dose administration of the likewise nonacidic compounds celecoxib and rofecoxib at clinically recommended doses of 200 mg and 25 mg, respectively (30) . Though not investigated, it is obvious from the present data that the recommended dosage regime of 500 or 1000 mg dipyrone up to four times daily (1 , 23) is associated with a constant 70–90% inhibition of COX-2. This finding may also explain previous studies suggesting a peripheral site of dipyrone's analgesic action. Accordingly, dipyrone has been demonstrated to counter peripheral inflammatory hyperalgesia and edema in the rat carrageenan footpad model (4 , 44 , 45) , an inflammatory condition critically dependent on COX-2-derived PGs (26) . In fact, PGs produced during peripheral inflammatory states may significantly increase the excitability of nociceptive nerve fibers, thereby contributing to the activation of "sleeping" nociceptors and the development of burning pain (for a review, see ref. 46 ). As such, it appears reasonable that at least part of the peripheral antinociceptive action of dipyrone arises from prevention of this type of sensitization. However, on the basis of its substantial COX-2-inhibitory action and its homogeneous distribution, dipyrone is also expected to antagonize central COX-2-mediated hyperalgesia in the dorsal horn of the spinal cord. Here, COX-2 is expressed constitutively and becomes up-regulated briefly after a peripheral trauma (47) with the produced PGs thereby, conferring a reduction in the inhibitory tone of the neutrotransmitter glycine onto neurons in the superficial layers and consequently a disinhibition of spinal nociceptive transmission (48) .

Our data concerning nonselective inhibition of the COX enzymes are in line with the results of Campos et al. (19) who used purified enzymes. However, relative high IC50 values (426 µmol/L for both COX-1 and COX-2) were reported in that investigation. A possible reason for this may lie in the use of the inactive prodrug dipyrone in an enzymatic assay of relatively short (10 min) duration. In the same paper, IC50 values for COX-1 inhibition in bovine aortic endothelial cells and fresh human platelets were reported to be far above pharmacological concentrations. The authors concluded that dipyrone might elicit a selective COX-2 inhibition in vivo (19) . In fact, this hypothesis could explain why use of dipyrone, but not of aspirin and other NSAIDs, does not exhibit an increased risk of gastrointestinal bleeding (20 , 21) . However, using the whole blood assay with experimental conditions more closely related to the situation in vivo, we measured a profound suppression of COX-1-derived TxB2. Previous data suggest that only an excess of 95% inhibition of serum TxB2 significantly affects platelet function (49) . In our hands, inhibition of COX-1-derived TxB2 was <95% in the 500 mg group. Suppression of TxB2 by slightly >95% was observed between 0.75 and 3 h after dosing with 1000 mg dipyrone, suggesting a short-term suppression of platelet function only. Compared with single-dose administration of 100 mg aspirin that causes persistent suppression of platelet TxB2 for 24 h (50) , the effect of 1000 mg dipyrone on TxB2 was short-lived. On the other hand, the degree of COX-1 inhibition by dipyrone was more pronounced than was 75 mg diclofenac given as a retarded formulation (51) and similar to that elicited by 400 mg ibuprofen (B. Hinz and K. Brune, unpublished observation).

Given the gastrointestinal safety of dipyrone shown in epidemiological studies (20 , 21) and its apparently contradictory impact on COX-1, it is suggested that physicochemical factors (lack of acidity) rather than differences in COX-1 inhibition may be responsible for dipyrone’s favorable gastric tolerability compared with acidic NSAIDs. In fact, there are at least two major components contributing to NSAIDs’ ulcerogenic action in the stomach, namely, a topical irritant effect on the epithelium and the ability to suppress PG synthesis (52 53 54 55) . Topical irritant properties are confined to acidic NSAIDs, which accumulate in gastric epithelial cells because of the phenomenon of "ion trapping" (11) . In this context, NSAIDs have been suggested to produce mucosal injury by uncoupling oxidative mitochondrial phosphorylation in epithelial cells resulting in diminished cellular ATP production, cellular toxicity due to calcium and reactive oxygen species, and subsequent increased mucosal permeability (54) . The uncoupling property of NSAIDs resides within their carboxylic or enolic acid groups. Accordingly, modifications of the carboxyl group (e.g., flurbiprofen dimer or nitroxybutyl modification of flurbiprofen) render the molecule inactive as an uncoupler of oxidative phosphorylation (54) . Further evidence against a sole role of PG inhibition in conferring the gastrointestinal toxicity of NSAIDs may be derived from observations in COX-1 knockout mice, which do not spontaneously develop gastric ulcers but still develop gastric erosions in response to oral administration of indomethacin (56) . In the case of dipyrone, this notion is supported by a study (15) showing that treatment of human volunteers three times a day with 1000 mg dipyrone for 3 days profoundly diminished PGE2 concentrations in gastric fluid without causing a substantial degree of adverse gastric effects.

Despite epidemiological evidence for a good gastrointestinal tolerability of dipyrone, no large-scale randomized gastrointestinal outcome trial has been performed in patients receiving long-term dipyrone. In a small-group endoscopic study, a 2 wk treatment of healthy volunteers with dipyrone yielded no statistically significant difference in the incidence of gastric and duodenal mucosal lesions either when dipyrone at two different dose levels (3 g/day and 1.5 g/day) was compared with placebo or when the lower dipyrone dose was compared with acetaminophen (1.5 g/day) (57) . However, 3 of 12 volunteers treated with the high dose of dipyrone developed relevant lesions, suggesting a dose-dependent mucosa-damaging effect in some individuals probably due to the somewhat higher and sustained inhibition of COX-1. Moreover, other mechanisms such as diminished glutathione metabolism (58) or inhibition of the nitric oxide/cyclic guanosine monophosphate pathway and nitric oxide synthase activity (59) , which have been suggested to contribute to high-dose dipyrone-elicited weak mucosal lesions in rats, appear feasible.

Collectively, the fast and pronounced inhibition of COX-2 in human blood monocytes supports the view that a significant portion of dipyrone's analgesic action may be due to peripheral mechanisms. Moreover, the observed COX-1 inhibition similar to that observed with various traditional NSAIDs, together with the fact that dipyrone does not elicit significant gastrointestinal toxicity, implies a combined contribution of COX-1 inhibition and drugs’s physicochemical factors in conferring gastrointestinal ulcerations and bleeding. In view of the high analgesic potency of dipyrone, its good gastrointestinal tolerability, and its widespread prescription or over-the-counter use in many countries (particularly Latin America, Asia, Eastern and Central Europe), further clinical studies to address peripheral analgesic and anti-inflammatory mechanisms appear to be warranted.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from Sanofi-Aventis (Frankfurt/Main, Germany).

Received for publication January 9, 2007. Accepted for publication March 1, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Levy, M., Zylber-Katz, E., Rosenkranz, B. (1995) Clinical pharmacokinetics of dipyrone and its metabolites. Clin. Pharmacokinet. 28,216-234[Medline]
  2. Noda, A., Goromaru, T., Tsubone, N., Matsuyama, K., Iguchi, S. (1976) In vivo formation of 4-formylaminoantipyrine as a new metabolite of aminopyrine. Chem. Pharm. Bull. 24,1502-1505[Medline]
  3. Volz, M., Kellner, H. M. (1980) Kinetics and metabolism of pyrazolones (propyphenazone, aminopyrine and dipyrone). Br. J. Clin. Pharmacol. 10(Suppl. 2),299S-308S[Medline]
  4. Brune, K., Alpermann, H. (1983) Non-acidic pyrazoles: inhibition of prostaglandin production, carrageenan oedema and yeast fever. Agents Actions 13,360-363[CrossRef][Medline]
  5. Tatsuo, M. A., Carvalho, W. M., Silva, C. V., Miranda, A. E., Ferreira, S. H., Francischi, J. N. (1994) Analgesic and antiinflammatory effects of dipyrone in rat adjuvant arthritis model. Inflammation 18,399-405[CrossRef][Medline]
  6. 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[CrossRef][Medline]
  7. Gelgor, L., Cartmell, S., Mitchell, D. (1992) Intracerebroventricular micro-injections of non-steroidal anti-inflammatory drugs abolish reperfusion hyperalgesia in the rat’s tail. Pain 50,323-329[CrossRef][Medline]
  8. Neugebauer, V., Schaible, H. G., He, X., Lucke, T., Gundling, P., Schmidt, R. F. (1994) Electrophysiological evidence for a spinal antinociceptive action of dipyrone. Agents Actions 41,62-70[CrossRef][Medline]
  9. Shimada, S. G., Otterness, I. G., Stitt, J. T. (1994) A study of the mechanism of action of the mild analgesic dipyrone. Agents Actions 41,188-192[CrossRef][Medline]
  10. 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[CrossRef][Medline]
  11. Brune, K., Rainsford, K. D., Schweitzer, A. (1980) Biodistribution of mild analgesics. Br. J. Clin. Pharmacol. 10(Suppl. 2),279S-284S[Medline]
  12. Levy, M., Brune, K., Zylber-Katz, E., Cohen, O., Caraco, Y., Geisslinger, G. (1998) Cerebrospinal fluid prostaglandins after systemic dipyrone intake. Clin. Pharmacol. Ther. 64,117-122[CrossRef][Medline]
  13. Eldor, A., Zylber-Katz, E., Levy, M. (1984) The effect of oral administration of dipyrone on the capacity of blood platelets to synthesize thromboxane A2 in man. Eur. J. Clin. Pharmacol. 26,171-176[CrossRef][Medline]
  14. Weithmann, K. U., Alpermann, H. G. (1985) Biochemical and pharmacological effects of dipyrone and its metabolites in model systems related to arachidonic acid cascade. Arzneimittelforschung/Drug Res. 35,947-952
  15. Frolich, J. C., Rupp, W. A., Zapf, R. M., Badian, M. J. (1986) The effects of metamizol on prostaglandin synthesis in man. In 100 Years of Pyrazolone Drugs (Brune, K., ed). Agents Actions (Suppl.) 19,155-166
  16. Geisslinger, G., Peskar, B. A., Pallapies, D., Sittl, R., Levy, M., Brune, K. (1996) The effects on platelet aggregation and prostanoid biosynthesis of two parenteral analgesics: ketorolac tromethamine and dipyrone. Thromb. Haemost. 76,592-597[Medline]
  17. Peskar, B. M., Kleine, A., Pyras, F., Muller, M. K. (1983) Gastrointestinal toxicity: role of prostaglandins and leukotrines. Med. Toxicol. 1(Suppl. 1),39-43
  18. Lanz, R., Polster, P., Brune, K. (1986) Antipyretic analgesics inhibit prostaglandin release from astrocytes and macrophages similarly. Eur. J. Pharmacol. 130,105-109[CrossRef][Medline]
  19. Campos, C., de Gregorio, R., Garcia-Nieto, R., Gago, F., Ortiz, P., Alemany, S. (1999) Regulation of cyclooxygenase activity by metamizol. Eur. J. Pharmacol. 378,339-347[CrossRef][Medline]
  20. Laporte, J. R., Carne, X. (1987) Blood dyscrasias and the relative safety of non-narcotic analgesics. Lancet 1,809[Medline]
  21. 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[CrossRef][Medline]
  22. Patrignani, P., Panara, M. R., Greco, A., Fusco, O., Natoli, C., Iacobelli, S., Cipolline, F., Ganci, A., Creminon, C., Maclouf, J., et al (1994) Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases. J. Pharmacol. Exp. Ther. 271,1705-1712[Abstract/Free Full Text]
  23. Novalgin. Fachinformation [product information] 2004 Aventis Pharma Deutschland GmbH Frankfut am Main, Germany.
  24. Dembinska-Kiec, A., Zmuda, A., Krupinska, J. (1976) Inhibition of prostaglandin synthetase by aspirin-like drugs in different microsomal preparations. Adv. Prostaglandin Thromboxane Res. 1,99-103[Medline]
  25. Chandrasekharan, N. V., Dai, H., Roos, K. L., Evanson, N. K., Tomsik, J., Elton, T. S., Simmons, D. L. (2002) COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc. Natl. Acad. Sci. U. S. A. 99,13926-13931[Abstract/Free Full Text]
  26. 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) Pharmacological analysis of cyclooxygenase-1 in inflammation. Proc. Natl. Acad. Sci. U. S. A. 95,13313-13318[Abstract/Free Full Text]
  27. Dinchuk, J. E., Liu, R. Q., Trzaskos, J. M. (2003) COX-3: in the wrong frame in mind. Immunol. Lett. 86,121[CrossRef][Medline]
  28. Schwab, J. M., Beiter, T., Linder, J. U., Laufer, S., Schulz, J. E., Meyermann, R., Schluesener, H. J. (2003) COX-3—a virtual pain target in humans?. FASEB J. 17,2174-2175[Free Full Text]
  29. Qin, N., Zhang, S. P., Reitz, T. L., Mei, J. M., Flores, C. M. (2005) Cloning, expression, and functional characterization of human cyclooxygenase-1 splicing variants: evidence for intron 1 retention. J. Pharmacol. Exp. Ther. 315,1298-1305[Abstract/Free Full Text]
  30. Hinz, B., Dormann, H., Brune, K. (2006) More pronounced inhibition of cyclooxygenase 2, increase in blood pressure, and reduction of heart rate by treatment with diclofenac compared with celecoxib and rofecoxib. Arthritis Rheum. 54,282-291[CrossRef][Medline]
  31. Patrono, C. (2001) Measurement of cyclooxygenase isozyme inhibition in humans: exploring the clinical relevance of biochemical selectivity. Clin. Exp. Rheumatol. 19(Suppl. 25),S45-S50[Medline]
  32. Weiss, R., Brauer, J., Goertz, U., Petry, R. (1974) Comparative study on the problem of absorption and metabolism of the pyrazolone derivative metamizole, in man after oral and intramuscular administration. Arzneimittelforschung/Drug Res. 24,345-348
  33. Asmardi, G., Jamali, F. (1983) High-performance liquid chromatography of dipyrone and its active metabolite in biological fluids. J. Chromatogr. 277,183-189[CrossRef][Medline]
  34. Asmardi, G., Jamali, F. (1985) Pharmacokinetics of dipyrone in man; role of the administration route. Eur. J. Drug Metab. Pharmacokinet. 10,121-125[Medline]
  35. Vlahov, V., Badian, M., Verho, M., Bacracheva, N. (1990) Pharmacokinetics of metamizol metabolites in healthy subjects after a single oral dose of metamizol sodium. Eur. J. Clin. Pharmacol. 38,61-65[CrossRef][Medline]
  36. Ergun, H., Frattarelli, D. A., Aranda, J. V. (2004) Characterization of the role of physicochemical factors on the hydrolysis of dipyrone. J. Pharm. Biomed. Anal. 35,479-487[CrossRef][Medline]
  37. Pigoso, A. A., Uyemura, S. A., Santos, A. C., Rodrigues, T., Mingatto, F. E., Curti, C. (1998) Influence of nonsteroidal anti-inflammatory drugs on calcium efflux in isolated rat renal cortex mitochondria and aspects of the mechanisms involved. Int. J. Biochem. Cell Biol. 30,961-965[CrossRef][Medline]
  38. Galati, G., Tafazoli, S., Sabzevari, O., Chan, T. S., O’Brien, P. J. (2002) Idiosyncratic NSAID drug induced oxidative stress. Chem. Biol. Interact. 142,25-41[CrossRef][Medline]
  39. Kaufman, D. W., Kelly, J. P., Levy, M., Shapiro, S. (1991) The drug etiology of agranulocytosis an aplastic anemia. Monographs in Epidemiology and Biostatistics Vol. 18 Oxford Univ. Press Oxford, UK.
  40. Anonymous, (1986) The International Agranulocytosis and Aplastic Anemia Study. Risks of agranulocytosis and aplastic anemia: a first report of their relation to drug use with special reference to analgesics. J. Am. Med. Assoc. 256,1749-1757[Abstract]
  41. Ibanez, L., Vidal, X., Ballarin, E., Laporte, J. R. (2005) Agranulocytosis associated with dipyrone (metamizol). Eur. J. Clin. Pharmacol. 60,821-829[CrossRef][Medline]
  42. Andrade, S. E., Martinez, C., Walker, A. M. (1998) Comparative safety evaluation of non-narcotic analgesics. J. Clin. Epidemiol. 51,1357-1365[CrossRef][Medline]
  43. Hackenthal, E. (1997) Paracetamol and metamizol in the treatment of chronic pain syndromes. Schmerz. 11,269-275[CrossRef][Medline]
  44. Lorenzetti, B. B., Ferreira, S. H. (1985) Mode of analgesic action of dipyrone: direct antagonism of inflammatory hyperalgesia. Eur. J. Pharmacol. 114,375-381[CrossRef][Medline]
  45. Mazario, J., Herrero, J. F. (1999) Antinociceptive effects of metamizol (dipyrone) in rat single motor units. Neurosci. Lett. 274,179-182[CrossRef][Medline]
  46. Hinz, B., Brune, K. (2007) Antipyretic analgesics: nonsteroidal antiinflammatory drugs, selective COX-2 inhibitors, paracetamol and pyrazolinones. Handb. Exp. Pharmacol. 177,65-93[Medline]
  47. Beiche, F., Scheuerer, S., Brune, K., Geisslinger, G., Goppelt-Struebe, M. (1996) Up-regulation of cyclooxygenase-2 mRNA in the rat spinal cord following peripheral inflammation. FEBS Lett. 390,165-169[CrossRef][Medline]
  48. Ahmadi, S., Lippross, S., Neuhuber, W. L., Zeilhofer, H. U. (2002) PGE2 selectively blocks inhibitory glycinergic neurotransmission onto rat superficial dorsal horn neurons. Nat. Neurosci. 5,34-40[CrossRef][Medline]
  49. Reilly, I. A., FitzGerald, G. A. (1987) Inhibition of thromboxane formation in vivo and ex vivo: implications for therapy with platelet inhibitory drugs. Blood 69,180-186[Abstract/Free Full Text]
  50. Patrignani, P., Filabozzi, P., Patrono, C. (1982) Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J. Clin. Invest. 69,1366-1372[Medline]
  51. Hinz, B., Rau, T., Auge, D., Werner, U., Ramer, R., Rietbrock, S., Brune, K. (2003) Aceclofenac spares cyclooxygenase 1 as a result of limited but sustained biotransformation to diclofenac. Clin. Pharmacol. Ther. 74,222-235[CrossRef][Medline]
  52. McCormack, K., Brune, K. (1987) Classical absorption therapy and the development of gastric mucosal damage associated with the non-steroidal anti-inflammatory drugs. Arch. Toxicol. 60,261-269[CrossRef][Medline]
  53. Price, A. H., Fletcher, M. (1990) Mechanisms of NSAID-induced gastroenteropathy. Drugs 40(Suppl. 5),1-11[Medline]
  54. Somasundaram, S., Rafi, S., Hayllar, J., Sigthorsson, G., Jacob, M., Price, A. B., Macpherson, A., Mahmod, T., Scott, D., Wrigglesworth, J. M., Bjarnason, I. (1997) Mitochondrial damage: a possible mechanism of the "topical" phase of NSAID induced injury to the rat intestine. Gut 41,344-353[Abstract/Free Full Text]
  55. Wallace, J. L. (1997) Nonsteroidal anti-inflammatory drugs and gastroenteropathy: the second hundred years. Gastroenterology 112,1000-1016[CrossRef][Medline]
  56. Langenbach, R., Morham, S. G., Tiano, H. F., Loftin, C. D., Ghanayem, B. I., Chulada, P. C., Mahler, J. F., Lee, C. A., Goulding, E. H., Kluckman, K. D., et al (1995) Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 83,483-492[CrossRef][Medline]
  57. Bianchi Porro, G., Ardizzone, S., Petrillo, M., Caruso, I., Montrone, F. (1996) Endoscopic assessment of the effects of dipyrone (metamizol) in comparison to paracetamol and placebo on the gastric and duodenal mucosa of healthy adult volunteers. Digestion 57,186-190[CrossRef][Medline]
  58. Sanchez, S., Martin, M. J., Ortiz, P., Motilva, V., Alarcon de la Lastra, C. (2002) Effects of dipyrone on inflammatory infiltration and oxidative metabolism in gastric mucosa: comparison with acetaminophen and diclofenac. Dig. Dis. Sci. 47,1389-1398[CrossRef][Medline]
  59. Sanchez, S., Martin, M. J., Ortiz, P., Motilva, V., Herrerias, J. M., Alarcon de la Lastra, C. (2002) Role of prostaglandins and nitric oxide in gastric damage induced by metamizol in rats. Inflamm. Res. 51,385-392[CrossRef][Medline]




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