Cytochrome P450 1A2 is the most important enzyme for hepatic metabolism of the metamizole metabolite 4-methylaminoantipyrine.

AIMS: Metamizole (dipyrone) is a prodrug not detectable in serum or urine after oral ingestion. The primary metabolite, 4-methylaminoantipyrine (4-MAA), can be N-demethylated to 4-aminoantipyrine (4-AA) or oxidized to 4-formylaminoantipyrine (4-FAA) by cytochrome P450 (CYP)-dependent reactions. We aimed to identify the CYPs involved in 4-MAA metabolism and to quantify the effect of CYP inhibition on 4-MAA metabolism. METHODS: We investigated the metabolism of 4-MAA in vitro using CYP expressing supersomes and the pharmacokinetics of metamizole in the presence of CYP inhibitors in male subjects. RESULTS: The experiments in supersomes revealed CYP1A2 as the major CYP for 4-MAA N-demethylation and 4-FAA formation with CYP2C19 and CYP2D6 contributing to N-demethylation. In the clinical study, we investigated the influence of ciprofloxacin (CYP1A2 inhibitor), fluconazole (CYP2C19 inhibitor) and the combination ciprofloxacin/fluconazole on the pharmacokinetics of metamizole in n = 12 male subjects in a randomized, placebo-controlled, double-blind study. The geometric mean ratios for the area under the concentration-time curve of 4-MAA after/before treatment were 1.17 (90% CI 1.09-1.25) for fluconazole, 1.51 (90% CI 1.42-1.60) for ciprofloxacin and 1.92 (90% CI 1.81-2.03) for ciprofloxacin/fluconazole. Fluconazole increased the half-life of 4-MAA from 3.22 hours by 0.47 hours (95% CI 0.13-0.81, P < .05), ciprofloxacin by 0.69 hours (95% CI 0.44-0.94, P < .001) and fluconazole/ciprofloxacin by 2.85 hours (95% CI 2.48-3.22, P < .001). CONCLUSION: CYP1A2 is the major CYP for the conversion of 4-MAA to 4-AA and 4-FAA. The increase in 4-MAA exposure by the inhibition of CYP1A2 and by the combination CYP1A2/CYP2C19 may be relevant for dose-dependent adverse reactions of 4-MAA.

¿Estamos sobredosificando el metamizol por vía parenteral? / Are we overdosing parenteral metamizole?

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Metamizole (Dipyrone) and the Liver: A Review of the Literature.

Metamizole, also known as dipyrone, was introduced to the market nearly a century ago. Due to its excellent analgesic, antipyretic, and spasmolytic properties combined with its mostly favorable gastrointestinal tolerability, the drug was extensively applied worldwide during the first decades after its market introduction. Although rare, agranulocytosis is a well-known adverse event of metamizole and led to its withdrawal from the market in a number of countries beginning in the 1960s. Nevertheless, metamizole is still a frequently used drug worldwide either legally (by prescription in some countries, over the counter in other countries) or without official approval (especially by immigrants knowing the drug from their home countries) or even illegally (due to its growing application as an adulterant in illicit drugs). Metamizole undergoes extensive metabolism in the liver and cases of potential metamizole-associated hepatotoxicity have been described. Here, the literature is extensively reviewed for the first time regarding hepatic effects associated with the use of metamizole.

Non-immunological toxicological mechanisms of metamizole-associated neutropenia in HL60 cells.

Metamizole is an analgesic and antipyretic, but can cause neutropenia and agranulocytosis. We investigated the toxicity of the metabolites N-methyl-4-aminoantipyrine (MAA), 4-aminoantipyrine (AA), N-formyl-4-aminoantipyrine (FAA) and N-acetyl-4-aminoantipyrine (AAA) on neutrophil granulocytes and on HL60 cells (granulocyte precursor cell line). MAA, FAA, AA, and AAA (up to 100 µM) alone were not toxic for HL60 cells or granulocytes. In the presence of the myeloperoxidase substrate H2O2, MAA reduced cytotoxicity for HL60 cells at low concentrations (<50 µM), but increased cytotoxicity at 100 µM H2O2. Neutrophil granulocytes were resistant to H2O2 and MAA. Fe2+ and Fe3+ were not toxic to HL60 cells, irrespective of the presence of H2O2 and MAA. Similarly, MAA did not increase the toxicity of lactoferrin, hemoglobin or methemoglobin for HL60 cells. Hemin (hemoglobin degradation product containing a porphyrin ring and Fe3+) was toxic on HL60 cells and cytotoxicity was increased by MAA. EDTA, N-acetylcystein and glutathione prevented the toxicity of hemin and hemin/MAA. The absorption spectrum of hemin changed concentration-dependently after addition of MAA, suggesting an interaction between Fe3+ and MAA. NMR revealed the formation of a stable MAA reaction product with a reaction pathway involving the formation of an electrophilic intermediate. In conclusion, MAA, the principle metabolite of metamizole, increased cytotoxicity of hemin by a reaction involving the formation of an electrophilic metabolite. Accordingly, cytotoxicity of MAA/hemin could be prevented by the iron chelator EDTA and by the electron donors NAC and glutathione. Situations with increased production of hemin may represent a risk factor for metamizole-associated granulocytopenia.

N-demethylation of N-methyl-4-aminoantipyrine, the main metabolite of metamizole.

Metamizole is an old analgesic used frequently in some countries. Active metabolites of metamizole are the non-enzymatically generated N-methyl-4-aminoantipyrine (4-MAA) and its demethylation product 4-aminoantipyrine (4-AA). Previous studies suggested that 4-MAA demethylation can be performed by hepatic cytochrome P450 (CYP) 3A4, but the possible contribution of other CYPs remains unclear. Using human liver microsomes (HLM), liver homogenate and HepaRG cells, we could confirm 4-MAA demethylation by CYPs. Based on CYP induction (HepaRG cells) and CYP inhibition (HLM) we could identify CYP2B6, 2C8, 2C9 and 3A4 as major contributors to 4-MAA demethylation. The 4-MAA demethylation rate by HLM was 280 pmol/mg protein/h, too low to account for in vivo 4-MAA demethylation in humans. Since peroxidases can perform N-demethylation, we investigated horseradish peroxidase and human myeloperoxidase (MPO). Horse radish peroxidase efficiently demethylated 4-MAA, depending on the hydrogen peroxide concentration. This was also true for MPO; this reaction was saturable with a Km of 22.5 µM and a maximal velocity of 14 nmol/min/mg protein. Calculation of the entire body MPO capacity revealed that the demethylation capacity by granulocyte/granulocyte precursors was approximately 600 times higher than the liver capacity and could account for 4-MAA demethylation in humans. 4-MAA demethylation could also be demonstrated in MPO-expressing granulocyte precursor cells (HL-60). In conclusion, 4-MAA can be demethylated in the liver by several CYPs, but hepatic metabolism cannot fully explain 4-MAA demethylation in humans. The current study suggests that the major part of 4-MAA is demethylated by circulating granulocytes and granulocyte precursors in bone marrow.

Pharmacokinetic profiles of the active metamizole metabolites in healthy horses.

Metamizole (MT) is an analgesic and antipyretic drug labelled for use in humans, horses, cattle, swine and dogs. MT is rapidly hydrolysed to the active primary metabolite 4-methylaminoantipyrine (MAA). MAA is formed in much larger amounts compared with other minor metabolites. Among the other secondary metabolites, 4-aminoantipyrine (AA) is also relatively active. The aim of this research was to evaluate the pharmacokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.) and intramuscular (i.m.) routes in healthy horses. Six horses were randomly allocated to two equally sized treatment groups according to a 2 × 2 crossover study design. Blood was collected at predetermined times within 24 h, and plasma was analysed by a validated HPLC-UV method. No behavioural changes or alterations in health parameters were observed in the i.v. or i.m. groups of animals during or after (up to 7 days) drug administration. Plasma concentrations of MAA after i.v. and i.m. administrations of MT were detectable from 5 min to 10 h in all the horses. Plasma concentrations of AA were detectable in the same range of time, but in smaller amounts. Maximum concentration (Cmax ), time to maximum concentration (Tmax ) and AUMC0-last of MAA were statistically different between the i.v. and i.m. groups. The AUCIM /AUCIV ratio of MAA was 1.06. In contrast, AUC0-last of AA was statistically different between the groups (P < 0.05) with an AUCIM /AUCIV ratio of 0.54. This study suggested that the differences in the MAA and AA plasma concentrations found after i.m. and i.v. administrations of MT might have minor consequences on the pharmacodynamics of the drug.

Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS.

A reliable and simple method for the detection and quantification of residues of 14 non-steroidal anti-inflammatory drugs and a metamizole metabolite in swine muscle was developed using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The samples were extracted with acetonitrile (ACN) in solid-liquid extraction followed by a low-temperature partitioning (LLE-LTP) process at -20 ± 2°C. After evaporation to dryness, the residue was reconstituted with hexane and a mixture of water:acetonitrile (1:1). LC separation was achieved on a reversed-phase (RP18) column with gradient elution using water (phase A) and ACN (phase B) both containing 1 mmol l(-)(1) ammonium acetate (NH4COO) with 0.025% acetic acid. Analysis was carried out on a triple-quadrupole tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode using an electrospray interface in negative and positive mode in a single run. Method validation was performed according to the criteria of Commission Decision No. 2002/657/EC. The matrix effect and linearity were evaluated. Decision limit (CCα), detection capability (CCß), accuracy and repeatability of the method are also reported. The proposed method proved to be simple, easy and adequate for high-throughput analysis and was applied to routine analysis by the Brazilian Ministry of Agriculture, Livestock and Food Supply.

Pharmacokinetic investigations of the marker active metabolites 4-methylamino-antipyrine and 4-amino-antipyrine after intramuscular injection of metamizole in healthy piglets.

Metamizole (MT) is a pyrazolone nonsteroidal anti-inflammatory drug labelled for humans and animals. The aim of this study was to assess the pharmacokinetics of its active metabolites 4-methylamino-antipyrine (MAA) and 4-amino-antipyrine (AA) in male piglets after a single intramuscular injection of MT. Eight healthy male piglets were administered MT (100 mg/kg) intramuscularly. Blood was sampled at scheduled time intervals, and drug plasma concentrations evaluated by a validated HPLC method. MAA and AA plasma concentrations were quantitatively detectable from 0.25 to 48 h and 0.50 to 72 h, respectively, in 6 of 8 and 7 of 8 animals. The average maximum concentrations of MAA and AA were of 47.59 and 4.94 mg/mL, respectively. The average half-lives were 8.57 and 13.3 h for MAA and AA, respectively. This study showed that the amount of MAA and AA produced in piglets is different to that in the animal species previously investigated. Further studies are necessary to understand whether these differences in MAA and AA plasma concentrations between animal species necessitate diverse therapeutic drug dosing.

Pyrazolones metabolites are relevant for identifying selective anaphylaxis to metamizole.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common cause of hypersensitivity reactions, with pyrazolones the most frequent drugs inducing selective reactions. Immediate selective hypersensitivity to pyrazolones is thought to be mediated by specific-IgE. Sensitivity of in vitro diagnostic tests is low and this may be due to the incomplete characterization of the structures involved. Here we investigated whether main metabolites of metamizole (dipyrone) in human could be involved in the immune response using the basophil activation test (BAT). We studied subjects with confirmed selective immediate hypersensitivity to metamizole and performed BAT with metamizole and its metabolites: 4-methylamino-antipyrine (MAA), 4-aminoantipyrine (AA), 4-acetylamino-antipyrine (AAA) and 4-formylamino-antipyrine (FAA). BAT results showed an increase of positive results from 37.5% to 62.5% using metamizole plus metabolites as compared with the BAT carried out only with the parent drug, demonstrating that metamizole metabolites have a role in the reaction and can induce specific basophil activation in patients with immediate hypersensitivity to this drug. Our findings indicate that pyrazolone metabolites are useful for improving the in vitro diagnosis of allergic reactions to metamizole.

Effects of metabolites of the analgesic agent dipyrone (metamizol) on rostral ventromedial medulla cell activity in mice.

The molecular mechanism of action of dipyrone, a widely used antipyretic and non-opioid analgesic drug, is still not fully understood. Actions upon peripheral inflamed tissues as well as the central nervous system, especially upon the PAG-RVM axis, have been suggested. Dipyrone is a prodrug and its activity is due to its immediate conversion to its active metabolites. We tested the effect of two recently discovered metabolites of dipyrone, the arachidonoyl amides of 4-methylaminoantipyrine and 4-aminoantipyrine, on the neurons of the rostral ventromedial medulla (RVM), which are part of the descending pathway of antinociception. These compounds reduced the activity of ON-cells and increased the activity of OFF-cells. Both CB1 and TRPV1 blockade reversed these effects, suggesting that the endocannabinoid/endovanilloid system takes part in the analgesic effects of dipyrone.

Gender and functional CYP2C and NAT2 polymorphisms determine the metabolic profile of metamizole.

Metamizole is a pain-killer drug that has been banned in some countries because of its toxicity, but it is still used in many countries due to its effective analgesic and antispasmodic properties. Although large variability in the biodisposition and adverse effects of metamizole are known, factors underlying this variability are poorly understood. We analyzed the urinary recovery of metabolites, as well as the association of these profiles with genetic and non-genetic factors, in a group of 362 healthy individuals. Gender and functional polymorphisms are strongly related to metabolic profiles. N-demethylation of the active metabolite MAA is diminished in carriers of the CYP2C19*2 allele and in NAT2-slow acetylators. Acetylation of the secondary metabolite AA is decreased in men, in drinkers and in NAT2-slow acetylators with a differential effect of NAT2*5 and NAT2*6 alleles. The formylation of MAA is diminished in older subjects and in carriers of defect CYP2C9 and CYP2C19 alleles. Two novel arachidonoyl metabolites were identified for the first time in humans. Women and NAT2-slow acetylators show higher concentrations, whereas the presence of the rapid CYP2C19*17 allele is associated with lower concentrations of these metabolites. All genetic associations show a gene-dose effect. We identified for the first time genetic and non-genetic factors related to the oxidative metabolism of analgesic drug metamizole, as well as new active metabolites in humans. The phenotypic and genetic factors identified in this study have a potential application as biomarkers of metamizole biotransformation and toxicity.

Dipyrone metabolite 4-MAA induces hypothermia and inhibits PGE2 -dependent and -independent fever while 4-AA only blocks PGE2 -dependent fever.

BACKGROUND AND PURPOSE: The antipyretic and hypothermic prodrug dipyrone prevents PGE2 -dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects. EXPERIMENTAL APPROACH: Male Wistar rats were treated i.p. with indomethacin (2 mg·kg(-1) ), dipyrone, 4-methylaminoantipyrine (4-MAA), 4-aminoantipyrine (4-AA) (60-360 mg·kg(-1) ), 4-formylaminoantipyrine, 4-acethylaminoantipyrine (120-360 mg·kg(-1) ) or vehicle 30 min before i.p. injection of LPS (50 µg·kg(-1) ), Tsv (150 µg·kg(-1) ) or saline. Rectal temperatures were measured by tele-thermometry and dipyrone metabolite concentrations determined in the plasma, CSF and hypothalamus by LC-MS/MS. PGE2 concentrations were determined in the CSF and hypothalamus by elisa. KEY RESULTS: In contrast to LPS, Tsv-induced fever was not followed by increased PGE2 in the CSF or hypothalamus. The antipyretic time-course of 4-MAA and 4-AA on LPS-induced fever overlapped with the period of the highest concentrations of 4-MAA and 4-AA in the hypothalamus, CSF and plasma. These metabolites reduced LPS-induced fever and the PGE2 increase in the plasma, CSF and hypothalamus. Only 4-MAA inhibited Tsv-induced fever. The higher doses of dipyrone and 4-MAA also induced hypothermia. CONCLUSIONS AND IMPLICATIONS: The presence of 4-MAA and 4-AA in the CSF and hypothalamus was associated with PGE2 synthesis inhibition and a decrease in LPS-induced fever. 4-MAA was also shown to be an antipyretic metabolite for PGE2 -independent fever induced by Tsv suggesting that it is responsible for the additional antipyretic mechanism of dipyrone. Moreover, 4-MAA is the hypothermic metabolite of dipyrone.

Monitoring of four dipyrone metabolites in communal wastewater by solid phase extraction liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry.

Liquid chromatography quadrupole time-of-flight spectrometry (LC-Q-TOF-MS), equipped with electrospray ionization (ESI), was developed for the determination of the main metabolites of dipyrone - 4-aminoantipyrine (4-AA), 4-acetylaminoantipyrine (4-AAA), 4-formylaminoantipyrine (4-FAA) and 4-methylaminoantipyrine (4-MAA) in communal wastewater after reversed-phase solid phase extraction (SPE) in the low to several µg/l concentration range. Samples originated from conventional wastewater treatment plant (WWTP) using activated sewage sludge as well as from a pilot-scale WWTP operating in mixed mode (activated sewage sludge and cascade biofilms reactors with biofilms growing on fix beds and roots of greenhouse plants). Results of the present study confirmed the outcomes of our previous report according to which, 4-FAA was the most persistent metabolite, while 4-AAA and 4-MAA could be determined in the highest and lowest concentration, respectively. Moreover, the study of intraday variation of the concentration of these metabolites revealed that the concentration of 4-AA, 4-AAA and 4-FAA registered a 46%-75% increase in the samples collected at noon compared to those collected at 6 AM. Chlorination did not affect considerably the removal efficiency (about 15%) of these metabolites in samples collected for 3 months consecutively before and after disinfection. Both wastewater treatment techniques efficiently removed 4-AAA (between 80 and 96%). However, in the summer season, the removal efficiency of conventional WWTP using open-air aerated tanks is lower by 30%, (on average) than in the cold season. The concentration of the investigated metabolites showed increased concentrations in the winter season confirming the intake habits of the population from this popular analgesic and antipyretic drug.

Eficacia de la asociación paracetamol-metamizol vs. paracetamol-dexketoprofeno en manejo de dolor agudo postoperatorio / No disponible

Objetivo: El uso de farmacos con mecanismos diferentes combinados entre si para el tratamiento del dolor, en concreto del dolor agudo postoperatorio, forma parte fundamental de un tipo de analgesia llamada multimodal. El objetivo de este trabajo es evaluar la eficacia de la asociacion de paracetamol mas metamizol y compararla con la asociacion de paracetamol mas dexketoprofeno en dolor agudo postoperatorio. Métodos: Disenamos un estudio prospectivo de intervencion en el que se incluyeron 42 pacientes sometidos bajo anestesia general a cirugias de probable bajo nivel algesico y duracion < 120 minutos. Todos ellos fueron tratados con un gramo de paracetamol intraoperatorio y, posteriormente en reanimacion, cuando la puntuacion en la escala numerica simple fue mayor de 3, recibieron el farmaco del grupo al que fueron asignados (metamizol: grupo M; dexketoprofeno: grupo D), evaluando las variaciones en la puntuacion de dicha escala. Se examinaron en ambos grupos las variables demograficas, las variables relacionadas con la anestesia, aquellas relacionadas con la cirugia, las variaciones en la escala numerica simple (ENS), la aparicion de complicaciones y las incidencias durante el proceso. Resultados: 20 pacientes fueron estudiados en el grupo M y 22 pacientes en el grupo D. Los dos grupos siguieron una distribucion similar en cuanto a variables demograficas, antecedentes medicos, tipo y duracion de la cirugia a la que se sometieron. En ambos grupos se observo una disminucion clinicamente relevante en la puntuacion de la escala numerica simple (p < 0,05). Sin embargo, no existieron diferencias en la disminucion de ENS al comparar los dos grupos entre si (p > 0,05). Conclusión: Nuestros resultados sugieren que la combinación de paracetamol y metamizol como terapia combinada para el manejo de dolor agudo postoperatorio es eficaz y equiparable a la combinación de paracetamol y dexketoprofeno, pudiendo constituir, por tanto, una alternativa a esta última (AU)

Objective: The use of drugs with different mechanisms, in combination for the treatment of pain, particularly acute postoperative pain, is a main part of the multimodal analgesia. The aim of this study was to evaluate the efficacy of metamizol plus paracetamol and compare it with the association of paracetamol plus dexketoprofen in acute postoperative pain. Methods: We designed a prospective interventional study that included 42 patients undergoing general anesthesia for probably low algesic and duration of surgery < 120 minutes. All were treated with one grame of intraoperative paracetamol and then, in the resuscitation unit, when the score on the simple numeric scale was > 3, they received the drug of the group that they were assigned (metamizol: group M; dexketoprofen: group D), with evaluation of variations in the scale score. We examined in both groups demographic variables, variables related to anesthesia and surgery, simple numeric scale (NSE) changes, complications and the incidents during the process. Results: 20 patients were studied in the M group and 22 patients in D group. Both groups had a similar distribution in terms of demographic variables, medical history, type and duration of surgery. Both group showed a clinically relevant decrease in the score of the simple numeric scale (p < 0.05). No differences were in the decrease of NSE when we compared the two groups together (p > 0.05). Conclusion: Our results suggest that the combination of paracetamol plus metamizol in combined therapy for management of acute postoperative pain is effective and comparable to the combination of paracetamol plus dexketoprofen and can constitute an alternative therapy (AU)

Simultaneous determination of dipyrone metabolites in rat hypothalamus, cerebrospinal fluid and plasma samples by LC-MS/MS.

BACKGROUND: After oral administration dipyrone is rapidly hydrolyzed to 4-methylaminoantipyrine, which is absorbed and further metabolized to 4-formylaminoantipyrine and to 4-aminoantipyrine, which is acetylated by a polymorphic N-acetyltransferase system to 4-acetylaminoantipyrine. To evaluate the presence of dipyrone metabolites in different rat matrices after intraperitoneal administration, an analytical method was developed and validated. METHODOLOGY: The four main dipyrone metabolites were extracted from plasma, cerebrospinal fluid and hypothalamus samples by LLE prior to LC-MS/MS. RESULTS: Standard calibration graphs for all metabolites were linear (r > 0.99). The intra- and inter-day precision and accuracy values were both inferior to 15%. CONCLUSION: This method is simple and specific for studying dipyrone metabolites after intraperitoneal administration.

Effects of nabumetone and dipyrone on experimentally induced gastric ulcers in rats.

Nabumetone and dipyrone are non-acidic, nonsteroidal anti-inflammatory drugs. Both of them are known to have weak inhibitory effects of cyclooxygenases. Gastric side effects represent the most common adverse drug effects of the widely used nonsteroidal anti-inflammatory drugs. The gastric effects of these drugs may be comparable in experimental ulcer models. In the present study, the gastric ulcerogenic activity of nabumetone and dipyrone were investigated on stress- and diethyldithiocarbamate-induced experimental ulcer models by determining the ulcer index and gastric mucus secretion in rats. It was found that diethyldithiocarbamate increased both ulcer index and mucus secretion. Nabumetone inhibited dose-dependently the increase of diethyldithiocarbamate-induced mucus secretion. Dipyrone inhibited both stress- and diethyldithiocarbamate-induced ulcer index and mucus secretion. Nabumetone inhibited stress-induced ulcer index at 25-mg/kg dose but stimulated dose-dependently mucus secretion. These effects may be attributed to their non-acidic structures and weak inhibitory effects on gastric mucosal cyclooxygenases.

Síndrome de Kounis secundario a reacción alérgica a metamizol / Kounis syndrome secondary to an allergic reaction to metamizole

Durante las reacciones anafilácticas pueden ocurrir eventos cardiovasculares graves como el vasoespasmo coronario o el infarto agudo de miocardio. Esta causa de cardiopatía isquémica es conocida aunque poco frecuente. Presentamos el caso de un paciente que sufrió un episodio anginoso tras una reacción anafiláctica por la administración de metamizol, objetivándose en la coronariografía ausencia de lesiones significativas(AU)

Severe cardiovascular events, such as coronary vasospasm or acute myocardial infarction can occur during anaphylactic reactions. Although rare, this cause of ischaemic heart disease is known. We present the case of a patient who suffered an angina episode after an anaphylactic reaction due tot administering metamizole, with no significant lesions observed in the coronary catheterisation(AU)

Novel bioactive metabolites of dipyrone (metamizol).

Dipyrone is a common antipyretic drug and the most popular non-opioid analgesic in many countries. In spite of its long and widespread use, molecular details of its fate in the body are not fully known. We administered dipyrone orally to mice. Two unknown metabolites were found, viz. the arachidonoyl amides of the known major dipyrone metabolites, 4-methylaminoantipyrine (2) and 4-aminoantipyrine (3). They were identified by ESI-LC-MS/MS after extraction from the CNS, and comparison with reference substances prepared synthetically. The arachidonoyl amides were positively tested for cannabis receptor binding (CB(1) and CB(2)) and cyclooxygenase inhibition (COX-1 and COX-2 in tissues and as isolated enzymes), suggesting that the endogenous cannabinoid system may play a role in the effects of dipyrone against pain.

Investigation of the microbial degradation of phenazone-type drugs and their metabolites by natural biofilms derived from river water using liquid chromatography/tandem mass spectrometry (LC-MS/MS).

The degradation of the pharmaceuticals phenazone and metamizole, two pyrazolone-derivates in widespread use, using biofilms created by natural organisms from the national park Unteres Odertal, Germany, were investigated. An analytical method based on LC-MS/MS was optimised to determine the substances phenazone and methylaminoantipyrine (MAA), the hydrolysis product of metamizole (also known as dipyrone), as well as their metabolites 1,5-dimethyl-1,2-dehydro-3-pyrazolone (DP), acetaminoantipyrine (AAA), formylaminoantipyrine (FAA) and 4-aminoantipyrine (AA). Performance characteristics of the method were evaluated in terms of recovery, standard deviation, coefficient of variation, method detection limits (MDL) and method quantification limits (MQL). Degradation studies of phenazone and MAA were conducted using a laboratory-scale continuous flow biofilm reactor fed with different nutrient media and with variable hydraulic retention times of 24 and 32 h. MAA was degraded rapidly to FAA and AA, while phenazone was not degraded under the prevailing conditions even after 32 h. By operating the bioreactor in batch mode to study the phenazone degradation potential of the biofilm under limiting nutrient conditions, an elimination rate of 85% phenazone was observed, but because of the slow elimination rate and aerobic conditions, the metabolite DP was not detected. In additional batch experiments using bacterial isolates from the natural biofilm to decompose phenazone, some bacterial strains were able to form DP from phenazone in marginal concentrations over the sampling period of eight weeks. Obviously, the microorganisms need a reasonably long time to adapt their metabolisms to enable the removal of phenazone from water samples.

Quantification of 4-methylaminoantipyrine, the active metabolite of dipyrone, in human plasma.

BACKGROUND: Dipyrone (metamizole) is a nonsteroidal anti-inflammatory drug used as an analgesic and antipyretic in both pediatric and adult patients. Dipyrone hydrolyses to 4-methylaminoantipyrine (MAA) in the stomach before absorption. There are several HPLC methods available for analysis of MAA from human plasma but no method has yet been developed on liquid chromatography-mass spectrometry (LC-MS) or LC tandem MS (LC-MS/MS), which are much more specific and sensitive techniques. METHODOLOGY: A high-performance LC-MS method for the quantification of 4-methylaminoantipyrine from human plasma has been developed, validated and applied to a pharmacokinetic study of 500 mg oral dose dipyrone. Following liquid-liquid extraction, the analyte was first separated on a reverse phase column using isocratic mobile phase and then analyzed by MS in selected ion monitoring mode using [M+H](+) ions, m/z 218.2 for 4-methylaminoantipyrine and 231.3 for 4-isopropylantipyrine (internal standard). RESULTS: The method exhibited a linear range from 0.2 to 10.0 µg/ml when only 100 µl human plasma sample was used. The lower limit of detection was 0.04 µg/ml (160 pg on column). The recovery was 80%. The accuracy and precision were obtained over the calibration curve range and were well within the limits specified under guidelines for bioanalytical method validation. The compound was stable under the experimental conditions. CONCLUSION: The method was demonstrated to be, simple, sensitive and rapid. It can be easily adopted in laboratories with access to LC-MS or MS/MS and applied to sample analysis in clinical settings where a large number of samples are generated.