Pharmacokinetic profiles of the active metamizole metabolites after four different routes of administration in healthy dogs.

Metamizole (MT), an analgesic and antipyretic drug, is rapidly hydrolyzed to the active primary metabolite 4-methylaminoantipyrine (MAA) and relatively active secondary metabolite 4-aminoantipyrine (AA). The aim of this study was to assess the pharmacokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.), intramuscular (i.m.), oral (p.o.), and rectal (RC) routes in dogs. Six dogs were randomly allocated to an open, single-dose, four-treatment, four-phase, unpaired, crossover study design. Blood was collected at predetermined times within 24 hr, and plasma was analyzed by a validated HPLC-UV method. Plasma concentrations of MAA and AA after i.v., i.m., p.o., and RC administrations of MT were detectable from 5 (i.v. and i.m.) or 30 (p.o. and RC) min to 24 hr in all dogs. The highest concentrations of MAA were found in the i.v., then i.m., p.o., and RC groups. Plasma concentrations of AA were similar for i.v., i.m., and RC, and the concentrations were approximately double those in the PO groups. The AUCEV/IV ratio for MAA was 0.75 ± 0.11, 0.59 ± 0.08, and 0.32 ± 0.05, for i.m., p.o., and RC, respectively. The AUCEV/IV ratio for AA was 1.21 ± 0.33, 2.17 ± 0.62, and 1.08 ± 0.19, for i.m., p.o., and RC, respectively. Although further studies are needed, rectal administration seems to be the least suitable route of administration for MT in the dog.

Pharmacokinetic profiles of the two major active metabolites of metamizole (dipyrone) in cats following three different routes of administration.

This study was performed to determine pharmacokinetic profiles of the two active metabolites of the analgesic drug metamizole (dipyrone, MET), 4-methylaminoantipyrine (MAA), and 4-aminoantipyrine (AA), after intravenous (i.v., intramuscular (i.m.), and oral (p.o.) administration in cats. Six healthy mixed-breed cats were administered MET (25 mg/kg) by i.v., i.m., or p.o. routes in a crossover design. Adverse clinical signs, namely salivation and vomiting, were detected in all groups (i.v. 67%, i.m. 34%, and p.o. 15%). The mean maximal plasma concentration of MAA for i.v., i.m., and p.o. administrations was 148.63 ± 106.64, 18.74 ± 4.97, and 20.59 ± 15.29 µg/ml, respectively, with about 7 hr of half-life in all routes. Among the administration routes, the area under the plasma concentration curve (AUC) value was the lowest after i.m. administration and the AUCEV/i.v . ratio was higher in p.o. than the i.m. administration without statistical significance. The plasma concentration of AA was detectable up to 24 hr, and the mean plasma concentrations were smaller than MAA. The present results suggest that MET is converted into the active metabolites in cats as in humans. Further pharmacodynamics and safety studies should be performed before any clinical use.

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.

Spectroscopic, electrochemical DNA binding and in vivo anti-inflammatory studies on newly synthesized Schiff bases of 4-aminophenazone.

4-Aminophenazone (Ap-1) Schiff bases i.e., 4-{(3,4,5-trimethoxybenzylidine) amino}phenazone (Ap-2), 4-{(2-chlorobenzylidine) amino}phenazone (Ap-3) and 4-{(4-chlorobenzylidine)amino} phenazone (Ap-4) were synthesized and characterized by different spectroscopic techniques. Interaction of these compounds with ds.DNA was investigated through UV-Visible spectroscopy, fluorescence spectroscopy and cyclic voltammetry at stomach (4.7) and blood (7.4) pH under 37 °C (human body temperature). Instrumental findings were further quantified both kinetically and thermodynamically. Results obtained through these techniques inferred intercalative mode of binding of all the compounds with DNA. The binding constant data, "Kb", and free energy change, ΔG, indicated comparatively greater binding affinity and more spontaneity of binding of compounds with DNA at stomach pH (4.7), respectively. However, among these compounds, Ap-4 showed comparatively greater binding at both the pH. Formation of compound-DNA complex was further confirmed through the decrease in diffusion rates after the addition of DNA. The in vivo anti-inflammatory activity of the compounds was evaluated using the carrageenan-induced hind paw edema method. The results revealed that among all the compounds, Ap-4 showed greater percentage of edema inhibition compared to standard drug.

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.

CREBH determines the severity of sulpyrine-induced fatal shock.

Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and in vivo knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock.

High-performance liquid chromatographic assay for metamizol metabolites in rat plasma: application to pharmacokinetic studies.

In order to evaluate the pharmacokinetics of metamizol in the presence of morphine in arthritic rats, after subcutaneous administration of the drugs, an easy, rapid, sensitive and selective analytical method was proposed and validated. The four main metamizol metabolites (4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine) were extracted from plasma samples (50-100µl) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography with diode-array detection. Standard calibration graphs for all metabolites were linear within a range of 1-100µg/ml (r(2)≥0.99). The intra-day coefficients of variation (CV) were in the range of 1.3-8.4% and the inter-day CV ranged from 1.5 to 8.4%. The intra-day assay accuracy was in the range of 0.6-9.6% and the inter-day assay accuracy ranged from 0.9 to 7.5% of relative error. The lower limit of quantification was 1µg/ml for all metabolites using a plasma sample of 100µl. Plasma samples were stable at least for 4 weeks at -20°C. This method was found to be suitable for studying metamizol metabolites pharmacokinetics in arthritic rats, after simultaneous administration of metamizol and morphine, in single dose.

Effect of 4-aminoantipyrine on oxidative stress induced by glutathione depletion in single human erythrocytes using a microfluidic device together with fluorescence imaging.

The effects of 4-aminoantipyrine (AAP) on oxidative stress induced by glutathione (GSH) depletion in single human erythrocytes were investigated using microfluidic technique and fluorescence imaging. Most cell-based toxicity evaluations on GSH are performed with bulk experiments based on analysis of cell populations. This work established a single-cell toxicity evaluation method to statistically analyze the GSH amount in single erythrocytes incubated with AAP in different concentrations. The experimental conditions of cell flow rate and cell concentration were optimized. The GSH contents in erythrocytes decreased with increasing dose of AAP. At low concentration, AAP had a little effect on GSH; while at high concentration, AAP led to GSH depletion reaching a maximum of 14.53%. The depletion of GSH leads to a significant shift to a more oxidizing intracellular environment. This study provides basic data for presenting the effect of AAP on GSH in erythrocytes and is helpful for understanding its toxicity during the blood transportation process. In addition, it will also complement studies on the environmental risk assessment of AAP pollution.

Impairment of the metabolism of dipyrone in asymptomatic carriers of the hepatitis-B virus does not occur in rapid acetylators.

OBJECTIVE: We previously found that, compared with healthy subjects. asymptomatic hepatitis-B virus (HBV) carriers displaying slow acetylator phenotype demonstrate a significant prolongation of the elimination half-life of 4-methylaminoantipyrine (MAA) and a decrease in the clearance of formation of 4-aminoantipyrine (AA) and 4-formylaminoantipyrine (FAA). However, the formation of 4-acetylaminoantipyrine (AAA) was unchanged. The present study was designed to examine the effect of the asymptomatic HBV carrier state on the metabolism of dipyrone. as a model drug, in rapid acetylators. METHODS: The plasma and urine concentrations of the metabolites of dipyrone were measured in eight asymptomatic HBV carriers and eight healthy subjects who had normal liver function tests, all displaying the rapid acetylation phenotype and genotype, after the administration of a 1.0-g oral dose of dipyrone. RESULTS: The following pharmacokinetic parameters were evaluated: peak plasma concentration, time to peak plasma concentration, elimination rate constant, area under the plasma concentration-time curve (0-->infinity), amount excreted (0-->infinity), renal and non-renal clearances for MAA and the clearances of formation for AA, FAA and AAA. No significant differences were found between the two subject groups. CONCLUSION: The effect of hepatic viral carrier state on drug metabolism may vary according to metabolic pathways and genetic polymorphism.

Cerebrospinal fluid and plasma concentrations of dipyrone metabolites after a single oral dose of dipyrone.

OBJECTIVE: Dipyrone is a veteran analgesic and antipyretic drug. After oral administration it is rapidly converted by hydrolysis to 4-methylaminoantipyrine (MAA), which is further metabolized to 4-formylaminoantipyrine (FAA), 4-aminoantipyrine (AA) and 4-acetylaminoantipyrine (AAA). It is still debated whether the site of dipyrone action is in the central nervous system or in the periphery. The purpose of this study was to assess whether dipyrone metabolites cross the blood-brain barrier (BBB) when administered systemically. METHODS: Twenty-eight patients undergoing diagnostic lumbar puncture (LP) were randomly assigned to receive two 0.5-g dipyrone tablets either 30 min, 1, 1.5, 2, 4, 6, 8 h or 12 h before the lumbar tap. A 5-ml blood sample was drawn concomitantly. RESULTS: All four metabolites were found in the cerebrospinal fluid (CSF). Their appearance in the CSF lagged but followed that found in the plasma. Mean CSF/plasma ratios were 0.40 (for samples taken between 0.5-2 h) and 0.83 (for samples taken between 4-12 h) for MAA, 0.62 for AA, 0.55 for FAA and 0.40 for AAA (for all samples). Significant correlation was found between plasma and CSF concentrations for MAA, AA, FAA and AAA. CONCLUSION: The concentration-time course of dipyrone metabolite CSF concentrations are in agreement with that of their plasma concentrations and the analgesic effect of dipyrone.

Determination of antipyrine metabolites in human plasma by solid-phase extraction and micellar liquid chromatography.

A rapid solid-phase extraction (SPE) procedure was developed for the quantitative isolation of three important antipyrine (dipyrone) metabolites from human plasma: 4-formylaminoantipyrine (FAA), 4-aminoantipyrine (AA) and 4-methylaminoantipyrine (MAA). Separation and quantitation were performed using micellar liquid chromatography (MLC) with a 0.1 mol l-1 sodium dodecyl sulfate (SDS)-2.5% pentanol mobile phase and UV detection at 262 nm. The metabolites were well resolved in less than 5 min using an octadecyl silica-bonded stationary phase. The extraction procedure involved passing 0.3 ml of plasma sample through a disposable SPE cartridge packed with C18 bonded porous silica. The adsorbed metabolites were removed from the cartridge with methanol. The eluent was evaporated to dryness and the residue was reconstituted with mobile phase and injected into the chromatographic system. The cartridge blank interferent peaks, the effects on reproducibility of sample loading in the cartridge and volume needed for desorption of metabolites were evaluated. The concentration of metabolites ranged between 2.4 and 4 micrograms ml-1. The present procedure yields recoveries for the three metabolites ranging from 93 to 100%. The relative standard deviation (Sr) ranged between 1.2 and 13.6%. Limits of detection (LODs) were 10.5, 11.5 and 17.0 ng ml-1 for FAA, AA and MAA, respectively.

Effect of fetal intravascular 4-aminoantipyrine infusions on myometrial activity (contractures) at 125 to 143 days' gestation in the pregnant sheep.

Myometrial activity of low amplitude and long duration--contractures--is present throughout gestation in the pregnant ewe and other species. This activity differs from the contractions of labor and delivery. Between 125 and 143 days' gestation, 4-aminoantipyrine infused into the fetus at rates that produced maternal uterine vein plasma concentrations of 4-aminoantipyrine of 5.95 +/- 1.23 (mean +/- SEM, n = 5) mg X 100 ml-1 markedly depressed both total myometrial electromyographic activity to 28.6% and frequency of contractures to 30.5% of preinfusion values. By 60 minutes of infusion, maternal uterine vein plasma 13,14-dihydro-15-keto prostaglandin F2 alpha concentration was reduced to 14% of the resting level. Two conclusions were drawn from this study. First, prostaglandins are involved in the regulation of contractures. Second, when infusions of 4-aminoantipyrine are used to quantify uterine or umbilical blood flow, the possibility exists that changes will occur in physiologic systems that are modified by prostaglandins.

Plasma kinetics of dipyrone metabolites in rapid and slow acetylators.

The pharmacokinetics of the dipyrone metabolites 4-methylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 4-formylaminoantipyrine (FAA) and 4-acetylaminoantipyrine (AAA) were evaluated following the administration of a single oral 1.0 g dose of dipyrone to 23 healthy volunteers. Twelve were slow and 11 were rapid acetylators as previously determined by dapsone phenotyping. For MAA and FAA the mean peak plasma concentrations were 10.5 +/- 2.8 micrograms/ml and 2.1 +/- 0.8 micrograms/ml and the half-lives were 3.3 +/- 1.0 and 10.1 +/- 1.8 h, respectively. No significant difference was found between rapid and slow acetylators in MAA and FAA kinetics. For AA, the mean peak plasma concentrations were 2.7 +/- 0.6 and 1.6 +/- 0.7 micrograms/ml (p less than 0.01), the peak times 6.7 +/- 2.1 and 3.1 +/- 1.1 h (p less than 0.01) and the half-lives were 5.5 +/- 1.0 and 3.8 +/- 1.2 h in slow and rapid acetylators, respectively. For AAA, the mean peak plasma concentrations were 1.6 +/- 0.4 and 4.4 +/- 1.1 micrograms/ml (p less than 0.01) and the peak time 16.1 +/- 5.1 and 10.0 +/- 2.6 h (p less than 0.01) in slow and rapid acetylators, respectively. There was no difference in the elimination half-life between the two groups (10.6 +/- 2.2 h). Thus, it has been demonstrated that the AAA/AA ratio is an indicator of the acetylation phenotype, as it is closely correlated with that determined by dapsone (r = 0.895, p less than 0.0005).

The unusual occurrence of 4-aminoantipyrine (4-aminophenazone) in human biological fluids.

During toxicology screening of human biological fluids, 4-aminoantipyrine (4-AAP) was detected in 19 patients and identified by two-dimensional TLC and UV spectrophotometry with whole blood concentrations of 4-AAP ranging from 0.4 to greater than 4.9 mg/dL. Of the 19 patients, 15 had received prior treatment at a Mexican health-care facility before transfer to this medical center. Since 4-AAP is a known metabolite of aminopyrine, it is likely that the patients had ingested this anti-inflammatory, analgesic agent in Mexico; however, the clinical histories in this respect were vague. Recognition of the presence of 4-AAP in biological fluids is important, since the parent drug may produce fatal agranulocytosis and the compound may complicate the detection of other compounds that simultaneously partition into the weak-base fraction in toxicology screening.

[Quantitative thin-layer chromatographic estimation of aminophenazone (I.N.N.), 4-methylaminophenazone and 4-aminophenazone in plasma (author's transl)]. / Quantitative dünnschichtchromatografische Bestimmung von Aminophenazon (I.N.N.), 4-Methylaminophenazon und 4-Aminophenazon im Plasma.

A TLC method for measurement of aminophenazone (1) and of its degradation products 4-methylaminophenazone (2) and 4-aminophenazone (3) in plasma was described. After chloroform extraction amd separation on Silufol plates the substances were stained by ferric chloride/potassium hexacyanotoferrate(III) and the area of the spots was measured. Concentrations from 1 to 25 micrograms 1, 2 and 3 per ml plasma could be estimated.