Metabolism of metamizol in early stages of the incubated hen's egg

In a previous paper we introduced a model for studying the metabolism of xenobiotics in the incubated hen's egg. This model is characterized by application of the xenobiotic into the yolk sac and identification of metabolites in the allantoic fluid (AF). Depending on the stage of development, the incubated hen's egg can be used both conservatively--in the sense of an animal experiment--and alternatively as a complementary method (appr. first half of incubation period). In the present investigation we used the sodium salt of [(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-lH-pyrazol-4-yl)-N-methylamino] methanesulfonic acid (1; metamizol, dipyrone) as model substance. Concentrations of 1 up to 32 mg/egg did not affect the viability of the incubated eggs. After inoculation of 1 (10 mg/egg) on the sixth day of incubation (DI) eight compounds were identified in the AF on DI 11 (TLC, GC, GC-MS, synthesis, MS, 13C NMR): 4-methylamino-(2), 4-amino-(3), 4-hydroxy-(4), 4-acetylamino-(5), 4-N-acetyl-N-methylamino-(6), 4-N-hydroxyacetyl-N-methylamino-(7), 4-N-hydroxyoxalyl-N-methylamino-(8) and 4-N-formyl-N-methylamino-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol- 3-one (-antipyrine; 9), respectively. Main metabolites are 2 and 5; 4 and 7 occur as sulfate or glucuronide conjugates. Compound 7 is also eliminated in it's free form. To our knowledge 7 has been detected as a new metabolite of 1 for the first time. The metabolic route of 7 was elucidated after application of 6. Compounds 8 and 9 are artefacts of 7. The data presented here demonstrate the usefulness of the developed model also in early stages of the incubated hen's egg. Consequently, the model is suitable as a complementary method for xenobiotic metabolism.     

Drug metabolism studies with the incubated hen's egg. Identification of 2,3,5-trihydroxybenzoic acid as a metabolite of gentisic acid

Previous investigations have shown that an ex vivo model on the basis of the incubated hen's egg represents a potential alternative to animal experimentation for xenobiotic metabolism studies. This model is characterized by administration of the xenobiotic into the yolk sac and identification of metabolites in the allantoic fluid. In the present study, gentisic acid (2,5-dihydroxybenzoic acid, 2,5-DHBA, CAS 490-79-9) was used as a test substance. The oxidative fate of this salicylate metabolite in human and animal species could not be completely elucidated until now. Inoculation of 2,5-DHBA into the incubated hen's egg in concentrations up to 8 mg/egg did not affect embryo viability. For the metabolism studies a dose of 2 mg/egg was chosen and 2,5-DHBA was identified as the main metabolite in both the free and conjugated form (glucuronidel/sulfate). Two minor metabolites were detected in addition. The first of these could be identified as 2,3,5-trihydroxybenzoic acid (2,3,5-THBA) and the second one as 2-hydroxy-5-methoxybenzoic acid (2-H-5-MBA). 2,3,5-THBA has been characterized as a metabolite of 2,5-DHBA in a vertebrate species for the first time. Since 2,3,5-THBA can be methylated to the guaiacol derivative, 2,5-dihydroxy-3-methoxybenzoic acid (2,5-DH-3-MBA) can not to be excluded as a further possible metabolite. The metabolites were identified by comparative TLC with the authentic reference substances as well as additionally by GC and GC-MS analysis of the trimethylsilyl derivatives. The structures of the synthesized reference substances were confirmed by MS, 1H and 13C NMR spectral data.     

Inhibition of spinal reflexes by acetylsalicylate and metamizol (dipyrone) in rats

The effects of acetylsalicylate and metamizol on spinal monosynaptic reflexes were tested in spinal rats. Adult rats were anesthetized with ketamine, artificially ventilated, and spinalized at the C1 level. A laminectomy was performed in the lumbosacral region. Following electrical stimulation of the sciatic nerve by single pulses, the reflex potentials were recorded from the ipsilateral L5 ventral root. Acetylsalicylate was administered orally via nasogastric tube and metamizol intramuscularly. Acetylsalicylate (50 and 100 mg/kg) and metamizol (15 mg/kg) significantly decreased the amplitude of the reflex response (p < 0.05). But the 10-mg/kg metamizol dose did not significantly decrease the amplitude of the reflex response. The cyclooxygenase products of arachidonic acid may play an important role in regulating the reflex potential.     

Evaluation of the endogenous cannabinoid system in mediating the behavioral effects of dipyrone (metamizol) in mice

Dipyrone is a common nonopioid analgesic and antipyretic, which, in many countries, is available over the counter and is more widely used than paracetamol or aspirin. However, the exact mechanisms by which dipyrone acts remain inconclusive. Two novel arachidonoyl-conjugated metabolites are formed in mice following the administration of dipyrone that are dependent on the activity of fatty acid amide hydrolase (FAAH), which also represents the major catabolic enzyme of the endogenous cannabinoid ligand anandamide. These arachidonoyl metabolites not only inhibit cyclooxygenase (COX-1/COX-2) but also bind to cannabinoid receptors at low micromolar concentrations. The relative contributions of cannabinoid receptors and FAAH in the overall behavioral response to dipyrone remain untested. Accordingly, the two primary objectives of the present study were to determine whether the behavioral effects of dipyrone would (a) be blocked by cannabinoid receptor antagonists and (b) occur in FAAH mice. Here, we report that thermal antinociceptive, hypothermic, and locomotor suppressive actions of dipyrone are mediated by a noncannabinoid receptor mechanism of action and occurred after acute or repeated administration irrespective of FAAH. These findings indicate that FAAH-dependent arachidonoyl metabolites and cannabinoid receptors are not requisites by which dipyrone exerts these pharmacological effects under noninflammatory conditions.     

Formation and excretion of dipyrone metabolites in man

Summary The formation and urinary excretion of the dipyrone metabolites, methylaminoantipyrine (MAA), aminoantipyrine (AA), formylaminoantipyrine (FAA) and acetylaminoantipyrine (AAA) were determined following administration of a single oral 1.0 g dose of dipyrone to 12 healthy volunteers. The AAA/AA plasma ratio showed that 3 subjects were slow and 9 were rapid acetylators. Pharmacokinetic parameters were determined separately for each group.A good correlation was found between the plasma and urine AAA/AA ratios. The renal clearance of the four metabolites was similar for both phenotypes. A significant difference in the rate of formation of dipyrone metabolites was found for AA, 0.25 (slow) vs 0.1 ml·min^–1·kg^–1 (rapid), and for AAA 0.75 (slow) vs 7.53 ml·min^–1·kg^–1 (rapid). There were comparable differences between slow and rapid acetylators in the AUC and the urinary excretion extrapolated to infinity for AA and AAA.The present results show that the kinetics of dipyrone metabolites in plasma and urine can provide a useful measure of the activity of the enzymes involved in their production.     

Differential effect of acetylsalicylic acid and dipyrone on prostaglandin production in human fibroblast cultures.

Human skin fibroblasts incubated with arachidonic acid in culture show basal release of prostaglandins. They produce the same prostaglandins after stimulation with bradykinin. Basal release of prostaglandins I2 (6-oxo-PGF1 alpha), F2 alpha and E2 is inhibited dose-dependently by both acetylsalicylic acid (ASA) and dipyrone (P less than 0.05). The examined dose-range was 10(-7) to 10(-4) M for both drugs. During the first 5 min after removal of the drugs from the incubation medium, bradykinin-stimulated release remains dose-dependently inhibited (P less than 0.001) in ASA-, but not in dipyrone-treated cultures. The difference between the effects of ASA and of dipyrone is highly significant (P less than 0.0001), whereas the dipyrone-treated cultures are not different from controls. The findings are consistent with cyclo-oxygenase inhibition by ASA as well as by dipyrone. However, the data demonstrate rapid reversibility of the effect of dipyrone. This suggests that in contrast to ASA, dipyrone does not inhibit cyclo-oxygenase by binding covalently to the enzyme.     

Plasma kinetics of dipyrone metabolites in rapid and slow acetylators

Summary 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 µg/ml and 2.1±0.8 µg/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 µg/ml (p<0.01), the peak times 6.7±2.1 and 3.1±1.1 h (p<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 µg/ml (p<0.01) and the peak time 16.1±5.1 and 10.0±2.6 h (p<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<0.0005).     

Biochemical and pharmacological effects of dipyrone and its metabolites in model systems related to arachidonic acid cascade

The metabolites of dipyrone (metamizol, Novalgin) were compared with appropriate standard drugs for their influences on the pathways of the arachidonic acid metabolism. The drugs in this study had no significant effects on the lipoxygenase pathway in human neutrophils in vitro. The dipyrone metabolites 4-methylaminoantipyrine (MAAP) and 4-aminoantipyrine (AAP) inhibited prostaglandin synthesis in the 10(-3) to 10(-4) mol/l range thus being comparable to acetylsalicylic acid (ASA), whereas the two additional metabolites 4-acetylaminoantipyrine (AAAP) and 4-formylaminoantipyrine (FAAP) were practically inactive. This result is in accordance with the effects of the metabolites on the formation of oedema in the arthritis rat model, and supports published data showing that MAAP and AAP are the metabolites responsible for the clinical effects of dipyrone. Further systems in our study depending at least partially on the prostaglandin pathway were the release of antiaggregatory activity from rat aortae in vitro and the aggregation of human platelets induced by arachidonic acid in vitro. MAAP exhibits antiaggregatory activity (IC50 5 x 10(-6) mol/l), whereas the inhibitory effect on the vascular antiaggregatory release is much weaker. Compared to normals platelet aggregability ex vivo is enhanced in arthritic rats, but could significantly be lowered again by treatment of the rats with MAAP. A further system studied was the release of 6-keto-PGF1 alpha from rat mucosa in vitro and ex vivo. In vitro there is inhibition to be found with MAAP as well as with ASA. Ex vivo, however, dipyrone or MAAP slightly stimulates mucosal 6-keto-PGF1 alpha rather than inhibiting it, whereas ASA exerts inhibition, as expected.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biotransformation and toxicity of xenobiotics in the chick embryo. 1. The embryonated hen's egg as an in vivo model and sodium salicylate as substrate

After application of sodium salicylate (1) on d 6 of incubation into the yolk sac of the embryonated hen's egg (Gallus domesticus) 3 compounds were detected in the allantois fluid (AF) dependent on embryo age. Compounds were isolated and identified (M.p., MS, TLC, HPLC) as salicylic acid (1a), o-pyrocatechuic acid (2,3-dihydroxy benzoic acid; 2) and gentisic acid (2,5-dihydroxy benzoic acid; 3), respectively. 1a, 2 and 3 are eliminated from chick embryo in both the free and the glucuronidated form. The toxicity of 1 which was inoculated on d 6 of incubation increased between d 10 and 18 of the embryo age (LD50 318 and 281 mg X kg-1 egg weight, respectively, parallel to the rise of building and excretion of dihydroxy benzoic acid metabolites (increase from 0 to 78.5% of total metabolites).     

Structure-antigastrin activity relationships of new (R)-4-benzamido-5-oxopentanoic acid derivatives.

New (R)-4-benzamido-5-oxopentanoic acid derivatives were synthesized by a stereoconservative procedure and evaluated in vitro for their capacity to inhibit the binding of [125I](BH)-CCK-8 to either rat peripheral (CCK-A) or central (CCK-B) CCK receptors, or the binding of [3H]pentagastrin to rabbit gastric glands, as well as to inhibit, in vivo, the acid secretion induced by pentagastrin infusion in the perfused rat stomach. The parent compound of this series (lorglumide) is the first nonpeptidic, potent and selective antagonist of the CCK-A receptor. Chemical manipulations of the structure of lorglumide led to the discovery of selective antagonists of the CCK-B/gastrin receptors. Structure-activity relationships are discussed. Some of these new derivatives exhibit different affinities with rabbit gastric gland cells and rat cortex membranes, suggesting that the stomach gastrin receptor (arbitrarily termed CCK-B1 receptor) is not as closely related to the CCK central receptor (termed CCK-B2) as previously hypothesized. The antigastric activity of the most potent compound of the series, i.e. (R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro[4.5]decan- 8-yl)-5-oxopentanoic acid (compound 28, CR 2194) was further evaluated in vivo: in the first hour after administration the compound inhibits acid secretion induced by pentagastrin infusion, in both cat and dog (in the cat with gastric fistula and in the dog with Heidenhain pouch), with ID50s (mg/kg) of 15.5 (iv) (cat), 8.7 (IV) (dog) and 24.2 (oral) (Heidenhain dog). The characteristics of CR 2194, that is, the selectivity for the gastrin receptor, the simple nonpeptidic molecular structure, and the activity after oral administration, indicate that this compound is a useful tool in the study of the biological effects of gastrin and a potential agent for diagnostic or therapeutic use.     

Characterization of human urinary metabolites of the antimalarial piperaquine.

Five metabolites of the antimalarial piperaquine (PQ) (1,3-bis-[4-(7-chloroquinolyl-4)-piperazinyl-1]-propane) have been identified and their molecular structures characterized. After a p.o. dose of dihydroartemisinin-piperaquine, urine collected over 16 h from two healthy subjects was analyzed using liquid chromatography (LC)/UV, LC/tandem mass spectrometry (MS/MS), Fourier transform ion cyclotron resonance (FTICR)/MS, and H NMR. Five different peaks were recognized as possible metabolites [M1, 320 m/z; M2, M3, and M4, 551 m/z (PQ + 16 m/z); and M5, 567 m/z (PQ + 32 m/z)] using LC/MS/MS with gradient elution. The proposed carboxylic M1 has a theoretical monoisotopic molecular mass of 320.1166 m/z, which is in accordance with the FTICR/MS (320.1168 m/z) findings. The LC/MS/MS results also showed a 551 m/z metabolite (M2) with a distinct difference both in polarity and fragmentation pattern compared with PQ, 7-hydroxypiperaquine, and the other 551 m/z metabolites. We suggest that this is caused by N-oxidation of PQ. The results showed two metabolites (M3 and M4) with a molecular ion at 551 m/z and similar fragmentation pattern as both PQ and 7-hydroxypiperaquine; therefore, they are likely to be hydroxylated PQ metabolites. The molecular structures of M1 and M2 were also confirmed using H NMR. Urinary excretion rate in one subject suggested a terminal elimination half-life of about 53 days for M1. Assuming formation rate-limiting kinetics, this would support recent findings that the terminal elimination half-life of PQ has been underestimated previously.     

Characterization of fimasartan metabolites in human liver microsomes and human plasma

1.?The metabolites of fimasartan (FMS), a new angiotensin II receptor antagonist, were characterized in human liver microsomes (HLM) and human subjects.

2.?We developed a method for a simultaneous quantitative and qualitative analysis using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning. To characterize metabolic reactions, FMS metabolites were analyzed using quadrupole-time of flight mass spectrometer in full-scan mode.

3.?The structures of metabolites were confirmed by comparison of chromatographic retention times and mass spectra with those of authentic metabolite standards.

4.?In the cofactor-dependent microsomal metabolism study, the half-lives of FMS were 56.7, 247.9 and 53.3?min in the presence of NADPH, UDPGA and NADPH?+?UDPGA, respectively.

5.?The main metabolic routes in HLM were S-oxidation, oxidative desulfuration, n-butyl hydroxylation and N-glucuronidation.

6.?In humans orally administered with 120?mg FMS daily for 7 days, the prominent metabolites were FMS S-oxide and FMS N-glucuronide in the 0–8-h pooled plasma sample of each subject.

7.?This study characterizes, for the first time, the metabolites of FMS in humans to provide information for its safe use in clinical medicine.     

Amino acid conjugates: metabolites of 2-propylpentanoic acid (valproic acid) in epileptic patients.

In this study, spectroscopic and chromatographic evidence is presented for the identification and characterization of the metabolites, valproyl glutamate (2-propylpentanoyl glutamate, VPA-GLU) and valproyl glutamine (2-propylpentanoyl glutamine, VPA-GLN) in the urine, serum, and cerebrospinal fluid (CSF) of patients on valproic acid (VPA) therapy. Moreover, the identification of valproyl glycine (2-propylpentanoyl glycine, VPA-GLY) in the serum and urine of patients on VPA, albeit in trace concentrations, is also reported here. The three amino acid conjugates excreted in urine accounted for about 1% of the VPA dose in four patients who were on VPA therapy chronically and had reached steady state. VPA-GLU was quantitatively the most prominent metabolite (0.66-13.1 microg/mg creatinine) compared with VPA-GLN (0.78-9.93 microg/mg creatinine) and VPA-GLY (trace-1.0 microg/mg creatinine) in overnight urine samples of all patients studied (n = 29). The relatively low serum concentrations of the three amino acid conjugates of VPA in six patients suggest that the metabolites are readily excreted once formed. In contrast, whereas VPA GLY was absent in the CSF of one patient on VPA, the concentrations of VPA-GLU and VPA-GLN in this CSF sample were 9 and 5 times, respectively, their corresponding serum concentrations.     

Studies on the metabolism of Inabenfide (a new plant growth regulator) in rats. I. Characterization of metabolites in rats

1. The metabolism of 4'-chloro-2'-(alpha-hydroxybenzyl) isonicotinanilide (Inabenfide, IBF) was studied in the rat. After intraperitoneal administration of IBF to rats, eight metabolites were detected in urine by g.l.c.-mass spectrometry and a stable isotope technique. 2. The major metabolites were hydroxylated IBF, and minor metabolites were dihydrodiol IBF, methylated catechol IBF, IBF ketone, IBF N-oxide and an amine derivative. 3. Of these metabolites, IBF ketone was produced by oxidation of the carbinol between the benzene rings, which is an interesting metabolite since similar oxidation between benzene rings is not well known. 4. Metabolism involving an NIH shift of chlorine and an epoxide-diol pathway for IBF are presented.