Identifying Metabolites of Meclonazepam by High-Resolution Mass Spectrometry Using Human Liver Microsomes, Hepatocytes, a Mouse Model, and Authentic Urine Samples.

Meclonazepam is a benzodiazepine patented in 1977 to treat parasitic worms, which recently appeared as a designer benzodiazepine and drug of abuse. The aim of this study was to identify metabolites suitable as biomarkers of drug intake in urine using high-resolution mass spectrometry, authentic urine samples, and different model systems including human liver microsomes, cryopreserved hepatocytes, and a mice model. The main metabolites of meclonazepam found in human urine were amino-meclonazepam and acetamido-meclonazepam; also, minor peaks for meclonazepam were observed in three of four urine samples. These observations are consistent with meclonazepam having a metabolism similar to that of other nitro containing benzodiazepines such as clonazepam, flunitrazepam, and nitrazepam. Both metabolites were produced by the hepatocytes and in the mice model, but the human liver microsomes were only capable of producing minor amounts of the amino metabolite. However, under nitrogen, the amount of amino-meclonazepam produced increased 140 times. This study comprehensively elucidated meclonazepam metabolism and also illustrates that careful selection of in vitro model systems for drug metabolism is needed, always taking into account the expected metabolism of the tested drug.

Metabolic profiling of praziquantel enantiomers.

Praziquantel (PZQ), prescribed as a racemic mixture, is the most readily available drug to treat schistosomiasis. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based metabolomics was employed to decipher the metabolic pathways and enantioselective metabolic differences of PZQ. Many phase I and four new phase II metabolites were found in urine and feces samples of mice 24h after dosing, indicating that the major metabolic reactions encompassed oxidation, dehydrogenation, and glucuronidation. Differences in the formation of all these metabolites were observed between (R)-PZQ and (S)-PZQ. In an in vitro phase I incubation system, the major involvement of CYP3A, CYP2C9, and CYP2C19 in the metabolism of PZQ, and CYP3A, CYP2C9, and CYP2C19 exhibited different catalytic activity toward the PZQ enantiomers. Apparent Km and Vmax differences were observed in the catalytic formation of three mono-oxidized metabolites by CYP2C9 and CYP3A4 further supporting the metabolic differences for PZQ enantiomers. Molecular docking showed that chirality resulted in differences in substrate location and conformation, which likely accounts for the metabolic differences. In conclusion, in silico, in vitro, and in vivo methods revealed the enantioselective metabolic profile of praziquantel.

Isolation and identification of 8-hydroxypraziquantel as a metabolite of the antischistosomal drug praziquantel.

Praziquantel, a broad spectrum anthelmintic drug, is extensively metabolized in the liver, yielding mainly monohydroxylated and dihydroxylated phase-I metabolites. However, the exact chemical structure of most metabolites is still unknown. One of these unidentified phase-I metabolites was isolated from human urine by high performance liquid chromatography using an isocratic separation method. This metabolite was identified as 8-hydroxypraziquantel. For the structure elucidation, electrospray ionization-mass spectrometry, 1H and 13C NMR spectroscopy have been used.

Determination of a chemopreventive agent, Oltipraz, in rat plasma and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the determination of a chemopreventive agent, Oltipraz, in rat plasma and urine. The sample preparation was simple; 2 volumes of acetonitrile were added to deproteinize the biological sample. A 50-microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase, acetonitrile : 0.5 mM ammonium acetate (55: 45, v/v for rat plasma and 45 : 55, v/v for rat urine), was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 305 nm. The retention times for Oltipraz in rat plasma and urine were approximately 5.8 and 8.6 min, respectively. The detection limits of Oltipraz in rat plasma and urine were 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 4.65%) in concentration ranges from 0.02 (0.05) to 10 microg/ml for rat plasma and urine. No interference from endogenous substances was found.

A simple kinetic spectrophotometric method for the determination of oxamniquine in formulations and spiked biological fluids.

A simple and sensitive kinetic method for the determination of oxamniquine in pharmaceutical preparations and biological fluids was developed. The procedure is based upon a kinetic investigation of the oxidation reaction of the drug with alkaline potassium permanganate at room temperature for a fixed time of 20 min. The absorbance of the colored manganate ions was measured at 610 nm. Alternatively, the decrease in the absorbance of potassium permanganate after addition of the drug was measured at 525 nm. The absorbance concentration plots in both procedures were rectilinear over the range 0.5-4 microg ml(-1). The concentration of oxamniquine is calculated using the corresponding calibration equation for the fixed-time method. The determination of oxamniquine by fixed-concentration and rate-constant methods was feasible with the calibration equations obtained but the fixed time method had been found to be more applicable. Both procedures were applied to the determination of oxamniquine in formulations. The results obtained were in good agreement with those obtained using the official method. The fixed time method of 20 min was further applied to spiked human urine and plasma, the recoveries (%) were 100.94 +/- 0.57 and 98.07 +/- 0.88 for urine and plasma, respectively, at 610 nm, and 97.51 +/- 1.27 and 95.69 +/- 1.23 for urine and plasma, respectively, at 525 nm.

Fluorimetric determination of oxamniquine in biological fluids.

A highly sensitive and specific fluorimetric method was developed for the determination of oxamniquine in biological fluids (urine and plasma). The proposed method is based on the reduction of oxamniquine using zinc/calcium chloride to obtain its nitroso derivative. The latter is then allowed to react with 2-cyanoacetamide to get a highly fluorescent product. The different experimental parameters affecting the intensity of the fluorescence were carefully studied and incorporated into the procedure. Under the described conditions, the method is applicable over the concentration range of 0.08-0.88 microgram/ml with a minimum detectability (S/N = 2) of 8 ng/ml. The percentage recoveries from spiked urine and plasma were 99.75 +/- 1.58 and 97.46 +/- 0.44%, respectively.

Formation of a mutagenic drug metabolite by intestinal microorganisms.

A new broad-spectrum antiparasitic agent, 4-isothiocyano-4'-nitrodiphenylamine, is devoid of mutagenic activity in vitro, either alone or in the presence of activating enzymes of rat liver. However, six species of mammals receiving this drug excrete as as yet unidentified mutagenic metabolite. Several observations suggested that one or several constituents of the enteric bacterial flora, rather than the metabolic activities of the host, are involved in the formation of this mutagen. Unequivocal demonstration for such a mechanism was provided by germ-free rats that do not form this metabolite, in contrast to their conventional littermates. Only a relatively moderate and apparently quite selective reduction in the total number of microorganisms of the intestinal flora is needed to elminate this mutagenic transformation. For example, following administration of a single dose of erythromycin or erythromycylamine, conversion of the isothiocyanate to a mutagen can be prevented completely, while antiparastitic activity is maintained. There is no obligatory association between chemotherapeutic activity and the formation of the mutagenic metabolite, and these two activities can be dissociated completely. This suggests a new approach for increasing the safety of pharmacological agents.

The metabolism of oxamniquine - a new schistosomicide.

The metabolism of oxamniquine, 6-hydroxymethyl-7-nitro-2-isopropylaminomethyl-1,2,3,4-tetrahydroquinoline, has been studied in the mouse, rat, hamster, rabbit, rhesus monkey, dog and man. Urinary excretion is a major route of elimination in man. The compound is converted to two metabolites, the major one arising from oxidation of the 6-hydroxymethyl group to a carboxyl group and the other by oxidation of the side chain to give the 2-carboxylic acid. There is a species at the dose levels used since both acidic metabolites were found in appreciable quantities only in the urine of mouse, rabbit, hamster and dog. The 2-carboxylic acid was not found in the urine of rhesus monkey and rat and occurred in only trace amounts in human urine.