Worsening of the Toxic Effects of (±)Cis-4,4'-DMAR Following Its Co-Administration with (±)Trans-4,4'-DMAR: Neuro-Behavioural, Physiological, Immunohistochemical and Metabolic Studies in Mice.

4,4'-Dimethylaminorex (4,4'-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1-60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4'-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers' co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.

An Innovative Approach to Characterize Clinical ADME and Pharmacokinetics of the Inhaled Drug Nemiralisib Using an Intravenous Microtracer Combined with an Inhaled Dose and an Oral Radiolabel Dose in Healthy Male Subjects.

An innovative open-label, crossover clinical study was used to investigate the excretion balance, pharmacokinetics, and metabolism of nemiralisib-an inhaled phosphoinositide 3-kinase delta inhibitor being developed for respiratory diseases. Six healthy men received a single intravenous microtracer of 10 µg [14C]nemiralisib with a concomitant inhaled nonradiolabeled 1000 µg dose followed by an oral 800 µg dose of [14C]nemiralisib 14 days later. Complementary methods including accelerator mass spectrometry allowed characterization of a range of parameters including oral absorption (Fabs), proportion of nemiralisib escaping gut wall metabolism (Fg), hepatic extraction (Eh), fraction of dose absorbed from inhaled dose (Flung), and renal clearance. Intravenous pharmacokinetics of nemiralisib were characterized by low blood clearance (10.0 l/h), long terminal half-life (55 hours), and high volume of distribution at steady state (728 l). Nemiralisib exhibited moderate inhaled and oral bioavailability (38% and 35%) while Flung was 29%. Absorption and first-pass parameters were corrected for blood renal clearance and compared with values without correction. Any swallowed nemiralisib was relatively well absorbed (Fabs, 0.48) with a high fraction escaping gut wall metabolism and low extraction by the liver (Fg and Eh being 0.83 and 0.10, respectively). There were no major human plasma metabolites requiring further qualification in animal studies. Both unchanged nemiralisib and its oxidative/conjugative metabolites were secreted in bile, with nemiralisib likely subject to further metabolism through enterohepatic recirculation. Direct renal clearance and metabolism followed by renal clearance were lesser routes of elimination. SIGNIFICANCE STATEMENT: A number of innovative features have been combined into one small clinical study enabling a comprehensive description of the human pharmacokinetics and metabolism of an inhaled molecule. Design elements included an intravenous 14C tracer administration concomitant with an inhalation dose that enabled derivation of parameters such as fraction absorbed (Fabs), the proportion of drug escaping first-pass extraction through the gut wall and liver (Fg and Fh) and hepatic extraction (Eh). Entero-test bile sampling enabled characterization of biliary elimination pathways.

Analysis of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA) as a new potential biomarker of exposure to vinclozolin in urine.

Vinclozolin (V) is a fungicide with anti-androgenic properties whose metabolism is not fully understood, and data on urinary elimination of either V or its metabolites are limited. Therefore the kinetics of urinary elimination of V and its metabolites, after an oral dose in adult male rats were investigated. A single oral dose of V (100 mg/kg) suspended in corn oil was administered to male adult Wistar rats, and urine was collected at different times after dosing. V and its metabolites were extracted from urine, then enzymatically hydrolyzed using ß-glucuronidase/sulfatase of H. pomatia, and analyzed by HPLC/DAD. Urinary pharmacokinetic parameters were calculated using the analyte concentrations adjusted by creatinine levels. V and its metabolites 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA, formerly denoted as M5), 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3,5-dichloroaniline (M3), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) were efficiently detected. The mean urine concentrations of V and M1 metabolite were fitted to a two-compartmental model for pharmacokinetic analysis. DTMBA approximately represented 88% of the total excreted metabolites, it was easily detected up to 168 h after dosing and its half-lives were 21.5 and 74.1 h, respectively. M1 was the second most abundant metabolite and was detected up to 144 h after being void. V and M3 were detected before 48 h, and M2 exhibited the lowest levels during the first 8 h after dosing. DTMBA, the most abundant V metabolite is quickly eliminated by urine, it is chemically stable, specific and could represent a useful alternative to be used as a biomarker of exposure to V.

Absorption, distribution and excretion of the anti-tuberculosis drug delamanid in rats: Extensive tissue distribution suggests potential therapeutic value for extrapulmonary tuberculosis.

Delamanid (OPC-67683, Deltyba™, nitro-dihydro-imidazooxazoles derivative) is approved for the treatment of adult pulmonary multidrug-resistant tuberculosis. The absorption, distribution and excretion of delamanid-derived radioactivity were investigated after a single oral administration of 14 C-delamanid at 3 mg/kg to rats. In both male and female rats, radioactivity in blood and all tissues reached peak levels by 8 or 24 h post-dose, and thereafter decreased slowly. Radioactivity levels were 3- to 5-fold higher in lung tissue at time to maximum concentration compared with plasma. In addition, radioactivity was broadly distributed in various tissues, including the central nervous system, eyeball, placenta and fetus, indicating that 14 C-delamanid permeated the brain, retinal and placental blood barriers. By 168 h post-dose, radioactivity in almost all the tissues was higher than that in the plasma. Radioactivity was also transferred into the milk of lactating rats. Approximately 6% and 92% of radioactivity was excreted in the urine and feces, respectively, indicating that the absorbed radioactivity was primarily excreted via the biliary route. No significant differences in the absorption, distribution and excretion of 14 C-delamanid were observed between male and female rats. The pharmacokinetic results suggested that delamanid was broadly distributed to the lungs and various tissues for a prolonged duration of time at concentrations expected to effectively target tuberculosis bacteria. These data indicate that delamanid, in addition to its previously demonstrated efficacy in pulmonary tuberculosis, might be an effective therapeutic approach to treating extrapulmonary tuberculosis. Copyright © 2017 John Wiley & Sons, Ltd.

An efficient data-filtering strategy for easy metabolite detection from the direct analysis of a biological fluid using Fourier transform mass spectrometry.

RATIONALE: High-throughput analyses require an overall analytical workflow including not only a robust and high-speed technical platform, but also dedicated data-processing tools able to extract the relevant information. This work aimed at evaluating post-acquisition data-mining tools for selective extraction of metabolite species from direct introduction high-resolution mass spectrometry data. METHODS: Investigations were performed on spectral data in which seven metabolites of vinclozolin, a dicarboximide fungicide containing two chloride atoms, were previously manually identified. The spectral data obtained from direct introduction (DI) and high-resolution mass spectrometry (HRMS) detection were post-processed by plotting the mass defect profiles and applying various data-filtering methods based on accurate mass values. RESULTS: Exploration of mass defect profiles highlighted, in a specific plotting region, the presence of compounds containing common chemical elements and pairs of conjugated and non-conjugated metabolites resulting from classical metabolic pathways. Additionally, the judicious application of mass defect and/or isotope pattern filters removed many interfering ions from DI-HRMS data, greatly facilitating the detection of vinclozolin metabolites. Compared with previous results obtained by manual data treatment, three additional metabolites of vinclozolin were detected and putatively annotated. CONCLUSIONS: Tracking simultaneously several specific species could be efficiently performed using data-mining tools based on accurate mass values. The selectivity of the data extraction was improved when the isotope filter was used for halogenated compounds, facilitating metabolite ion detection even for low-abundance species. Copyright © 2016 John Wiley & Sons, Ltd.

Identification of xenobiotic metabolites from biological fluids using flow injection analysis high-resolution mass spectrometry and post-acquisition data filtering.

RATIONALE: Concern for public health entails the need to evaluate the degree of exposure of population to toxicants. To do this, robust high-throughput approaches are required to be able to perform a large number of analyses in cohort studies. In this study, a data-filtering procedure was applied to mass spectral data acquired by direct analysis of biological fluids leading to rapid detection of metabolites in a model xenobiotic system. METHODS: Flow injection analysis (FIA) coupled to negative electrospray ionization (ESI)-LTQ Orbitrap Fourier transform mass spectrometry was used to directly analyze urine of rats treated with vinclozolin. Tandem mass spectrometry (MS/MS) experiments were subsequently performed for confirmation of a new metabolite structure. The isotope filtering based on the difference between accurate masses of (35)Cl and (37)Cl was applied to the raw data for the specific detection of ions containing at least one chlorine atom. RESULTS: Seven metabolites of vinclozolin were manually identified thanks to the characteristic isotope pattern of dichlorinated compounds. A new metabolite of vinclozolin was detected for the first time and identified as a sulfate conjugate. The application of an isotope-filtering procedure allowed the selective extraction of pertinent signals from the data. The processed mass spectrum was greatly simplified, significantly facilitating the detection of the seven metabolites previously identified. CONCLUSIONS: The use of FIA-HRMS in combination with dedicated bio-informatics data processing is shown to be an efficient approach for the rapid detection of metabolites in biological fluids. This is a very promising high-throughput approach for rapid characterization of the exposure status to xenobiotics.

Metabolism and disposition of 14C-labeled peliglitazar in humans.

The metabolism and disposition of dual (14)C-labeled peliglitazar, a dual α/γ peroxisome proliferator-activated receptor activator, was investigated in 10 healthy male subjects with and without bile collection (groups 1 and 2) after a single 10-mg oral dose. Serial blood samples, urine, and feces (0-240 h) as well as bile samples (3-8 h after dosing from group 2 subjects) were collected. The maximum plasma concentration (C(max)) of drug was reached at approximately 1 h and the elimination half-life (t(1/2)) was approximately 3.5 h. The exposure to drug metabolites (C(max) and area under the plasma concentration versus time curve) was not significantly different between the two groups. The parent compound and its 1-O-ß-acyl-glucuronide conjugate were the major components in plasma; other circulating metabolites, including several other glucuronide conjugates, were minor components at all time points. The major portion of the radioactive dose was recovered in feces (94% for group 1 and 32% for group 2). Approximately 24% of the radioactive dose was recovered in the bile from group 2 subjects, nearly all of which was assigned as glucuronides of peliglitazar and its oxidative metabolites (M14, M14a, M14b, M15, M15a, M15b, and M17). In contrast, fecal samples contained peliglitazar and its oxidative metabolites resulting from aliphatic/aryl hydroxylation, and O-demethylation. These results suggested that the major clearance pathway of peliglitazar was through biliary elimination of glucuronide conjugates, which were hydrolyzed to peliglitazar and its oxidative metabolites in the intestines before excretion.

Qualitative confirmation procedure for ephedrines as acetonide derivatives in doping urine samples by gas chromatography/electron ionization mass spectrometry.

Ephedrines are sympathomimetic amines which have central nervous system stimulating properties and, for this reason, some of them are forbidden in sport by the World Antidoping Agency (WADA). They are screened and quantitated in urine by several published techniques and confirmed by gas chromatography/mass spectrometry (GC/MS). In this paper, a simple and easy confirmation procedure for norpseudoephedrine, norephedrine, ephedrine and pseudoephedrine in human urine by GC/electron ionization (EI)-MS is described. After the addition of diphenylamine as internal standard, a liquid-liquid extraction procedure under alkaline conditions with tert-butyl methyl ether was applied to the samples. The analytes were derivatized with acetone and pyridine to form the correspondent oxazolidine derivatives (acetonide). The EI mass spectra of all the studied substances have many diagnostic ions with relative abundance in accordance with WADA requirements and show great structural information content. The fragmentation of theses derivatives is discussed.

Biotransformation of vinclozolin in rat precision-cut liver slices: comparison with in vivo metabolic pattern.

Vinclozolin is a dicarboxymide fungicide that presents antiandrogenic properties through its two hydrolysis products M1 and M2, which bind to the androgen receptor. Because of the lack of data on the biotransformation of vinclozolin, its metabolism was investigated in vitro in precision-cut rat liver slices and in vivo in male rat using [ (14)C]-vinclozolin. Incubations were performed using different concentrations of substrate, and the kinetics of formation of the major metabolites were studied. Three male Wistar rats were fed by gavage with [ (14)C]-VZ. Urine was collected for 24 h and analyzed by radio-HPLC for metabolic profiling. Metabolite identification was carried out on a LCQ ion trap mass spectrometer. In rat liver slices and in vivo, the major primary metabolite has been identified as 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide (M5) and was mainly present as glucuronoconjugates. M5 is produced by dihydroxylation of the vinyl group of M2. Other metabolites have been identified as 3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione (M4), a dihydroxylated metabolite of vinclozolin, which undergoes further conjugation to glucuronic acid, and 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3,4-dihydroxy-butanoic acid (M6), a dihydroxylated metabolite of M1.

NMR analysis of male fathead minnow urinary metabolites: a potential approach for studying impacts of chemical exposures.

The potential for profiling metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy was used for the assignment of metabolites in urine from unexposed fish. Because fathead minnow urine is dilute, we lyophilized these samples prior to analysis. Furthermore, 1D 1H NMR spectra of unlyophilized urine from unexposed male fathead minnow and Sprague-Dawley rat were acquired to qualitatively compare rat and fish metabolite profiles and to provide an estimate of the total urinary metabolite pool concentration difference. As a small proof-of-concept study, lyophilized urine samples from male fathead minnows exposed to three different concentrations of the antiandrogen vinclozolin were analyzed by 1D 1H NMR to assess exposure-induced changes. Through a combination of principal components analysis (PCA) and measurements of 1H NMR peak intensities, several metabolites were identified as changing with statistical significance in response to exposure. Among those changes occurring in response to exposure to the highest concentration (450 microg/L) of vinclozolin were large increases in taurine, lactate, acetate, and formate. These increases coincided with a marked decrease in hippurate, a combination potentially indicative of hepatotoxicity. The results of these investigations clearly demonstrate the potential utility of an NMR-based approach for assessing chemical exposures in male fathead minnow, using urine collected from individual fish.

Analysis of 3,5-dichloroaniline as a biomarker of vinclozolin and iprodione in human urine using liquid chromatography/triple quadrupole mass spectrometry.

The fungicides vinclozolin and iprodione are widely used in agriculture. These pesticides are dicarboximide fungicides containing the common moiety 3,5-dichloroaniline (3,5-DCA). It has been suggested that low-level exposures to such compounds may be associated with adverse health effects such as endocrine disruption. In this study a method using liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) was developed for the analysis of 3,5-DCA as a biomarker of exposure to these fungicides in human urine. The urine samples were treated by basic hydrolysis to degrade the fungicides, their metabolites and conjugates to 3,5-DCA. The 3,5-DCA was then extracted using toluene and derivatized using pentafluoropropionic anhydride (PFPA). Analysis of the derivative was carried out using selected reaction monitoring (SRM) in the negative ion mode. Quantification of the derivative was performed using [(13)C(6)]-labeled 3,4-DCA as an internal standard with good precision and linearity in the range 0.1-200 ng/mL urine. The limit of detection was determined to be 0.1 ng/mL. The metabolites in urine were found to be stable during storage at -20 degrees C. To validate 3,5-DCA as a biomarker the method was applied in a human experimental exposure to iprodione and vinclozolin. Two healthy volunteers received 200 microg single oral doses of each pesticide followed by urine sampling during 72-120 h post-exposure. Between 78-107% of the dose was recovered as 3,5-DCA in the urine after exposure.

Subchronic urinary bladder effects of muraglitazar in male rats.

Muraglitazar, a PPARalpha/gamma dual agonist, was dosed orally to rats once daily for 13 weeks to evaluate urinary and urothelial changes of potential relevance to urinary bladder tumorigenesis. Groups of 17 young or aged rats per sex were fed a normal or 1% NH4Cl-supplemented diet and were dosed with 0, 1, or 50 mg/kg muraglitazar. Lithogenic ions and sediment were profiled from freshly voided urine samples collected 24 h after dosing, and drug exposures were measured. Urinary citrate, oxalate, and epidermal growth factor (EGF) were assayed from 18-h urine collections. Urothelium was assessed by light microscopy, scanning electron microscopy, and BrdU and TUNEL immunohistochemistry. When fed a normal diet, urine pH was higher in males (above 6.5). Urine volume/body weight was greater in females. Urine soluble/total calcium and magnesium and phosphorus/creatinine ratios were lower in male rats fed a normal diet. Urine citrate levels were decreased and oxalate was increased in young male rats treated with 50 mg/kg muraglitazar compared to age/sex/diet-matched controls. No changes in urine sediment were detected 24 h after dosing. In young male rats treated with 50 mg/kg on normal diet, multifocal urothelial necrosis and proliferation were observed, whereas urothelial apoptosis and urine EGF levels were unchanged compared to age/sex/diet-matched controls. Urothelial necrosis and proliferation were not correlated to systemic or urinary drug exposures and were prevented by dietary acidification. These data suggest that muraglitazar-associated changes in urine composition predispose to urothelial cytotoxicity and proliferation in the urinary bladder of young male rats and that urine sediment must be profiled at multiple daily timepoints to fully qualify drug-induced changes in urine composition.

Comparative metabolism of radiolabeled muraglitazar in animals and humans by quantitative and qualitative metabolite profiling.

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) activator, has both glucose- and lipid-lowering effects in animal models and in patients with diabetes. This study describes the in vivo and in vitro comparative metabolism of [(14)C]muraglitazar in rats, dogs, monkeys, and humans by quantitative and qualitative metabolite profiling. Metabolite identification and quantification methods used in these studies included liquid chromatography/mass spectrometry (LC/MS), LC/tandem MS, LC/radiodetection, LC/UV, and a newly described mass defect filtering technique in conjunction with high resolution MS. After oral administration of [(14)C]muraglitazar, absorption was rapid in all species, reaching a concentration peak for parent and total radioactivity in plasma within 1 h. The most abundant component in plasma at all times in all species was the parent drug, and no metabolite was present in greater than 2.5% of the muraglitazar concentrations at 1 h postdose in rats, dogs, and humans. All metabolites observed in human plasma were also present in rats, dogs, or monkeys. Urinary excretion of radioactivity was low (<5% of the dose) in all intact species, and the primary route of elimination was via biliary excretion in rats, monkeys, and humans. Based on recovered doses in urine and bile, muraglitazar showed a very good absorption in rats, monkeys, and humans. The major drug-related components in bile of rats, monkeys, and humans were glucuronides of muraglitazar and its oxidative metabolites. The parent compound was a minor component in bile, suggesting extensive metabolism of the drug. In contrast, the parent drug and oxidative metabolites were the major components in feces, and no glucuronide conjugates were found, suggesting that glucuronide metabolites were excreted in bile and hydrolyzed in the gastrointestinal tract. The metabolites of muraglitazar resulted from both glucuronidation and oxidation. The metabolites in general had greatly reduced activity as PPARalpha/gamma activators relative to muraglitazar. In conclusion, muraglitazar was rapidly absorbed, extensively metabolized through glucuronidation and oxidation, and mainly eliminated in the feces via biliary excretion of glucuronide metabolites in all species studied. Disposition and metabolic pathways were qualitatively similar in rats, dogs, monkeys, and humans.

Glucuronidation as a major metabolic clearance pathway of 14c-labeled muraglitazar in humans: metabolic profiles in subjects with or without bile collection.

The metabolism and disposition of 14C-labeled muraglitazar (Pargluva), a novel dual alpha/gamma peroxisome proliferator-activated receptor activator, was investigated in eight healthy male subjects with and without bile collection (groups 1 and 2) after a single 20-mg oral dose. Bile samples were collected for 3 to 8 h after dosing from group 2 subjects in addition to the urine and feces collection. In plasma, the parent compound was the major component, and circulating metabolites, including several glucuronide conjugates, were minor components at all time points. The exposure to parent drug (Cmax and area under the plasma concentration versus time curve) in subjects with bile collection was generally lower than that in subjects without bile collection. The major portion of the radioactive dose was recovered in feces (91% for group 1 and 51% for group 2). In addition, 40% of the dose was recovered in the bile from group 2 subjects. In this 3- to 8-h bile, the glucuronide of muraglitazar (M13, 15% of dose) and the glucuronides of its oxidative metabolites (M17a,b,c, M18a,b,c, and M20, together, 16% of dose) accounted for approximately 80% of the biliary radioactivity; muraglitazar and its O-demethylated metabolite (M15) each accounted for approximately 4% of the dose. In contrast, fecal samples only contained muraglitazar and its oxidative metabolites, suggesting hydrolysis of biliary glucuronides in the intestine before fecal excretion. Thus, the subjects with and without bile collection showed different metabolic profiles of muraglitazar after oral administration, and glucuronidation was not observed as a major pathway of metabolic clearance from subjects with the conventional urine and fecal collection, but was found as a major elimination pathway from subjects with bile collection.

Structural elucidation of human oxidative metabolites of muraglitazar: use of microbial bioreactors in the biosynthesis of metabolite standards.

Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor activator, is currently in clinical development for treatment of type 2 diabetes. This study describes the structural elucidation of the human oxidative metabolites of muraglitazar through the use of a combination of microbial bioreactors, NMR and accurate mass analyses, and organic synthesis. Plasma, urine, and feces were collected from six healthy subjects following oral administration of 14C-labeled muraglitazar (10 mg, 100 microCi) and pooled samples were analyzed. Approximately 96% of the recovered radioactive dose was found in the feces and 3.5% in the urine. The parent compound represented >85% of the radioactivity in plasma. The fecal radioactivity was distributed among 16 metabolites (M1-M12, M14-M16, and M8a) and the parent drug, of which hydroxylation and O-demethylation metabolites (M5, M10, M11, M14, and M15) represented the prominent human metabolites. The urinary radioactivity was distributed into several peaks including muraglitazar glucuronide (M13) and the parent drug. Low concentrations of metabolites in human samples prevented direct identification of metabolites beyond liquid chromatographic (LC)-mass spectrometric analysis. Microbial strains Cunninghamella elegans and Saccharopolyspora hirsuta produced muraglitazar metabolites that had the same high performance liquid chromatography retention times and the same tandem mass spectrometric (MS/MS) properties as the corresponding human metabolites. The microbial metabolites M9, M10, M11, M14, M15, and M16 were isolated and analyzed by NMR. Based on these LC-MS/MS and NMR analyses, and organic synthesis, the structures of 16 human oxidative metabolites were identified. The oxidative metabolism of muraglitazar was characterized by hydroxylation, O-demethylation, oxazolering opening, and O-demethylation/hydroxylation, as well as O-dealkylation and carboxylic acid formation. This study demonstrated the utility of microbial bioreactors for the identification of metabolites.

Determination and validation of a simple high-performance liquid chromatographic method for simultaneous assay of iprodione and vinclozolin in human urine.

A method based on solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) was developed for the simultaneous determination of 3-(3,5-diclorophenyl)-5-ethenyl-5-methyl-2,4-oxazolidinedione (vinclozolin) and 3-(3,5-diclorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide (iprodione) in human urine. Urine samples containing vinclozolin and iprodione were collected by solid phase extraction using C(18) cartridges. The chromatographic separation was achieved on a Spherisorb ODS2 (250 mm x 4.6 mm, 5 microm) column with an isocratic mobile phase of acetonitrile-water (60:40, v/v). Detection was UV absorbance at 220 nm. The calibration graphs were linear from 30 to 1000 ng/mL for the two fungicides. Intra- and inter-day R.S.D. did not exceed 2.9%. The quantitation limit was 50 ng/mL for vinclozolin and 30 ng/mL for iprodione, respectively.

Glucuronidation of 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetic acid (TA-1801A) in humans: species differences in liver and intestinal microsomes.

The metabolism of ethyl 2-(4-chlorophenyl)-5-(2-furyl)-4-oxazoleacetate (TA-1801), a potent hypolipidemic agent, was studied in humans after oral administration and compared with that found in rats, rabbits, and dogs previously. Hydrolysis of the ethyl ester to produce metabolite M1 (TA-1801 active form; TA-1801A) is the first metabolic step and the subsequent biotransformation includes the glucuronidation to form the metabolite M4 and the oxidation to form the metabolites M2 and M3. The metabolism of TA-1801 in humans was qualitatively similar to that in the experimental animals studied, although species differences were seen in the amount of metabolites. M4, the glucuronide of TA-1801A was the most abundant metabolite in human urine (24.3% of the dose). In vitro studies using human liver and jejunum microsomes indicated that the TA-1801A glucuronosyltransferase activity in human jejunum microsomes was 2-fold higher than that in liver microsomes. With regard to the interspecies differences in the TA-1801A glucuronosyltransferase activities, the intrinsic clearance for the TA-1801A glucuronidation in liver microsomes was in the following order: rabbit>monkey>human=rat=dog. In jejunum microsomes, the intrinsic clearance for the TA-1801A glucuronidation was in the following order: human>monkey>rabbit>rat=dog. These results suggest that the species differences in the intestinal TA-1801A glucuronidation contribute to the species differences in the excretion rate of TA-1801A glucuronide into the urine.

Detection and assay of cis- and trans-isomers of 4-methylaminorex in urine, plasma and tissue samples.

The 4-methylaminorex (4-MAX) is an amphetamine-related psychostimulant drug that has appeared on the clandestine market with a street name of "U4Euh". This compound exists as four stereoisomers, trans-4R,5R, trans-4S,5S, cis-4R,5S and cis-4S,5R, of which the cis forms have been classified as Schedule I substances in the US. The increasing variety of designer drugs has highlighted the importance of detection, identification, and quantitative measurement of these drugs, including 4-MAX, in biological samples. In the present study, the isomers of 4-MAX were detected in urine of rats treated with the drugs by some but not all of the on-site immunoassays tested, mainly as amphetamine or methamphetamine. To facilitate identification of 4-MAX by laboratories specialized in drug analysis, the electron-ionization mass spectrum and TLC data for underivatized 4-MAX using a routine laboratory drug-screening procedure is provided. In addition, a GC/MS method is described for the quantitative determination of cis- and trans-4-MAX as tert-butyldimethylsilyl-derivatives in plasma, urine and tissue.

Disposition of radiolabeled ifetroban in rats, dogs, monkeys, and humans.

Ifetroban is a potent and selective thromboxane receptor antagonist. This study was conducted to characterize the pharmacokinetics, absolute bioavailability, and disposition of ifetroban after i.v. and oral administrations of [14C]ifetroban or [3H]ifetroban in rats (3 mg/kg), dogs (1 mg/kg), monkeys (1 mg/kg), and humans (50 mg). The drug was rapidly absorbed after oral administration, with peak plasma concentrations occurring between 5 and 20 min across species. Plasma terminal elimination half-life was approximately 8 h in rats, approximately 20 h in dogs, approximately 27 h in monkeys, and approximately 22 h in humans. Based on the steady-state volume of distribution, the drug was extensively distributed in tissues. Absolute bioavailability was 25, 35, 23, and 48% in rats, dogs, monkeys, and humans, respectively. Renal excretion was a minor route of elimination in all species, with the majority of the dose being excreted into the feces. After a single oral dose, urinary excretion accounted for 3% of the administered dose in rats and dogs, 14% in monkeys, and 27% in humans, with the remainder excreted in the feces. Extensive biliary excretion was observed in rats with the hydroxylated metabolite at the C-14 position being the major metabolite observed in rat bile. Ifetroban was extensively metabolized after oral administration. Approximately 40 to 50% of the radioactivity in rat and dog plasma was accounted for by parent drug whereas, in humans, approximately 60% of the plasma radioactivity was accounted for by ifetroban acylglucuronide.

The absolute bioavailability and metabolic disposition of the novel antimigraine compound zolmitriptan (311C90).

AIMS: Two open studies in healthy volunteers were conducted to determine the absolute bioavailability and metabolic disposition of zolmitriptan (311C90), a novel 5HT1D agonist for the acute treatment of migraine. METHODS: After an initial test i.v. infusion, bioavailability was assessed by comparison of AUC after an i.v. infusion (3.5 mg) and an oral tablet (10 mg), in six men and six women using a randomised, crossover design. Disposition was studied by administration of a 25 mg capsule, labelled with 100 microCi [14C]-zolmitriptan, to five men and one woman on a single occasion. RESULTS: Zolmitriptan was well tolerated by both i.v. and oral routes. Adverse events were mostly mild, consistent with earlier studies and characteristic of this class of drug. Reports were similar in nature and number after both oral and i.v. dosing. Mean +/- s.d. oral bioavailability was 0.49 +/- 0.24 (0.38 +/- 0.16 in men and 0.60 +/- 0.28 in women). After oral dosing, Cmax and AUC values in women were approximately double those in men. Relative to zolmitriptan concentrations, metabolite concentrations were higher after oral dosing than after i.v., and higher in men compared with women. Half-life was significantly longer after oral dosing (mean 22%, 95% CI 6-35%). Mean +/- s.d. values for CL, V2 and t1/2,z after i.v. dosing (all subjects) were 8.7 +/- 1.7 ml min-1 kg-1, 122 +/- 321 and 2.30 +/- 0.59 h respectively. Following administration of 25 mg [14C]-zolmitriptan, 91.5% of the dose was recovered in 7 days, 64.4 +/- 6.5% in urine and 27.1 +/- 6.0% in faeces. Less than 10% was recovered unchanged in urine, with 31.1 +/- 6.4% recovered as the inactive indole acetic acid metabolite. Most of the faecal material was unchanged zolmitriptan, representing unabsorbed drug. Plasma concentrations of [14C] were slightly higher than those of the summed concentrations of known analytes zolmitriptan, the active N-desmethyl metabolite (183C91), the inactive N-oxide (1652W92) and indole acetic acid (2161W92) metabolites, which accounted for 86% of total plasma radioactivity. No other significant metabolites were detected in plasma. Some minor additional metabolites were detected in urine, none of which contributed more than 5% of the dose. CONCLUSIONS: The data suggest that zolmitriptan undergoes first-pass metabolism and this is more extensive in men than in women. Zolmitriptan has suitable bioavailability for an acute oral migraine treatment and there are no significant unidentified metabolites in man.