Integrated histopathological and urinary metabonomic investigation of the pathogenesis of microcystin-LR toxicosis.

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Pharmacokinetics, metabolism and excretion of an inhibitor of inducible nitric oxide synthase, L-NIL-TA, in dog.

1. The pharmacokinetics, metabolism and excretion of L-NIL-TA, an inducible nitric oxide synthase inhibitor, were investigated in dog. 2. The dose of [14C]L-NIL-TA was rapidly absorbed and distributed after oral and intravenous administration (5 mg kg-1), with Cmax of radioactivity of 6.45-7.07 microg equivalents g-1 occurring at 0.33-0.39-h after dosing. After oral and intravenous administration, radioactivity levels in plasma then declined with a half-life of 63.1 and 80.6-h, respectively. 3. Seven days after oral and intravenous administrations, 46.4 and 51.5% of the radioactive dose were recovered in urine, 4.59 and 2.75% were recovered in faeces, and 22.4 and 22.4% were recovered in expired air, respectively. The large percentages of radioactive dose recovered in urine and expired air indicate that [14C]L-NIL-TA was well absorbed in dogs and the radioactive dose was cleared mainly through renal elimination. The mean total recovery of radioactivity over 7 days was approximately 80%. 4. Biotransformation of L-NIL-TA occurred primarily by hydrolysis of the 5-aminotetrazole group to form the active drug L-N6-(1-iminoethyl)lysine (NIL or M3), which was further oxidized to the 2-keto acid (M5), the 2-hydroxyl acid (M1), an unidentified metabolite (M2) and carbon dioxide. The major excreted products in urine were M1 and M2, representing 22.2 and 21.2% of the dose, respectively.

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry.

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.

Comparison of celecoxib metabolism and excretion in mouse, rabbit, dog, cynomolgus monkey and rhesus monkey.

1. The metabolism and excretion of celecoxib, a specific cyclooxygenase 2 (COX-2) inhibitor, was investigated in mouse, rabbit, the EM (extensive) and PM (poor metabolizer) dog, and rhesus and cynomolgus monkey. 2. Some sex and species differences were evident in the disposition of celecoxib. After intravenous (i.v.) administration of [14C]celecoxib, the major route of excretion of radioactivity in all species studied was via the faeces: EM dog (80.0%), PM dog (83.4%), cynomolgus monkey (63.5%), rhesus monkey (83.1%). After oral administration, faeces were the primary route of excretion in rabbit (72.2%) and the male mouse (71.1%), with the remainder of the dose excreted in the urine. After oral administration of [14C]celecoxib to the female mouse, radioactivity was eliminated equally in urine (45.7%) and faeces (46.7%). 3. Biotransformation of celecoxib occurs primarily by oxidation of the aromatic methyl group to form a hydroxymethyl metabolite, which is further oxidized to the carboxylic acid analogue. 4. An additional phase I metabolite (phenyl ring hydroxylation) and a glucuronide conjugate of the carboxylic acid metabolite was produced by rabbit. 5. The major excretion product in urine and faeces of mouse, rabbit, dog and monkey was the carboxylic acid metabolite of celecoxib.

Determination of free levels of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry.

A liquid chromatography combined with tandem mass spectrometry assay for the determination of free levels of the highly protein bound drug phenytoin (5,5-diphenylhydantoin) in human plasma is described. The assay was demonstrated to be reliable, accurate and precise, and specific for phenytoin. The procedure involves isolation of the unbound drug from the drug/protein complex by ultrafiltration. Liquid-liquid extraction was employed to extract the resultant ultrafiltrate. PHT was separated on a 50 x 3 mm reversed-phase column using isocratic mobile phase conditions that yielded a run time of 1.5 min, enabling high throughput sample analysis. Linearity was obtained over the range 5.00 to 500 ng/ml. Both between-run and within-run coefficients of variation were less than 15% and accuracy's across the assay range were all within 100 +/- 10%. The assay was successfully implemented to support a clinical interaction study with phenytoin.

Pharmacokinetics, tissue distribution, metabolism, and excretion of celecoxib in rats.

The pharmacokinetics, tissue distribution, metabolism, and excretion of celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide, a cyclooxygenase-2 inhibitor, were investigated in rats. Celecoxib was metabolized extensively after i.v. administration of [(14)C]celecoxib, and elimination of unchanged compound was minor (less than 2%) in male and female rats. The only metabolism of celecoxib observed in rats was via a single oxidative pathway. The methyl group of celecoxib is first oxidized to a hydroxymethyl metabolite, followed by additional oxidation of the hydroxymethyl group to a carboxylic acid metabolite. Glucuronide conjugates of both the hydroxymethyl and carboxylic acid metabolites are formed. Total mean percent recovery of the radioactive dose was about 100% for both the male rat (9.6% in urine; 91.7% in feces) and the female rat (10.6% in urine; 91.3% in feces). After oral administration of [(14)C]celecoxib at doses of 20, 80, and 400 mg/kg, the majority of the radioactivity was excreted in the feces (88-94%) with the remainder of the dose excreted in the urine (7-10%). Both unchanged drug and the carboxylic acid metabolite of celecoxib were the major radioactive components excreted with the amount of celecoxib excreted in the feces increasing with dose. When administered orally, celecoxib was well distributed to the tissues examined with the highest concentrations of radioactivity found in the gastrointestinal tract. Maximal concentration of radioactivity was reached in most all tissues between 1 and 3 h postdose with the half-life paralleling that of plasma, with the exception of the gastrointestinal tract tissues.

Effects of LY295427, a low-density lipoprotein (LDL) receptor up-regulator, on LDL receptor gene transcription and cholesterol metabolism in normal and hypercholesterolemic hamsters.

The action of LY295427 [(3alpha,4alpha, 5alpha)-4-(2-propenylcholestan-3-ol)], a compound that derepresses low-density lipoprotein receptor (LDL-R) expression in a cell-based model, was examined in hamsters. It was found that the compound does not have an effect in normal chow-fed hamsters, in which LDL-R levels are not repressed, but exerts a marked hypocholesterolemic effect (>70% decrease) in cholesterol-coconut oil-fed hamsters, in which LDL-R is repressed. In this model, there is a dose-response for cholesterol lowering with an approximate ED50 value of 40 mg/kg/day and an inverse relationship between serum cholesterol and serum LY295427 levels. LDL-R mRNA is increased (2-fold) and liver cholesterol ester content is decreased (>90%). Unlike the 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor lovastatin, the decreased serum cholesterol is confined to the non-high-density lipoprotein fraction. Furthermore, LY295427 does not affect cholesterol biosynthesis, and it does not have a significant effect on cholesterol absorption. These data suggest that LY295427 acts in the hypercholesterolemic hamster by derepressing LDL-R transcription, thereby enhancing cholesterol clearance from the blood. The results with LY295427 suggest that compounds that act to increase LDL-R may represent a novel approach in the pharmacotherapy for hypercholesterolemia.

Plasma protein binding of celecoxib in mice, rat, rabbit, dog and human.

The plasma protein binding of celecoxib was determined for animals and humans using in vitro and ex vivo methods. Eight, healthy, human volunteers (three male, five female, 20-39 years) received celecoxib (600 mg) BID for 7 days, blood samples were collected and concentrations of bound and unbound celecoxib determined. The fraction of bound drug in the volunteers was constant (97.4 +/- 0.1%) at total celecoxib plasma concentrations ranging from 0.01 to 4.02 microg/mL. The ex vivo plasma protein binding of celecoxib in the animals was concentration-independent up to approximately 12, 8 and 10 microg/mL for mouse, rat and dog, respectively. The plasma protein binding of celecoxib after a single oral dose of 10 and 300 mg/kg to mice was 98.3 +/- 0.2%, of 1 and 400 mg/kg to rats was 98.3 +/- 0.2% and of 1 and 100 mg/kg to dogs was 98.5 +/- 0.1%. The percent binding of celecoxib to plasma proteins in vitro was slightly lower than those values determined ex vivo. The in vitro binding of celecoxib to plasma protein was constant over the concentrations of 0.1-10 microg/mL for all species, except rat.

Oxidative and reductive metabolism of 9-cis-retinoic acid in the rat. Identification of 13,14-dihydro-9-cis-retinoic acid and its taurine conjugate.

9-cis-Retinoic acid (9-cis-RA), a hormone that binds and activates all known retinoid receptor subtypes, is structurally similar to all-trans-retinoic acid and may share common metabolic fates. Both oral and intravenous doses of 9-cis-RA to rats led to hydroxylation and ketone formation at carbon-4. 9-Cis-RA also isomerized in vivo to 13-cis-retinoic acid, 9-cis, 13-cis-retinoic acid, and all-trans-retinoic acid. After administration of [11-3H]9-cis-RA, the proportion of plasma radioactivity that was volatile increased over time, which suggested that beta-oxidative chain-shortening of 9-cis-RA might occur. An equimolar mixture of [1-13C2H3]9-cis-RA and 9-cis-RA was administered to rats for stable-isotope-labeled metabolite production. A chromatographic peak that had a lambdamax = 290 nm vs. 348 nm for the parent compound, had a retention time similar to the parent, and yielded a 1:1 positive-ion isotope cluster at m/z 303/307 in its mass spectrum. NMR analysis revealed 9-cis and 13,14-dihydro configurations, indicating that 9-cis-RA can be metabolized in rat by reduction to 13,14-dihydro-9-cis-RA. An earlier-eluting HPLC peak that exhibited a lambdamax at 290 nm, and a negative-ion-MS isotope cluster at m/z 408/412 was observed during separations of rat liver extracts. LC/MS/MS analysis revealed product ions for this peak diagnostic for carboxylic acid taurine conjugates. In rats, reduction of 9-cis-RA to 13,14-dihydro-9-cis-RA may represent an initial step leading to beta-oxidation, although available data demonstrate it is conjugated with taurine to form a novel metabolite.

Disposition of zatosetron, a serotonin (5-HT3) receptor antagonist, in humans.

Zatosetron is being tested clinically as an antianxiety agent; it is a highly selective antagonist of the serotonin 5-HT3 receptor, with minimal agonist activity. The disposition of [14C]zatosetron was studied in five healthy men after a single oral dose (46.2 mg). Serum levels of radioactivity and parent drug peaked in 3-8 hr. About 15% more radioactivity was measured in red blood cells than in plasma. In serum, the parent compound represented about 85% of the radioactivity, zatosetron-N-oxide represented 10%, and N-desmethyl-zatosetron and 3-hydroxy-zatosetron each represented 2-3%. The t1/2 of zatosetron was 25-37 hr. About 75% of zatosetron added to human plasma became reversibly bound to protein. Concentrations of zatosetron in saliva were generally 10-50% higher than those in serum. About 80% of the administered radioactivity was eliminated in urine and 20% in feces; radioactivity was measurable in the excreta for 10-12 days after drug administration. The major route of metabolism of zatosetron was a stereoselective N-oxidation to form 8-alpha-methyl, 8-beta-oxo zatosetron (zatosetron N-oxide). In urine, approximately 45% of the radioactivity was unchanged zatosetron, 35% was zatosetron N-oxide, 10% was N-desmethyl-zatosetron, and 5% was 3-hydroxy-zatosetron. In feces, 30% of the radioactivity was unchanged zatosetron, and 70% was N-desmethyl-zatosetron. Overall, approximately 60% of the administered zatosetron was metabolized in humans. In a separate multiple-dose study, the disposition of zatosetron was found to be similar to that in the single-dose study.

Biotransformation of the antiviral agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and characteristics of a mesoionic ribose metabolite.

The biotransformation of the antiinfluenza agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896, I) was studied. In addition to a urea metabolite (II) formed by transformation of the cyanamide functionality, another highly polar metabolite was found in mouse urine and in BSC-1, MDCK, and other cell culture incubations of [14C]LY217896. Using 13C-labeled LY217896 together with NMR and MS techniques, this highly polar metabolite was identified as a ribose derivative (III), which apparently exists in a mesoionic form (i.e. positive and negative charges within the same ring system). It was also found that this ribose is formed from LY217896 and ribose-1-phosphate in a reaction catalyzed by the enzyme purine nucleoside phosphorylase, but that the reverse reaction (cleavage of the ribose) is not observed under the conditions used. When tested in vitro using the same assay as that used to measure the antiviral activity of LY217896, this ribose and the urea metabolite exhibit essentially no activity. The presence of a ribose has been implicated in the activity of antiviral compounds such as ribavirin and anticancer agents like 2-aminothiadiazole and tiazofurin, which are structurally similar to LY217896. These activities have been postulated to involve either mono- or triphosphorylated forms, or NAD-type analogs. Possible implications of the formation of this mesoionic ribose metabolite for the mechanism of antiviral activity of LY217896 are discussed.

Characterization of LY233569 on 5-lipoxygenase and reperfusion injury of ischemic myocardium.

LY233569 produced concentration-dependent inhibition of isolated guinea pig 5-lipoxygenase (5-LPO) and 5-LPO activity of human polymorphonuclear leukocytes in vitro; IC50 values were 0.4 and 0.1 microM, respectively. LY233569 also inhibited (IC50 approximately 1.8 microM) zymosan-stimulated production of leukotriene B4 in canine whole blood but had little or no concomitant effect on the production of thromboxane B2. Concentrations of LY233569 as high as 10 microM did not inhibit production or scavenge superoxide from activated human neutrophils. In normal anesthetized dogs, infusion of LY233569 (0.11 mg/kg/min, i.v.) for 6 hr produced persistent inhibition (approximately 80%) of leukotriene B4 production in blood challenged ex vivo with zymosan; the plasma concentration (approximately 4 microM) of LY233569 was consistent with in vitro data illustrating selective and maximal inhibition of 5-LPO. In subsequent experiments, myocardial infarct size was measured after 1 hr of occlusion of the circumflex coronary artery and 5 hr of reperfusion. Continuous infusion of LY233569 (0.11 mg/kg/min, i.v.) had little or no effect on base-line systolic arterial pressure, cardiac rate and the pressure rate product when compared with time-related changes observed in control dogs. LY233569 infusion also did not alter the degree of ST-segment deviation or the intensity and duration of cardiac arrhythmias associated with coronary artery occlusion and reperfusion. Resultant myocardial infarct sizes were 45 +/- 5% of the left ventricle placed at risk in control dogs and 43 +/- 4% in dogs given LY233569. Myeloperoxidase activity of infarcted myocardium did not differ between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenic and cell transformation activities of representative phosphorothioate esters in vitro.

Five alkyl and five aryl phosphorothioates are ranked relative to parathion in effectiveness as base-pair mutagens in the Ames mutagenic assay. Three in each series were mutagenic. Two commercial insecticidal phosphates, included for comparison, were mutagenic. The mutagenic phosphorothioates contained a strong electron-withdrawing and/or a good leaving group, together with two other groups small enough to permit nucleophilic attack by a biomacromolecule on the electrophilic phosphorus atom. All but one of the phosphorothioates [i.e., O,O,O-tris(2,2,2-trifluoro)ethyl phosphorothioate, VI] required metabolic activation for mutagenicity to be manifested; hence most of the phosphorothioates per se evidently are not ordinarily sufficiently electrophilic to be mutagenic but must instead be transformed to more electrophilic oxygen-containing products. In evaluation for cell-transformation properties, methyl parathion was inactive, in contrast to VI. The phosphorothioates that were novel were synthesized by formation of the phosphite from the appropriate alcohol or phenol, followed by reaction of the phosphite with sulfur.

Thiono compounds. 4. In vitro mutagenic and antineoplastic activity of TEPA and thio-TEPA.

Tris (1-aziridinyl) phosphine oxide (TEPA) and tris (1-aziridinyl) phosphine sulfide (thio-TEPA) induced base pair mutations in the Ames mutagenic assay. Thio-TEPA required metabolic activation while TEPA was active without metabolic activation. Growth of a human vaginal carcinoma (A431), a human breast carcinoma (MDA-MB-231), and a human cervical carcinoma (HeLa) were inhibited in soft agar in vitro at concentrations which induced mutagenesis in the Ames Assay. A fourth line, JEG choriocarcinoma, was sensitive to the antigrowth properties of both drugs at concentrations below that which induced mutagenesis. These data suggest that as more antineoplastic agents become available, and as mean survival times increase, knowledge of the relative in vitro sensitivity of a patient's neoplasm to a specific antineoplastic drug (i.e., dose required for growth inhibition) as a function of its mutagenic index might be useful for prediction of clinical remission, as well as the risk of secondary neoplasm induction.

Mechanism of action of methyldopa in the rat. Role of 3-O-methylated metabolites.

We studied the effect of 3-O-methyl-methyldopa (OMMD), the 3-O-methylated metabolite of the antihypertensive drug methyldopa (alpha-methyldopa, AMD), on blood pressure in the spontaneously hypertensive rat. OMMD lowered blood pressure in a dose-related manner when given orally or intraperitoneally. Its action lasted longer than that of AMD, and daily oral administration produced a cumulative fall in blood pressure. Oral and intraperitoneal OMMD produced similar reductions of blood pressure and similar tissue OMMD levels. After intraperitoneal injection of different doses, levels of OMMD measured in brain, spinal cord, and plasma correlated with the magnitude of the antihypertensive effect. No AMD was detected in tissues after either route of administration, which suggests that the antihypertensive effect was not based on demethylation of OMMD to AMD. Peripheral inhibition of the enzyme, aromatic amino acid decarboxylase (AAAD), failed to suppress OMMD's effect on blood pressure; in contrast, central inhibition of AAAD did decrease OMMD's antihypertensive effect. These observations suggest that 3-O-methylated metabolites may participate in the antihypertensive effect of AMD.