Bioactivation of diclofenac via benzoquinone imine intermediates-identification of urinary mercapturic acid derivatives in rats and humans.

The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.     

Bioactivation of diclofenac in vitro and in vivo: correlation to electrochemical studies

Diclofenac is widely used in the treatment of, for example, arthritis and muscle pain. The use of diclofenac has been associated with hepatotoxicity, which has been linked to the formation of reactive metabolites. Diclofenac can be metabolized to 4'-OH- and 5-OH-diclofenac, both of which are able to form quinone imines capable of reacting with, for example, GSH and nucleophilic groups in proteins. Electrochemistry has been shown to be a suitable tool for mimicking some types of oxidative drug metabolism and for studying the formation of reactive metabolites. In these studies, the electrochemical oxidation of diclofenac to a +16 Da metabolite was shown to be identical to a synthetic standard of 5-OH-diclofenac. Furthermore, two different experimental designs were investigated with respect to the electrochemical oxidation of 4'-OH- and 5-OH-diclofenac. In the first approach, the oxidized sample was collected in an aqueous solution of GSH, whereas in the other approach, GSH was added to the sample before the oxidation was performed. From these electrochemical oxidations, a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac were observed and characterized by MS/MS. This allowed the development of sensitive LC-MS methods in order to detect the GSH conjugates from in vivo (rat bile) and in vitro (human liver microsomes (HLM), rat liver microsomes (RLM), and rat hepatocytes) samples. A wide range of mono-, di-, and triglutathionyl conjugates were detected in the in vitro and in vivo samples. It was also observed that 5-OH-diclofenac formed GSH conjugates with RLM and HLM without addition of NADPH, whereas GSH conjugate formation of 4'-OH-diclofenac was NADPH-dependent. This indicated that 5-OH-diclofenac was prone to auto-oxidation. The oxidation potentials of the two hydroxy metabolites were determined by cyclic voltammetry. A difference of 69 mV was observed between the two oxidation potentials, which in part may explain the extent of auto-oxidation for 5-OH-diclofenac. In conclusion, it was shown that electrochemical oxidation was capable of mimicking the metabolic hydroxylation of diclofenac to 5-OH-diclofenac. Furthermore, electrochemical oxidation was used to generate a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac and a number of these conjugates were also detected in metabolism studies with microsomes (HLM/RLM) and freshly isolated rat hepatocytes, and in vivo in rat bile.     

Characterization of glutathione conjugates of reactive metabolites of 3'-hydroxyacetanilide, a nonhepatotoxic positional isomer of acetaminophen

3'-Hydroxyacetanilide (AMAP) is a nonhepatotoxic regioisomer of acetaminophen (APAP) that nonetheless does form reactive metabolites which bind to hepatic proteins. Because differences in the nature of reactive metabolites formed from AMAP and APAP may explain differences in their propensity to cause hepatotoxicity, characterization of the reactive metabolites of AMAP was undertaken. The naturally occurring sulfhydryl-containing tripeptide glutathione (GSH) was used to trap the reactive metabolites. Four mono-GSH conjugates and one di-GSH conjugate of oxidative AMAP metabolites were characterized by 1H NMR and soft ionization (LSIMS or FAB) mass spectral techniques, as well as by comparison of liquid chromatographic and spectral characteristics with synthetic standards. Two isomeric mono-GSH conjugates of 2-acetamidohydroquinone (2-AcHQ) are formed as well as a bis-GSH conjugate. A mono-GSH conjugate of 3',4'-dihydroxyacetanilide (3-OH-APAP) also was formed. Thus, these GSH conjugates most likely arise by reaction of GSH with 2-acetamido-p-benzoquinone (2-APBQ) and 4-acetamido-o-benzoquinone (4-AOBQ), respectively, as oxidation products of the known AMAP metabolites 2-AcHQ and 3-OH-APAP. Finally, a GSH conjugate of 3'-methoxy-4'-hydroxy-acetanilide (3-OMe-APAP) was detected in bile of mice administered AMAP. This conjugate probably arises by oxidation of 3-OMe-APAP, another known metabolite of AMAP. The presumed oxidation product, N-acetyl-3-methoxy-p-benzoquinone imine (MAPQI), was synthesized and found to react with GSH to give the same GSH conjugate as that detected in bile and in incubations of 3-OMe-APAP with mouse liver microsomes plus GSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of biliary conjugates of 4,4'-methylenedianiline in male versus female rats

4,4′-Methylenedianiline (4,4′-diaminodiphenylmethane; DAPM) is an aromatic diamine used in the production of numerous polyurethane foams and epoxy resins. Previous studies in rats revealed that DAPM initially injures biliary epithelial cells of the liver, that the toxicity is greater in female than in male rats, and that the toxic metabolites of DAPM are excreted into bile. Since male and female rats exhibit differences in the expression of both phase I and phase II enzymes, our hypothesis was that female rats either metabolize DAPM to more toxic metabolites or have a decreased capacity to conjugate metabolites to less toxic intermediates. Our objective was thus to isolate, characterize, and quantify DAPM metabolites excreted into bile in both male and female bile duct-cannulated Sprague Dawley rats. The rats were gavaged with [¹⁴C]-DAPM, and the collected bile was subjected to reversed-phase HPLC with radioisotope detection. Peaks eluting from HPLC were collected and analyzed using electrospray MS and NMR spectroscopy. HPLC analysis indicated numerous metabolites in both sexes, but male rats excreted greater amounts of glutathione and glucuronide conjugates than females. Electrospray MS and NMR spectra of HPLC fractions revealed that the most prominent metabolite found in bile of both sexes was a glutathione conjugate of an imine metabolite of a 4′-nitroso-DAPM. Seven other metabolites were identified, including acetylated, cysteinyl-glycine, glutamyl-cysteine, glycine, and glucuronide conjugates. While our prior studies demonstrated increased covalent binding of DAPM in the liver and bile of female compared to male rats, in these studies, SDS-PAGE with autoradiography revealed 4-5 radiolabeled protein bands in the bile of rats treated with [¹⁴C]-DAPM. In addition, these bands were much more prominent in female than in male rats. These studies thus suggest that a plausible mechanism for the increased sensitivity of female rats to DAPM toxicity may be decreased conjugation of reactive DAPM metabolites, leading to greater levels of protein adduct formation.     

Metabolism related toxicity of diclofenac in yeast as model system

Diclofenac is a widely used drug that can cause serious hepatotoxicity, which has been linked to metabolism by cytochrome P450s (P450). To investigate the role of oxidative metabolites in diclofenac toxicity, a model for P450-related toxicity was set up in Saccharomyces cerevisiae. We expressed a drug-metabolizing mutant of cytochrome P450 BM3 (BM3 M11) in yeast. Importantly, BM3 M11 yielded similar oxidative metabolite profiles of diclofenac as human P450s. It was found that yeast strains expressing BM3 M11 grew significantly slower when exposed to diclofenac than strains without BM3 M11. Furthermore, the amount of reactive oxygen species (ROS) after incubation with diclofenac was higher in strains expressing BM3 M11 than in strains without this enzyme, confirming that P450 activity increases diclofenac toxicity. Interestingly, 4′- and 5-hydroxydiclofenac had no effect on cell growth or ROS formation in cells expressing BM3 M11, although hydroxydiclofenac-derived quinone imines were identified in these strains by detection of their glutathione conjugates. This suggests that 4′- and 5-hydroxydiclofenac, as well as their quinone imines, are not involved in toxicity in yeast. Rather, the P450-related toxicity of diclofenac is caused by primary metabolites such as arene oxides resulting in hydroxydiclofenac or radical species formed during decarboxylation.     

Differences in the metabolism of the antitumour agents amsacrine and its derivative CI-921 in rat and mouse.

1. Rats and mice were treated with the antitumour agent CI-921 (I), and parent compound amsacrine, with all biliary metabolites being analysed. 2. In both rat and mouse the major biliary metabolites of amsacrine are the 5'- and 6'-glutathione (GSH) conjugates, with no C9-GSH conjugate being detected. 3. 5'- and 6'-GSH conjugates of I are also formed in both species. However, two additional products were detected and their structures confirmed by liquid secondary ion mass spectrometry and 1H-n.m.r. spectrometry, and by comparison with synthetic standards. 4. Additional metabolites of I are the C9-GSH conjugate and the 4-hydroxymethyl derivative which, either as the aglycone or glucuronide, is the predominant product in rat bile (comprising 56% of the dose eliminated over 3.5 h). 5. Relative amounts of the C9-GSH conjugate to the 5'- and 6'-GSH conjugates to the 4-hydroxymethyl derivatives, were 24:65:10 in mouse bile and 2:8:90 in rat bile. 6. These differences indicate first, likely enzyme involvement in the formation of the C9-GSH conjugate of I, and second, in comparison with amsacrine, alternative pathways which may decrease formation of the reactive quinone diimine intermediate of I and consequent hepatotoxicity.     

Development and evaluation of an electrochemical method for studying reactive phase-I metabolites: correlation to in vitro drug metabolism

A reactive metabolite may react covalently with proteins or DNA to form adducts that ultimately may lead to a toxic response. Reactive metabolites can be formed via, for example, cytochrome P450-mediated phase 1 reactions, and in this study, we report the development and evaluation of an electrochemical method for generating reactive metabolites. Paracetamol was used as a test compound to develop the method. The stability of the electrochemically generated N-acetyl-p-benzoquinoneimine (NAPQI) from paracetamol was investigated at 37 degrees C at pH 5.0, 7.4, and 9.0. The highest stability of NAPQI was observed at pH 7.4. The reaction rate between NAPQI and glutathione (GSH) was studied with cyclic voltammetry. NAPQI reacted quantitatively with GSH within 130 ms. The reactivity of NAPQI toward other nucleophiles was investigated, and for the reaction with N-acetyltyrosine, a time-dependent formation of a conjugate with N-acetyltyrosine was observed from 0 to 4 min. The applicability of the method was evaluated with compounds that were able to form quinone imines (amodiaquine), quinones (3-tert-butyl-4-hydroxyanisole and p-cresol), imine methides (3-methylindole; trimethoprim), quinone methides (3,5-di-tert-butyl-4-hydroxytoluene), and nitrenium ions (clozapine). The compounds were oxidized in an analytical electrochemical cell, and the formed reactive metabolites were trapped with GSH. The samples were then analyzed by LC-MS and LC-MS/MS. For comparison, all compounds were incubated with GSH in rat and human liver microsomes, and the formation of GSH conjugates was compared with that observed by electrochemical oxidation. Furthermore, the electrochemical method was used to synthesize a GSH conjugate of clozapine, which made it possible to obtain structural information by NMR. In summary, a high degree of similarity was observed between the conjugates identified from electrochemical oxidation and GSH conjugates identified from incubation with liver microsomes. In conclusion, we have developed a method that is useful for studies on reactive metabolites and furthermore can be scaled up for the synthesis of GSH conjugates for NMR.     

Synthesis and characterization of some new phase II metabolites of the alkylator bendamustine and their identification in human bile, urine, and plasma from patients with cholangiocarcinoma.

The alkylating agent bendamustine is currently in phase III clinical trials for the treatment of hematological malignancies and breast, lung, and gastrointestinal tumors. Renal elimination mainly as the parent compound is thought to be the primary route of excretion. Because polar biliary conjugates were expected metabolites of bendamustine, three cysteine S-conjugates were synthesized, purified by quantitative high-performance liquid chromatography (HPLC), and characterized by NMR spectroscopy and mass spectrometry (MS). HPLC assays with MS, as well as fluorescence detection of bile, urine, and plasma after single-dose intravenous infusion of 140 mg/m(2) bendamustine in five subjects with cholangiocarcinoma, indicated the existence of these phase II metabolites, which were identified as cysteine S-conjugates by comparison with the previously characterized synthetic reference standards. The sum of the three cysteine S-conjugates of bendamustine was determined in human bile and urine to be 95.8 and 26.0%, respectively, expressed as mean percentage of the sum of the parent compound and identified metabolites. The percentage of administered dose recovered in urine as cysteine S-conjugates ranged from 0.9 to 4.1%, whereas the total percentage of the administered dose excreted in urine as the parent drug and seven metabolites ranged from 3.8 to 16.3%. The identification of cysteine S-conjugates provide evidence that a major route of bendamustine metabolism in humans involves conjugation with glutathione. Results indicate the importance of phase II conjugation in the elimination of bendamustine, besides phase I metabolism and hydrolytic degradation, and require further investigation.     

Formation and reversibility of S-linked conjugates of N-(1-methyl-3,3-diphenylpropyl)isocyanate, an in vivo metabolite of N-(1-methyl-3,3-diphenylpropyl)formamaide, in rats

The metabolic disposition of N-(1-methyl-3,3-diphenylpropyl) formamide was studied in rats. The water-soluble metabolites, N-acetyl-S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]cysteine and S-[N-(1-methyl-3,3-diphenylpropylcarbamoyl)]glutathione, were identified in urine and bile, respectively, of rats doses with the secondary formamide. The structures of these metabolites were confirmed by comparison with synthetic standards and by using liquid chromatography mass spectrometry and fast atom bombardment mass spectrometry. Synthetic standards of these metabolites were obtained by reacting the N-(1-methyl-3,3-diphenylpropyl)isocyanate with glutathione or N-acetylcysteine in methanolic solutions. The isocyanate was obtained in high yield by reacting 1-methyl-3,3-diphenylpropylamine with trichloromethyl chloroformate. The S-linked conjugates released the isocyanate in mild alkali, but were stable under acidic conditions. The released isocyanate was characterized by comparison with the synthetic standard using GC/MS and HPLC. A mechanism is proposed for the base-catalyzed elimination of the isocyanate from the thiol conjugates.     

Reactive intermediates formed during the peroxidative oxidation of anisidine isomers

The ortho isomer of anisidine (2-methoxyaniline) causes urinary bladder tumors in both mice and rats while the para isomer (4-methoxyaniline) is inactive. Since the urinary bladder contains substantial peroxidase activity, we investigated the peroxidative metabolism of both o- and p-anisidine using horseradish peroxidase as a model enzyme. Both isomers were excellent reducing cofactors for the oxidized state of horseradish peroxidase (HRP), resulting in one-electron oxidation to free radicals. Using high-pressure liquid chromatography, we observed that HRP oxidized p-anisidine to a diimine metabolite which subsequently hydrolyzed to form a quinone imine. Also observed was a dimeric metabolite with an azo bond. Both the diimine and quinone imine metabolites were reactive toward nucleophiles. The quinone imine formed a conjugate with glutathione and was also reduced by glutathione or ascorbic acid. Higher concentrations of substrate (greater than 1 mM) led to the formation of polymeric products (tetramer). Similar metabolites (diimine, quinone imine, azo dimer, polymers) were observed with o-anisidine. Using tritium-labeled anisidine, we observed substantial metabolism-dependent covalent binding of both isomers to protein and DNA. These results demonstrate that horseradish peroxidase dependent metabolism of anisidine isomers yields similar metabolites, although some differences in reactivity of the respective intermediates with nucleophiles were observed.     

Glucuronides of hydroxylated metabolites of amitriptyline and nortriptyline isolated from rat bile

Polar conjugates were isolated from the bile of rats given amitriptyline (AT, unlabeled or labeled with 14C), nortriptyline (NT), or 10-hydroxy (10-OH) derivatives of the drugs. The procedure involved extraction on a column of polystyrene resin, elution with methanol, and separation by preparative TLC followed by reversed phase HPLC. Individual metabolites were characterized by NMR spectroscopy and fast atom bombardment mass spectrometry and by enzymatic or acid deconjugation with subsequent identification of aglycones and glucuronic acid. Conversely, they were compared with conjugates obtained from hydroxy compounds by incubation with rat liver microsomes and UDP-glucuronic acid. Glucuronides isolated from the bile of rats given AT were derived from 2-OH-AT, (E)- and (Z)-10-OH-AT, 2-hydroxy-3-methoxy- (or 3-hydroxy-2-methoxy) AT, 10, 11-(OH)2-AT, and some of the N-demethylated analogues of these compounds. In most cases, 10-OH compounds form two diastereoisomeric glucuronides produced from the enantiomeric alcohols; 10, 11-(OH)2 metabolites occur as cis- and trans-isomers that are conjugated with glucuronic acid. Administration of synthetic (E)- and (Z)-10-OH-AT and -NT leads to the excretion of their glucuronides along with conjugates formed after demethylation and/or introduction of a second OH group. NT gives rise to 2-OH-NT glucuronide besides those conjugates derived from (E)-10-OH-NT. No glutathione conjugates could be detected.     

鉴定大鼠注射绿原酸后体内的代谢产物

绿原酸为多种中药注射液的主要成分, 本文采用超高效液相色谱-四极杆飞行时间质谱法 (UPLC/Q- TOF MS) 鉴定大鼠注射给予绿原酸后胆汁、尿、粪和血浆中的代谢产物。利用碰撞能量梯度 (MS^E) 和质量亏损过滤 (MDF) 技术, 在大鼠胆汁、尿、粪和血浆中共检测到35种代谢产物。胆汁中主要代谢产物为O-甲基绿原酸谷胱甘肽结合物, 其排泄量超过胆汁中全部代谢物的80%, 尿中主要为原形、O-甲基结合物、水解代谢产物及葡糖醛酸结合物, 粪中主要为O-甲基结合物及其半胱氨酸结合物, 血浆中主要为原形化合物。绿原酸及其代谢产物经尿和粪便排泄比例相近。实验结果表明, 绿原酸在大鼠体内代谢广泛, 主要途径之一是与谷胱甘肽结合, 提示绿原酸的烯酮双键具有亲电性, 可能与蛋白的巯基共价结合, 导致过敏性不良反应, 应予以关注。     

Studies on the fate of the glutathione and cysteine conjugates of acetaminophen in mice

Studies were conducted in mice to examine the origin and fate of the amino acid-containing conjugates of acetaminophen (APAP). Collection of bile containing [14C]APAP metabolites (mainly the glutathione conjugate) in common duct-cannulated mice given a 250 mg/kg oral dose of the drug reduced by greater than 70% the urinary excretion of the cysteine and mercapturic acid conjugates of APAP. This confirmed previous reports which indicated that these urinary metabolites originated from the glutathione conjugate excreted in bile. The urinary excretion of cysteine and mercapturic acid conjugates was not altered, however, by ligation of the common bile duct in mice given APAP. Thus, biliary excretion of the glutathione conjugate is not obligatory for the appearance of cysteine and mercapturic acid conjugates in urine. Intravenous administration of purified glutathione conjugate to mice having a bile-duct cannula indicated that this conjugate did not appear in bile but appeared in urine primarily in the form of the cysteine conjugate. An identical pattern of excretion was observed after an iv dose of the purified cysteine conjugate of APAP to bile duct-cannulated mice. These results indicated that, if the glutathione conjugate leaves the liver via the blood, it is rapidly converted to the cysteine conjugate which is eliminated in urine. This conversion takes place at multiple sites in the body and evidence is presented to implicate both intestine and kidney in the process. The appearance of a small amount of glutathione conjugate in urine (16%) after an iv dose of the cysteine conjugate indicates that formation of the glutathione of APAP can occur by a route that does not involve direct conjugation of reactive metabolites of the drug with glutathione.     

CYP2C9 genotypes and diclofenac metabolism in Spanish healthy volunteers

OBJECTIVE: This study analyzed the frequency of CYP2C9 variant alleles and evaluated the impact of CYP2C9 genotype on diclofenac metabolism in a Spanish population. METHODS: Diclofenac hydroxylation capacity was studied in a population of 102 healthy volunteers. After a single oral dose of 50 mg diclofenac the 0- to 8-h urinary concentrations of diclofenac and its main metabolites, 4'-hydroxy (OH), 3'-OH and 5-OH diclofenac were analyzed by high-performance liquid chromatography. CYP2C9 genotyping for the variant alleles CYP2C9*2 and *3 was carried out with PCR-RFLP. RESULTS: The frequencies of CYP2C9*1, *2, and *3 alleles were 0.74 (95%CI: 0.68-0.80), 0.16 (95%CI: 0.11-0.21) and 0.10 (95%CI: 0.06-0.15), respectively, among the 102 Spaniards studied. The diclofenac/4'-OH diclofenac urinary ratio, but not the diclofenac/3'-OH diclofenac and diclofenac/5-OH diclofenac ratios, was related to CYP2C9 genotype. The diclofenac/4'-OH ratio was significantly higher among subjects with CYP2C9*1/*3 (0.83+/-0.4, n=14, 95% CI for the difference: 0.02-0.4) and CYP2C9*2/*3 (1.10+/-0.5, n=4, 95% CI for the difference: 0.16-0.8) genotypes compared to CYP2C9*1/*1 (0.62+/-0.3, n=59) and approximately threefold higher (1.8) in the only subject homozygous for CYP2C9*3 variant. CONCLUSIONS: The frequencies of CYP2C9*1, *2, and *3 alleles in the Spanish population reported here were similar to those found in the previously studied white European populations, and different of the previously reported in another Spanish population. CYP2C9*3 allele seems to influence the 4'-hydroxylation of diclofenac, although there is a large overlapping in the urinary metabolic ratio between the genotype groups studied     

Identification of biliary metabolites of m-dichlorobenzene in rats.

At least 12 metabolites were observed in the bile of rats administered with m-dichlorobenzene in the preliminary experiments by HPLC. Some of them were assumed to be conjugates containing phenyl and dihydro-hydroxyphenyl moieties with glutathione or cysteine by enzymatic and thermal reactions. To isolate these metabolites, the bile collected from 20 rats administered with 500 mg/kg of m-dichlorobenzene and 300 mg/kg of cysteine was separated into five fractions by HPLC. Eighteen metabolites were isolated from these fractions by TLC and HPLC. The chemical structure of the major metabolite excreted was determined as trans-2,4-dichloro-6-(glutathion-S-yl)cyclohexa-2,4-dien+ ++-1-ol by 13C-NMR, 1H-NMR, FAB-MS, and some reaction experiments. The metabolite excreted in the secondary largest amount was identified as its positional isomer, trans-3,5-dichloro-6-(glutathion-S-yl)cyclohexa-2,4-dien+ ++-1-ol. Their diastereomers were also observed in the bile. trans-2,4-Dichloro-6-(cystein-S-yl)cyclohexa-2,4-dien- 1-ol and its positional isomer, which were possibly derived from glutathione conjugates above, were also identified. 3,5-Dichlorophenyl conjugates with glutathione or cysteine and 3,5-dichlorophenyl mercapturic acid, and their 2,4-dichlorophenyl isomers, were excreted. Three monochlorophenol conjugates, of which chemical structures were still not established, were present in the bile.     

Stimulation of biliary glutathione secretion by sulfonylureas

In isolated perfused rat livers, infusion of the sulfonylureas, glyburide (2.5 microM) and tolbutamide (0.5 mM), stimulated by 2-fold the rate of biliary glutathione secretion. This increase was mainly the result of an apparent increase in the rate of reduced glutathione release by the liver since oxidized glutathione levels in the bile remained unchanged. Sulfonylurea infusion into perfused livers did not alter the rate of glutathione release in the perfusate, indicating that sinusoidal release was not perturbed. N-Benzylimidazole (0.2 mM), an inhibitor of cytochrome P-450, blocked the tolbutamide-mediated increase in biliary release of glutathione. However, the cytochrome P-450 inhibitor did not alter the glyburide-induced increase in biliary glutathione secretion. Glyburide infusion into perfused livers also decreased tissue oxidized glutathione content without altering the total tissue levels of glutathione. The stimulation of biliary glutathione release by sulfonylureas is probably the result of excretion of labile conjugates of glutathione and sulfonylurea metabolites. Although the precise identity of these metabolites is presently unknown, formyltolbutamide and hydroxyglyburide formed during metabolism of tolbutamide and glyburide, respectively, may be the prime candidates for forming labile glutathione conjugates.     

Lumiracoxib metabolism in male C57bl/6J mice: characterisation of novel in vivo metabolites

1.?The pharmacokinetics and metabolism of lumiracoxib in male C57bl/6J mice were investigated following a single oral dose of 10?mg/kg.

2.?Lumiracoxib achieved peak observed concentrations in the blood of 1.26?+?0.51?μg/mL 0.5?h (0.5–1.0) post-dose with an AUC_inf of 3.48?+?1.09?μg?h/mL. Concentrations of lumiracoxib then declined with a terminal half-life of 1.54?+?0.31?h.

3.?Metabolic profiling showed only the presence of unchanged lumiracoxib in blood by 24?h, while urine, bile and faecal extracts contained, in addition to the unchanged parent drug, large amounts of hydroxylated and conjugated metabolites.

4.?No evidence was obtained in the mouse for the production of the downstream products of glutathione conjugation such as mercapturates, suggesting that the metabolism of the drug via quinone–imine generating pathways is not a major route of biotransformation in this species. Acyl glucuronidation appeared absent or a very minor route.

5.?While there was significant overlap with reported human metabolites, a number of unique mouse metabolites were detected, particularly taurine conjugates of lumiracoxib and its oxidative metabolites.     

Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), in rats. Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.     

Cytotoxic effects of polychlorinated biphenyl hydroquinone metabolites in rat hepatocytes

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that exhibit various toxic effects in animals and exposed human populations. The molecular mechanisms of PCB toxicity have been attributed to the toxicological properties of its metabolites, such as hydroquinones, formed by cytochrome‐P‐450 oxidation. The effects of PCB hydroquinone metabolites towards freshly isolated rat hepatocytes were investigated. Hydroquinones can be oxidized to semiquinones and/or quinone metabolites. These metabolites can conjugate glutathione or can oxidize glutathione as a result of redox cycling. This depletes hepatocyte glutathione, which can inhibit cellular defence mechanisms, causing cell death and an increased susceptibility to oxidative stress. However in the following, glutathione‐depleted hepatocytes became more resistant to the hydroquinone metabolites of PCBs. This suggested that their glutathione conjugates were toxic and that there was a third type of quinone toxicity mechanism which involved a hydrogen peroxide‐accelerated autoxidation of the hydroquinones to form toxic electrophilic quinone and semiquinone–glutathione conjugates. Copyright © 2009 John Wiley & Sons, Ltd.     

The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent

An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry (microLC-microESI-MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH+ to [MH - 129]+ transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH+ to [MH - 129]+ transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen-GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).