Liver Microsomal Biotransformation Research Articles

Biomimetic Metabolism of Kaurenoic Acid Validated by Microsomal Reactions

The cytochrome P450 is a remarkable family of enzymes with widespread occurrence in animals and plants. In humans, they catalyze phase I metabolism reactions, such as oxidations and hydroxylations, playing a significant role in xenobiotics detoxification. Although mechanistic details of the catalytic cycle of cytochrome P450 are known, and human isoforms have their crystal structure solved, mimicking cytochrome P450 reactions using in vitro systems is still challenging. For this reason, biological assays using isolated enzymes or microsomal fractions are typically required to study the oxidative metabolism of new drugs before approval by regulatory agencies. Here we demonstrate that a series of metalloporphyrin catalysts generate the same oxidative product in vitro as a liver microsomal assay. The bioactive diterpene kaurenoic acid was chosen as a substrate model because it is a component of widely used phytomedicines in Brazil, such as guaco syrups and copaiba waxes. We evaluated the influence of reaction parameters such as catalyst type, oxygen donor type, solvent, and catalyst concentration on substrate consumption. Three products had their structures proposed by mass spectrometry, and among them, a dihydroxylated oxidation product was detected as a major metabolite after liver microsomal biotransformation and using porphyrin catalysts. We envision that the use of metalloporphyrins could be a useful strategy for biomimetic oxidation of kaurenic terpenes.

Metabolite identification, tissue distribution, excretion and preclinical pharmacokinetic studies of ET-26-HCl, a new analogue of etomidate.

ET-26-HCl, a novel anaesthetic agent with promising pharmacological properties, lacks extensive studies on pharmacokinetics and disposition in vitro and in vivo. In this study, we investigated the metabolic stability, metabolite production and plasma protein binding (PPB) of ET-26-HCl along with its tissue distribution, excretion and pharmacokinetics in animals after intravenous administration. Ultra-high performance liquid chromatography–tandem quadrupole time-of-flight mass spectrometry identified a total of eight new metabolites after ET-26-HCl biotransformation in liver microsomes from different species. A hypothetical cytochrome P450-metabolic pathway including dehydrogenation, hydroxylation and demethylation was proposed. The PPB rate was highest in mouse and lowest in human. After intravenous administration, ET-26-HCl distributed rapidly to all tissues in rats and beagle dogs, with the highest concentrations in fat and liver. High concentrations of ET-26-acid, a major hydroxylation metabolite of ET-26-HCl, were found in liver, plasma and kidney. Almost complete clearance of ET-26-HCl from plasma occurred within 4 h after administration. Only a small fraction of the parent compound and its acid form were excreted via the urine and faeces. Taken together, the results added to a better understanding of the metabolic and pharmacokinetic properties of ET-26-HCl, which may contribute to the further development of this drug.

Toxicokinetics of α-zearalenol and its masked form in rats and the comparative biotransformation in liver microsomes from different livestock and humans

Alpha-zearalenol (α-ZEL) and its masked form α-zearalenol-14 glucoside (α-ZEL-14G) have much higher oestrogenic activity than zearalenone. Owing to very limited toxicokinetic and metabolic data, no reference points could be established for risk assessment. To circumvent it, the toxicokinetic, metabolic profiles, and phenotyping of α-ZEL and α-ZEL-14G were comprehensively investigated in this study. As a result, the plasma concentrations of α-ZEL and α-ZEL-14G were all below LOQ after oral administration, while after iv injection, both could be significantly bio-transformed into various metabolites. A complete hydrolysis of α-ZEL-14G contributed to α-ZEL overall toxicity. Additionally, 31 phase I and 10 phase II metabolites of α-ZEL, and 9 phase I and 5 phase II metabolites were identified for α-ZEL-14G. For α-ZEL, hydroxylation, dehydrogenation, and glucuronidation were the major metabolic pathways, while for α-ZEL-14G, it was deglycosylation, reduction, hydroxylation, and glucuronidation. Significant metabolic differences were observed for α-ZEL and α-ZEL-14G in the liver microsomes of rats, chickens, swine, goats, cows and humans. Phenotyping studies indicated that α-ZEL and α-ZEL-14G were mediated by CYP 3A4, 2C8, and 1A2. Moreover, the deglycosylation of α-ZEL-14G was critically mediated by CES-I and CES-II. The acquired data would provide fundamental perspectives for risk evaluation of mycotoxins and their modified forms.

Metabolism and Disposition of Siponimod, a Novel Selective S1P1/S1P5 Agonist, in Healthy Volunteers and In Vitro Identification of Human Cytochrome P450 Enzymes Involved in Its Oxidative Metabolism.

Siponimod, a next-generation selective sphingosine-1-phosphate receptor modulator, is currently being investigated for the treatment of secondary progressive multiple sclerosis. We investigated the absorption, distribution, metabolism, and excretion (ADME) of a single 10-mg oral dose of [14C]siponimod in four healthy men. Mass balance, blood and plasma radioactivity, and plasma siponimod concentrations were measured. Metabolite profiles were determined in plasma, urine, and feces. Metabolite structures were elucidated using mass spectrometry and comparison with reference compounds. Unchanged siponimod accounted for 57% of the total plasma radioactivity (area under the concentration-time curve), indicating substantial exposure to metabolites. Siponimod showed medium to slow absorption (median Tmax: 4 hours) and moderate distribution (Vz/F: 291 l). Siponimod was mainly cleared through biotransformation, predominantly by oxidative metabolism. The mean apparent elimination half-life of siponimod in plasma was 56.6 hours. Siponimod was excreted mostly in feces in the form of oxidative metabolites. The excretion of radioactivity was close to complete after 13 days. Based on the metabolite patterns, a phase II metabolite (M3) formed by glucuronidation of hydroxylated siponimod was the main circulating metabolite in plasma. However, in subsequent mouse ADME and clinical pharmacokinetic studies, a long-lived nonpolar metabolite (M17, cholesterol ester of siponimod) was identified as the most prominent systemic metabolite. We further conducted in vitro experiments to investigate the enzymes responsible for the oxidative metabolism of siponimod. The selective inhibitor and recombinant enzyme results identified cytochrome P450 2C9 (CYP2C9) as the predominant contributor to the human liver microsomal biotransformation of siponimod, with minor contributions from CYP3A4 and other cytochrome P450 enzymes.

Liver microsomal biotransformation of albendazole in deer, cattle, sheep and pig and some related wild breeds

Albendazole (ABZ) biotransformation was studied in vitro in liver microsomes of adult noncastrated male farm animals (ram, buck, bull and boar), castrated adult males (wether, billy and hog), and free living males (fallow buck, red deer stag, mouflon ram, roe buck and wild boar). Liver microsomal fractions were incubated with either ABZ or racemic albendazole sulphoxide (ABZSO). ABZ was extensively metabolized to the (+) and (-) enantiomers of ABZSO, whereas ABZSO underwent a slow oxidation to albendazole sulphone (ABZSO2) in all species. In all species both ABZSO enantiomers were detected. The chiral ratio, (+)-ABZSO/(-)-ABZSO, was greater than one in farm animals, mouflon and wild boar, and less than one in three species of deer. For total ABZ sulphoxidation, deer like species had lower values compared to the other species. Mouflon ram and ram had lower total sulphoxidation rates compared to wethers, as well as ABZ suphoxidation towards (+)-ABZSO. No significant difference occurred comparing ABZSO formation in mouflon ram and ram, but ABZSO2 formation rate in mouflon ram was higher than in rams and wethers. Roe deer stag, fallow buck and red deer stag did not differ in both total-ABZSO and (-)-ABZSO synthesis rates and roe deer stag and fallow buck did not differ in synthesis rates of (+)-ABZSO and ABZSO2. The bull differed from other species in all metabolites studied, except for red deer stag and boar in (-)-ABZSO synthesis rate. The extent of ABZSO sulphonation to ABZSO2 in bull microsomes was more than twice that of other species.

Liver microsomal biotransformation of nitro‐aryl drugs: mechanism for potential oxidative stress induction

Toxic effects of several nitro-aryl drugs are attributed to the nitro-reduction that may be suffered in vivo, a reaction that may be catalysed by different reductases. One of these enzymes is NADPH-cytochrome P450 reductase, which belongs to the cytochrome P450 oxidative system mainly localized in the endoplasmic reticulum of the hepatic cell. This system is responsible for the biotransformation of oxidative lipophilic compounds, so that oxidative and reductive metabolic pathways of lipophilic nitro-aryl drugs can take place simultaneously. Because of the affinity of nitro-aryl drugs (xenobiotics) for the endoplasmic reticulum, we propose this subcellular organelle as a good biological system for investigating the toxicity induced by the biotransformation of these or another compounds. In this work we used rat liver microsomes to assess the oxidative stress induced by nitro-aryl drug biotransformation. Incubation of microsomes of rat liver with nifurtimox and nitrofurantoin in the presence of NADPH induced lipoperoxidation, UDP-glucuronyltransferase activation and an increase in the basal microsomal oxygen consumption. Nitro-aryl-1,4-dihydropyridines did not elicit these prooxidant effects; furthermore, they inhibited lipoperoxidation and oxygen consumption induced by Fe3+/ascorbate. Nifurtimox and nitrofurantoin modified the maximum absorption of cytochrome P450 oxidase and inhibited p-nitroanisole O-demethylation, an oxidative reaction catalysed by the cytochrome P450 system, signifying that oxidation may proceed in a similar way to that described for nitro-aryl-1,4-dihydropyridines. Thus the balance between lipophilic nitro-aryl drug oxidation and reduction may be involved in the potential oxidative stress induced by biotransformation.

Queuing for cardiac surgery

Siponimod, a next-generation selective sphingosine-1-phosphate receptor modulator, is currently being investigated for the treatment of secondary progressive multiple sclerosis. We investigated the absorption, distribution, metabolism, and excretion (ADME) of a single 10-mg oral dose of [¹⁴C]siponimod in four healthy men. Mass balance, blood and plasma radioactivity, and plasma siponimod concentrations were measured. Metabolite profiles were determined in plasma, urine, and feces. Metabolite structures were elucidated using mass spectrometry and comparison with reference compounds. Unchanged siponimod accounted for 57% of the total plasma radioactivity (area under the concentration–time curve), indicating substantial exposure to metabolites. Siponimod showed medium to slow absorption (median <i>T</i>_max: 4 hours) and moderate distribution (Vz/F: 291 l). Siponimod was mainly cleared through biotransformation, predominantly by oxidative metabolism. The mean apparent elimination half-life of siponimod in plasma was 56.6 hours. Siponimod was excreted mostly in feces in the form of oxidative metabolites. The excretion of radioactivity was close to complete after 13 days. Based on the metabolite patterns, a phase II metabolite (M3) formed by glucuronidation of hydroxylated siponimod was the main circulating metabolite in plasma. However, in subsequent mouse ADME and clinical pharmacokinetic studies, a long-lived nonpolar metabolite (M17, cholesterol ester of siponimod) was identified as the most prominent systemic metabolite. We further conducted in vitro experiments to investigate the enzymes responsible for the oxidative metabolism of siponimod. The selective inhibitor and recombinant enzyme results identified cytochrome P450 2C9 (CYP2C9) as the predominant contributor to the human liver microsomal biotransformation of siponimod, with minor contributions from CYP3A4 and other cytochrome P450 enzymes.

P3G186 - Detection of hydroxyl and 1 hydroxyethyl free radicals using spin traps followed by gas chromatographic-mass spectrometric detection. Application to studies on ethanol BIO-transformation in liver microsomes or highly purified nuclei

P3G186 - Detection of hydroxyl and 1 hydroxyethyl free radicals using spin traps followed by gas chromatographic-mass spectrometric detection. Application to studies on ethanol BIO-transformation in liver microsomes or highly purified nuclei

The role of different cytochrome P450 isoforms in in vitro chloroform metabolism.

The two CHCl3 activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and beta-naphthoflavone). The present results provide direct evidence that CHCl3 concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl3 concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10-15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl3 only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl3 oxidative bioactivation do not participate in CHCl3 reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl3 activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity.

Enantioselectivity in the Rabbit Liver Microsomal Biotransformation of Styrene and Phenylpropenes

The rabbit liver microsomal P-450 catalyzed oxidation of styrene (1a) and isomeric phenylpropenes, trans-1-phenylpropene (1b), cis-1-phenylpropene (1c) and 3-phenylpropene (1d), was investigated and the enantioselectivity of the epoxidation of the olefinic double bond was determined by checking the enantiomeric excesses of the corresponding first formed epoxides (2). These enantiomeric excesses were always modest, ranging between 7% of (1S,2S)-(2b) and 22% of (1R,2R)-(2c). In the case of (1d) a nonenantioselective hydroxylation at the benzylic-allylic C(3) was also oberved. The ratio between this hydroxylation and olefin epoxidation of (Id) was 1:2.

Evidence for hydroxyl free radical formation during paraquat but not for nifurtimox liver microsomal biotransformation. A dimethyl-sulfoxide scavenging study.

The effect of several experimental conditions on methane (CH4) production from dimethylsulfoxide (DMSO) in incubation mixtures containing liver microsomes and NADPH generating systems was studied. The process was heat sensitive in part but a significant fraction was non-enzymatic in nature. CH4 formation from DMSO was not significantly modified by 2-diethylaminoethyl-2,2-diphenylvalerate. HC 1 (SKF 525A) or EDTA 1 mM and significantly enhanced under an atmosphere of (CO 80% + O2 20%) rather than under air. A marked increase in CH4 production was observed when paraquat (PQ) was included in incubation mixtures but not when nifurtimox (Nfx) was added. Results support the hypothesis of hydroxyl free radical (.OH) formation during PQ biotransformation but cast doubts about its production for the case of Nfx. The low temperature gas chromatographic separation of d3-CH4 from CH4 described opens the future possibility for detecting trace formation of .OH in vivo, without interference from fecal CH4 formation by administering d6-DMSO to animals and collecting exhaled gases produced, in chambers containing the entire animal.

Effect of chronic ethanol administration on bromobenzene liver toxicity in the rat

Female Sprague-Dawley rats were pair-fed a nutritionally adequate liquid diet containing either ethanol or isocaloric carbohydrate for 3 weeks. In vitro studies showed that chronic ethanol pretreatment preferentially increased the liver microsomal biotransformation of bromobenzene to p-bromophenol (via the toxic 3,4-epoxide) rather than to o-bromophenol (via the nontoxic 2,3-epoxide) and could thus potentiate bromobenzene hepatotoxicity. Bromobenzene administration (500 mg/kg body weight, ip), after an overnight fast, was associated in ethanol-pretreated rats with greater and accelerated liver glutathione depletion and subsequent decrease in liver cytochrome P-450 content than in controls. As assessed histologically and by determination of the rise in activities of serum enzyme markers of liver necrosis, chronic ethanol pretreatment, however, mainly resulted in earlier onset and resolution of bromobenzene-induced liver necrosis, with only a mild increase in the maximal severity of liver lesions. These results suggest that the twofold increase in liver microsomal bromobenzene 3,4-epoxidation by ethanol, being much less than that seen after phenobarbital pretreatment in our animal model and in that of others, is apparently not sufficient to markedly affect the severity of bromobenzene-induced liver toxicity.

Impairment of drug metabolism in man by allopurinol and nortriptyline.

Normal medical students received either allopurinol or nortriptyline. To quantitate changes in rates of drug metabolism produced by allopurinol or nortriptyline administration, the plasma half-lives of antipyrine or bishydroxycoumarin were determined both before and 24 hours after the last dose of allopurinol or nortriptyline. Allopurinol and nortriptyline each markedly prolonged the plasma half-lives of antipyrine and bishydroxycoumarin; large individual variations in prolongation times were observed. In rats, allopurinol and nortriptyline each reduced the activity of hepatic microsomal drug-metabolizing enzymes and slightly lowered the level of cytochrome P-450. These results in rats support the interpretation that in man prolongation of antipyrine and bishydroxycoumarin half-lives after allopurinol or nortriptyline pretreatment is produced by reduced rates of drug biotransformation in liver microsomes. Lowering of the usual dose and close monitoring of blood levels of drugs given simultaneously with allopurinol or nortriptyline are recommended to avoid the potential danger of drug accumulation due to slowed metabolism.