Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos

Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 °C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at −20 °C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau’s medium for 48 h at 28 °C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound.

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A Complete Study of Farrerol Metabolites Produced in Vivo and in Vitro

Molecules ◽

10.3390/molecules24193470 ◽

2019 ◽

Vol 24 (19) ◽

pp. 3470

Author(s):

Yin ◽

Ma ◽

Liang ◽

Wang ◽

Sun ◽

...

Keyword(s):

High Performance ◽

Liver Microsomes ◽

Rat Liver Microsomes ◽

Flight Mass Spectrometry ◽

Multiple Data ◽

Glucuronide Conjugation ◽

Complete Study ◽

Glucose Conjugation

Although farrerol, a characteristically bioactive constituent of Rhododendron dauricum L., exhibits extensive biological and pharmacological activities (e.g., anti-oxidant, anti-immunogenic, and anti-angiogenic) as well as a high drug development potential, its metabolism remains underexplored. Herein, we employed ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry coupled with multiple data post-processing techniques to rapidly identify farrerol metabolites produced in vivo (in rat blood, bile, urine and feces) and in vitro (in rat liver microsomes). As a result, 42 in vivo metabolites and 15 in vitro metabolites were detected, and farrerol shown to mainly undergo oxidation, reduction, (de)methylation, glucose conjugation, glucuronide conjugation, sulfate conjugation, N-acetylation and N-acetylcysteine conjugation. Thus, this work elaborates the metabolic pathways of farrerol and reveals the potential pharmacodynamics forms of farrerol.

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

Royal Society Open Science ◽

10.1098/rsos.191666 ◽

2020 ◽

Vol 7 (2) ◽

pp. 191666

Author(s):

Lu Yu ◽

Xu Chen ◽

Wen Sheng Zhang ◽

Liang Zheng ◽

Wen Wen Xu ◽

...

Keyword(s):

Tissue Distribution ◽

Intravenous Administration ◽

High Performance ◽

Anaesthetic Agent ◽

Parent Compound ◽

Liver Microsomes ◽

Metabolite Identification ◽

Pharmacokinetic Studies

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.

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Identification of Metabolites of Eupatorin in Vivo and in Vitro Based on UHPLC-Q-TOF-MS/MS

Molecules ◽

10.3390/molecules24142658 ◽

2019 ◽

Vol 24 (14) ◽

pp. 2658 ◽

Cited By ~ 3

Author(s):

Luya Li ◽

Yuting Chen ◽

Xue Feng ◽

Jintuo Yin ◽

Shenghao Li ◽

...

Keyword(s):

High Performance ◽

Liver Microsomes ◽

Intestinal Flora ◽

Metabolite Identification ◽

Rat Plasma ◽

Rat Liver Microsomes ◽

Online Data ◽

Tof Ms

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.

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Screening and Identification of the Main Metabolites of Schisantherin a In Vivo and In Vitro by Using UHPLC-Q-TOF-MS/MS

Molecules ◽

10.3390/molecules25020258 ◽

2020 ◽

Vol 25 (2) ◽

pp. 258 ◽

Cited By ~ 1

Author(s):

Wei Feng ◽

Ling-Yu Zhou ◽

Rui-Feng Mu ◽

Le Gao ◽

Bing-Yuan Xu ◽

...

Keyword(s):

Metabolic Pathways ◽

High Performance ◽

Liver Microsomes ◽

Rat Liver Microsomes ◽

Sprague Dawley ◽

First Time ◽

Schisantherin A ◽

Tof Ms

Schisantherin A is an active ingredient originating from Schisandra chinensis (Turcz.) which has hepatoprotective and anti-oxidation activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on male Sprague Dawley rats of Schisantherin A were tested, respectively. The metabolites of Schisantherin A were identified using ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). Based on the method, 60 metabolites were successfully identified and structurally characterized including 48 phase-I and 12 phase-II metabolites. Among the metabolites, 45 metabolites were reported for the first time. Moreover, 56 and eight metabolites were detected in urine and bile and 19 metabolites were identified in rats’ plasma. It demonstrated that hepatic and extra-hepatic metabolic pathways were both involved in Schisantherin A biotransformation in rats. Five in vitro metabolites were structurally characterized for the first time. The results indicated that the metabolic pathways mainly include oxidation, reduction, methylation, and conjugation with glucuronide, taurine, glucose, and glutathione groups. This study provides a practical strategy for rapidly screening and identifying metabolites, and the results provide basic data for future pharmacological and toxicology studies of Schisantherin A and other lignin ingredients.

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In Vitro Metabolism of E2, G2: Novel Bile Acid-Coupling Camptothecin Analogues, in Rat Liver Microsomes

Recent Patents on Anti-Cancer Drug Discovery ◽

10.2174/1574892816666210204122028 ◽

2021 ◽

Vol 16 ◽

Author(s):

Xiangli Zhang ◽

Qin Shen ◽

Yi Wang ◽

Leilei Zhou ◽

Qi Weng ◽

...

Keyword(s):

Bile Acid ◽

Phase I ◽

Rat Liver ◽

Deoxycholic Acid ◽

Liver Microsomes ◽

Intrinsic Clearance ◽

Rat Liver Microsomes ◽

Camptothecin Analogues

Background: E2 (Camptothecin - 20 (S) - O- glycine - deoxycholic acid), and G2 (Camptothecin - 20 (S) - O - acetate - deoxycholic acid) are two novel bile acid-derived camptothecin analogues by introducing deoxycholic acid in 20-position of CPT(camptothecin) with greater anticancer activity and lower systematic toxicity in vivo. Objective: We aimed to investigate the metabolism of E2 and G2 by Rat Liver Microsomes (RLM). Methods: Phase Ⅰ and Phase Ⅱ metabolism of E2 and G2 in rat liver microsomes were performed respectively, and the mixed incubation of phase I and phase Ⅱ metabolism of E2 and G2 was also processed. Metabolites were identified by liquid chromatographic/mass spectrometry. Results: The results showed that phase I metabolism was the major biotransformation route for both E2 and G2. The isoenzyme involved in their metabolism had some difference. The intrinsic clearance of G2 was 174.7mL/min. mg protein, more than three times of that of E2 (51.3 mL/min . mg protein), indicating a greater metabolism stability of E2. 10 metabolites of E2 and 14 metabolites of G2 were detected, including phase I metabolites (mainly via hydroxylations and hydrolysis) and their further glucuronidation products. Conclusion: These findings suggested that E2 and G2 have similar biotransformation pathways except some difference in the hydrolysis ability of the ester bond and amino bond from the parent compounds, which may result in the diversity of their metabolism stability and responsible CYPs(Cytochrome P450 proteins).

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Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

Journal of Xenobiotics ◽

10.4081/xeno.2011.e4 ◽

2011 ◽

Vol 1 (1) ◽

pp. 4 ◽

Cited By ~ 10

Author(s):

Hansen W. Murcia ◽

Gonzalo J. Díaz ◽

Sandra Milena Cepeda

Keyword(s):

Cytochrome P450 ◽

Aflatoxin B1 ◽

Enzyme Activities ◽

High Performance ◽

Biochemical Characterization ◽

Liver Microsomes ◽

Cytochrome P450 Enzymes ◽

Catalytic Rate

Cytochrome P450 enzymes (CYP) are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1), a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC). Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified) and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD) and 7-methoxyresorufin- O-deethylase (MROD) activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.

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Inhibitory Effect of Resveratrol on the Pharmacokinetics of Ticagrelor in Vivo and Vitro

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2020-0512 ◽

2021 ◽

Author(s):

Peng Wang ◽

Xiao-Xia Hu ◽

Ying-hui Li ◽

Nan-Yong Gao ◽

Guo-quan Chen ◽

...

Keyword(s):

Rat Liver ◽

Liver Microsomes ◽

In Vitro Study ◽

Inhibitory Effect ◽

Control Group ◽

Rat Liver Microsomes ◽

Sprague Dawley ◽

Group B

This study was to evaluate the effect of resveratrol on the pharmacokinetics of ticagrelor in rats and the metabolism of ticagrelor in human CYP3A4 and liver microsomes. Eighteen Sprague-Dawley rats were randomly divided into three groups: group A (control group), group B (50mg/kg resveratrol), and group C (150mg/kg resveratrol ). After 30 minutes administration of resveratrol, a single dose of ticagrelor (18mg/kg) was administered orally. The vitro experiment was performed to examine the influence of resveratrol on ticagrelor metabolism in CYP3A4*1, human, and rat liver microsomes. Serial biological samples were assayed by validated UHPLC-MS/MS methods. In vivo study, the AUC and Cmax of ticagrelor in group B and C appeared to be significantly higher than the control group, while Vz/F and CLz/F of ticagrelor in group B and C were significantly decreased. In vitro study, resveratrol exhibited an inhibitory effect on CYP3A4*1, human and rat liver microsomes. The IC50 values of resveratrol were 56.75μM，69.07μM and 14.22μM, respectively. Our results indicated that resveratrol had a inhibitory effect on the metabolism of ticagrelor in vitro and vivo. It should be paid more attention to the clinical combination of resveratrol with ticagrelor and ticagrelor plasma concentration should be monitored to avoid the occurrence of adverse reaction.

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Diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A)—In vivo and in vitro effects of metabolism by rat liver microsomes—Formation of an oxygenated complex

Biochemical Pharmacology ◽

10.1016/0006-2952(72)90389-9 ◽

1972 ◽

Vol 21 (17) ◽

pp. 2373-2383 ◽

Cited By ~ 97

Author(s):

John B. Schenkman ◽

Beverley J. Wilson ◽

Dominick L. Cinti

Keyword(s):

Rat Liver ◽

Liver Microsomes ◽

Rat Liver Microsomes ◽

In Vitro Effects

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Inhibition and Induction by Poziotinib of Different Rat Cytochrome P450 Enzymes In Vivo and in an In Vitro Cocktail Method

Frontiers in Pharmacology ◽

10.3389/fphar.2020.593518 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jinhui Wang ◽

Feifei Chen ◽

Hui Jiang ◽

Jia Xu ◽

Deru Meng ◽

...

Keyword(s):

Cytochrome P450 ◽

Rat Liver ◽

Clinical Investigation ◽

Liver Microsomes ◽

Pharmacokinetic Parameters ◽

Rat Liver Microsomes ◽

Cytochrome P450 Enzymes ◽

Significant Difference

Poziotinib is an orally active, irreversible, pan-HER tyrosine kinase inhibitor used to treat non-small cell lung cancer, breast cancer, and gastric cancer. Poziotinib is currently under clinical investigation, and understanding its drug-drug interactions is extremely important for its future development and clinical application. The cocktail method is most suitable for evaluating the activity of cytochrome P450 enzymes (CYPs). As poziotinib is partially metabolized by CYPs, cocktail probes are used to study the interaction between drugs metabolized by each CYP subtype. Midazolam, bupropion, dextromethorphan, tolbutamide, chlorzoxazone, phenacetin, and their metabolites were used to examine the effects of poziotinib on the activity of cyp1a2, 2b1, 2d1, 2c11, 2e1, and 3a1/2, respectively. The in vitro experiment was carried out by using rat liver microsomes (RLMs), whereas the in vivo experiment involved the comparison of the pharmacokinetic parameters of the probes after co-administration with poziotinib to rats to those of control rats treated with only probes. UPLC-MS/MS was used to detect the probes and their metabolites in rat plasma and rat liver microsomes. The in vitro results revealed that the half-maximal inhibitory concentration values of bupropion and tolbutamide in RLMs were 8.79 and 20.17 μM, respectively, indicating that poziotinib showed varying degrees of inhibition toward cyp2b1 and cyp2c11. Poziotinib was a competitive inhibitor of cyp2b1 and cyp2c11, with Ki values of 16.18 and 17.66 μM, respectively. No time- or concentration-dependence of inhibition by poziotinib was observed toward cyp2b1 and cyp2c11 in RLMs. Additionally, no obvious inhibitory effects were observed on the activity of cyp1a2, cyp2d1, cyp2e1, and cyp3a1/2. In vivo analysis revealed that bupropion, tolbutamide, phenacetin, and chlorzoxazone showed significantly different pharmacokinetic parameters after administration (p < 0.05); there was no significant difference in the pharmacokinetic parameters of dextromethorphan and midazolam. These results show that poziotinib inhibited cyp2b1 and cyp2c11, but induced cyp1a2 and cyp2e1 in rats. Thus, poziotinib inhibited cyp2b1 and cyp2c11 activity in rats, suggesting the possibility of interactions between poziotinib and these CYP substrates and the need for caution when combining them in clinical settings.

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