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Objectives: The purpose of this study was to evaluate the mutagenic potential of fluoxetine and fluoxetine-galactomannan. Methods: Chromosomal aberration test and Salmonella typhimurium/microsome mutagenicity assay. Results: The results showed that fluoxetine (250 µg/mL) can cause chromosomal breaks of treated leukocytes and increase the frequency of reversion of the tester strains of S. typhimurium / microsome assay only at the highest concentration (5 mg/mL), while fluoxetine encapsulated in galactomannan did not cause these changes (leukocytes and S. typhimuriums strains). Conclusion: In summary, fluoxetine showed a mutagenic effect detectable only at high concentrations in both eukaryotic and prokaryotic models. Furthermore, the fluoxetine/galactomannan complex, in this first moment, prevented the mutagenicity attributed to fluoxetine, emphasizing that the present encapsulation process can be an alternative in preventing these effects in vitro.
Objetivos: avaliar o potencial mutagênico da fluoxetina e da fluoxetina-galactomanana. Métodos: Teste de aberração cromossômica e ensaio de mutagenicidade de Salmonella typhimurium /microssoma. Resultados: a fluoxetina (250 µg/mL) pode causar quebras cromossômicas de leucócitos tratados e aumentar a frequência de reversão das cepas testadoras de S. typhimurium /microssoma apenas na concentração mais alta (5 mg/mL), enquanto a fluoxetina encapsulada em galactomanano não causou essas alterações (leucócitos e cepas de S. typhimurium). Conclusão: a fluoxetina mostrou um efeito mutagênico detectável apenas em altas concentrações em modelos eucarióticos e procarióticos. Além disso, o complexo fluoxetina/galactomanan, neste primeiro momento, evitou a mutagenicidade atribuída à fluoxetina, ressaltando que o presente processo de encapsulamento pode ser uma alternativa na prevenção desses efeitos in vitro.
Subject(s)
Fluoxetine , Chromosome Aberrations , Salmonella typhimurium , Chromosome Breakage , Microsomes , Mutagenicity TestsABSTRACT
To investigate the metabolites of a new synthetic cannabinoid 3,3-dimethyl-2-[1-(4-cyanobutyl)indazole-3-formamimino]methyl butyrate (4CN-MDMB-BUTINACA) in vitro, a human liver microsome incubation model was established to analyze the metabolic biotransformation of synthetic cannabinoids using ultra-high performance liquid chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry. Nontarget metabolomic results showed that the metabolites of 4CN-MDMB-BUTINACA included hydroxylation, ester hydrolysis, ester hydrolysis with hydroxylation reaction, pentane oxidation and ester hydrolysis with pentane oxidation reaction, among which M1-a, M2 and M4 were potential metabolic markers. The research results provide a theoretical basis and technical support for the biomonitoring and metabolic characterisation of the cannabinoid 4CN-MDMB-BUTINACA.
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OBJECTIVE To inv estigate the in vitro inhibitory effects of acteoside on cytochrome P 450（CYP）enzymes in liver microsomes of rats. METHODS Using probe substrates method ，acteoside（0.1，0.3，1，3，10，30 μmol/L）was incubated with probe substrates phenacetin ，mephentoin，diclofenac，coumarin，dextromethorphan and testosterone （substrates of CYP 1A2， CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）in liver microsomes of rats. Another blank control group and positive inhibitor group [ α-naphthoflavone，ticlopidine，sulfabendazole，pilocarpine，quinidine and ketoconazole （inhibitors of CYP 1A2，CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）] were set up. Using indapamide as the internal standard ， the contents of corresponding metabolites （acetaminophen， 4-hydroxymephenytoin， 4-hydroxydiclofenac，7-hydroxycoumarin，dextran，6 β-hydroxytestosterone） were detected by ultra high performance liquid chromatography-tandem mass spectrometry . The IC 50 values were calculated by GraphPad v 8.0 software. By computer molecular docking technology ，acteoside and positive inhibitors were molecularly docked with the CYP enzyme ，and the binding mode and strength of the two molecules were analyzed. RESULTS The IC 50 values of acteoside to CYP 1A2 and CYP 2A6 enzymes were more than 30 μmol/L，and those of acteoside to CYP 2D6，CYP2C19，CYP2C9 and CYP 3A4 enzymes were 24.87，21.52，12.56 and 7.55 μmol/L，respectively. The hydrogen bond and hydrophobic force could form between acteoside and CYP 3A4 enzyme，and the hydrogen bond and electrostatic interaction could form between ketoconazole and CYP 3A4 enzyme. The binding free energy of acteoside and ketoconazole to CYP 3A4 enzyme were － 10.2 and － 12.4 kcal/mol （1 kcal/mol＝4.19 kJ），respectively. CONCLUSIONS Acteoside shows moderate inhibitory effect on CYP 3A4 enzyme in liver microsomes of rats ，and its affinity is equivalent to that of positive inhibitor ；the compound shows weak inhibitory effect on other 5 CYP enzymes.
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Cytochrome P450s (CYP450) is a superfamily of phase I metabolic enzymes, which participates in more than 90% of drug oxidation. The induction or inhibition of CYP450s is the main mechanism of drug-drug interaction. In recent years, in vitro metabolism studies conducted through isolated organs, cells, or enzyme systems have developed rapidly, due to their precision and simplicity. Therefore, profiles of the in vitro metabolism studies of traditional Chinese medicines can infer the possible metabolic pathways of drugs, predict the potential drug interactions, and may enhance the rational use of drugs in clinic. This article reviews the in vitro inhibitory effects of traditional Chinese medicine, ingredients, and extracts on the activities of CYP450 enzymes in the liver microsomes, which can provide a reference for further researches on the interaction between Chinese medicine and chemical medicine.
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OBJECTIVE：To study the inhibitor y effects of cajanonic acid A on 5 kinds of cytochrome P 450（CYP）enzyme，in human liver microsomes in vitro . METHODS ：By Cocktail probe substrate method ，50.0，15.0，5.0，1.5，0.5，0.15，0.05 μmol/L cajanonic acid A were added into liver microsomes ， and incubated with mixed probe substrates [including phenacetin ， dextromethorphan，omeprazole，testosterone and toluenesulfonbutylurea （probe substrates of CYP 1A2，CYP2D6，CYP2C19， CYP3A4，CYP2C9，respectively）]. On the basis of setting up blank group and positive control group [ α-naphthalene brass ， quinidine，（+）-N-3-benzyl vanillin ，ketoconazole and sulfabendazole （specific inhibitors of CYP 1A2，CYP2D6，CYP2C19， CYP3A4，CYP2C9，respectively）]，using puerarin as internal standard ，UPLC-MS/MS method was adopted to determine the contents of corresponding metabolites （acetaminophen， dextrophane， 5-hydroxy omeprazole ， 6 β-hydroxytestosterone， hydroxytolbutamide）. The determination was performed on ACQUITY UPLC ® BEH C 18 column，with mobile phase consisted of 0.01％ formic acid aqueous solution- 0.01％ acetonitrile formic acid （gradient elution ）at the flow rate of 0.4 mL/min. The column temperature was 40 ℃，and the sample size was 2 μL. An electrospray ionization source was used to conduct positive and negative ion scanning in the multiple reaction monitoring mode. The data acquisition range was m/z 100-1 200，the collision gas was argon ， the atomized gas was nitrogen ，the gas flow rate of the cone hole was 50 L/h，the desorption gas flow rate was 800 L/h，the capillary voltage under positive and negative mode was 2.0， 1.5 kV，and the ion source temperature was 120 ℃，110 ℃， respectively. The desolvent temperature were 400 ℃ and 450 ℃ ， respectively. Non linear regression analysis was performed by using Graphpad Prism 5.0 software and IC 50 wascalculated. RESULTS ：The linear ranges of above metabolifes were 0.26-8.35，0.36-34.56，0.10-3.09, 3.67-117.37，0.15-4.88 μmol/L（R2＞0.99）. The limits of quantitation were 0.26，0.36， 0.10，3.67，0.15 μmol/L，respectively. The IC 50 values of specific inhibitors in positive control group to CYP 1A2，CYP2D6， CYP2C19，CYP3A4 and CYP 2C9 in human liver microsomes were all within the acceptable range reported in the literature. The IC50 values of cajanonic acid A to CYP 1A2，CYP2D6 and CYP 3A4 in human liver microsomes were all more than 50 μmol/L，and the IC 50 values of CYP 2C9 and CYP 2C19 were 4.94 and 18.00 μmol/L，respectively. CONCLUSIONS ：Cajanonic acid A has no inhibitory effect on CYP 1A2，CYP2D6 and CYP 3A4，but has a certain inhibitory effect on CYP 2C9 and CYP 2C19.
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The purpose of this study was to investigate the effect of apigenin on UGT1 A1 enzyme activity and to predict the potential drug-drug interaction of apigenin in clinical use. First,on the basis of previous experiments,the binding targets and binding strength of apigenin to UGT1 A1 enzyme were predicted by computer molecular docking method. Then the inhibitory effect of apigenin on UGT1 A1 enzyme was evaluated by in vitro human liver microsomal incubation system. Molecular docking results showed that apigenin was docked into the active region of UGT1 A1 enzyme protein F,consistent with the active region of bilirubin docking,with moderate affinity. Apigenin flavone mother nucleus mainly interacted with amino acid residues ILE343 and VAL345 to form hydrophobic binding Pi-Alkyl. At the same time,the hydroxyl group on the mother nucleus and the amino acid residue LYS346 formed an additional hydrogen bond,which increased the binding of the molecule to the protein. These results suggested that the flavonoid mother nucleus structure had a special structure binding to the enzyme protein UGT1 A1,and the introduction of hydroxyl groups into the mother nucleus can increase the binding ability. In vitro inhibition experiments showed that apigenin had a moderate inhibitory effect on UGT1 A1 enzyme in a way of competitive inhibition,which was consistent with the results of molecular docking. The results of two experiments showed that apigenin was the substrate of UGT1 A1 enzyme,which could inhibit the activity of UGT1 A1 enzyme competitively,and there was a risk of drug interaction between apigenin and UGT1 A1 enzyme substrate in clinical use.
Subject(s)
Humans , Apigenin/chemistry , Bilirubin/chemistry , Drug Interactions , Glucuronosyltransferase/metabolism , Hydrogen Bonding , Microsomes, Liver/drug effects , Molecular Docking SimulationABSTRACT
@#A new method based on rat liver microsomes and chromatographic fingerprint comparison was established to investigate the possible herb-drug interactions between Liuwei Dihuang Pills(LDP)and nifedipine(NIF). LDP and NIF were incubated simultaneously with rat liver microsomes at 37 °C for 60 min in a shaking water bath. The separation was achieved on a C18 column using 0. 1% formic acid solution and acetonitrile as mobile phase with a liner gradient program. The flow rate was set at 1. 0 mL/min. Detection was achieved by UV light at 240 nm. To evaluate the interactions between LDP and NIF, the similarity of the fingerprinting chromatograms before and after co-incubation was calculated by similarity evaluation system for chromatographic fingerprint of TCM. Furthermore, pharmacokinetic experiments in rat suggested that there was no significant difference in the pharmacokinetic parameters of the typical components in LDP.
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OBJECTIVE: To study the enzymatic kinetics of TM-2 in rat, Beagle dog and human liver microsomes by LC-MS/MS. METHODS: TM-2 was incubated with liver microsomal incubation system. LC-MS/MS method was established for quantitative analysis of TM-2 with cabazitaxel as internal standard. The enzyme kinetics parameters Vmax and Km was calculated by the GraphPad Prism 5.0 software. RESULTS: A rapid and sensitive LC-MS/MS method was developed to study the enzyme kinetics of TM-2 in rat, Beagle dog and human liver microsome. The corresponding enzymatic kinetic parameters in rat, Beagle dog and human were as follows: Vmax values were 16.3, 354.6 and 154.8 nmol•min-1•mg(protein)-1, respectively; Km values were 25.7, 313.8 and 89.4 μmol• L-1, respectively; CLint values were 0.63, 1.13 and 1.73 mL•min-1•mg(protein)-1, respectively. CONCLUSION: The result indicates that there are species differences in the activity of metabolic enzyme and the affinity of TM-2 to the metabolic enzyme. Enzyme kinetic parameters obtained of TM-2 provide important parameters for the further study.
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OBJECTIVE: To investigate the hepatic toxicity of 8 monomers in Polygonum multiflorum using a combination of UDPglucuronic acid transferase 1A1(UGT1A1 enzyme). METHODS: Bilirubin was used as the substrate for UGT1A1. Incubation method in RLM in vitro was adopted to test the apparent inhibition constants(Ki) of different components. Furthermore the structure-activity relationship between the 8 components and UGT1A1 was analyzed. RESULTS: The inhibition effects on UGT1A1 enzyme of the 8 components were in the following sequence: emodin-8-O-glc > emodin > citreorosein > (+) -catechin > gallic acid > physcion > rhein > emodin-6-O-glc. Moreover, there was a structure-activity relationship, and it was presumed that the 6-position hydroxyl group is an active and necessary group. CONCLUSION: The established method in vitro is stable and feasible. Experimental results shows that the enzyme inhibition has structural selectivity, which provides an experimental basis for predicting the enzyme inhibition activity of the analogues of components of Polygonum multiflorum.
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The aim of this study is to establish the in vitro methods for the study of induction and inhibition on CYP450 by drugs, and to validate the analytical method and incubation system. A method for the simultaneous determination of eight metabolites of seven subtypes of CYP450 enzymes probe substrates in human liver microsomes (HLM) was established and validated. The incubation system was optimized to confirm the incubation time and protein concentration of HLM, the enzyme activity of seven subtypes of CYP450 enzymes in HLM was determined, and the inhibition effects on each CYP450s were checked by positive controls. The method for the simultaneous determination of three metabolites of subtypes of CYP450 enzymes was established and validated in human primary cultured hepatocytes (HPCH) using the incubation medium. The enzyme activity of three subtypes of CYP450 enzymes in HPCH was determined, and the total RNA was extracted from HPCH after incubation. The expression of CYP450 enzymes were measured by Taqman fluorescence probe method. The induction effects on each CYP450s were examined using the positive controls. The established methods for the determination of metabolites of probe substrates were fully validated, and the results were conformed to the requirements of bioanalytical method validation. The induction and inhibition effects on each CYP450s were checked by positive controls. The established in vitro methods for the study of drug induction and inhibition on CYP450 were simple and reliable, which could be used in the investigation of enzyme induction or inhibition properties of new drug candidates and to evaluation the metabolic interactions of concomitant medication in clinical.
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Objective@#To investigate the clinical and laboratory features of patients with liver disease and positive anti-liver/kidney microsomal-1 (anti-LKM-1) antibody, and to provide a reference for clinical diagnosis and differential diagnosis.@*Methods@#The clinical data of patients with positive anti-LKM-1 antibody who were treated in our hospital from 2006 to 2016 were collected, and clinical and laboratory features were analyzed and compared. An analysis was also performed for special cases.@*Results@#The measurement of related autoantibodies was performed for about 100 thousand case-times, and 15 patients were found to have positive anti-LKM-1 antibody. Among the 15 patients, 7 were diagnosed with type 2 autoimmune hepatitis (AIH) with an age of 11.0 ± 9.0 years and were all adolescents with acute onset; 8 were diagnosed with hepatitis C with an age of 51.5 ± 9.0 years, among whom 7 were middle-aged patients and 1 was a child aged 12 years, and all of them had an insidious onset. Compared with the patients with hepatitis C, the AIH patients had significantly higher levels of alanine aminotransferase (1 003.9 ± 904.3 U/L vs 57.0 ± 84.1 U/L, P < 0.05), aspartate aminotransferase (410.7 ± 660.3 U/L vs 34.9 ± 42.9 U/L, P < 0.05), and total bilirubin (98.0 ± 191.0 μmol/L vs 15.4 ± 6.0 μmol/L, P < 0.05). There was a reduction in immunoglobulin G after the treatment with immunosuppressant, compared with the baseline. Of all 8 patients with hepatitis C, 6 received antiviral therapy with interferon and ribavirin, and 5 out of them achieved complete response, among whom 4 had a reduction in the level of anti-LKM-1 antibody after treatment; however, a 12-year-old child developed liver failure after interferon treatment and died eventually.@*Conclusion@#Positive anti-LKM-1 antibody is commonly seen in patients with type 2 AIH or hepatitis C, but there are differences between these two groups of patients in terms of age, disease onset, liver function, and the level of anti-LKM-1 antibody. The hepatitis C patients with a confirmed diagnosis and exclusion of autoimmune hepatitis can achieve good response to interferon under close monitoring, even if anti-LKM-1 antibody is positive. As for adolescent patients with hepatitis C and positive anti-LKM-1 antibody, the possibility of AIH should be excluded.
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OBJECTIVE: To establish a UPLC-Q-TOF-MS method to characterize the metabolites of phillygenin in human liver microsomal incubation system for the first time. METHODS: The chromatography separation was performed on a C18 reversed phase LC column (Phenomenex Kinetex C18, 2.1 mm×100 mm, 2.6 μm). The mobile phase consisted of water-formic acid (100:0.1, V/V) and acetonitrile and a gradient elution program was adopted at the flow rate of 400 μL·min-1. The mass spectral analysis was performed in a positive electrospray ionization mode, and the turbo spray temperature was 550℃. The full MS experiment was run with a scan range from m/z100 to m/z 1 000. RESULTS: The possible fragmentation pathways of phillygein were speculated in a positive electrospray ionization mode, and eight metabolites was identified in human liver microsomal incubation system. CONCLUSION: The UPLC-Q-TOF-MS method is very convenient and efficient for detecting phillygein in human liver mirosomes. The developed method is suitable for the metabolism research of phillygein in human liver microsomes, which providing valuable reference for pharmacokinetic study of phillygenin.
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OBJECTIVE: To identify the metabolites of imperatorin in rat urine, feces and bile after oral administration as well as the transformation products of imperatorin after incubation with rat liver microsomes with HPLC-QTrap-MS technology. METHODS: The combination use of HPLC-QTrap-MS scanning mode including multiple ion monitoring-information dependent acquisition-enhanced product ion (MRM/MIM-IDA-EPI)mode, precursor scan-enhanced resolution-information dependent acquisition-enhanced product ion (PREC-ER-IDA-EPI)mode and enhanced product ion (EPI) mode were performed for the identification of the metabolites. Based on the simultaneous appearance of [M+H]+ and [M+NH4]+ in the spectrum of PREC, the molecular weight could be unambiguously identified. The structures of compounds were then identified by the fragment ions generated from these three modes. RESULTS: With the HPLC-QTrap-MS method, 32 metabolites in urine sample, 14 metabolites in faces sample, 6 metabolites in bile sample and 17 transformation products from the rat liver microsomes sample were detected. CONCLUSION: Imperatorin is metabolized mainly in liver and excreted through kidney. The metabolic profiles of imperatorin in vivo and in vitro have good correlation.
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To investigate the effect of clinical dose of Realgar-Indigo Naturais formula (RIF) and large-dose of Realgar on main drug-metabolizing enzymes CYP450s of rat liver, as well as its regulatory effect on mRNA expression. Wistar rats were administrated orally with tested drugs for 14 days. A Cocktail method combined with HPLC-MS/MS was used in the determination of 4 cytochrome P450 isozymes (CYP1A2, CYP2B, CYP3A and CYP2C) in liver of the rats, and the mRNA expression levels of the above subtypes were detected by real-time fluorescent quantitative PCR. The results showed that RIF can significantly induce CYP1A2 and CYP2B enzyme activity, and inhibit CYP3A enzyme activity. This result was consistent with the mRNA expression. However, its single compound showed weaker or even contrary phenomenon. Different doses of Realgar also showed significant inconsistencies on CYP450 enzymes activity and mRNA expression. These phenomena may be relevant with RIF compatibility synergies or toxicity reduction. The results can also prompt drug interactions when RIF is combined with other medicines in application.
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OBJECTIVE: To study the metabolic characteristics of 5-bromo-2-fluorobenzonitrile in vitro and compare the differences between rats and human,and for the purpose of providing data for poison effect research and extrapolating poison effect of 5-bromo-2-fluorobenzonitrile from animals to human being. METHODS: Equilibrium dialysis method was used to analyze the protein binding ratio of 5-bromo-2-fluorobenzonitrile in the plasma of rats and humans in the groups of low dose,medium dose and high dose which were treated with mass concentration of 5-bromo-2-fluorobenzonitrile at 500,5 000 and 50 000 μg / L respectively. Metabolic incubation systems of SD rat microsomes and human liver microsomes were established in vitro. When the mass concentration of 5-bromo-2-fluorobenzonitrile in the systems was 800 μg / L,the concentration of liver microsome was 0. 5 g / L; after being incubated for 0,10,30,60 and 90 min with the involvement of the regeneration system of nicotinamide-adenine dinucleotide phosphate in the incubation systems,the metabolic reaction was stoped. The residual amounts of 5-bromo-2-fluorobenzonitrile were analyzed and metabolic half-life of 5-bromo-2-fluorobenzonitrile incubating with liver microsomes in vitro was figured out. RESULTS: Protein binding ratio of 5-bromo-2-fluorobenzonitrile in the groups of low dose,medium dose and high dose were( 83. 5 ± 0. 9) %,( 88. 8 ± 0. 3) % and( 88. 6 ± 0. 3) % in rats plasma,and( 85. 2 ± 0. 1) %,( 89. 0 ± 0. 1) % and( 91. 1 ± 0. 4) % in human plasma. Both in rat plasma and human plasma,the protein binding ratio of 5-bromo-2-fluorobenzonitrile in the groups of medium dose and high dose were significantly increased than that in the low-dose group( P < 0. 01). In human plasma,the protein binding ratio of 5-bromo-2-fluorobenzonitrile in the high-dose group significantly increased than that in the medium-dose group( P < 0. 01). In the groups of low dose and high dose,the protein binding ratio of 5-bromo-2-fluorobenzonitrile in human plasma significantly increased than that in rats plasma( P < 0. 01). Absolute differences in protein binding ratio of 5-bromo-2-fluorobenzonitrile between the rat plasma and the human plasma were no more than 2. 5% in the same dose groups. Metabolic half-life of 5-bromo-2-fluorobenzonitrile incubating with rats and human liver microsomes and control solution in vitro were respectively( 58. 6 ± 1. 6),( 59. 2 ± 1. 5) and( 65. 0 ± 6. 3) min,which shows no significant differences( P < 0. 05). CONCLUSION: The potein binding ratio and metabolism of 5-bromo-2-fluorobenzonitrile in liver microsomes in rat plasma is similar to those in human plasma. Both in the plasmas of rats and humans,5-bromo-2-fluorobenzonitrile has high protein binding ratio,and 5-bromo-2-fluorobenzonitrile is not metabolized in liver microsomes of either rats or humans.
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To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol(4-NP) and β-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18.38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes(RLM) system, while the inhibitory effect was not obvious in human liver microsomes(HLM) system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes (based on genkwanin, IC₅₀=8.76, 10.36 μmol•L⁻¹). The inhibition types were non-competitive inhibition(RLM) and uncompetitive inhibition(HLM). In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.
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To investigate the metabolic stability and parameters in vitro of lanceolatin B in liver microsomes of rats, human, Beagle dogs, and monkeys, and to identify the phaenotypes of CYP enzymes of lanceolatin B by using the liver microsome incubation system in vitro. After incubated with different species of liver microsomes, lanceolatin B was quantified by UPLC-MS/MS method to evaluate its metabolic stability and metabolic kinetic parameters in vitro. Lanceolatin B was incubated with specific inhibitors of CYP450 isoforms (CYP2E1, 2C19, 1A2, 2D6, 2C9, 3A4, and 2A1) to determine the phaenotypes of metabolic enzymes. The results showed that lanceolatin B was metabolized in the liver microsomes of rats and monkeys but not in the human and Beagle dogs. Their in vitro half-life T1/2 and intrinsic clearance rate CLint in rat and monkey liver microsomes were 11.57,8.07 min, and 0.12,0.17 mL•min⁻¹•mg⁻¹ without significant difference. The results of metabolic phenotyping indicated that CYP1A2 was mainly involved in the metabolism of lanceolatin B. There existed a difference in the metabolism of lanceolatin B in different types of liver microsomes. Several of CYP450 isoforms metabolized lanceolatin B together in liver microsomes of rats, in which CYP1A2 was the major enzyme mainly responsible for the metabolism of lanceolatin B.
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OBJECTIVE: To identify the specific cytochrome P450 (CYP) enzymes involved in the metabolism of dipfluzine hydrochloride (Dip) in the rat liver microsomes. METHODS: The rat liver microsomes were prepared and incubated with Dip. The Dip metabolites (M1, M2, M4 and M5) were identified by LC-MS/MS, and the CYP isoenzymes were identified by the combination of the selective CYP inhibitor study, correlation analysis and a panel of recombinant rat CYP expereiment, respectively. RESULTS: The results from the experiments of selective CYP inhibitors, correlation analysis and recombinant rat CYP isoenzymes indicated that CYP2A1, CYP3A and CYP2C11 contributed to the formation of M1 and M5 in the rat liver microsomes. CYP3A, CYP2A1, CYP1A2, CYP2C11 and CYP2E1 metabolized Dip to M2. CYP3A, CYP2A1, CYP2E1 and CYP2C11 contributed to M4 formation. And the recombinant rat CYP researches further indicated that CYP3A2 exhibited more activity than CYP3A1. CONCLUSION: CYP3A and CYP2A1 are the major CYP isoenzymes responsible for catalyzing Dip to the four metabolites formation in the rat liver microsomes.
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OBJECTIVE: To identify the metabolites oi Pulsatilla oleanolic acid 3-α-L-rhamnopyranosyl (1→2) [β-D-glucopyr-anosyl-(1→4)]-α-L-pyran arabinoside (B7) in rat plasma, bile, urine, feces, and in vitro liver microsomes incubation system. METHODS: An ultra-high performance liquid chromatography combined with electrospray ionization quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed and successfully applied to the study on the metabolites of B7 in rat plasma, bile, urine, feces, and in vitro liver microsomes incubation system after oral administration. RESULTS: A total of 23 metabolites were identified, of which 12 metabolites were present in rat liver microsomes incubation system. CONCLUSION: Deglycosylation, demethylation glucuronidation, and hydroxylation are the major metabolic transformation forms of B7 in rats in vivo and in vitro.
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OBJECTIVE: To evaluate the effects of dipfluzine hydrochloride (Dip) on CYP450s activities in vitro and in vivo in rats. METHODS: Markers were incubated in the normal rat liver microsomes with Dip (0-200 μmol·L-1) and the concentration of metabolites of the markers were determined by LC-MS/MS, and then the ratios were calculated to evaluate the effects of Dip on the CYP450s activities. Dip was administered by orally to the male SD rats at doses of 30, 60 and 90 mg·kg-1 body weight and phenobarbital was administered at doses of 120 mg·kg-1 body weight for 14 d. At the fifteenth day, the rats were orally administered the Cocktail probe markers and blood samples were collected via medial angle of eye at different time. The concentrations of markers were determined by LC-MS/MS and the drug-time curve was plotted, by which the pharmacokinetic parameters were calculated. And then the rat liver microsomes were prepared and the probe markers were added into the incubation samples. The concentrations of the probe markers and its metabolites were determined and the metabolism ratios were calculated. The effects of Dip on CYP450s activities were evaluated by comparing the following outcomes between the experimental groups and the control group: the relative liver weight, the concentrations of protein, the contents of CYP450 enzymes, the drug concentration-time curves, the pharmacokinetic parameters and the metabolism ratios of the probes. RESULTS: In the normal rat liver microsomes, Dip had the inhibitive effects on CYP2D1, CYP2C6 and CYP2C11, and the IC50 were 8.85, 20.93 and 69.45 μg·mL-1, respectively. Dip had no effect on the relative weight of livers, the protein concentrations and the CYP450 content for the rats after they were fed on Dip for 14 d, but these indexes were raised remarkably when phenobarbital was administered by orally to rats. The results displayed that the low-dose Dip inhibited the activity of CYP2D1 or induced the activity of CYP2C11 in rats. Moderate-dose Dip showed the abilities to inhibit CYP2D1, but induce CYP2C11 and CYP3A. High-dose Dip had the certain inhibitive effects to CYP2C6 and CYP2D1, and had the inductive effects on CYP2C11 and CYP3A. CYP1A2, CYP2C11, CYP2C12, CYP2D1 and CYP3A were all induced after administration of phenobarbital. CONCLU SION: The results of liver microsomes incubation and blood plasma from the normal and Dip-induced rats all show that Dip inhibit the activities of CYP2C6 and CYP2D1, however, Dip inhibit CYP2C11 in vitro and induce it in vivo.