ABSTRACT
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.
ABSTRACT
Uridine-diphosphate glucuronosyltransferase 1A1 (UGT1A1) is an important conjugative enzyme in mammals that is responsible for the conjugation and detoxification of both endogenous and xenobiotic compounds. Strong inhibition of UGT1A1 may trigger adverse drug/herb-drug interactions, or result in metabolic disorders of endobiotic metabolism. Therefore, both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have recommended assaying the inhibitory potential of drugs under development on the human UGT1A1 prior to approval. This review focuses on the significance, progress and challenges in discovery and characterization of UGT1A1 inhibitors. Recent advances in the development of UGT1A1 probes and their application for screening UGT1A1 inhibitors are summarized and discussed in this review for the first time. Furthermore, a long list of UGT1A1 inhibitors, including information on their inhibition potency, inhibition mode, and affinity, has been prepared and analyzed. Challenges and future directions in this field are highlighted in the final section. The information and knowledge that are presented in this review provide guidance for rational use of drugs/herbs in order to avoid the occurrence of adverse effects UGT1A1 inhibition, as well as presenting methods for rapid screening and characterization of UGT1A1 inhibitors and for facilitating investigations on UGT1A1-ligand interactions.
ABSTRACT
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.
ABSTRACT
Cocktail probe substrates approach is a fast, sensitive and high through put method to determine cytochrome P450 enzymes activity. It has been widely used to screen early drug development, analyze drug metabolism types and confirm the metabolism pathways, study drug-drug interactions, optimize clinical regimen, evaluate post marketing drugs and help liver/kidney pathological studies. This article reviewed characteristics of Cocktail probe substrates, focused on the application to traditional Chinese medicine to CYP450 system as follows: the metabolic pathway research of Chinese herb active ingredients; processing way and compatibility of medical herbs affect CYP450; find out the metabolic characteristic of Chinese patent medicine, study in pharmacy of national minority; do research in liver protective effect of traditional Chinese medicine and evaluate traditional Chinese medicine syndromes in animal models. This article make a summary of existing research results and also make a comparison of cocktail probe substrates approach application to western medicine and Chinese medicine.
ABSTRACT
OBJECTIVE: To study the effect of Danshen injection and Xinkeshu tablets on the metabolism of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in rabbits by cocktail probe substrates method. METHODS: Sixteen healthy fasted male rabbits were simultaneously given cocktail probe substrates including caffeine, tolbutamide, omeprazole, dextromethorphan and midazolam. The metabolic rates of cocktail probe substrates were determined by LC MS/MS methods at some specific time following the administration. Then the rabbits were randomly assigned to receive either Danshen injection or Xinkeshu tablets. The whole self-control trial was consisted of 3 cycles. The low, middle and high dosage (0.5, 1 and 2 mL · kg-1) of Danshen injection group would be administrated in 3 cycles in sequence. The low, middle and high dosage (90, 180 and 360 mg · kg-1) of Xinkeshu tablets group would be administrated in 3 cycles in sequence. Each cycle includes wall-out period of 7 d followed by administration of the 2 herbal medicines for consecutive 7 d. On the 15th day the cocktail probe substrates were administrated and the metabolic rates were determined. The differences of the metabolic rates between after and before admininstration of the 2 herbal medicines were assessed by one-way analysis of variance (ANOVA). RESULTS: After taking the agents of Danshen injection and Xinkeshu tablets, the metabolic rates of the five cocktail probe substrates had a tendency to decline compared with the before-administration. CONCLUSION: The outcome in vivo is in accordance with that in vitro. Danshen injection and Xinkeshu tablets can inhibit the metabolism of five CYPs (1A2, 2C9, 2C19, 2D6 and 3A4) activities in rabbits. At sometimes precautions are needed to co-administrate either Danshen injection or Xinkeshu tablets for-possible herb-drug interactions.
ABSTRACT
OBJECTIVE: To study the induction effect of bencycloquidium bromide (BCQB) on rat liver cytochrome P450 enzymes. METHODS: Rats were divided into solvent control group, positive control (phenobarbital) group and BCQB group. The rats in BCQB groups were intranasally administered with 1, 3, and 9 mg · kg-1 BCQB, respectively. After multiple-dose administration, the rats were sacrificed, and the livers were weighed and prepared to microsomes. Then the liver coefficients were calculated, and the total content of CYP450 enzymes in liver microsomes was determined by spectrophotometer. HPLC-MS/MS method was adopted and validated to simultaneously determine the productive velocity of 6β-hydroxyl testosterone and paracetamol after incubation of rat liver microsomes. The activities of CYP1A1/2 and CYP3A1/2 were measured according to the productive velocity. RESULTS: The liver coefficients, total content of CYP450 enzymes, and activity of CYP1A1/2 were significantly different (P0.05) between solvent control group and BCQB groups. The activity of CYP3A1/2 in BCQB groups was not higher than solvent control group. CONCLUSION: Phenobarbital has induction effect on rat liver CYP450 enzymes. The test system can be used to evaluate the induction effect of BCQB on rat liver CYP450 enzymes. BCQB has no induction effect on liver CYP450 enzymes in rats.