Gukang Capsules are often used in combination with drugs to treat fractures, osteoarthritis, and osteoporosis. Cytochrome P450(CYP450) mainly exists in the liver and participates in the oxidative metabolism of a variety of endogenous and exogenous substances and serves as an important cause of drug-metabolic interactions and adverse reactions. Therefore, it is of great significance to study the effect of Gukang Capsules on the activity and expression of CYP450 for increasing its clinical rational medication and improving the safety of drug combination. In this study, the Cocktail probe method was used to detect the changes in the activities of CYP1A2, CYP3A2, CYP2C11, CYP2C19, CYP2D4, and CYP2E1 in rat liver after treatment with high-, medium-and low-dose Gukang Capsules. The rat liver microsomes were extracted by the calcium chloride method, and protein expression of the above six CYP isoform enzymes was detected by Western blot. The results showed that the low-dose Gukang Capsules could induce CYP3A2 and CYP2D4 in rats, medium-dose Gukang Capsules had no effect on them, and high-dose Gukang Capsules could inhibit them in rats. The high-dose Gukang Capsules did not affect CYP2C11 in rats, but low-and medium-dose Gukang Capsules could induce CYP2C11 in rats. Gukang Capsules could inhibit CYP2C19 in rats and induce CYP1A2 in a dose-independent manner, but did not affect CYP2E1. If Gukang Capsules were co-administered with CYP1A2, CYP2C19, CYP3A2, CYP2C11, and CYP2D4 substrates, the dose should be adjusted to avoid drug interactions.
Rats ; Animals ; Cytochrome P-450 CYP1A2/metabolism* ; Cytochrome P-450 CYP2C19 ; Cytochrome P-450 CYP2E1/pharmacology* ; Rats, Sprague-Dawley ; Cytochrome P-450 Enzyme System/metabolism* ; Microsomes, Liver ; Liver ; Cytochrome P-450 CYP3A/metabolism*

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.
Animals ; Benzofurans ; Coumarins ; Dogs ; Glucuronides ; Glucuronosyltransferase/metabolism* ; Kinetics ; Mice ; Microsomes, Liver/metabolism* ; Phenotype ; Rats ; Species Specificity ; Swine ; Swine, Miniature/metabolism*

The bilirubin metabolism mediated by the phase Ⅱ metabolizing enzyme UGT1A1 in the liver was evaluated to study the potential hepatotoxicity risk based on investigation on the inhibitory effect of rhein and its metabolites on the UGT1A1 enzyme in Rhei Radix et Rhizoma. Firstly, in vitro liver microsomes incubation was used to initiate the phase Ⅱ metabolic reaction to investigate the inhibitory effect of rheinon UGT1A1 enzyme. Secondly, the phase Ⅰ and phase Ⅱ metabolic reactions were initiated to investigate the hepatotoxicity risk of rhein metabolites. It was found that the rhein and its phase Ⅱ metabolites had no significant inhibitory effect on UGT1A1 enzyme, but its phase Ⅰ metabolites significantly reduced UGT1A1 enzyme activity. Based on the metabolites analysis, it is speculated that the rhein phase Ⅰ metabolite rheinhydroxylate and its tautomers have certain hepatotoxicity risks, while the toxicity risk induced by the prototype and phase Ⅱ metabolites of rheinglucoside, rheinglucuronic acid and rhein sulfate is small.
Anthraquinones/toxicity* ; Chemical and Drug Induced Liver Injury ; Drugs, Chinese Herbal/toxicity* ; Glucuronosyltransferase/metabolism* ; Humans ; Liver/enzymology* ; Microsomes, Liver/drug effects* ; Rhizome

To evaluate the hepatotoxicity risks of physcion on the basis of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1A1(UGT1A1 enzyme). The monomers were added into the rat liver microsomes to test the hepatotoxicity by using bilirubin as UGT1A1 enzyme substrate, with apparent inhibition constant K_i as the evaluation index. Liver microsome incubation in vitro was adopted to initiate phase Ⅱ metabolic reaction and investigate the inhibitory effect of physcion. Then the phase Ⅰ and Ⅱ metabolic reactions were initiated to investigate the comprehensive inhibition of metabolites and prototype components. The results showed that when only the phase Ⅱ reaction was initiated, physcion directly acted on the UGT1A1 enzyme in a prototype form, exhibited weak inhibition and the inhibition type was mixed inhibition; When the phase Ⅰ and Ⅱ reactions were initiated simultaneously, the inhibitory effects of physcion on UGT1A1 enzyme became strong and the inhibition type was mixed inhibition, suggesting that physcion had phase Ⅰ and Ⅱ metabolic processes, and the metabolites had strong inhibitory effect on UGT1A1 enzyme. This experiment preliminarily proved that the metabolites of physcion may be the main components to induce hepatotoxicity.
Animals ; Chemical and Drug Induced Liver Injury ; Emodin ; analogs & derivatives ; toxicity ; Glucuronosyltransferase ; metabolism ; Kinetics ; Microsomes, Liver ; drug effects ; Rats

The mass spectrometry-based metabolomics method was used to systematically investigate the formation of celastrol metabolites,and the effect of celastrol on endogenous metabolites. The mice plasma,urine and feces samples were collected after oral administration of celastrol. Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS) was applied to analyze the exogenous metabolites of celastrol and its altered endogenous metabolites. Mass defect filtering was adopted to screen for the exogenous metabolites of celastrol. Multivariate statistical analysis was used to identify the endogenous metabolites affected by celastrol. Celastrol and its eight metabolites were detected in urine and feces of mice,and 5 metabolites of them were reported for the first time. The hydroxylated metabolites were observed in the metabolism of both human liver microsomes and mouse liver microsomes. Further recombinant enzyme experiments revealed CYP3 A4 was the major metabolic enzyme involved in the formation of hydroxylated metabolites. Urinary metabolomics revealed that celastrol can affect the excretion of intestinal bacteria-related endogenous metabolites,including hippuric acid,phenylacetylglycine,5-hydroxyindoleacetic acid,urocanic acid,cinnamoylglycine,phenylproplonylglycine and xanthurenic acid. These results are helpful to elucidate the metabolism and disposition of celastrol in vivo,and its mechanism of action.
Animals ; Chromatography, High Pressure Liquid ; Humans ; Mass Spectrometry ; Metabolomics ; Mice ; Microsomes, Liver ; metabolism ; Triterpenes ; metabolism ; pharmacokinetics

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.
Apigenin/chemistry* ; Bilirubin/chemistry* ; Drug Interactions ; Glucuronosyltransferase/metabolism* ; Humans ; Hydrogen Bonding ; Microsomes, Liver/drug effects* ; Molecular Docking Simulation

Pharmacological activities and adverse side effects of ginkgolic acids (GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA (17 : 1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA (17 : 1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA (17 : 1) metabolism were human CYP1A2, CYP3A4, UGT1A6, UGT1A9, and UGT2B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA (17 : 1) in HepG2 cells occurred in a time- and dose-dependent manner. Further investigation showed that GA (17 : 1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1A- and CYP3A-mediated metabolism.
Animals ; Cells, Cultured ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Ginkgo biloba ; chemistry ; Glucuronosyltransferase ; metabolism ; Hepatocytes ; chemistry ; drug effects ; enzymology ; metabolism ; Humans ; Kinetics ; Liver ; chemistry ; drug effects ; enzymology ; metabolism ; Microsomes, Liver ; chemistry ; drug effects ; enzymology ; metabolism ; Plant Extracts ; chemistry ; metabolism ; toxicity ; Rats ; Rats, Sprague-Dawley ; Salicylates ; chemistry ; metabolism ; toxicity

This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group (crude drug 1.25 g·kg⁻¹), D. huoshanense high dose group (crude drug 7.5 g·kg⁻¹), and phenobarbital positive control group (0.08 g·kg⁻¹). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1A1, CYP1A2 and CYP2B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin (a probe drug for CYP1A1) and bupropion (a probe drug for CYP2B) between D. huoshanense group and control group. The metabolism of phenacetin (a probe drug for CYP1A2) showed a statistical difference in rate Vmax, and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CLint in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1A2-catalyzed drugs, which needs to be considered in clinical practice.
Animals ; Cytochrome P-450 CYP1A1 ; metabolism ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Dendrobium ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Mice ; Mice, Inbred C57BL ; Microsomes, Liver ; drug effects ; enzymology ; Random Allocation

Our study aims to determine the metabolism and excretion of novel pulmonary-targeting docetaxel liposome (DTX-LP) using the in vitro and in vivo animal experimental models. The metabolism and excretion of DTX-LP and intravenous DTX (DTX-IN) in New Zealand rabbits were determined with ultraperformance liquid chromatography tandem mass spectrometry. We found DTX-LP and DTX-IN were similarly degraded in vitro by liver homogenates and microsomes, but not metabolized by lung homogenates. Ultra-performance liquid chromatography tandem mass spectrometry identified two shared DTX metabolites. The unconfirmed metabolite M(un) differed structurally from all DTX metabolites identified to date. DTX-LP likewise had a similar in vivo metabolism to DTX-IN. Conversely, DTX-LP showed significantly diminished excretion in rabbit feces or urine, approximately halving the cumulative excretion rates compared to DTX-IN. Liposomal delivery of DTX did not alter the in vitro or in vivo drug metabolism. Delayed excretion of pulmonary-targeting DTX-LP may greatly enhance the therapeutic efficacy and reduce the systemic toxicity in the chemotherapy of non-small cell lung cancer. The identification of M(un) may further suggest an alternative species-specific metabolic pathway.
Animal Experimentation ; Carcinoma, Non-Small-Cell Lung ; Chromatography, Liquid ; Drug Therapy ; Feces ; In Vitro Techniques ; Liposomes* ; Liver ; Lung ; Lung Neoplasms ; Mass Spectrometry ; Metabolic Networks and Pathways ; Metabolism* ; Microsomes ; Models, Animal ; Rabbits* ; Tandem Mass Spectrometry

Ferulic acid (FA) is an active component of herbal medicines. One of the best documented activities of FA is its antioxidant property. Moreover, FA exerts antiallergic, anti-inflammatory, and hepatoprotective effects. However, the metabolic pathways of FA in humans remain unclear. To identify whether human CYP or UGT enzymes are involved in the metabolism of FA, reaction phenotyping of FA was conducted using major CYP-selective chemical inhibitors together with individual CYP and UGT Supersomes. The CYP- and/or UGT-mediated metabolism kinetics were examined simultaneously or individually. Relative activity factor and total normalized rate approaches were used to assess the relative contributions of each major human CYPs towards the FA metabolism. Incubations of FA with human liver microsomes (HLM) displayed NADPH- and UDPGA-dependent metabolism with multiple CYP and UGT isoforms involved. CYPs and UGTs contributed equally to the metabolism of FA in HLM. Although CYP1A2 and CYP3A4 appeared to be the major contributors in the CYP-mediated clearance, their contributions to the overall clearance are still minor (< 25%). As a constitute of many food and herbs, FA poses low drug-drug interaction risk when co-administrated with other herbs or conventional medicines because multiple phase I and phase II enzymes are involved in its metabolism.
Coumaric Acids ; chemistry ; metabolism ; Cytochrome P-450 Enzyme System ; chemistry ; metabolism ; Drugs, Chinese Herbal ; metabolism ; Glucuronosyltransferase ; chemistry ; metabolism ; Humans ; Kinetics ; Medicine, Chinese Traditional ; Microsomes, Liver ; chemistry ; enzymology