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Objective::To investigate the effect of polyethylene glycol 400 (PEG400) on rat bile excretion of baicalin and its main metabolite [baicalein 6-O-β-D-glucuronide (B6G)], and to analyze its mechanism of action. Method::Rats were randomly divided into baicalin+ water group and baicalin+ PEG400 group, the anesthesia was induced by intraperitoneal injection of 10% chloral hydrate (dose of 4 mL·kg-1) to prepare a rat bile duct intubation model. After the rats were fully awake, rats were given baicalin aqueous solution and baicalin PEG400 solution with dose of 168 mg·kg-1 for baicalin, respectively. And bile was collected from 0 h to 12 h after administration. UPLC-MS/MS was used to determine the concentration of drug excreted through bile at different time periods. Thermo Hypersil GOLD C18 column was used with acetonitrile (A)-0.1% formic acid solution (B) as the mobile phase for gradient elution (0-9 min, 90%-27%B; 9-10 min, 27%-90%B; 10-12 min, 90%B), the flow rate was 0.3 mL·min-1, the column temperature was 30 ℃, the injection volume was 5 μL. The mass spectra were obtained in positive ion mode with electrospray ionization (ESI). The effects of PEG400 on the activities and expressions in rat liver of uridine diphosphate glucuronyltransferase (UGT) 1A8 and UGT1A9 were studied in vitro incubation assay and enzyme linked immunosorbent assay (ELISA). Result::Compared with the baicalin+ water group, in the baicalin+ PEG400 group, the bile cumulative excretions of baicalin and B6G increased by 1.8 times and 2.1 times within 12 h, respectively. PEG400 increased the enzyme activities of UGT1A8 and UGT1A9 by 2.0 times and 1.5 times, and their concentrations in liver were increased by 2.2 times and 1.3 times, respectively. Conclusion::PEG400 can significantly increase the bile excretion of baicalin and its main metabolite B6G by enhancing the activities and expressions of UGT1A8 and UGT1A9, and its promoting effect on bile excretion of B6G is greater than that of baicalin, which provides a basis for the rational clinical application of PEG400 and the design of new dosage forms of flavonoids such as baicalin.
Résumé
Objective: To investigate and characterize the O-glucuronidation pathways of the S-stereoisomer of shikonin (alkannin). Methods: Liquid chromatography and mass spectrometry were employed for the detection of akannin and its glucuronide. The incubation of alkannin in human liver microsomes (HLM), human kidney microsomes (HKM) and recombinant human UDP-glucuronyltransferases (UGT) were employed for the study of metabolism profile, the involved UGT isoforms and kinetic analysis. Recombinant human UGT screening, correlation study and chemical inhibition experiments were used for elucidation the selectivity of UGT isoform towards alkannin. Results: In the incubation of alkannin in HLM with the presence of UDPGA, a single UGT metabolite was detected. The screening of the recombinant human UGTs found that UGT1A9 high selectively catalyzed the glucuronidation of alkannin. Kinetic analysis revealed the kinetic of alkannin in HLM, HKM and recombinant UGT1A9 all followed substrate inhibition model and the Km values were 3.75-4.50 μmol/L. The glucuronidation of alkannin and propofol, a probe substrate of UGT1A9, in 12 individual HLM showed really good correlation, the correlation coefficient R2 was 0.88. Chemical inhibition experiments indicated that HLM magnolol and niflumic acid showed obvious inhibition to alkannin glucuronidation; Testerone, celastrol, and nilotinib did not inhibit alkannin glucuronidation. Conclusion: This study finds that UGT metabolism is an important metabolism pathway of alkannin in human, and alkannin is a highly selective probe substrate of human UGT1A9.
Résumé
OBJECTIVE:To investigate the effects of UGT1A6 and UGT1A9 gene polymorphisms on blood concentration of valproic acid in Han epileptic patients.METHODS:Totally 107 Chinese Han epileptic patients were selected from outpatient department of our hospital during Jan.2014-Apr.2015.They were given valproic acid monotherapy treatment for 3 months to 6 years.The steady state concentration ofvalproic acid was detected by EMIT.UGT1A6 (rs2070959,rs6759892) and UGT1A9 (rs13418420,rs2741045,rs2741049,rs6731242,rs72551330) genotypes were detected by MALDI-TOF-MS.The correlation of gene polymorphism with con centration dose ratios (CDR) of valproic acid was investigated.RESULTS:UGT1A9 rs72551330 mutation had not been detected,and the frequency of genotypes in other 6 sites were all in line with Hardy-Weinberg balance (P＞0.05).The CDR of valproic acid in pa tients with UGT1A6 rs2070959,rs6759892 mutation (AG+GG or TG+GG type) were significantly lower than those with wild homozy gote (AA or TT type),with statistical significance (P＜ 0.05).There was no statistical significance in CDR of valproic acid among patients with UGT1A9 rs13418420,rs2741045,rs2741049 and rs6731242 wild homozygote and mutation (P＞0.05).CONCLUSIONS:UGT1A6 rs2070959,rs6759892 gene polymorphisms of Han epileptic patients are associated with blood concentration of valproic acid,and the patients with UGT1A6 rs2070959,rs6759892 mutation need more dose ofvalproic acid.
Résumé
UDP-glucuronosyltransferase 1A9 (UGT1A9) is a major phase II enzyme responsible for elimination of drugs and endogenous molecules. Clinical data have shown increased elimination of UGT1A9 substrates in pregnant women or oral contraceptive users, but the role of estrogen in the regulation of UGT1A9 expression remains unknown. In this study, we investigated the effect of 17-estradiol (E2) on UGT1A9 expression and the role of ERin the transcriptional regulation of UGT1A9. E2 significantly increased UGT1A9 promoter activity in HepG2 cells in the presence of ER. UGT1A9 induction by E2 was abrogated by antiestrogen ICI182,780 in HepG2 cells that constitutively express ER. Results from transient transfection of ERmutants into HepG2 cells demonstrated that mutation at DNA-binding domain of ERabrogates increased UGT1A9 promoter activity by E2. Deletion and mutation assays of UGT1A9 promoter revealed a putative ERE located within -2262/-1987 region. Examination of healthy human liver tissues revealed significantly higher UGT1A9 expression in women as compared to men. Together, these findings provide a mechanistic basis for the previous clinical reports and may shed a light on identifying sources for inter-individual variability in UGT1A9-mediated drug metabolism.