Shuqing Chen

PXR-mediated transcriptional activation of CYP3A4 by cryptotanshinone and tanshinone IIA

Danshen (Radix Salvia miltiorrhiza) is a famous Traditional Chinese Medicine used widely for the treatment of coronary heart disease and cerebrovascular disease. Diterpenoid tanshinones including tanshinone I, tanshinone IIA and cryptotanshinone are the major bioactive components from Danshen herb. Previous reports have demonstrated that Danshen extracts could induce the expression of CYP3A in rodents, however, the constituents responsible for Danshen-mediated CYP3A induction and the underlying molecular mechanisms remain unknown. The discovery of a family of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR) and glucocorticoid receptor (GR) gives insight into the molecular explanation of CYP3A induction by xenobiotics. In the present study, interactions between Danshen constituents and human PXR were evaluated using a reporter gene assay. Our observations showed that Danshen ethanol extract could activate human PXR and induce the CYP3A4 reporter construct in HepG2 cells. Tanshinone IIA and cryptotanshinone were identified as efficacious PXR agonists, and cryptotanshinone activated the CYP3A4 promoter more strongly than tanshinone IIA. Furthermore, CAR and GR were also involved in the induction of CYP3A4 expression by tanshinones, though their roles seemed not as important as PXR. Treatment of LS174T cells with cryptotanshinone or tanshinone IIA resulted in a significant increase of CYP3A4 mRNA, which was consistent with the results from the reporter gene assay. Collectively, activation of PXR and the resultant CYP3A4 induction mediated by cryptotanshinone and tanshinone IIA provide a molecular mechanism for previously observed CYP3A induction by Danshen extracts, and our findings also suggest that caution should be taken when Danshen products are used in combination with therapeutic drugs metabolized by CYP3A4.

Identification of novel pregnane X receptor activators from traditional Chinese medicines

AIM OF THE STUDY To investigate the ability of traditional Chinese medicines (TCMs) and their bioactive compounds to activate pregnane X receptor (PXR) signalling pathway. MATERIALS AND METHODS We screened ethanol extracts of 28 commonly used TCMs for their capability to induce cytochrome P450 3A4 (CYP3A4) via PXR signalling pathway using a cell-based reporter gene assay combined with RT-PCR analysis. In addition, 34 bioactive components from these TCMs were examined for their potential to activate PXR. RESULTS Our observations showed that 22 ethanol extracts and 8 compounds could activate human PXR and induce CYP3A4 reporter construct in HepG2 cells. Among them, Ginkgo biloba, Ligusticum chuanxiong, Chinese angelica, prepared Rehmannia root, Epimedium brevicornum, Atractylodes macrocephala, Schisandra chinensis, Paeonia lactiflora, Ophiopogon japonicus, Polygonum multiflorum, Coptis chinensis, Artemisia scoparia, Trichosanthes kirilowii, Silybum marianum, Gardenia fruit and Lycium chinense could strongly trans-activate PXR. Moreover, ligustilide, schisantherin A, berberine hydrochloride and trans-resveratrol were identified for the first time as efficacious PXR agonists. CONCLUSIONS Twenty-two TCM ethanol extracts and eight bioactive compounds could activate PXR signalling pathway and induce CYP3A4 reporter gene. Therefore, caution should be taken when these PXR activators are used in combination with prescribed drugs metabolized by CYP3A4.

Genetic Variants of Human UGT1A3: Functional Characterization and Frequency Distribution in a Chinese Han Population

UDP-glucuronosyltransferase 1A3 (UGT1A3) contributes to glucuronidation of many important endogenous compounds and xenobiotics, including some flavonoids. Recently, a total of six single nucleotide polymorphisms (SNPs) have been identified in the human UGT1A3 gene. Among them, four SNPs (A17G, Q6R; T31C, W11R; C133T, R45W; and T140C, V47A) cause amino acid substitutions. Variants caused by these SNPs showed an activity change in estrone metabolism, whereas their activities toward other substrates were not examined. In the present study, three common flavonoids, quercetin, luteolin, and kaempferol, were used as substrates for glucuronidation by wild-type and variant UGT1A3s. Our results demonstrated that the activities of three variants, UGT1A3.2, UGT1A3.3, and UGT1A3.5, were remarkably lower than that of UGT1A3.1. In contrast, UGT1A3.4 exhibited an increase in glucuronidation efficiency of approximately 4 times and a clear preference to quercetin 7- and 3-hydroxyl groups. The frequency distributions of UGT1A3 alleles and SNPs in UGT1A3 in a Chinese Han population were statistically different from the reported value in German-Caucasians (p < 0.05). UGT1A3 variants have an altered glucuronidation activity toward quercetin, luteolin, and kaempferol and may alter human susceptibility to flavonoid exposure.

Structure–metabolism relationships for the glucuronidation of flavonoids by UGT1A3 and UGT1A9

Objectives  This study tries to find structure–metabolism relationships between flavonoids and human UGT1A3 and UGT1A9.

Effects of intrauterine undernutrition on the expression of CYP3A23/3A1, PXR, CAR and HNF4α in neonate rats

Cytochrome P‐450 3A (CYP3A) together with its nuclear receptors plays a critical role in drug metabolism. The present study investigated the effects of undernutrition in utero on hepatic mRNA and protein expression of the enzyme CYP3A23/3A1 and nuclear receptors including pregnane X receptor (PXR; NR1I2), constitutive androstane receptor (CAR; NR1I3) and nuclear factor‐4alpha (HNF4α; HNF4A) in neonatal rats. At gestational day 2, pregnant rats were randomly divided into two groups: nourished (fed ad libitum) and undernourished (50% of nourished group). The pups delivered by nourished rats were designated as the normal‐birth‐weight group (NBW, n=15) and those delivered by undernourished rats were designated as the low‐birth‐weight group (LBW, n=15). Hepatic mRNA expression was detected by quantitative real‐time PCR and the corresponding protein expression was examined by immunohistochemistry (IHC). Compared with NBW pups, LBW pups tended to have lower mRNA expression levels of CYP3A23/3A1, PXR and CAR but higher levels of HNF4α. Only the CAR mRNA expression differences were significant (p<0.05). mRNA expression of CYP3A23/3A1 correlated with that of HNF4α in both the LBW(r=0.808, p=0.007) and NBW (r=0.452, p=0.012) groups. CYP3A23/3A1 and CAR protein expression differed between the two groups (CYP3A23/3A1, χ2=7.87, p=0.005; CAR, χ2=12.069, p=0.001). In conclusion, these findings suggest that undernutrition may influence the mRNA expression of CAR and protein expression of both CYP3A23/3A1 and CAR in neonatal rats. Since CYP3A23/3A1 and CAR are critically involved in drug metabolism, these results may have clinical implications for optimal medication in LBW children. Copyright

Population pharmacokinetics of ciclosporin in Chinese children with aplastic anemia: effects of weight, renal function and stanozolol administration.

Aim:To develop a population pharmacokinetic model for the immunosuppressant ciclosporin in Chinese children with aplastic anemia and to identify covariates influencing ciclosporin pharmacokinetics.Methods:A total of 102 children with either acquired or congenital aplastic anemia aged 8.8±3.6 years (range 0.9–17.6 years) were included. Therapeutic drug monitoring (TDM) data for ciclosporin were collected. The population pharmacokinetic model of ciclosporin was described using the nonlinear mixed-effects modeling (NONMEM) VI software. The final model was validated using bootstrap and normalized prediction distribution errors.Results:A one-compartment model with first-order absorption and elimination was developed. The estimated CL/F was 15.1, which was lower than those of children receiving stem cell or kidney transplant reported in the West (16.9–29.3). The weight normalized CL/F was 0.45 (range: 0.27–0.70) Lh−1·kg−1. The covariate analysis identified body weight, serum creatinine and concomitant administration of the anabolic steroid stanozolol as individual factors influencing the CL/F of ciclosporin.Conclusion:Our model could be used to optimize the ciclosporin dosing regimen in Chinese children with aplastic anemia.

Stereoselective glucuronidation of carvedilol by Chinese liver microsomes

ObjectiveTo study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes.MethodsThe metabolites of CARV were identified by a hydrolysis reaction with β-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylisothiocyanate.ResultsTwo CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of Km and Vmax for (S)-CARV and (R)-CARV enantiomers were (118±44) μmol/L, (2500±833) pmol/(min·mg protein) and (24±7) μmol/L, (953±399) pmol/(min·mg protein), respectively.ConclusionThese results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.