Investigation of metabolic kinetics and reaction phenotyping of ligustrazin by using liver microsomes and recombinant human enzymes / 药学学报

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.

Effect of shenfu injection on CYP450s of rat liver / 药学学报

The paper is to report the study of the effect of Shenfu injection on the enzyme activity of liver CYP450 and its mRNA level of rat liver. Microsome of rat liver was prepared after intravenous administration of Shenfu injection for 7 days. The enzyme activity was quantified by Cocktail method. Meanwhile, the mRNA expression of CYP1A2, CYP2B1/2, CYP2C11 and CYP3A1 in the liver was detected by RT-PCR. Shenfu injection obviously induced the enzyme activities of CYP2B and CYP2C. Meantime Shenfu injection decreased the enzyme activities of CYP1A2 and CYP3A. The mRNA levels of CYP2B and CYP2C were also induced in rats treated with Shenfu injection. But it obviously inhibited the mRNA level of CYP1A2 and CYP3A. Since the enzyme activity and mRNA level were obviously changed after administration, the potential effect of drug-drug interaction should be concerned.

Effect of Siwu decoction and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats / 中国中药杂志

To study the effect of Siwu decoction (SWD) compound and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats. Rats were orally administered with SWD and water decoction combined with other medicines for two weeks, and then sacrificed. Their livers were perfused with normal saline to prepare liver micrisomes. Mixed probe and liver microsome in vitro incubation method were adopted to detect the effect of SWD on hepatic cytochrome P450. The real-time quantitative polymerase chain reaction (Q-PCR) was used to detect the effect of SWD on the expression of hepatic cytochrome P450. Compared with the control group, the SWD compound group showed higher CYP1A2 enzymatic activity (P < 0.05); Rehmanniae-paeoniae, angelicae-paeoniae, angelicae-rhizome, paeoniae-rhizome groups had lower CYP1A2 and CYP2C19 enzymatic activities (P < 0.05); And the compound group, the single component group and the combination group showed lower CYP2B6 enzymatic activities (P < 0.05). The compound could up-regulated the mRNA expression of CYP2B1 (P < 0.05); And the four single components could down-regulated the mRNA expression of CYP2B1 (P < 0.05). SWD compound had the effect in inducing CYP1A2 enzymatic activity. The rehmanniae-paeoniae group and the angelicae-paeoniae group had identical enzymatic activity with the control group, but significant down-regulation in CYP1A2 enzymatic activity after being combined with paeoniae. The compound and its combined administration showed the inhibitory effect on CYP2B6 enzymatic activity, particularly being combined with angelicae. The compound showed identical effect with the four single components in terms of CYP1A2 mRNA expression and enzymatic activity.

Evaluation of P-glycoprotein mediated in vitro loperamide biliary excretion with sandwich-cultured rat hepatocytes model / 药学学报

An in vitro P-glycoprotein mediated drug biliary excretion model (B-Clear model) was developed and validated using sandwich-cultured rat hepatocytes (SCRH) and a model substrate rhodamine 123 (Rh123). SCRH formed functional bile canalicular networks after 5 days of culture. Rh123 (10 micromol x L(-1)) was then incubated with the SCRH in standard Ca+ Hanks buffer or Ca(2+)-free buffer. The cumulative cell uptake and canalicular efflux of Rh123 under Ca2+ and Ca(2+)-free conditions were measured with a LC-MS/MS method. The biliary excretion index (BEI) and instinct biliary clearance (CL(bile, int)) were calculated. To assess the effect of known P-gp inhibitors on the efflux of Rh123, cyclosporine A (CyA), tariquidar (TQD) or quinidine (QND) (10, 50 and 100 micromol x L(-1)) was pre-incubated separately with SCRH for 30 min, then co-incubated with Rh123. The BEI and CL(bile, int) of Rh123 obtained from the SCRH model were (17.8 +/- 1.3) % and (10.7 +/- 0.9) mL x min(-1) x kg(-1), respectively. All the three P-gp inhibitors showed a dose-dependent inhibition on the bile clearance of Rh123, indicating that the B-Clear model with SCRH was functional properly. The biliary excretion of loperamide (LPAD) and the role of P-gp were further investigated with this validated model. The BEI and CL(bile, int) for LPAD (20 micromol x L(-1)) were obtained after it was incubated with SCRH for 30 min, and found to be (12.9 +/- 1.2)% and (6.1 +/- 0.3) mL x min(-1) x kg(-1) respectively. The dose-dependent inhibition on LPAD biliary excretion by CyA, TQD or QND confirmed the major role of P-gp in LPAD canalicular efflux. The results suggested that the B-Clear model with SCRH would be a useful tool for evaluation of P-gp mediated efflux and drug-drug interaction.

Studies on callus culture of Akebia trifoliata / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the inductive factors of effect on Akebia trifoliata and establish culture method for A. trifoliata callus.</p><p><b>METHOD</b>To study the possible effective factors of culture condition by comparing with different explantation, nutrient medium, pH, temperature, illumination, growth substance of plant and its ratio.</p><p><b>RESULT</b>The inductivity of leaves was the highest about 87.5%, followed with the stem section and leafstalk; The inductivity of nutrient medium such as MS, B5 callus was higher than the ones like H, SH and the White callus amended one; It was found that low-grade Phvalue benefits the growth of callus. The experiment result showed that different pH showed little difference in quality. The best condition of culture was 25 degrees C in temperature.</p><p><b>CONCLUSION</b>The best culture condition for callus was the leaves as explantation. The A. trifoliata callus culture's best inductive condition was MS +2, 4-D 4.0 mg x L(-1) + NAA 1.0 mg x L(-1) + KT 1.0 mg x L(-1) (pH 5.8), cultural temperature was 25 degrees C, cultivation was dark.</p>