[Optimization of midazolam dosage and pharmacokinetics of CYP3A probe substrate in rats].

The study was conducted to evaluate the pharmacokinetics of midazolam (MDZ) under different oral dosages in rats, and determine the optimum oral dosage of MDZ, a CYP3 A probe substrate in vivo. Male Sprague-Dawley rats were given a single oral dosages of MDZ at 1,2,5,10,15 or 20 mg·kg(-1). Plasma concentrations of MDZ and its major metabolite 1-hydroxyl midazolam (1-OH MDZ) were determined at different time points using a validated LC-MS/MS method. Pharmacokinetic parameters were calculated using non-compartmental model. The C(max), AUC(0-t) and AUC0(∞) of MDZ and 1-OH MDZ were linearly increased over the dose range of 1-5 mg·kg(-1) (r > 0.99), but not at the dose of 15 or 20 mg·kg(-1). The AUC/Dose at 1-10 mg·kg(-1) were not significant different, but that of 15 or 20 mg·kg(-1) were significantly higher. No significant sedative reaction was observed in all the rats at dosages of 1-5 mg·kg(-1), however loss of righting reflex was observed in rats receiving dosages of 10-20 mg·kg(-1). In conclusion, the optimized oral dose of MDZ was 1-5 mg·kg(-1) when MDZ is used as the CYP3 A probe substrate in rats.

In vivo to in vitro effects of six bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) on the CYP3A/P-glycoprotein-mediated absorption and metabolism of tacrolimus.

We recently reported that Wuzhi tablet (WZ; Schisandra sphenanthera extract) can inhibit P-glycoprotein (P-gp)-mediated efflux and CYP3A-mediated metabolism of tacrolimus (FK506) and thus increase the blood concentrations of FK506. Major active lignans of WZ include schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, and schisantherin A. Whether and how these six lignans affect the pharmacokinetics of FK506 remains unclear. Therefore, this study aimed to investigate the effects of these lignans on the first-pass absorption and metabolism of FK506 and the involved mechanisms in vitro and in vivo. The results showed that whole-blood concentrations of FK506 were increased to different degrees following coadministration of the six lignans, respectively. Schisandrol B showed the strongest effect on the increase of the area under the concentration-time curve, the oral bioavailability, the gut processes affecting availability, and the hepatic availability of FK506. The reduction of intestinal first-pass effect contributed most to the increase in oral bioavailability of FK506 when coadministered with schisandrol B. In vitro transport experiment showed that schisandrin A, schisandrin B, and schisandrol B inhibited P-gp-mediated efflux of FK506. In vitro metabolism study showed that the inhibitory effect of these six lignans on FK506 metabolism was dose-dependent. In conclusion, the exposure of FK506 in rats was increased when coadministered with these lignans, and schisandrol B showed the strongest effect. Lignans of WZ inhibited P-gp-mediated efflux and CYP3A-mediated metabolism of FK506, and the reduction of intestinal first-pass affected by the lignans was the major cause of the increased FK506 oral bioavailability.

Effect of Wuzhi tablet (Schisandra sphenanthera extract) on the pharmacokinetics of cyclosporin A in rats.

In our previous reports, Wuzhi tablet (an herbal preparation of ethanol extract of Wuweizi (Schisandra sphenanthera)) can significantly increase the blood concentration of tacrolimus and paclitaxel in rats by inhibiting the CYP3A-mediated metabolism and the P-gp-mediated efflux. Cyclosporin A (CsA), a well-known immunosuppressant agent, is also a substrate of CYP3A and P-gp. Therefore, this study aimed to investigate whether and how WZ affects pharmacokinetics of CsA in rats. The AUC0-48 h and Cmax of CsA were increased by 40.1% and 13.1%, respectively, with a single oral co-administration of WZ and high dose of CsA (37.8 mg/kg). Interestingly, after a single oral co-administration of WZ and low dose of CsA (1.89 mg/kg), the AUC0-36 h and Cmax of CsA were dramatically increased by 293.1% (from 1103.2 ± 293.0 to 4336.5 ± 1728.3 ng.h/mL; p < 0.05) and 84.1% (from 208.5 ± 67.9 to 383.1 ± 92.5 ng/mL; p < 0.05), respectively. The CL/F was decreased from 1.7 L/h/kg to 0.5 L/h/kg. Thus, the effect of WZ on high dose of CsA was not significant, but pharmacokinetic parameters of CsA at low dose were significantly influenced by co-administration of WZ. The herb-drug interaction should be taken into consideration at this situation.

Effect of Tacrolimus on the pharmacokinetics of bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) and the potential roles of CYP3A and P-gp.

We recently reported that Wuzhi tablet (WZ), a preparation of the ethanol extract of Wuweizi (Schisandra sphenanthera), had significant effects on blood concentrations of Tacrolimus (FK506) in renal transplant recipients and rats. The active lignans in WZ are schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, schisantherin A, and schisantherin B. Until now, whether the pharmacokinetics of these lignans in WZ would be affected by FK506 remained unknown. Therefore, this study aimed to investigate whether and how FK506 affected pharmacokinetics of lignans in WZ in rats and the potential roles of CYP3A and P-gp. After a single oral co-administration of FK506 and WZ, the blood concentration of lignans in WZ was decreased by FK506; furthermore, the AUC of schisantherin A, schisandrin A, schisandrol A and schisandrol B was only 64.5%, 47.2%, 55.1% and 57.4% of that of WZ alone group, respectively. Transport study in Caco-2 cells showed that these lignans were not substrates of P-gp, suggesting decreased blood concentration of lignans by FK506 was not via P-gp pathway. Metabolism study in the human recombinant CYP 3A showed that these lignans had higher affinity to CYP3A than that of FK506, and thus had a stronger CYP3A-mediated metabolism. It was concluded that the blood concentrations of these lignans were decreased and their CYP3A-mediated metabolisms were increased in the presence of FK506 since these lignans had higher affinity to CYP3A.

CAR-mediated up-regulation of CYP3A4 expression in LS174T cells by Chinese herbal compounds.

The constitutive androstane receptor (CAR) is an orphan nuclear receptor which has been shown to participate in the activation of human CYP3A4, which metabolizes more than 50% of clinically used drugs. We investigated the effects of an array of compounds isolated from herbal medicines such as Rheum palmatum (Da Huang), Peucedanum praeruptorum Dunn (Qian Hu), Cortex Mori Radicis (Sang Bai Pi), Radix Asteris (Zi Wan), Salvia miltiorrhiza (Dan Shen), Polygonum cuspidatum Sieb. et Zucc (Hu Zhang), and Ginkgo biloba (Yin Xing) on the CAR-mediated transactivation of CYP3A4. The effect of herbal compounds on CYP3A4 expression was measured using a CYP3A4 luciferase reporter gene assay in transiently transfected human intestinal LS174T cells. The gene expression, protein expression, and catalytic activity of CYP3A4 in LS174T cells transfected with CAR were determined by using real-time PCR, Western blot analysis, and LC-MS/MS-based substrate assay. The study found that in CAR-transfected cells, praeruptorin A, C, and D significantly induced CYP3A4 luciferase activity, mRNA expression, and functional activity through the CAR-mediated pathway; conversely, induction was not found in untransfected cells. Our findings suggest that these herbal compounds can significantly up-regulate the CYP3A4 gene via the CAR-mediated pathway, which has important implications in herb-drug interactions.

Mechanistic understanding of the different effects of Wuzhi Tablet (Schisandra sphenanthera extract) on the absorption and first-pass intestinal and hepatic metabolism of Tacrolimus (FK506).

We recently reported that the blood concentrations of Tacrolimus (FK506) in rats were markedly increased following the intake of a Chinese herbal preparation, Wuzhi Tablet (WZ, Schisandra sphenanthera extract). In order to identify the underlying mechanisms of the increase in FK506 level, we investigated the effects of WZ on the absorption and first-pass intestinal and hepatic metabolism of FK506 in vitro and in vivo. When co-administered with WZ, the AUC(0-infinity) value after oral FK506 dosing was increased by 2.1 fold, the oral bioavailability (F(oral)) was increased from 5.4% to 13.2% (p=0.0002), and the (F(abs) x F(G)) was 111.4% (p<0.01), much greater than that when FK506 was given alone. However, the F(H) was only 21.2% greater than that when FK506 was given alone, which indicates that the reduction of intestinal first-pass effect was the major cause of the increased FK506 oral bioavailability when co-administered with WZ. In the Caco-2 cell transport study, the transport ratio of FK506 with WZ extract was significantly lower than that of FK506 alone, which suggested WZ extract inhibited P-gp-mediated efflux of FK506. Furthermore, 100 microM of WZ extract almost completely inhibited FK506 metabolism in rat and human liver microsomes, indicating WZ extract potently inhibited the CYP3A-mediated metabolism of FK506. In conclusion, WZ inhibited P-gp-mediated efflux and CYP3A-mediated metabolism of FK506, and the reduction of intestinal first-pass effect by WZ was the major cause of the increased FK506 oral bioavailability.