Studies On Pharmacokinetics Of Wuzhi Capsule And Interactions Of Tacrolimus And Wuzhi Capsule

Traditional Chinese medicine (TCM), which has a long history in China and other Asian countries, has already been proven to have a certain advantage of chronic disease treatment that hardly leads to adverse or toxic effects and gradually taken seriously by North America and Europe. In recent years, under the influence of the "back to nature" trend of thought in the world, the use of TCM increased dramatically. Most of them are often co-administered with chemical synthetic drugs, especially for people with HIV infection or cancer who are taking a higher proportion of TCM for improving psychical and physiological conditions. The past, most people think of TCM derived from natural plants, safe and non-toxic, unlike the chemical drugs as vulnerable to drug interactions. In fact, TCM contains a variety of bio-active ingredients, whose chemical composition is similar with commonly used chemical drugs, thus can play a role in physiological regulation together through synergistic or antagonistic effects and is also possible to interact with any type of drug. Although most people paid attention to drug-drug interaction in the past years, recently, there are an increased number of reports that interactions between TCM and chemical drugs resulted in decreased efficacy and toxicity, which has aroused widespread concern in the medical community.Here, we investigated quality control of Nan-wuweizi (TCM) and developed quantitative analysis of tacrolimus, one of common clinical immunosuppressants, and Wuzhi-capsule (WZC) in vivo by HPLC and LC-MS/MS for understanding pharmacokinetic interactions of both drugs combination. The results are as follows:1. A simple and sensitive high performance liquid chromatography method with photodiode array detection (HPLC-DAD) was developed for simultaneous determination of eight bioactive constituents (schisandrin, schisandrol B, schisantherin A, schisanhenol, anwulignan, deoxyshisandrin, schisandrin B and schisandrin C) in the ripe fruit of Schisandra sphenanthera and its traditional Chinese herbal preparations Wuzhi capsule by optimizing the extraction, separation and analytical conditions of HPLC-DAD. The chemical fingerprint of S. sphenanthera was established using raw materials of 15 different origins in China. The chromatographic separations were obtained by an Agilent Eclipse XDB-C18 reserved-phase column (250mm×4.6mm i.d.,5μm) using gradient elution with water-formic acid (100:0.1, v/v) and acetonitrile, at a flow rate of 1.0 mLmin-1, an operating temperature of 35℃, and a wavelength of 230 nm. The constituents were confirmed by (+) electrospray ionization LC-MS. The new method was validated and was successfully applied to simultaneous determination of components in 13 batches of Wuzhi-capsule. The results indicate that this multi-component determination method in combination with chromatographic fingerprint analysis is suitable for quantitative analysis and quality control of S. sphenanthera and its preparation. It is also concluded that the quality of Wuzhi-capsule is stable.2. A rapid sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of multiple bioactive lignan constituents of Wuzhi capsule in rat plasma. The extraction, separation, and analytical conditions were optimized. Five constituents of the Wuzhi-capsule (schisandrin, schisandrol B, schisantherin A, schisanhenol, and deoxyshisandrin) were determined by the LC-MS/MS method. Liquid-liquid extraction with methyl tert-butyl ether was carried out using bifendate as the internal standard. The five bioactive constituents were separated on a Zorbax SB-C18 reserved-phase column (100mm×2.1mm i.d.,3.5μm) by isocratic elution using amobile phase consisting of acetonitrile, methanol, and 0.1% aqueous formic acid (72:18:10, v/v/v) at aflow rate of 0.3 mL/min. The total run time was only 3.5 min. All analytes showed good linearity over a wide concentration range (r2> 0.99) and their lower limit of quantification was 0.5 ng/mL. The average extraction recovery of the five analytes from rat plasma was more than 85%, and the intra-day and inter-day accuracy and precision of the assay were less than 15%. Our method was successfully used for pharmacokinetic study of the five components in the Wuzhi capsule. Meantime, quantitative analysis of tacrolimus was developed in rat plasma. The method meets the current FDA criteria for bioanalytical method validation and successfully applied to pharmacokinetic interactions with Wuzhi capsule. We also developed a sensitive, selective, convenient and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of five lignan constituents of Wuzhi capsule in human plasma and their pharmacokinetics in healthy Chinese volunteers, which provided reference for clinical reasonable medication.3. Quantitative analysis method of tacrolimus was developed in rat plasma, which meets the current FDA criteria for bioanalytical method validation and successfully applied to pharmacokinetic interactions with Wuzhi capsule. The results showed pharmacokinetic parameters of tacrolimus (Cmax,59.42±30.32ng/mL; Tmax,1.54±0.15h; AUC0-t, 239.71±28.86 ng h/mL) in rat plasma after single administration of Wuzhi capsule were remarkably enhanced five times, compared to administration of tacrolimus alone(Cmax, 18.87±10.29ng/mL; Tmax,0.38±0.21h; AUC0-t, 40.98±37.07 ng h/mL). After 12d administration of tacrolimus, the pharmacokinetic parameters of tacrolimus (Cmax, 43.16±10.61 ng/mL; Tmax,0.32±0.09h; AUC0-t,89.21±26.39 ng h/mL) were obtained among the rats orally administered tacrolimus and Wuzhi capsule on the 12th day. Compared to blank group, the pharmacokinetic parameters improved or extended a little, but compared to single administration of tacrolimus and Wuzhi capsule, the pharmacokinetic parameters have somewhat decreased. These results implied that Wuzhi capsule at in vivo different dose and times has synergistic or antagonistic effects on tacrolimus efficacy. Its mechanism may be more complicated, when the collaboration is stronger than inhibition, showed synergy. However, it is affirmative that Wuzhi capsule at single dose makes significant synergies effects on the pharmacokinetics of tacrolimus. The above validated method was successfully applied to the pharmacokinetic study of tacrolimus. The SD rats were randomly divided into groups. Different dosage Wuzhi capsule was orally administrated at a dosage of 1.2mg/kg tacrolimus for each group. The pharmacokinetic parameters of tacrolimus were calculated on the plasma concentrations of tacrolimus in rats plasma samples. The results indicated that Cmax depends on Wuzhi capsule dosage; AUC with increasing doses of Wuzhi capsule gradually increased, until orally administrated dosage of 450mg/kg Wuzhi capsule, AUC increased to maximum825.34 ng h/mL, then decreased. The reason probably was that excessive stomach medicine is difficult to absorb in time along with increasing dosage.4. The absorption and metabolism of tacrolimus and major index component of ethanol extracts of Nan-wuweizi in vitro were systematically investigated through Caco-2 cell model and liver microsomes incubation, which determined the role of cytochrome P450 enzymes (CYP450) in their metabolic pathway for the knowledge of the metabolism mechanism and prediction of herb-drug interaction. The results showed that schisandrol B, deoxyschizandrin, schisantherin A and Wuzhi capsule solution can obviously inhibit uptake and efflux function of tacrolimus, especially deoxyschizandrin most. The transport of tacrolimus using Caco-2 cell monolayers demonstrated that its bioavailability was enhanced when Wuzhi capsule inhibited the P-gp efflux pump.5. The effects of Wuzhi capsule and its bioactive constituents on CYP3A4,CYP1A2,CYP2C9,CYP2C19 and CYP2D6 enzyme activity were investigated through liver microsomes incubation, which could further affect tacrolimus metabolism in the liver microsomes. The results showed Wuzhi capsule and eight bioactive constituents in the ripe fruit of Schisandra sphenanthera had different degrees of inhibition on five enzymes, among which main constituents showed stronger inhibition on CYP3A4 and CYP2C19. Our studies have further proved that Wuzhi capsule inhibited tacrolimus metabolism and enhanced its bioavailability through inhibition of CYP450.