The Pharmacokinetics/Pharmacodynamics Of Resveratrol And PK-PD Modeling Research

Part I The pharmacokinetics of Res in melanoma tumor bearing mouseObjective:To study the pharmacokinetics of resveratrol (Res), resveratrol-glucuronide (Res-G) and resveratrol-sulfate (Res-S) in C57BL/6melanoma tumor bearing mouse after single-and multiple-dose administration of Res, and select PK input for PK-PD modeling.Methods:1) An sensitive HPLC-MS/MS method was developed for simutaneous determination of Res, Res-G and Res-S in mouse plasma.2) single dose of Res (50,100mg/kg) were oral administered to C57BL/6mouse implanted with B16F10melanoma tumor, blood sample were collected just before administration of drug and0.083,0.167,0.333,0.5,1.0,1.5,2.0,3.0,4.0,6.0,8.0,12.0h thereafter, the separated plasma samples were analyzed with the established method and the WinNonlin software was used to calculate corresponding pharmacokinetic parameters.3) C57BL/6tumor-bearing mouse were treated with continuous oral dosing of Res (50、100mg/kg/d) for21d, blood sample were collected just before administration of drug and0.167,1.0,2.0,4.0h thereafter at day3,6,9,12,15,18and21. The WinNonlin software was used to simulate the concentration-time curve of Res, Res-G and Res-S during multiple dosing and calculate corresponding pharmacokinetic parametersResults:Resveratrol showed rapid absorption and elimation in C57BL/6tumor-bearing mouse after single-dose administration, the plasma concentration of resveratrol glucuronide was higher than that of resveratrol and resveratrol-sulfate. Increases in AUC and reductions in clearance rate were observed during multiple-dose administration, indicating the accumulation effect of Res and Res-G, Res-S.Conclusion:Resveratrol was rapidly metabolized after its oral absorption and extensively metabolized into its two major metabolites, and the two metabolites are the main forms in circulation. The progession of melanoma tumor may cause accumulation effecs of resveratrol and its main metabolite during multiple dose administration. Part II The pharmacodynamics of Res in melanoma tumor bearing mouseObjective:To study The dynamic changes of tumor volume,Tyrosinase, COX-2, Ape/Ref-1, VEGFA and Survivin during the progession of melanoma tumor and Res treatment for selection of PD endpoint.Methods:1) C57BL/6tumor-bearing mouse were treated with continuous oral dosing of0.5%CMC-Na, Res (50、100mg/kg/d) for21d, and sacrificed at day3,6,9,12,15,18and21. Tumor volume were measured by vernier caliper every three days.2) Recombinant plasmids were construeted as standard reference used for absolute Quantitive real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for mRNA expression of Tyrosinase, COX-2, Ape/Ref-1, VEGFA and Survivin. Enzyme-linked immunosorbent assays (ELISA) were used for analysis of protein expression levels in tumor tissue homogenate. Specific substrates were used for determination of enzyme activity of Tyrosinase and COX-2. the redox-active of Ape/Ref-1in nuclear extract was detected by using of DNA-Binding ELISA kit.Results:The tumor volume, as well as the enzyme activities and expression levels of Tyrosinase, COX-2and Survivin increased with time during the melanoma tumor progession in control group, the enzyme activities and expression levels of Ape/Ref-1increased at early time and decreasd thereafter, the mRNA and protein expression of VEGFA increase on day9-12. The administration of resveratrol could reduce the growth speed of tumor, inhibit the activities of Tyrosinase, COX-2and Ape/Ref-1,down regulate the mRNA and protein expression of COX-2, Ape/Ref-1, VEGFA and Survivin.Conclusion:Tyrosinase, COX-2, Ape/Ref-1, VEGFA and Survivin showed dynamic changes during the progession of melanoma tumor, treatment with resveratrol could inhibit the growthspeed of melanoma tumor and inhibit the effects indexs described above the regulation at different levels Part Ⅲ The pharmacokinetic-pharmacodynamic modeling of Res in melanoma tumor bearing mouseObjective:To establish a PK-PD combination model for anti-melanoma effects of Res, which could elucidate the contradiction between the pharmacokinetic and pharmacodynamics of resveratrol, as well as the mechanisms of resveratrol.Methods:1) The plasma concentration and AUC of resveratrol and its metabolites were respectively fitted to pharamacodynamic indexs for selection of PK input;2) PK-PD modeling for the effects of resveratrol treatment on tumor volume;3) The choosed PK input were respectively fitted to the activities, mRNA and protein levels of Tyrosinase, COX-2, APE/Ref-1, VEGFA and Survivin for selection of PD endpoint.4) PK-PD modeling of the choosed PK input and PD endpoint for quantitative description of the correlation between the pharmacokinetics and pharmacodynamics of resveratrol treatment.Results:1) the total plasma concentration of resveratrol and its metabolites were selected as PK input;2) Sigmoid Emax model combined with exponential equation was fitted to the PK-PD modeling for the effects of resveratrol treatment on tumor volume;3) Simple Emax model was fitted to the PK-PD modeling for the effects of resveratrol treatment on enzyme activity, mRNA and protein expression of COX-2;4) Sigmoid Emax model was fitted to the PK-PD modeling for the effects of resveratrol treatment on enzyme activity of Tyrosinase, as well as mRNA and protein expression of VEGFA and Survivin.Conclusion:1) Res-G and Res-S may play roles in the in vivo activities of Res, which could be attribute to the deconjugation reactions of metabolite in tumor tissue or their own activities;2) PK-PD models were developed for the effects of resveratrol treatment on enzyme activity of Tyrosinase, as well as mRNA and protein expression of VEGFA and Survivin, which could quantitative describe the correlation between the pharmacokinetics and pharmacodynamics of resveratrol and Res-G and Res-S.