PHARMACOKINETICS OF RESVERATROL, ITS MONOCONJUGATES AND ITS TRIMETHOXY ANALOG TMS

Abstract

Resveratrol (RES) has been associated with numerous pharmacological effects. Yet its pharmacokinetics is not clearly understood. It is known to get extensively metabolized into its sulfated and glucuronidated metabolites and has very low circulating RES concentrations in plasma. Although the concentrations of conjugated metabolites of RES have been reported to be much greater than that of RES, they have not been evaluated. This also becomes important in light of positive biological activities reported for sulfated metabolites of RES. Conjugation is a complex process which can sometimes be a reversible process and needs comprehensive evaluation to better understand RES and its metabolites' disposition. There has been a debate among the researchers regarding the differences in kinetics of preformed versus in vivo formed metabolites in the light of guidelines issued by regulatory bodies regarding metabolites in safety testing (MIST). We have addressed the above questions in this work, in addition to evaluating brain permeability of a potent RES analog, trimethoxy-trans-stilbene (TMS). Chapter 1 presents a detailed introduction, hypothesis and significance of my work. Chapter 2 includes the development and validation of a bioanalytical method for quantitation of RES and its metabolites on LC/MS/MS. We were able to develop and validate a robust bioanalytical method to quantitate RES and its four major metabolites resveratrol-4'-glucuronide (R4'G), resveratrol-3-glucuronide (R3G), resveratrol-4'-sulfate (R4'S) and resveratrol-3-sulfate (R3S). In Chapter 3, lung as a possible metabolizing organ for RES was evaluated. This study was performed in vivo in mouse model using multiple site of administration and single site of sampling approach. In vitro studies were also performed to confirm the in vivo results. Inter species differences in RES pulmonary metabolism were also studied. We observed lungs to be the major metabolizing organs for RES with inter species differences in its metabolism. Chapter 4 provides detailed pharmacokinetics of sulfated metabolites of RES, i.e. resveratrol-3-sulfate (R3S) and resveratrol-4'-sulfate (R4'S) in mouse model by both systemic and oral routes. In vitro studies were also conducted to test the desulfation in liver. Although we did not observe any significant RES in plasma, we observed from our in vitro studies that sulfated metabolites were desulfated in liver. Chapter 5 explains the detailed pharmacokinetics of glucuronidated metabolites of RES i.e. resveratrol-3-glucuornide (R3G) after both systemic and oral route. R3G was observed to undergo enterohepatic circulation. Explanation of R3G disposition in hepatocytes and enterocytes were proposed based on our own and reported results. In Chapter 6 we compared the differences in the kinetics of preformed versus in vivo formed metabolites using modeling and simulation approach. Individual models for disposition of RES, R3S and R3G were developed. These models were combined to give a global model for RES metabolism into R3S and R3G. Simulations were performed under two assumptions; preformed versus in vivo formed metabolite kinetics a) are same and b) they are not same. Our results reported that preformed and in vivo formed metabolite kinetics are not same at least for hydrophilic phase II metabolites. Chapter 7 includes method development and validation for quantitation of TMS in plasma and brain of mouse. Chapter 8 includes a steady state study to characterize the pharmacokinetic parameters of TMS, which was used to evaluate brain permeability of TMS. In summary we developed a robust bioanalytical method for direct quantitation of RES and its metabolites, found the lung to be a major metabolizing organ for RES, delineated complex kinetics of conjugated metabolites of RES, and showed differences in preformed versus in vivo formed metabolite kinetics and better brain permeability of TMS.