Characterizing the transport and disposition of resveratrol and its metabolites in the presence of MRP2, BCRP and enterohepatic circulation inhibitors

Abstract

My research deals with the interplay between metabolism and transport of resveratrol and its metabolites. It takes into account the role of uptake and efflux transporters and enterohepatic circulation in the disposition of resveratrol and conjugated metabolites of resveratrol. The issue of enzyme- and transporter-mediated drug-drug interaction (DDI) is also addressed. Chapter 1 presents an introduction to resveratrol, its biological activities as well as its interactions with enzymes and transporters. It provides a background for enzyme inhibition. It also explains the hypotheses and describes in short, the studies performed. Chapter 2 is based on P450 enzyme inhibition. In the first part of this chapter, we explored the ability of sandwich cultured cryopreserved human hepatocytes to predict inhibition parameters and drug-drug interaction (DDI) values. Two lots of cryopreserved human hepatocytes were used to predict inhibition parameters. The predicted DDI values were compared with those reported in literature and clinical studies and were found to be within 1.5 fold of those reported in clinical studies. The second part of this chapter focuses on the potential of resveratrol or resveratrol-3-glucuronide (R3G) to inhibit CYP2C8. CYP2C8 has been found to be inhibited by glucuronide metabolite such as gemfibrozil-O-β-glucuronide and clopidogrel-β-glucuronide. Hence, we examined the potential of resveratrol, R3G and resveratrol-3-sulfate (R3S) to inhibit CYP2C8. We found that resveratrol, R3G and R3S inhibited CYP2C8 in a reversible manner. Chapter 3 details studies performed in human cancer cell lines (HT-29 and Caco-2) to study the role of uptake transporters in the disposition of resveratrol and R3G. Western blotting was initially performed to examine the expression of OATP1B transporters in cancer cell lines. Uptake studies were performed in HT-29 and Caco-2 cell lines with atorvastatin as a positive control. Both, western blots and uptake studies were inconclusive in detecting the presence of OATP1B transporters. Our studies showed that resveratrol undergoes passive diffusion and sulfation in Caco-2 cell line. The uptake of R3G in Caco-2 cell line was not detectable. In chapter 4, we evaluated the impact of inhibition of efflux transporters on the disposition of resveratrol, R3G and R3S. Mrp2 and bcrp inhibition studies were performed in mice and resveratrol, R3G and R3S were monitored using LC-MS/MS. Non-compartmental analysis was performed to obtain pharmacokinetic parameters. We observed that the inhibition of efflux transporters had a greater impact on area under the curve (AUC) of R3S as compared to R3G and resveratrol. Resveratrol and R3G have been shown to undergo enterohepatic circulation (EHC). This occurs due to the action of gut bacterial β-glucuronidase. This enzyme converts the glucuronide metabolite into parent, which is reabsorbed into enterocytes. The impact of inhibition of gut bacterial β-glucuronidase due to antibiotics was studied in chapter 5. Elimination of gut microbiome was attempted by using a combination of neomycin and bacitracin. Non-compartmental analysis was performed on the observed data. There was no observable difference in the AUCs of resveratrol, R3G and R3S. Chapter 6 deals with simulations performed using an existing pharmacokinetic model to explain the data obtained upon transporter and EHC inhibition. The simulations showed that the inhibition of transporters seemed to decrease the elimination rate constant of R3G and R3S. In summary, we investigated the impact of transporters on pharmacokinetics of resveratrol and its major metabolites. We also investigated P450 inhibition in sandwich cultured human hepatocytes and the potential of resveratrol, R3G and R3S to inhibit rCYP2C8. We were able to show that inhibition of transporters does impact pharmacokinetics of R3S and R3G.