Nonsteroidal anti-inflammatory drugs (NSAIDs) are a widely used class of drugs, due in part to the effective anti-inflammatory and analgesic properties they exhibit. Unfortunately, NSAIDs also exhibit substantial gastrointestinal (GI) toxicity. The mechanisms underlying the ability of NSAIDs to cause ulceration in the stomach and proximal duodenum are well understood, and this injury can largely be prevented through the suppression of gastric acid secretion by proton pump inhibitors (PPIs) or histamine H2 receptor antagonists (H2RAs). In contrast, the pathogenesis of small intestinal injury induced by NSAIDs (i.e., NSAID-enteropathy) is poorly understood, and there are no proven-effective therapies. This is a major clinical concern as NSAID-induced enteropathy and bleeding occur more frequently than NSAID-induced gastropathy, and is associated with significantly higher rates of morbidity and mortality. There is clear evidence that indicates important contributions to NSAID-enteropathy by bile, enteric bacteria, and the enterohepatic circulation of NSAIDs. However, it is not clear which of these mechanisms is/are the primary driver(s) of intestinal damage and injury. There is also evidence that hydrogen sulfide (H2S) can protect the GI mucosa from ulceration and reduce the severity of NSAID-induced GI damage, although the mechanisms of H2S-induced intestinal protection remain to be determined. Therefore, the central aim of this thesis was to evaluate the roles of bile, enteric bacteria, and the enterohepatic circulation of NSAIDs in the pathogenesis of NSAID-enteropathy, and to investigate the ability of H2S to protect the small intestine from NSAID-induced damage. Chapter 1 is an introduction to the relevant literature and Chapter 2 is an outline of the thesis scope and objectives. In Chapter 3, I demonstrated that the co-administration of an H2S-releasing agent protected rats from NSAID-induced enteropathy, in part by preventing NSAID-induced dysbiosis and bile cytotoxicity. In Chapter 4 and 5, I established that the co-administration of PPIs and H2RAs exacerbated NSAID-enteropathy in part by causing intestinal dysbiosis and enhanced bile cytotoxicity. Lastly, I demonstrated that the small intestine-sparing effects of an H2S-releasing NSAID, ATB-346, are partly attributable to the reduced enterohepatic circulation of ATB-346 or the naproxen liberated from this drug (Chapter 5). In summary, the work presented in this thesis provided novel understanding of the complicated pathogenesis of NSAID-enteropathy by confirming that the nature of the bile, the enterohepatic circulation of NSAIDs, and the nature of the intestinal microbiota are of paramount importance. In addition, the results also demonstrated that hydrogen sulfide represents an effective preventative therapy for NSAID-enteropathy and that H2S-releasing NSAIDs, such as ATB-346, have remarkable preclinical safety. / Thesis / Doctor of Philosophy (PhD)

Orally administered drugs are generally absorbed by the small intestine and transported either to the lymphatic system or to the hepatic portal system. In general, lipid soluble drugs and vitamins are transported by the small intestine to the lymphatics, and water-soluble drugs are transported to the hepatic portal system. By avoiding the early hepatic first pass effect, the lymphatic transport system may increase drug bioavailability. In addition to its transport systems, the small intestine may affect drug bioavailability through drug uptake, intestinal first pass effect, recruitment of drugs by chylomicrons, formation and secretion of chylomicrons, and enterohepatic circulation. All of these factors should be considered when formulating orally administered lipophilic drugs. Our data also suggest that Caco-2 cells may serve as a valuable in vitro model to study the intestinal transport of orally administered drugs.

Orally administered drugs are generally absorbed by the small intestine and transported either to the lymphatic system or to the hepatic portal system. In general, lipid soluble drugs and vitamins are transported by the small intestine to the lymphatics, and water-soluble drugs are transported to the hepatic portal system. By avoiding the early hepatic first pass effect, the lymphatic transport system may increase drug bioavailability. In addition to its transport systems, the small intestine may affect drug bioavailability through drug uptake, intestinal first pass effect, recruitment of drugs by chylomicrons, formation and secretion of chylomicrons, and enterohepatic circulation. All of these factors should be considered when formulating orally administered lipophilic drugs. Our data also suggest that Caco-2 cells may serve as a valuable in vitro model to study the intestinal transport of orally administered drugs.

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. / Pharmaceutical Sciences

Chemotherapy, employing single-molecule or multidrug concoctions inspired by the diverse repository of plant chemicals, has been the mainstay of cancer treatment for years. However, isolating single molecules has proven to be expensive along with limited therapeutic window and toxicity. On the other hand, whole foods, while preserving the natural complex balance between their constituent phytochemicals and being non-toxic, have proven to impart better disease-fighting efficacies, thus leading to an increased focus on dietary interventions to both treat and prevent cancer. Owing to the complex interactions between their constituent phytochemicals, several dietary agents have been investigated for their therapeutic and preventive efficacies. However, due to lack of emphasis on confounding factors like bioavailability, absorption, metabolism, and excretion, essentially driven by phytocomplexity, incorporation of whole foods in therapeutic regimen has not been successful. This thesis exemplifies the need to investigate factors associated with the limitations in the current approach with respect to dietary agents. Bioactivity-guided fractionation of sweet potato greens extract (SPGE) led to the identification of ~100-fold more potent fraction in vitro. However, this efficacy could not be translated in vivo. We also studied whole ginger extract (GE) for its in vitro and in vivo prostate tumor growth-inhibitory and apoptosis-inducing effects. In addition, GE proved to be more efficacious as compared to its individual most-active constituents owing to the differences in their pharmacokinetic (PK) and bioavailability measurements. Hence, these studies emphasize the crucial role of synergistic/additive interactions among the constituents of whole foods in successful translation of their therapeutic benefits. Another factor that seeks further attention is the unique cellular mechanisms engaged by these phytochemicals to confer their remarkable effects. Phenolic compounds, the most-abundant of all phytochemicals, are well known for their antioxidant properties and act via reactive oxygen species (ROS)-mediated mechanisms. We however assert the underappreciated xenohormetic prooxidant role of phenolics, where cancer cell death is caused by induction of intolerable levels of ROS. We demonstrated that a Piper betel constituent, hydroxychavicol (HC), mediates cytotoxicity via ROS-induced DNA-damage. This thesis thus provides compelling grounds for future preclinical studies to validate their potential usefulness for cancer management.