Date of Completion

12-23-2015

Embargo Period

12-26-2016

Keywords

Mechanistic toxicology, transporters, knock-outs, toxicokinetics, NSAID, reactive metabolite

Major Advisor

Jose E. Manautou, Ph.D.

Associate Advisor

Xiao-bo Zhong, Ph.D.

Associate Advisor

Christopher L. Shaffer, Ph.D.

Field of Study

Pharmaceutical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Diclofenac (DCF) is commonly used in the treatment of arthritis and pain. Although mechanisms of injury following DCF administration have been characterized, the roles that transporters have as modulators of exposure and toxicity remain unclear. To that end, in vivo transporter knockout (KO) models were used to assess the toxicokinetics of DCF and its major metabolites. DCF and its reactive acyl glucuronide (DCF-AG) were shown to be substrates for the efflux transporter Bcrp. Biliary excretion of DCF and DCF-AG decreased in Bcrp KOs while only DCF-AG plasma levels increased compared to wild-type (WT) mice. DCF-AG was likewise determined to be a substrate for the efflux transporter Mrp3 evidenced by reduced plasma concentrations in Mrp3 KOs without changes in biliary excretion. Toxic DCF challenge resulted in increased intestinal injury in Mrp3 KOs compared to WTs. In vitro assays revealed that human MRP3 can transport DCF-AG with apparent high affinity compared to human MRP2. The active uptake of DCF-AG was shown to be mediated by human OATPs in an in vitro system, specifically OATP2B1 exhibited concentration-dependent kinetics. Mechanistic studies indicated that DCF-AG was able to induce oxidative stress through creation of reactive oxygen species and inhibition of superoxide dismutase. Furthermore, DCF-AG was shown to inhibit both COX-1 and COX-2, which may have contributed to intestinal injury. Lastly it was demonstrated that DCF-AG can inhibit human MRP4 that is upregulated during oxidative stress. In summary, the data presented herein demonstrate the multifactorial pathways by which DCF-AG is transported and contributes to tissue injury.

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