Multidrug Resistance-Associated Protein 3 Plays an Important Role in Protection against Acute Toxicity of Diclofenac

Abstract

Diclofenac (DCF) is a nonsteroidal anti-inflammatory drug commonly prescribed to reduce pain in acute and chronic inflammatory diseases. One of the main DCF metabolites is a reactive diclofenac acyl glucuronide (DCF-AG) that covalently binds to biologic targets and may contribute to adverse drug reactions arising from DCF use. Cellular efflux of DCF-AG is partially mediated by multidrug resistanceassociated proteins (Mrp). The importance of Mrp2 during DCFinduced toxicity has been established, yet the role of Mrp3 remains largely unexplored. In the present work, Mrp3-null (KO) mice were used to study the toxicokinetics and toxicodynamics of DCF and its metabolites. DCF-AG plasma concentrations were 90% lower in KO mice than in wild-type (WT) mice, indicating that Mrp3 mediates DCF-AG basolateral efflux. In contrast, there were no differences in DCF-AG biliary excretion between WT and KO, suggesting that only DCF-AG basolateral efflux is compromised by Mrp3 deletion. Susceptibility to toxicity was also evaluated after a single high DCF dose. No signs of injury were detected in livers and kidneys; however, ulcers were found in the small intestines. Furthermore, the observed intestinal injuries were consistently more severe in KO compared with WT. DCF covalent adducts were observed in liver and small intestines; however, staining intensity did not correlate with the severity of injuries, implying that tissues respond differently to covalent modification. Overall, the data provide strong evidence that (1) in vivo Mrp3 plays an important role in DCF-AG disposition and (2) compromised Mrp3 function can enhance injury in the gastrointestinal tract after DCF treatment.

Diclofenac and Its Acyl Glucuronide: Determination of In Vivo Exposure in Human Subjects and Characterization as Human Drug Transporter Substrates In Vitro

“…The sinusoidal efflux of DF-AG into blood, once formed in the liver, is largely dependent on Mrp3 (Lagas et al, 2009). Recently, it was reported that the plasma concentration of DF-AG is 90% lower in Mrp3 gene knockout mice (versus wild-type mice), confirming that basolateral efflux of DF-AG is mediated by Mrp3 (Scialis et al, 2015). These results support the fact that various transporters play an important role in the distribution and elimination of DF-AG.…”

Elucidation of the Mechanisms through Which the Reactive Metabolite Diclofenac Acyl Glucuronide Can Mediate Toxicity

“…The argument against covalent adduction as an injury mechanism was supported by our observation that covalent adducts were observed in the liver without apparent toxicity or morphologic changes following DCF administration (Scialis et al, 2015). A possible causal factor for enteropathy is oxidative stress through inhibition of superoxide dismutase (SOD), which is a critical cellular defense mechanism for coping with reactive oxygen species (ROS) (Fridovich, 1978).…”

“…We observed that mice lacking the efflux transporter Mrp3 were more susceptible to intestinal injury compared with WT (Scialis et al, 2015). Though the intestinal toxicity differences between WT and KO were unequivocal, the mechanisms behind those differences were uncertain.…”

“…3B). The cytotoxic DCF-AG concentrations are physiologically relevant as they reflect the biliary concentrations detected in mice after an administration of 75 mg/kg of DCF (Scialis et al, 2015). Importantly, the upper concentration of 1 mM in the cytotoxicity assay reflects the lowest DCF-AG biliary concentration observed in our previous toxicokinetic mouse study.…”

“…Our work with efflux transporters demonstrated Bcrp (Abcg2) and Mrp3 (Abcc3) have functional roles with respect to DCF and DCF-AG disposition (submission pending). Toxicodynamic studies conducted in Mrp3 knockout (KO) mice showed conclusively that KO mice developed increased intestinal damage compared with wild-type (WT) (Scialis et al, 2015). Despite the susceptibility differences between genotypes, the mechanisms for the increased injury remain unclear.…”