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

Abstract

We have previously reported that mice lacking the efflux transporter Mrp3 had significant intestinal injury after toxic diclofenac (DCF) challenge, and proposed that diclofenac acyl glucuronide (DCF-AG), as a substrate of Mrp3, played a part in mediating injury. Since both humans and mice express the uptake transporter OATP2B1 in the intestines, OATP2B1 was characterized for DCF-AG uptake. In vitro assays using human embryonic kidney (HEK)-OATP2B1 cells demonstrated that DCF-AG was a substrate with a maximal velocity (V max ) and K m of 17.6 6 1.5 pmol/min per milligram and 14.3 6 0.1 mM, respectively. Another key finding from our in vitro assays was that DCF-AG was more cytotoxic compared with DCF, and toxicity occurred within 1-3 hours of exposure. We also report that 1 mM DCF-AG caused a 6-fold increase in reactive oxygen species (ROS) by 3 hours. Investigation of oxidative stress through inhibition of superoxide dismutase (SOD) revealed that DCF-AG had 100% inhibition of SOD at the highest tested dose of 1 mM. The SOD and ROS results strongly suggest DCF-AG induced oxidative stress in vitro. Lastly, DCF-AG was screened for pharmacologic activity against COX-1 and COX-2 and was found to have IC 50 values of 0.620 6 0.105 and 2.91 6 0.36 mM, respectively, which represents a novel finding. Since cyclooxygenase (COX) inhibition can lead to intestinal ulceration, it is plausible that DCF-AG can also contribute to enteropathy via COX inhibition. Taken in context, the work presented herein demonstrated the multifactorial pathways by which DCF-AG can act as a direct contributor to toxicity following DCF administration.

Protective effect of cilastatin against diclofenac‐induced nephrotoxicity through interaction with diclofenac acyl glucuronide via organic anion transporters

“…In particular, both diclofenac and diclofenac acyl glucuronide exhibited cytotoxicity to renal primary proximal tubule cells and HEK293 cells (Figures a and a; Figures and ). In fact, diclofenac acyl glucuronide was more cytotoxic than diclofenac in the present study and in the previous studies (Oda et al, ; Scialis & Manautou, ). Transporters act as a key determinant of intracellular levels of substrates.…”

Description and Analysis of Glycosidic Residues in the Largest Open Natural Products Database

“… Examples of glycosidic molecules in ZINC “for sale” and “in vitro” subsets. ( a ) Diclofenac acyl glucuronide (ZINC ID ZINC35048352, part of the “for sale” and “in vitro” subsets), a metabolite of diclofenac that is suspected to mediate toxic side-effects of this drug [ 32 ]. The SRU detects the glucuronic acid moiety (in blue) added in metabolisation as a terminal circular sugar moiety.…”

Protective effects of Ajwa date extract against tissue damage induced by acute diclofenac toxicity

“…Diclofenac is absorbed from the gastrointestinal tract, leading to peak plasma concentrations 1.5–2 h after ingestion. Diclofenac is metabolised in the liver to hydroxydiclofenac, which undergoes conjugation to sulfate and glucuronic acid, facilitating excretion through the renal system 4 …”