Species and tissue differences in Diclofenac Glucuronidation

Abstract ID 95726 Poster Board 232 Purpose: Glucuronidation of Diclofenac by UDP-glucuronosyltransferase (UGT) 2B7 leads to the formation of its highly reactive acyl glucuronide. Although species differences in glucuronidation, especially in rodents, have been reported for other compounds, there is limited data on that of Diclofenac. This study aims to determine the impact of species and tissue differences on the Glucuronidation of Diclofenac in vitro. Method: Microsomes from different species (Rat, Monkey, Mouse, Dog, and Human) and tissues (Liver, Intestine, and Kidney) were used to assess the rate of Glucuronidation reaction using Diclofenac as a substrate. An in-vitro Phase II metabolism reaction was carried out using Uridine diphosphate Glucuronic acid (UDPGA) as a cofactor, Saccharolactone as a beta-glucuronidase inhibitor to drive the forward reaction to release the metabolite, Potassium phosphate Buffer (KPI) to mimic physiological conditions and alamethicin as a surfactant for the reaction. The protein concentration used was 0.05mg/ml which was incubated for 2 hours at 37OC with different substrate concentrations. The metabolites were quantified using Ultra Performance Liquid Chromatography (UPLC) and fitted into the Michaelis-Menten model to determine the enzyme kinetic parameters Km and Vmax for the different species and tissues. The results were compared to assess whether microsomes from different species and tissues have an impact on the rate of glucuronidation of Diclofenac. Results: The results showed higher rates of metabolism in the liver compared to that of the intestine and kidney. There were also differences in the rate of metabolism in the liver, with Mouse having the highest reaction rate (Km = 91.87 uM, Vmax = 7.22 nmol/min/mg) followed by Human (Km = 59.5 uM, Vmax = 6.66 nmol/min/mg), Dog (Km = 41.46 uM, Vmax = 5.05 nmol/min/mg), Monkey (Km = 17.87 uM, Vmax =3.88 nmol/min/mg) and Rat (Km = 24.03 uM, Vmax = 0.83 nmol/min/mg). Conclusion: The data demonstrates that the liver is the major organ for the metabolism of this drug although the intestine and kidney exhibited some level of metabolism as well. Also, it is evident that species difference has an impact on the Glucuronidation rate of Diclofenac. This will be crucial for predicting the pharmacokinetic and toxicity patterns in different species, tissues, and ultimately, in vivo. Keywords: Diclofenac, Glucuronidation, Species and tissue differences, UDP-glucuronosyltransferase (UGT), in vitro metabolism study