An investigation into the in vitro and in vivo fate of acyl glucuronides

Thomas Hammond1, Sophie Regan1, Xiaoli Meng1, Rosalind Jenkins1, Gerry Kenna2, Jean Sathish1, Dominic Williams1. 1University of Liverpool, Liverpool, United Kingdom, 2AstraZeneca, Alderley Park, United Kingdom.

Introduction: Tolmetin and diclofenac are members of the non-steroidal anti-inflammatory class of drugs (NSAIDs). NSAIDs are associated with idiosyncratic hepatotoxicity. This has been suggested to be a result of their metabolism to acyl glucuronide (AG) reactive metabolites. It is hypothesised that acyl glucuronides can elicit a hepatic idiosyncratic reaction through the initiation of an immune response. Presentation to the immune system through binding to endogenous protein, resulting in an autoimmune response is thought to be the mechanism responsible. Conclusive evidence, however, is lacking.

We have investigated the metabolism of NSAIDs into their AG metabolites, as well as their protein binding.

Methods: Tolmetin acyl glycuronide (T-AG) and diclofenac acyl glucuronide (D-AG) were incubated with HSA at increasing concentrations for 16 hours at 37°c, with AG covalent adduct formation assessed using LC-MS/MS technology (AB Sciex QTRAP 5500.)

T-AG was incubated with phosphate buffer at pH 7.4 at 37°C for 100 minutes, with isomeric degradation assessed using HPLC. Male Wistar rats were anaesthetized using 14% urethane, with the bile duct and carotid artery cannulated. Tolmetin was then administered orally, with bile and blood samples collected over 4 hours for pharmacokinetic analysis.

Results:

Table 1: Tolmetin and Diclofenac AG adduct formation with albumin following in vitro incubation at 37°C for 12 hours. G represents adducts formed through the glycation pathway, T represents adducts formed through the transacylation pathway. Adducts were preferentially found at the hydrophobic region of HSA (Sudlow sites I and II)

Following anesthesia, femoral veins of male Wistar rats were cannulated, and both T-AG and D-AG were administered through this route. Blood samples were collected, and albumin was isolated using preparative HPLC. No adducts were detected using an AB Sciex QTRAP 5500 LC-MS/MS.

Degradation kinetics of T-AG in phosphate buffer and biological matrices (including urine, bile and plasma,) as well as pharmacokinetics of tolmetin and T-AG in the conscious rat, focusing on urinary and biliary excretion, as well as plasma disposition are being investigated.

Summary: In vitro methods were established and applied to in vivo generated samples to allow for the understanding of the fate of these two NSAIDs and their AG metabolites.