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Title: In vitro and in vivo investigations into the interactions between the acyl glucuronide metabolite of diclofenac and serum albumin
Author: Hammond, Thomas
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2012
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Abstract:
Adverse drug reactions represent a major challenge to clinicians, healthcare systems, pharmaceutical companies and academia. With carboxylic acid drugs accounting for the most common class of drugs withdrawn from the market, the carboxylate pharmacophore has received much attention as a potential toxicophore. Direct glucuronidation of the carboxylate group, producing chemically unstable and protein reactive acyl glucuronide (AG) metabolites has received much attention as a bioactivation pathway responsible for generation of these off-target hypersensitivity and hepatotoxicity. It is the chemical instability and protein reactivity of AG metabolites that has led to their hypothesised ability to covalently modify proteins in vivo and subsequently stimulate inappropriate immune responses in susceptible patients. Despite this, whilst the reactivity of AGs has been shown in vitro, their reactivity has never been confirmed in any in vivo system, meaning their association with toxicity may be unjustified. The focus of this thesis was to investigate whether acyl glucuronides could identify covalent adducts to protein in vivo. To address this aim, the thesis first investigates the chemistry of interaction between acyl glucuronides and protein during in vitro investigation. 2mM 1-β diclofenac-AG was found to degrade spontaneously via acyl migration following incubation with 0.1M phosphate buffer pH 7.4 at 37°C with a degradation half-life of 0.78 hours, confirming diclofenac as amongst the most reactive AGs. Further incubations confirmed the action of human serum albumin (HSA) as a mild esterase, and the presence of plasma esterases acting to hydrolyse AGs. The covalent binding of diclofenac-AG to HSA was confirmed using both an alkaline hydrolysis as well as direct mass-spectrometric analyses of modified proteins. Covalent modification of lysine residues was specifically identified, and was found to be concentration and time dependent. Further in vitro incubation experiments revealed for the first time that the 1-β isomer of AGs is responsible for the formation of transacylation adducts, and confirmed previous suggestion that acyl migration is required for the extensive glycation of HSA. Following characterisation of the interaction of diclofenac-AG with HSA, investigations were undertaken in the rat to identify interactions of AGs with circulating rat serum albumin in vivo. In vitro incubations of diclofenac-AG revealed RSA contained fewer binding sites when compared to HSA. Further to this no covalent modification of RSA could be detected in vivo following intravenous administration of 60mg/kg diclofenac-AG. The rapid plasma clearance of diclofenac-AG (67.81 ± 12.83 ml min-1 kg-1) in the rat was shown to be 600 fold faster than that of diclofenac (12.00 ± 2.98 ml min-1 kg-1) following bolus intravenous administration. Use of a continuous intravenous infusion drug delivery system revealed an adaptive change in rats upon continuous infusion of diclofenac, resulting in enhanced plasma elimination of the drug, and induction of the ROS scavenging enzymes catalase and superoxide dismutase-2, without detection of hepatotoxicity. The final experiments in the thesis revealed for the first time the detection of glycation adducts to HSA extracted from volunteer patients receiving chronic diclofenac therapy. These were shown through the detection of glycation adducts in three out of six patients tested. Between 1 and 4 lysine residues were identified in patients, with modifications towards one or all of lysine residues 195, 199, 432 and 525. Transacylation adducts were detected towards lysine residues in all six patient samples analysed. Whilst identification of transacylation adducts reveals bioactivation of the carboxylic acid functional group, it is the identification of glycation adducts to albumin isolated from three of the six patients which reveals, for the first time, definitive evidence for AG reactivity in vivo. This reinforces concerns over the potential of AGs to act as haptens, and re-affirms the carboxylic acid structure as a site of bioactivation forming reactive metabolites.
Supervisor: Williams, Dominic; Sathish, Jean; Kenna, Gerry; Park, Kevin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.579317  DOI: Not available
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