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Title: Chemical synthesis and investigation of reactive drug metabolites
Author: Meng, Xiaoli
ISNI:       0000 0001 3394 1876
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2007
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Bioactivation of drugs to their chemically reactive metabolites (CRMs), often leading to protein-drug conjugates, is a widely-known hypothesis of adverse drug reactions. However the direct evidence of this hypothesis is lacking. Therefore highly pure CRMs are of great interest to both academia and industry, and substantial amounts of'highly pure CRMs are essential for the study of drug toxicity mechanisms. Thus the aims of this project are developing efficient methods to synthesize reactive drug metabolites in large quantity for thorough toxicological evaluation and investigating the chemical reactivity ofCRMs.towards proteins. Several efficient methods for the synthesis of CRMs of some important drugs have been developed. For example, acyl glucuronides, potentially reactive electrophilic metabolites implicated in adverse drug reactions, were obtained by selective acylation of benzyl or allyl glucuronate and subsequent deprotection under mild conditions. As demonstrated in chapter 2, highly pure acyl glucuronides (AGs) of various important drugs were synthesized through the selective acylation method, and their stabilities and reactivities were evaluated by LC-MS and NMR. Variable stabilities and reactivities of AGs were identified: AG of UK414, 495 is extremely unstable and underwent cyclisation to form a reactive imide at pH 7.4; whereas AG of UK505,749 is quite stable for several hours, then acyl migration rather than cyclisation occurred as shown by LC-MS. Furthermore, the chemical reactivities of the reactive imide towards various nucleophiles containing -NH2, -SH and -OH functional groups were investigated and the formed adducts were fully characterised. Benzyl penicillin (BP) is well-known to cause allergic reactions in 3-5% patients. Previous studies have shown that BP can covalently bind to albumin molecules and the resulting penicilloyl/albumin conjugates are considered to be the major antigenic determinants in penicillin allergy. Whether BP reacts directly with albumin or through penicillenic acid (PA), a highly reactive degradation product of penicillin, has been a debate for many years. Here we demonstrate a simple and sensitive method to characterize the modified protein as well as an efficient 4-step method to prepare the penicilllenic acid of benzyl penicillin for the study of penicillin allergy. Evaluation of protein reactivity of CRMs has been carried out both ill vitro and ill vivo. In chapter 3 and 4 we have investigated BP (or PA) modification of albumin in detail by ESIIMSIMS. BP and penicillenic acid have both shown high affinity binding to albumins ill vitro. 12 lysine residues on HSA and 7 lysine residues on RSA were found to be modified by both BP and PA, and PA appears to be more reactive than BP. In addition, BP or PA binding to albumins proved to be time and concentration dependent. Modification was also detected. in rats administered with a single intravenous dose of BP as well as multiple Lp. dose of BP. The modification also has been shown to vary in a dose- and time-dependent manner.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available