Metabolism of xenobiotic drug molecules can result in the formation of metabolites which are more chemically reactive than the parent drug from which they are derived. These reactive species have the potential to covalently modify biological macromolecules if they are not detoxified. The formation of drug-protein adducts carries a potential risk of clinical toxicities and idiosyncratic adverse drug reactions which can, in severe cases, result in hospitalisation and even death. Current methods for the evaluation of the risk for a drug to cause adverse drug reactions due to drug-protein binding rely on risk factors such as quantitative covalent binding value, structure, dose etc. The objective of this project was to develop methods for the detection of reactive metabolites directly bound to proteins, which could be used in future evaluations of the mechanisms of binding of candidates in drug development. Three compounds known to produce reactive metabolites, acetaminophen, SB-648969 and amodiaquine, were used as tool substrates. In vitro incubations with human liver microsomes and individual cytochrome P450 enzymes (as Supersomes ) were used to produce reactive metabolite species and binding with the incubation proteins evaluated. Analysis of the intact proteins, peptides generated via trypsin digestion of the incubation protein, and amino acids generated via digestion with pronase were evaluated using a combination of LC/MS and LC-MS/MS. Reactive metabolite trapping experiments with glutathione were used to provide information about the likely structure of the bound species and the specificity of binding, and were useful in the development of sensitive targeted precursor ion scanning and multiple reaction monitoring methods. [14C] radiolabelled acetaminophen and SB-649868 were used to assess the quantitative levels of binding (<5% modification of protein in both cases). Radiodetection using accelerator mass spectrometry (AMS) was used to evaluate the stoichiometry of binding and aid the identification of adducted peptides through retention time comparison. Chemical and electrochemical methods were utilised to produce stable solutions of N-acetyl-p-benzoquinone imine (NAPQI) and amodiaquine quinone imine (AQQI), reactive metabolites of acetaminophen and amodiaquine, respectively, which were bound to selected proteins and used as chromatographic and mass spectrometric standards. These methods were used to successfully identify an acetaminophen-modified peptide (T56) of cytochrome P450 CYP2E1. No modified proteins were observed for the SB-649868 incubations, however, examination of the AMS chromatograms for the incubations with acetaminophen and SB-649868 revealed a difference in the stoichiometry of binding, with one modified peptide observed with acetaminophen, and several for the incubations with SB-649868. The detection and identification of drug-protein adducts remains extremely challenging due to the low levels of any adducts observed, which can be exacerbated by binding on multiple sites of a protein; however this project has demonstrated that sensitive and selective LC/MS methods can be successfully developed to identify drug-protein adducts.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:740246 |
| Date | January 2013 |
| Creators | Squillaci, Bianca |
| Contributors | Pritchard, Robin |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/approaches-to-the-detection-of-adducts-formed-via-the-covalent-binding-of-reactive-metabolites-to-proteins(3b4ccc97-ea77-40d1-bb2b-2c4e70a1424d).html |
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