Toxicities of sulfur-based drugs have been attributed to formation of highly reactive sulfur oxo-acids and depletion of glutathione by the formation of reactive metabolites. Metabolic activation of these sulfur centers to conceivably toxic reactive metabolites (RMs) that can covalently modify proteins is considered the initial step in drug-induced toxicity. Despite considerable effort and research, detection and characterization of these RMs during drug development and therapy remains a challenge. Methimazole (MMI) and 6-propyl-2-thiouracil (PTU) are two commonly used antithyroid, sulfur-based drugs. Though effective, these drugs are associated with idiosyncratic toxicity. PTU has acquired a black box warning and physicians are calling for its withdrawal. RMs resulting from bioactivation of these drugs have been implicated in the aforementioned adverse reactions. Unfortunately, isolating and detecting RMs using traditional analytical techniques has not been successful due to their high reactivity and short life span, typically less than a minute.
Current approaches in drug metabolism studies use microsomal incubations to generate RMs, which are then trapped using nucleophiles. Antithyroid drugs, however, are known to deactivate enzymes involved in their oxidation. Moreover, due to the complex nature of biological matrices and low abundance of possible toxic conjugates, this technique results in poor selectivity and sensitivity. This study developed and optimized an analytical method based on coupling electrochemical redox reactions and mass spectrometry to generate, detect and identify RMs from antithyroid drugs. The metabolites were also compared to those that were generated using chemical oxidants and biological microsomes. Mimicry of enzymatic oxidation of the antithyroid drugs was carried out by electrochemically oxidizing them using a coulometric cell coupled on-line to electrospray ionization mass spectrometry (EC/ESI-MS). Oxidation of MMI and subsequent trapping with nucleophile resulted in formation of adducts with N-acetylcysteine, revealing reactive metabolites. The most-postulated metabolite, sulfenic acid, had never been isolated or detected until now, using electrochemistry on-line with electrospray ionization. The results showed that bioactivation of MMI proceeds predominantly through the S-oxide and not through formation of thiyl radicals. These same trapping experiments were also conducted with PTU, but no conjugates were detected. The lack of conjugates from PTU does not preclude formation of RMs, but asserts radical pathway might be dominant in EC oxidation. A double mixing stopped flow was used to investigate the kinetics and mechanism of reaction of the MMI and the biologically relevant hypochlorous acid (HOCl), a product of oxidation of chloride (Cl-) ions by myeloperoxidase. The products from the chemical oxidations were compared to the electrochemically generated metabolites, some differences were apparent. Human liver microsomes (HLM) were also used, to investigate oxidation of PTU. Oxidation of PTU, resulted in the supposedly toxic S-oxide, but this has never been isolated, save for speculation. A comparison of metabolites that were found with HLM to those generated electrochemically showed some degree of similarity. These results show that in vitro techniques such as chemical oxidations and electrochemistry coupled to mass spectrometry can be used to mimic oxidative metabolism and subsequent high throughput screening of reactive metabolites.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-4093 |
| Date | 10 June 2016 |
| Creators | Chipiso, Kudzanai |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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