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Idiosyncratic adverse drug reactions (IADRs) occur in a small but significant percentage of the population, are unpredictable, and frequently cause life-threatening events requiring intensive medical care. The antibiotic, trimethoprim-sulfamethoxazole (TMP-SMX) is generally considered a safe and effective drug but has a relatively high rate of IADRs. Through metabolism, drugs may be bioactivated, yielding reactive metabolites which covalently bind to proteins making them a target for immune-mediated responses, and in some cases, fulminant drug hypersensitivity. Although the liver is the dominant contributor to drug metabolism, other organ systems are known to be metabolically active. Interestingly, TMP-SMX IADRs are associated with serious lung injury and/or mild to serious skin rash. Historically, SMX has been suspected of being the source of IADRs associated with TMP-SMX due to the formation of a reactive nitroso intermediate but more recent studies indicate that incidences of IADRs in the case of TMP use alone are higher than the combination drug, suggesting that SMX is not the sole cause of TMP-SMX IADRs. In this study, we seek to evaluate TMP metabolism in lung and skin and to assess the reactivity of any metabolites formed. Ex vivo subcellular s9 fractions contain active drug metabolizing enzymes and with the addition of appropriate co-factors, both phase I and II metabolites can be produced in vitro. Using this system, we discovered differential TMP metabolism in lung and skin as compared to liver. Liver, lung, and skin s9 reaction products were analyzed by liquid chromatography/mass spectrometry (LC/MS) Waters triple quadrupole instrument. The reactivity of tissue-specific metabolites was assessed by adding glutathione, n-acetyl cysteine, and n-acetyl lysine to s9/TMP reaction products (trapping assays). Trapping assay products were analyzed using a high-resolution Waters quadrupole/time of flight (qTOF) mass spectrometer. Results of this analysis were fed into a pick-picking algorithm (XCMS) to identify unique features against several negative controls. Using these approaches, we observed glucuronide conjugate formation in lung and sulfate conjugation in skin. Along with this novel discovery, we identified 2 potentially reactive sulfate conjugates of 4-desmethyl trimethoprim. These findings indicate that primary metabolites formed in the liver can undergo further bioactivation in lung and skin, the sites of clinical presentation of TMP IADRs. These findings are key mechanistic insights and will advance efforts to understand the etiology of this currently idiopathic medical condition.

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Characterization of Trimethoprim Metabolism and Metabolite Reactivity in Human Liver, Lung, and Skin