Presenter Status
Staff
Abstract Type
Basic Research
Primary Mentor or Principal Investigator
Jennifer Goldman
Presentation Type
Poster
Start Date
20-5-2026 11:00 AM
End Date
20-5-2026 12:00 PM
Abstract Text
Background:
A commonly prescribed antibiotic, trimethoprim-sulfamethoxazole (TMP-SMX), is associated with several idiosyncratic adverse drug reactions (IADRs) including severe cutaneous adverse reactions (SCAR), drug induced liver injury (DILI), and life threatening acute respiratory distress syndrome (ARDS). Because the mechanism of these reactions is poorly understood, failure to prevent harmful drug reactions persists. The nature of the reactive metabolite, where it is formed, and what it binds are likely contributing to these tissue specific IADR phenotypes. The chemical structure of a reactive metabolite is not always predictive of reactivity. Quantitative methods to measure drug and metabolite reactivity are necessary to establish which metabolites are reactive, and to what extent. Despite the correlation between reactivity and IADRs, drug and drug metabolite levels in patients in and of themselves have not translated into biomarkers to predict IADRs.
Objectives/Goal:
We propose that establishing a comprehensive view of drug metabolism and reactivity is critical to understanding IADR etiology. Our approach includes determining the relative reactivity of drugs and drug metabolites to form protein adducts that adversely contribute to metabolite burden. We will do this by ranking each drug and metabolite by reactive burden to prioritize them for downstream immune signaling studies.
Methods/Design:
Fluorescent dansyl glutathione (dGSH) derivatives have been used to quantify the formation of reactive metabolites in several studies without the need for synthetic glutathione adduct standards or time-consuming method development. We will use this and a related trapping agent, CysGlu-Dan, which is a dansyl derivative that includes soft and hard nucleophilic moieties for a broader assessment of reactivity. To estimate the reactive burden (RB) of a given metabolite, we will multiply the fraction of metabolite modified by a trapping reagent with the average circulating concentration of each metabolite.
Results:
We have observed reactivity of 4-desmethyl-TMP (4-DM-TMP) with proteins and N-acetylcysteine in past assays. With this knowledge, we have begun optimizing our trapping protocol to quantify reactive burden with known reactive metabolites. Preliminary analysis of trapping efficiency of metabolites 4DM-TMP, 4-nitroso-sulfamethoxazole (SMX-NO), and carbamazepine 10,11 epoxide (CBZ-E) when incubated with 50uM and 500uM dGSH were performed on a high-resolution mass spectrometer. MS analysis has confirmed dGSH trapping of these representative metabolites. Additionally, we were able to show that reactivity of 4-DM-TMP is detectable relative to the known reactive metabolite SMX-NO when quantified through absorbance and fluorescence detection via HPLC after dGSH trapping.
Conclusions:
We have observed dGSH trapping of known reactive metabolites via high-resolution mass spectrometry and HPLC. We detected trapped reactive metabolites via HPLC with a minimum signal-to-noise ratio of 8.7. We plan to 1) compare different trapping assay quenching methods, 2) compare trapping efficiencies of CysGlu-Dan and dGSH, 3) compare calculated rates of formation as measured by absorbance or fluorescence, 4) rank all phase I metabolites of TMP and SMX by reactive burden as a function of HPLC peak area and circulating concentration. We hope to use the identified reactive metabolites to probe immune pathway responses in future work.
Investigating Reactive Metabolite Burden of Drugs Implicated in Adverse Drug Reactions
Background:
A commonly prescribed antibiotic, trimethoprim-sulfamethoxazole (TMP-SMX), is associated with several idiosyncratic adverse drug reactions (IADRs) including severe cutaneous adverse reactions (SCAR), drug induced liver injury (DILI), and life threatening acute respiratory distress syndrome (ARDS). Because the mechanism of these reactions is poorly understood, failure to prevent harmful drug reactions persists. The nature of the reactive metabolite, where it is formed, and what it binds are likely contributing to these tissue specific IADR phenotypes. The chemical structure of a reactive metabolite is not always predictive of reactivity. Quantitative methods to measure drug and metabolite reactivity are necessary to establish which metabolites are reactive, and to what extent. Despite the correlation between reactivity and IADRs, drug and drug metabolite levels in patients in and of themselves have not translated into biomarkers to predict IADRs.
Objectives/Goal:
We propose that establishing a comprehensive view of drug metabolism and reactivity is critical to understanding IADR etiology. Our approach includes determining the relative reactivity of drugs and drug metabolites to form protein adducts that adversely contribute to metabolite burden. We will do this by ranking each drug and metabolite by reactive burden to prioritize them for downstream immune signaling studies.
Methods/Design:
Fluorescent dansyl glutathione (dGSH) derivatives have been used to quantify the formation of reactive metabolites in several studies without the need for synthetic glutathione adduct standards or time-consuming method development. We will use this and a related trapping agent, CysGlu-Dan, which is a dansyl derivative that includes soft and hard nucleophilic moieties for a broader assessment of reactivity. To estimate the reactive burden (RB) of a given metabolite, we will multiply the fraction of metabolite modified by a trapping reagent with the average circulating concentration of each metabolite.
Results:
We have observed reactivity of 4-desmethyl-TMP (4-DM-TMP) with proteins and N-acetylcysteine in past assays. With this knowledge, we have begun optimizing our trapping protocol to quantify reactive burden with known reactive metabolites. Preliminary analysis of trapping efficiency of metabolites 4DM-TMP, 4-nitroso-sulfamethoxazole (SMX-NO), and carbamazepine 10,11 epoxide (CBZ-E) when incubated with 50uM and 500uM dGSH were performed on a high-resolution mass spectrometer. MS analysis has confirmed dGSH trapping of these representative metabolites. Additionally, we were able to show that reactivity of 4-DM-TMP is detectable relative to the known reactive metabolite SMX-NO when quantified through absorbance and fluorescence detection via HPLC after dGSH trapping.
Conclusions:
We have observed dGSH trapping of known reactive metabolites via high-resolution mass spectrometry and HPLC. We detected trapped reactive metabolites via HPLC with a minimum signal-to-noise ratio of 8.7. We plan to 1) compare different trapping assay quenching methods, 2) compare trapping efficiencies of CysGlu-Dan and dGSH, 3) compare calculated rates of formation as measured by absorbance or fluorescence, 4) rank all phase I metabolites of TMP and SMX by reactive burden as a function of HPLC peak area and circulating concentration. We hope to use the identified reactive metabolites to probe immune pathway responses in future work.
Comments
Poster Board Number: 35