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Reactive metabolites / GSH Trapping / Cyanide Trapping

Reactive metabolites have been linked to idiosyncratic toxicity which is a major concern for patient safety and has led to drug withdrawals and black box warnings. Early assessment of the likelihood of reactive metabolite formation will allow mitigation of associated risks.

Sygnature offers in vitro reactive metabolite trapping assays, the Glutathione (GSH) Trapping assay and the Cyanide Trapping assay, for early assessment of drug candidates. These assays do not require radiolabelled compound.

Metabolism of some drug compounds can generate chemically reactive metabolites which can react with cellular molecules such as proteins or DNA, leading to adverse reactions. This risk can be assessed with tailored microsomal incubations using glutathione or cyanide to covalently bond with electrophilic species, thereby ‘trapping’ them. Any detected glutathione or cyanide adducts can provide a flag for potential liabilities around toxicity. The samples are acquired with non-targeted high-resolution mass spectrometry and adducts are detected with high selectivity of characteristic fragment ions or isotope patterns, enabling detection down to trace levels. This approach gives a wide coverage and provides an accurate assessment of overall liabilities around potential reactivity in a time-efficient manner. Subsequent structural characterisation of key metabolites can be performed where relevant and feasible, which can help direct a program to eliminate chemical liability by structural modification.

Glutathione (GSH) Trapping Assay

Our fully validated Glutathione (GSH) Trapping assay is based upon microsomal incubation of parent compound in the presence of an excess of reduced glutathione. Reactive metabolites formed during the incubation are trapped by GSH and are detected using high resolution mass spectrometry. This assay is particularly appropriate for molecules with a potential to form soft-electrophiles, such as Michael-acceptor, as reactive metabolites (e.g clozapine).

Cyanide Trapping Assay

Our fully validated Cyanide Trapping assay is based upon microsomal incubation of parent compound in the presence of a cyanide source (KCN). Reactive metabolites formed during the incubation are trapped using a mixture of isotopically-labelled and unlabelled potassium cyanide and are detected using accurate-mass high-resolution mass spectrometry. The distinctive isotopic pattern increases confidence in the detection of low-level metabolites and helps reduce false positives. This assay is particularly appropriate for molecules with a potential to form hard-electrophiles, such as iminium ions generated by tertiary amines, as reactive metabolites (e.g. nicotine).

The reactive metabolites identification assays can be tailored to answer specific questions relevant to your project. We have a collaborative and flexible approach to maximise the impact of your data. If you would like to discuss these assays in more detail or have particular requirements, please do not hesitate to contact us using the contact form.

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