Towards the Use of Metabolic Volatiles in Breath for Determining Drug Response: Gstachamine as an Unlabeled Substrate to Measure CYP3A4 Activity

A potential substrate, gstachamine, was engineered for a future CYP3A4 assay. HepG2 cell clones overexpressing CYP3A4 were used to investigate both liquid and headspace phases after incubation. A specific N-dealkylated metabolite and butanone were detected using LC-MS and PTR-ToF-MS, respectively. As a volatile compound found in the low ng L⁻¹ levels in breath, butanone supports gstachamine's potential for CYP3A4-specific breath testing.

Breath analysis is a promising noninvasive diagnostic tool, but the clinical applicability of breath tests depends on several factors. A salient criterion pertains to the presence of substrates with the ability to produce detectable volatile metabolites during the metabolism. In this work, we evaluated the potential of two candidate compounds, namely gstachidine and gstachamine, for their use in metabolic breath analysis. Both substrates were evaluated for their toxicity and metabolic conversion in HepG2 cell clones overexpressing CYP3A4. Gstachidine was found to be toxic and did not produce any volatile metabolite. In contrast, gstachamine successfully generated butanone as a volatile metabolite, making it the first substrate to yield a stable VOC detectable exclusively at low ppb_V levels in breath. To characterize its biotransformation, we conducted time-dependent analyses, as well as CYP specificity, toxicity, and inhibition investigations regarding the production of N-dealkylated gstachamine or butanone. The results demonstrated that gstachamine had a high metabolic turnover and a strong CYP3A4-dependency in the production of the specific N-dealkylated metabolite. Furthermore, a substantial reduction in the production of both metabolites was observed in HepG2-CYP3A4 cells following treatment with CYP inhibitors 1-aminobenzotriazole or ketoconazole. The results suggest that gstachamine has potential for noninvasive CYP3A4 metabolism monitoring.

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