2. Academic Validation
  3. Tolterodine is a novel candidate for assessing CYP3A4 activity through metabolic volatiles to predict drug responses

Tolterodine is a novel candidate for assessing CYP3A4 activity through metabolic volatiles to predict drug responses

  • Sci Rep. 2025 Jan 20;15(1):2462. doi: 10.1038/s41598-025-86450-9.
Valentina Stock 1,  Rebecca Hofer 1,  Franziska Lochmann 1,  Vera Spanke 2,  Klaus R Liedl 2,  Jakob Troppmair 3,  Thierry Langer 4,  Hubert Gstach 4,  Christian Dank 4 5,  Chris A Mayhew 1,  Sarah Kammerer 1 6,  Veronika Ruzsanyi 7
Affiliations

Affiliations

  • 1 Institute for Breath Research, University of Innsbruck, Innrain 80/82, Innsbruck, 6020, Austria.
  • 2 Institute for Theoretical Chemistry, University of Innsbruck, Innrain 80/82, Innsbruck, 6020, Austria.
  • 3 Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innrain 66, Innsbruck, 6020, Austria.
  • 4 Department of Pharmaceutical Chemistry, University of Vienna, Althanstraße 14, Vienna, 1090, Austria.
  • 5 Institute of Organic Chemistry, University of Vienna, Währinger Straße 38, Vienna, 1090, Austria.
  • 6 Institute of Biotechnology, Molecular Cell Biology, Brandenburg University of Technology Cottbus- Senftenberg, 01968, Senftenberg, Germany.
  • 7 Institute for Breath Research, University of Innsbruck, Innrain 80/82, Innsbruck, 6020, Austria. Veronika.Ruzsanyi@uibk.ac.at.
PMID: 39828876 DOI: 10.1038/s41598-025-86450-9
Abstract

Cytochrome P450 (CYP) 3A4 plays a major role in drug metabolism. Its activity could be determined by non-invasive and cost-effective assays, such as breath analysis, for the personalised monitoring of drug response. For the first time, we identify an isotopically unlabelled CYP3A4 substrate, tolterodine that leads to the formation of a non-toxic volatile metabolite, acetone, which could potentially be applied to monitor CYP3A4 activity in humans. In vitro biotransformation of tolterodine by HepG2 cells overexpressing CYP3A4, CYP2D6 or CYP2C9 was investigated by LC-MS analysis of Cell Culture supernatant for the non-volatile metabolite, N-dealkylated tolterodine, and PTR-ToF-MS analysis of the headspace for acetone. The highest level of the N-dealkylated metabolite was produced by HepG2-CYP3A4. Concentration dependent effects of tolterodine were analysed, resulting in TC50 values of 414 µM and 375 µM for HepG2-CYP3A4 and reference cells, respectively. Acetone and N-dealkylated tolterodine levels increased continuously over 24 h in HepG2-CYP3A4. Treatment with either a pan-CYP inhibitor, 1-aminobenzotriazole, or a CYP3A4 Inhibitor, ketoconazole, considerably reduced the production of both metabolites in HepG2-CYP3A4 cells. These findings pave the way for the further development of non-invasive breath tests using unlabelled precursors to determine CYP Enzyme activity in individuals.

Keywords

Acetone; Breath test; CYP3A4; HepG2; Tolterodine; Volatile biomarkers.

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