Towards Personalised Medicine: use of volatile metabolites

Wider research context: Patient-specific toxic side effects and lack of efficacy often hamper the therapeutic benefit of drugs. The reason for this lies in the fact the degradation of drugs by so called enzymes works with varying efficiency in different individuals. The Cytochrom P450 (CYP) enzymes belong to the major enzyme families responsible for drug metabolism in the liver. The use of specific substrates as predictors of drug response would significantly advance this field, the benefits of which would be enormous. A test could assess a patients ability to metabolize a drug, leading to improved patient care through the prescription of suitable drugs that are administered at the correct dosage. Objectives: We will test different substrates with a key CYP enzyme, CYP3A4, for the production and use of unique volatile substrate to provide a measure of its activity. Volatiles that are not normally present in exhaled breath will permit the development of a strategy that would lead to considerable reductions in the dose of a substrate needed for the clinical implementation of a breath test, toxic side- effects and medical costs. Approach: We will employ an interdisciplinary research programme to involve molecular modeling for substrate selection and engineering, and the use of cell-cultures for in vitro testing and metabolic analyses. The selectivity and specificity of substrates will be tested in two liver-like cell-based systems. These will confirm the suitability of substrates, and guide what modifications are required to optimize kinetic parameters, rate of metabolism and volatility. State-of-the-art, high resolution analytical techniques (gas-chromatography mass spectrometry and proton transfer reaction time of flight mass spectrometry, ultra-high pressure liquid chromatography mass spectrometry) will be employed to facilitate the identification, quantification and monitoring of volatile and non-volatile metabolites. Innovation: The identification of unique biomarkers resulting from CYP3A4 metabolism provides the underpinning knowledge to develop non-invasive breath tests that can assess an individuals response to a drug. These tests can be used to assess the best therapeutic outcome at reduced costs. Importantly, the approach we are proposing can be adopted to other CYPs relevant in drug metabolism, e.g. 2D6, 2C9, and 2C19. Primary researchers involved: University of Innsbruck: Dr. Veronika Ruzsanyi and Prof. Klaus Liedl; Medical University of Innsbruck: Prof. Jakob Troppmair; University of Vienna: Prof. Thierry Langer; Brandenburg University of Technology Cottbus: Dr. Sarah Kammerer.

The effects of many drugs can vary greatly from patient to patient. In some patients, a drug may have little effect, while in others, it may cause adverse side effects. One reason for this is the different enzymes present in the body that break down drugs. Particularly important is the cytochrome P450 (CYP) family, which is responsible for metabolizing over 70% of all medications. This poses a challenge for personalized medicine, as there are currently no simple methods of determining a person's enzyme activity. New methods using specific biomarkers for individual CYP enzymes could help predict drug effects more accurately. The aim of the PREDICT project was to develop new substrates for use in a non-invasive breath test that would eliminate the need for complex and expensive isotopic labeling. To this end, the activity of the CYP3A4 enzyme was determined using biomarkers in an interdisciplinary research approach. In the project, suitable substrates were first selected using computer models that produce volatile substances during metabolization, which are not present in the breath or only in small quantities. These substrates were then tested in special cell cultures to identify the metabolic pathway and develop analytical methods for detecting and quantifying the metabolites. Targeted modifications of the substrates were made to optimize properties such as metabolic rate, volatility, and biomarker yield. High-precision modern analytical methods were employed to detect the markers. The initial results with tolterodine were successful, as it forms the non-toxic volatile substance acetone Targeted modifications then led to diisopromine as an alternative with improved metabolism and higher biomarker yield. However, since acetone also appears in breath as a result of other metabolic processes, further modifications were made to create the substrate gstachamine, which generates the more specific volatile marker butanone. Preliminary findings suggest that gstachamine may be suitable for non-invasive monitoring of CYP3A4 activity. Our approach makes it possible to determine the optimal drug dose for each patient individually. Moreover, this concept can be extended to other CYP enzymes, providing a more comprehensive understanding of how individuals respond to different drugs.