Phd in CHEMICAL SCIENCES

Thesis title: NMR-based Metabolomics applied to Precision Medicine

All measurable aspects of human biology are all based on analytical chemistry methods. One of the great challenges is not only getting the analytical chemistry right for measuring the individual parts but also having the appropriate informatics to interpretate data and link the analytical technologies in order to have a “clinically actionable” information. In this context, the analytical platforms are increasing interest in clinical field towards precision medicine development. Precision medicine seeks to maximize the quality of health care by individualizing the health-care processes of the uniquely evolving health status of each patient. Precision medicine can be split in precision prevention and precision treatment. Precision prevention has generally involved surveillance and management programs for individuals in order to tailor interventions according to behavior, habits or environmental exposure (e.g. occupational exposure). Precision treatment is most concerned with finding the most efficacious and least harmful pharmaceutical treatment tailored for each individual. Among the omics approaches, metabolomics is able to detect causes and metabolic biomarkers for several prominent diseases such as cancer or viral diseases. These findings are identifying previously unsuspected therapeutic targets and novel potential therapeutic strategies. Metabolomic is the systematic study of small-molecular-weight compounds in complex matrices and/or whole organisms as influenced by multiple factors. The knowledge regarding how genetics, susceptibility factors, timing, and degree of exposure can have an impact on metabolome could be essential for preventing early phenotypic changes and development of individualized drug therapy. New techniques, such as metabolite imaging, and improved analytical technologies are making metabolomics increasingly useful for a wider range of biomedical and pharmaceutical applications, especially in the field of precision medicine. Nuclear magnetic resonance (NMR)-based metabolomics is a well-established technique that has been applied to many areas of biomedical research. High-resolution NMR spectroscopy is a quantitative and non-destructive technique. It is a robust and reliable analytical method with paramount reproducibility and repeatability. In metabolomics studies either biofluids, cell or tissue extracts are used as main samples for metabolic fingerprints and in such of complex matrices, the univocal assignment of molecules is granted. For these reasons, NMR-based metabolomics is a powerful tool that enables us to find predictive biomarkers related to the environment interactions, or biomarkers related to disease or drug response. This PhD thesis addresses precision medicine through NMR-based metabolomics. In Chapter 1 an introduction regarding metabolomics in precision medicine is reported, while chapter 2 recapitulates the main aspects concerning NMR acquisition, data preprocessing and statistical analysis for metabolomics purposes. In Chapter 3, two studies (I and II) regarding precision prevention will be illustrated. Study I regard NMR-based metabolomics and metagenomic analyses of fecal matter of mice housed in different environmental conditions, standard and enriched. Enriched environment mice (motor, sensorial and social enrichment obtained by the housing conditions of mice) are characterized by an environment-specific microbial community, along with a specific metabolomic fingerprint with the decrease of bile salts and the increase of formate and acetate, which are also related to brain plasticity effects induced by enriched environment. Study II is a pilot study on urine samples of five male divers, where we performed a metabolomics analysis by 1H NMR and measured the concentration of different oxidative stress biomarkers by means of liquid chromatography tandem mass (LC-MS/MS) spectrometry, suggesting urinary hypoxanthine as a predictive biomarker sensitive to hyperbaric exposure. In Chapter 4, three studies (III, IV, V) regarding precision treatment will be presented. In study III, the metabolomic profiling of gut microbiota will be characterized in 11 patients affected by non-small cell lung cancer (NSCLC) treated with nivolumab. 2-Pentanone (ketone) and tridecane (alkane) were significantly associated with early progression, and on the contrary short chain fatty acids (SCFAs) (i.e., propionate, butyrate), lysine and nicotinic acid were significantly associated with long-term beneficial effects. This study contributes to the personalized management of cancer patients by the identification of microbiota-linked “indicators” of early progressor and long responder patients. Study IV aims at characterizing the urinary metabolic profile of 23 patients affected by hepatitis C virus (HCV)-induced severe liver fibrosis, defining the biomarkers associated to metabolic changes induced by direct acting antiviral (DAA) therapy and HCV-clearance. The urinary metabolic profile of patients with severe liver fibrosis was associated to pseudouridine, hypoxanthine, methylguanidine and dimethylamine, highlighting a profile related to oxidative damage, and to tyrosine and glutamine, related to a decreased breakdown of aromatic aminoacids and ammonia detoxification, respectively. 1-Methylnicotinamide, a catabolic intermediate of nicotinamide-adenine-dinucleotide, was significantly increased in HCV-patients and restored after HCV-clearance, probably due to the reduced hepatic inflammation. 3-hydroxy-3-methylbutyrate, an intermediate of leucine-catabolism which was permanently restored after HCV-clearance, suggested an improvement of skeletal muscle protein synthesis. Finally, 3-hydroxyisobutyrate and 2,3-dihydroxy-2-methylbutyrate, intermediates of valine-catabolism, glycine and choline increased temporarily during therapy, resulting as potential biomarkers of DAAs systemic effects. Study V aims at determining the urinary concentration of Ribavirin (RBV) an antiviral prodrug, its main metabolites and the response of HCV patients to RBV intake. RBV, its proactive and inactive metabolites were identified and quantified in urines of 18 HCV males with severe liver fibrosis. From the fourth week to the end of treatment, a group of ten HCV patients maintained high concentrations of proactive forms and low concentration of inactive form, likely due to circulating native drug or to the compounds originated by nucleosidases and adenosine deaminases. In conclusion, the application of NMR-based metabolomics allows for the identification of metabolites that can be used as possible biomarkers of metabolic pathways involved in exposure to different environments and related to the organism's response to diseases and pharmacological treatment. This work highlighted the potential of NMR in clinical practice, as a more resolutive, sensitive and accessible tool for clinical applications and point-of-care diagnosis. Thanks to these advances, NMR is a valuable candidate to join the other analytical tools currently used in clinical settings.