Periodic Reporting for period 2 - EUROPOL (EUROPOL) Reporting period: 2017-01-01 to 2019-08-31 Summary of the context and overall objectives of the project Nuclear Magnetic Resonance spectroscopy (NMR) and Magnetic Resonance Imaging (MRI) play unique roles in contemporary Science, from Physics, Chemistry and Biology, to clinical research and diagnosis. Further progress in NMR and MRI is hampered by sensitivities that are much lower than those of alternatives, such as mass-spectrometry and PET. The prospects of solving this problem by building “bigger machines” (e.g. relying on stronger magnets) are uncertain and may offer only a poor return, given the high level of maturity already achieved by NMR/MRI. EUROPOL challenges this status quo by combining NMR/MRI with Nuclear Hyperpolarization, which can increase signal intensities by up to x50,000. Two approaches are pursued, dynamic nuclear polarization (DNP) and para-hydrogen-driven polarization (PHIP), which have shown the greatest potential for biophysical, metabolomic, pre-clinical and clinical research. EUROPOL has assembled leading experts in the physics and engineering of magnetic resonance, in the synthetic chemistry essential for the success of these methods, and focuses on applications of hyperpolarized NMR in structural and cell biology, and in preclinical and clinical MRI applications. The most important aspect for society is possibility to image metabolites in an MRI setting. This has now been demonstrated in patients using hyperpolarised pyruvate. Possible applications are in cancer where a metabolic change is observed as consequence of treatment. Partners of EUROPOL are developing this application as a new potential diagnostic.Furthermore, EUROPOL delivers a range of compounds and methods may have a wider range of applications in NMR/MRI. This may enable unprecedented analytical applications in chemistry, biochemistry, protein biochemistry and cell biology.The research objectives of the EUROPOL project were:RO1: Methodological development of a broad-based portfolio of hyperpolarisation platforms and customised acquisition methods. RO2: Develop the enabling chemistry required to drive biological applications of hyperpolarised MR. RO3: High impact biomolecular studies, biological discoveries and biomedical applications, based on hyperpolarised NMR and MRI. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the final 24 months of the project, the fellows have completed - Establishment of cell-based cancer models forcussing on different leukemias through NMR (ESR1 - UoB).- 2H MRSI of thermally polarized [6,6’-2H2]glucose was used to map glycolytic flux in vivo which decreased with chemotherapy treatment in a murine tumour model (ESR2 - UCam).- A methodology for the study of fluorinated proteins through dissolution DNP has been developed and successfully tested with fluorinated Lysozyme (ESR3 – UNott).- Development of new techniques for collecting high-quality NMR spectra in both liquids and solids with a focus on incorporating DNP (ESR4 – Weizmann).- Demonstrated the multiple contrast sources applicable for detection of pancreatic tumors in murine models; and the detection of metabolites in biological conditions also applicable for a multitude of applications (ESR5 – Weizmann).- The design and synthesis of new improved UV-induced polarizing agents for dissolution DNP and matrix dependence of the magnetic properties of trityl radical at high magnetic field was performed (ESR6 – AMU).- Development of a benchtop spectrometer developed to operate up to 450 MHz. It achieves 90% and 50% the SNR value of a dedicated full rack spectrometer for 1H and 13C spectra, respectively (ESR7 – DTU).- Establishment of synthetic pathways for 13C-Labelled Succinic acid, 1-13C Sodium Propionate, 1-13C Sodium Pyruvate and L-[1-13C] ascorbic acid have been successfully established (ESR8 & ESR14)- Increased understanding of NAFLD pathogenesis, providing mechanistic information about steatosis and inflammation, as well as carbohydrate and lipid metabolism and testing suitability for DNP (ESR9 – UoB).- Implementation of a fast, spectroscopic imaging method for use on Bruker pre-clinical MRI scanners, imaging both DNP and para-hydrogen polarised 13C-pyruvate (ESR10 – UKLFR).- A new hyperpolarisation technique, which allows the rapid production of hyperpolarised tracers in water without an external polarizer (ESR11 – UKLFR).- Synthesis and optimization of thienopyridazines for the SABRE process. Phantoms and in vivo MRS recorded on 7T pre-clinical scanner. SABRE-Relay developed with a second metal catalyst (ESR12 – UoY).- Development of new sensitivity-enhanced NMR experiments that exploit fast chemical exchange with abundant water and/or dissolution DNP principle and their application to biomolecules (ESR13 – Weizmann).- The first chemical synthesis of L-[1-13C, U-D] lactate has been developed. To synthesize L-[1-13C, U-D] lactate, methods for the selectively deuteration of amino acids have been developed (ESR15 – CortecNet). - Optimisation of pyrimidine motif molecules with the SABRE mechanism, including 19F N-heterocyclic molecules. Mechanistic studies of these molecules with SABRE and SABRE-Relay by proton exchange. 9.4 T and 3 T phantom MRI and MRS recorded (ESR16 – UoY).- Demonstrated feasibility of materializing sensitivity enhancements provided by dissolution DNP to acquire 13C-13C 1D INADEQUATE experiment in a single scan at natural abundance of 13C isotope for an array of compounds (ESR17 – Weizmann). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The work carried out by the EUROPOL fellows are provided the following advances in the field: - 1H-MRSI detection of hyperpolarized low gyromagnetic ratio nuclei significantly increases the sensitivity of their detection (ESR2 - UCam).- Mapping glycolytic flux with DMI could replace some aspects of FDG-PET imaging in cancer patients Clinical translation may also benefit the healthcare system due to potentially lower tracer costs (ESR2 - UCam).- The experimental protocol of dissolution DNP of fluorinated molecules has been improved. This study was used to develop an experimental protocol for the study of fluorinated proteins by dissolution DNP (ESR3 – UNott). - The potential to provide transportable hyperpolarized substrates These radicals are non-expensive and with the possibility of transport/storage, it can reduce the cost of experiments significantly (ESR6 – AMU).- Pre-clinical hyperpolarization systems that grant accessibility to dissolution DNP and its benefits. Such systems offer the ability to monitor metabolic conversions in vivo, (ESR7 – DTU).- The field of fatty liver disease will contribute towards the study and development of effective therapeutics to treat NAFLD (ESR9 – UoB).- Closing of the gap between the potential of metabolic imaging with hyperpolarised tracers and clinical application (ESR10 – UKLFR).- Development of a new hyperpolarisation technique which may lead to new diagnostic applications (ESR11 – UKLFR).- Expected images in phantoms and in vivo using optimised conditions for biomolecules This could have diagnostic potential for disease states similar to DNP. (ESR12&ESR16 – UoY).- The developed deuteration methods will be useful in many other fields (pharmaceutical, electronic) (ESR15 – CortecNet).