Community Research and Development Information Service - CORDIS

H2020

SingMet Report Summary

Project ID: 655864
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - SingMet (Development of MRI contrast agents based on long-lived singlet states)

Reporting period: 2015-08-01 to 2017-07-31

Summary of the context and overall objectives of the project

"Cancer is one of the leading causes for death accounting to 8.8 million in 2015 (http://www.who.int/mediacentre/factsheets/fs297/en/). It is predicted that the number of yearly cancer cases increases from 14 million to about 24 million over the next two decades.
For developing cures new investigation possibilities and improved diagnostics at an early stage are necessary. Magnetic resonance imaging (MRI) is an excellent method for the detection of diseases. Its advantage is a spatial resolution down to tens of micrometer, good contrast in soft tissue and the possibility to investigate dynamics. The main drawback of MRI is the low sensitivity, limiting the resolution, detectable amounts of metabolites and tumor tissue. To overcome sensitivity problems contrast agents are being developed that are mainly based on gadolinium complexes and iron oxide for in vivo human use. Although these contrast agents have found broad applications, limitations exist: Gadolinium contrast agents need to accumulate in high concentrations (millimolar) in tissue which is not always feasible and iron oxide is often accumulated in the liver due to unfavourable pharmacokinetics.

In this project the goal is to develop contrast agents for MRI utilizing the concepts of hyperpolarization and singlet state nuclear magnetic resonance (NMR). Hyperpolarization enhances detectable NMR signals by over four orders of magnitude and singlet states can be utilized to conserve the hyperpolarization in NMR ""silent"" states. The enhanced signal can be read out at a later stage of time either by chemically breaking the molecules symmetry or by applying radiofrequency pulses. Thus, it becomes possible to detect NMR signals after minutes instead of seconds compared to state-of-the-art metabolic tracers. Moreover, this technique leads to nanomolar limits of detection, which is several orders of magnitude higher than conventional contrast agents. Chemical transformations that break the chemical symmetry and make stored hyperpolarization detectable may be due to enzymes in organisms and can be used as diagnostics for cancer cells. The overarching goal of the project is to develop new molecules that have potential to act as MRI contrast agents without the use of ionizing radiation (such as in positron emission tomography) or heavy metals with toxicity concerns. This will be achieved with hyperpolarized tracers with nanomolar detection limits upon hyperpolarization that undergo chemical transformation induced by enzymes."

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

1) Identifying molecules with long singlet lifetimes, that can be utilized as functional groups for metabolites.

The synthesis of molecules in which singlet states can be populated was approached from two different angles:
Molecules were investigated in which protons and 15N nuclei were utilized

15N nuclei:
Derivatives of triazolindione (TAD, see images for detailed structure), which contains two adjacent 15N nuclei, have been investigated. Synthesis of three chiral TAD derivatives and one dimer was performed (Figure 1). In these molecules long T1 times were found (up to 6 minutes for the deuterated compound) in high magnetic fields (B(0)=9.4T). However, singlet states could be populated but did not show long-lived character (1-2 minutes at 2mT). For the protonated and deuterated compound we speculate that the scalar relaxation of the second kind is a major relaxation mechanism that prevented the detection of the singlet state. Whereby the chlorinated compound contains two protonated methyl groups that are geometrically close to the two 15N nuclei and serves as a dominant relaxation source. For the dimer a singlet state could not be populated because the symmetry break was too small or not existent to populate a singlet state.

1H nuclei:
Bromoacrylic acid was identified as a moiety that contains two protons in which a singlet state can be populated even in protonated water/buffer and under atmospheric oxygen. BrAc was subsequently utilized to funcionalize glucose, alanine, serine, ethanol, propargylaclohol and the long-livety of the singelt state confirmed in aqueous buffer under oxygen. T1 times of 1-2 seconds for the molecules have been found whereby the singlet lifetime is on the order of 12-15 seconds (Fig 3 shows a comparision of the BrAc-Alanine and BrAc). This finding represents a major result since a universal spin tag has been identified and proven that several metabolites can be tagged with it. Although the singlet lifetimes are only on the order of 13C T1 times that are already being used in metabolic imaging experiments, we expect that the designed molecules can now be used for in vitro cell experiments as the singlet states are long-lived in cell media. Additionally, phosphoenol pyruvate (PEP) was found to allow for populating a long-lived singlet state due to an acrylate functionality similar to the one found in BrAc. PEP is a metabolite of the glycolysis which may be overexpressed in cancer cells and may therefore serve as a probe for cancer.


2) Hyperpolarization of molecules

In order to investigate hyperpolarization possibilities, the para-hydrogen induced polarization (PHIP) techniques was utilized. PHIP utilizes the singlet state of molecular para-hydrogen upon addition to an unsaturated bond to create hyperpolarized signals. Strategies were investigated with coworkers to hyperpolarize symmetric molecules with para-hydrogen with the future goal to generate symmetric molecules with metabolites that may be used as imaging agents. The proof-of-principle experiment showed a polarization of 9% which is well above the threshold of polarization for potential in vivo experiments (>2%). In parallel derivatives of amino acids (alanine and glycine) were synthesized that contain an unsaturated bond for hyperpolarization. The purpose is to hyperpolarize the unsaturated bond on a sidearm, transfer the polarization to the amino acid and cleave of the sidearm to yield hyperpolarized amino acids. First experiments showed that this strategy is successful in polarizing amino acids although signal enhancement factors of 3 were achieved compared to a signal at B(0)=11.7T. The future plan is to synthesize symmetric molecules that contain metabolites, can be hyperpolarized with para-hydrogen and have storage possibilities in singlet states and release hyperpolarized metabolites at a later point of time.

Overview of results:

1) Development of singlet tracers based on metabolites that contain long-lived singlet states in aqueous buffer solutions under oxygen (Publication in preparation)
2) Development of an efficent hyperpolarization strategy of symmetric tracers with parahydrogen (published work: (J. Eills et al. J Magn. Reson. 274, 163-172, 2017).)
3) Hyperpolarization of amino acids with para-hydrogen (disseminated at two conferences: ISMRM in Singapore in 2016 and the Euromar in Aarhus in 2016).

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 project lead to new insights in developing singlet tracers and to their potential applicability under physiological conditions which is to be investigated in the future. Hyperpolarization techniques were developed and improved to broaden the metabolic tracer spectrum. Overall, the project conducted during the fellowship has brought us one step closer to the design of hyperpolarized singlet tracers that can be used in future for an early cancer detection.

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