Synthèse de nouveaux nitroxides pour le diagnostic et l'étude de pathologie chez le petit animal via l'activité des protéases vue par IRM et effet Overhauser. – NITROMRI

Synthesis of nitroxides
The objective was to prepare nitroxides exhibiting changes in hcc after proteolysis of an attached peptide fragment. These changes must occur by simple chemical changes (protonation of some parts of the nitroxide, conformational changes..).
Before performing a tedious work on grafting a peptidic chain on a nitroxide, Electron PAramagnetic features of all nitroxides were first investigated.
Several nitroxides exhibiting protonable sites were prepared. Some of them exhibited significant shift upon protonation except they were not suitable for grafting a peptide. Some others were suitable for grafting but the change in hcc was too small for OMRI applications.
Thus, we moved to the preparation of variously functionnalized 5-membered ring nitroxides carrying phosphorylated group. Significant changes were observed in organic solvents upon conformation changes due to the bulkiness of the substituents. Unfortunately, this feature was cancelled in water.
Recently, we focused on the preparation of 6-membered ring nitroxides carrying phosphorylated group

OMRI development
In order to investigate in vivo and in vitro proteolysis, NMR sequence developments were carried out. The main goal was to acquire Overhauser-enhanced images in shorter times, to prevent overheating and for kinetic assessments. In this respect, fully-balanced steady-state free-precession in 3D was implemented and optimized.

Proteolysis investigations
The recent success with the newly prepared nitroxides has revived this task which is currently underway. In the meantime in order to test the OMRI setup we designed an Overhauser switch made of a nitroxide-labelled elastin. Proteolysis by elastases was investigated both by EPR and OMRI. This system proved suitable both in vitro and in vivo. Proteolysis by neutrophil elastase was investigated first in vitro and ex vivo with very promising results, since very low enzyme concentrations in the nanomolar range could be revealed.

An OMRI machine has been developed to investigate enzymatic activity in vivo in mice. The set-up has been adapted for the use of nitroxides exhibiting different EPR features (3 or 6 lines). Some basic enzymatic activity such as proteolysis of nitroxide-tagged elastin has been successfully investigated by OMRI, first in vitro to monitor neutrophil degradation, and then in vivo to monitor digestion.
A new nitroxide carrying phosphorus atom at the position ? has been prepared via a 15-step procedure.

This project should provide a new non invasive molecular imaging technique by magnetic resonance to study proteolysis in deep seated organs. This technique should also help to the understanding of normal proteolytic events and lead to new diagnostic protocols (early diagnostic of cancer) and to monitor the therapeutic response to treatments against uncontrolled proteolytic activities.
To our knowledge, no other in vivo proteolysis imaging technique suitable for deep organs observation is currently available and no similar approach combining DNP and MRI is yet under study.
This project showed the potential of the OMRI technique and that it was possible to prepare shift-nitroxide suitable to investigate the in vivo enzymatic activity.

Numbering of the articles corresponds to those listed in section E2.
Articles 1, 2 and 3 confirm and highlight the potential of the Overhauser-enhanced MRI technique to investigate in vivo enzymatic processes.
Results obtained in NITROMRI provided bases to develop the concept of Theranostic for a new family of drugs (articles 4 and 6).
Article 5 relates cybotactic effects on ?-phosphorylated nitroxides which are the key-molecules of the project. It was a way to make profitable the ANR funding.

1-Scientific background and objectives : The Human Genome Project revealed more than 500 proteases. The substrate specificity, the trigger events and sites of activation are still unknown for most of them. We know however that proteolysis is involved in many physiological situations like inflammation, coagulation, fibrinolysis or tissue turnover. Pathological proteolysis has been observed in many diseases like cystic fibrosis, emphysema, rheumatoid arthritis, bacterial, viral and parasitic infections, tumour and metastasis spreading or pancreatitis. In the intracellular compartment apoptosis also involves specific proteolysis. At present there is no non-invasive method to monitor proteolysis in deep-seated tissues suitable for humans or small animals. This project consequently aims at designing an imaging method for specific proteolysis activity based on Magnetic Resonance Imaging (MRI). Such a method would be a breakthrough for diagnosis and follow-up of treatments in many diseases. Moreover, this new tool would help understanding the activation and persistence of any given protease in correlation with many physiological events, development for instance, a much needed knowledge in fundamental protease research. 2-Description of the project, methodology : The project aims to develop a new method for monitoring the proteolysis in vivo using MRI technique enhanced by Dynamic Nuclear Polarization (DNP, Overhauser effect). The choice of the MRI technique relies on its capacities for exploring deep tissues, for its good resolution, and for its 3D accuracy. Enhancement due to the DNP will provide us with higher sensitivity and generate higher contrast between the tissues under proteolysis and the unaffected tissues. With DNP, calculations predict a 100-fold increase for the signal and preliminary in vitro experiment with our setup yielded a 54-fold increase of the signal. The project will span on four steps. First, peptides carrying nitroxides will be designed and prepared in such way that, after proteolysis, the released nitroxides will exhibit Electronic Paramagnetic Resonance (EPR) signature altered from the one of nitroxides attached to peptides. EPR studies will be performed with model nitroxides to determine the importance of EPR signature difference between free nitroxides and attached nitroxides in media mimicking biological environment. Second, the best candidates will be used to set the imaging system (DNP-MRI) in the way to obtain the highest sensitivity and to observe the highest contrasts on model systems mimicking biological conditions. Third, when the best conditions will be achieved, 3D imaging of the protease activity will be performed on in vitro and ex vivo models, and fourth, in vivo on small animals. 3-Expected results : The fields array of proteases involvement is very broad and so are the potential applications. The imaging technique (DNP-MRI) developed in this project will first be applied in three main fields: i) diagnosis, e.g., proteases/inhibitors balance for cystic fibrosis, or rheumatoid arthritis, or early detection of proteases in tumour surrounding, ii) monitoring the efficiency of anti-protease drugs, iii) fundamental research on proteolysis in normal and pathological situations. Consequently, the DNP-MRI technique has the potential to become a unique tool for studying, in a non-invasive manner, the pivotal and ill-known role of the proteolysis in physiological events or diseases whatever the organs or the body parts where it occurs.