Development of magnetic resonance and optical imaging probes and their in vivo validation in detecting enzymatic activity in infection – ENZYMAGE

We develop two structurally different chelating units for the design of enzyme-responsive imaging probes:
(i) based ??amino glycine derivatives for coupling of the lanthanide chelate to the self-immolative linker. In order to make the complexes applicable to optical detection when luminescent lanthanides are used.
(ii) 6-amino 2-methyl pyridine-like heterocycle as linker between the lanthanide chelate and the self-immolative arm.

We have presented the proof of concept for the PARACEST detection of enzymatic activity with molecular agents based on a self-immolative platform approach. The concept relies on coupling an enzyme-specific substrate to a lanthanide (LnIII)-chelating unit through a self-immolative spacer. While these agents share an identical LnIII-chelating unit and a self-immolative spacer, the choice of the substrate ensures enzyme specificity.
We synthesized and investigated two structurally different chelating units for the design of enzyme-responsive imaging probes:
a) The first one is based on ??amino glycine derivatives for coupling of the lanthanide chelate to the self-immolative spacer. We synthesized a series of Gd3+ and Yb3+ complexes derived from this platform, including those bearing a self-immolative arm and a sugar unit as selective substrate to ?-galactosidase, LnL1, the -NH2 amine derivatives formed after enzymatic cleavage, LnL2, and the model compounds, LnL3 and LnL4. The relaxivity change upon enzymatic cleavage is limited which prevents application of this system as enzyme-responsive T1 relaxation agent. The Yb3+ analogues show a PARACEST effect after enzymatic cleavage which can be exploited for the specific detection of enzymatic activity.
b) The second family used a 6-amino 2-methyl pyridine-like heterocycle as linker between the lanthanide chelate and the self-immolative arm. For optical detection, a pyridine unit as an organic chromophore was incorporated. We have synthesized two model ligands, L4 and L5. L4 has a pyridine-derivative arm that, for the purpose of the physico-chemical studies, models the self-immolative linker and the enzyme-specific substrate that will be attached to the agent in the real enzymatic probe. We have demonstrated that, when complexed to GdIII, they function as efficient T1 MRI agents. When the same ligands are complexed to YbIII or EuIII, the system works as a CEST MRI contrast agent, but also as a luminescent probe.

The main application fields of our imaging agents are:
- 1. Biomedical research, including rationalization of the parameters related to disease processes, thus strongly contributing to drug development. They are biological tools that can contribute to faster identification and testing of new therapeutic agents. They can allow for replacement of the invasive research techniques (histology) by time-effective, repeatable, real-time visualization of biologically relevant processes to ensure a reduction of animal use in research experiments.
- 2. Clinical diagnostics, in a future perspective. Our imaging agents can allow early detection of the disease and prediction of treatment responsiveness. They can offer the possibility of unambiguous and rapid identification of the nature of the infection based on the specific enzymatic response related to a given bacteria. This would be a huge progress with respect to the currently used bacterial cultures to identify the infectious agent which require at least 24 hours (for the most rapidly growing bacteria) up to 3 weeks (Mycobacterium tuberculosis, the cause of tuberculosis).

1. T. Chauvin, S. Torres, R. Rosseto, J. Kotek, B. Badet, P. Durand and É. Tóth, Lanthanide(III) complexes bearing a self-immolative arm: potential enzyme responsive contrast agents for magnetic resonance imaging, Chem. Eur. J, 2012, 18, 1408–1418.

While a large number of in vitro techniques are available for assessing the abnormal functioning of cells at the molecular level, the in vivo real-time visualization of those processes remains a major limitation and constitutes one of the main challenges in diagnostic imaging. Molecular imaging seeks the in vivo real time follow up of molecular events occurring in cells and organisms. We propose to create magnetic resonance and optical imaging agents which enable non-invasive, real-time, repeatable, in vivo visualization of pathology-related enzyme activities. The visualization, either by MRI or by optical imaging, corresponds to the in vivo activation of the imaging probes by the specific enzymes.
We propose a highly adjustable platform of enzyme activatable imaging probes which confers potentialities both regarding the type of imaging modality and the diversity of targeted enzymes. We will synthesize and characterize molecular imaging agents based on stable, non-toxic lanthanide chelates that can be used either for MR or OI detection, depending on the lanthanide chosen. Lanthanides have fundamentally different magnetic and optical properties, while being chemically similar, allowing the easy replacement of one by another. The enzymatic activation of the probes occurs via a self-immolative mechanism. The same platform can be applied for the development of both MRI and optical probes. In contrast to any enzyme-responsive potential agent proposed in the literature, this platform is adaptable to a broad range of biological/medical problems by opening the way to specifically target a large variety of enzyme activities. While the Ln3+ chelating unit and the self-immolative spacer can be identical for the entire family, the appropriate choice of the substrate ensures enzyme specificity.
MRI detection will be based either on “traditional” Gd-enhanced, or on paramagnetic chemical exchange saturation transfer (PARACEST) images. PARACEST agents are ideally suited for molecular imaging since the contrast can be switched on or off at will. The optical detection is based on the luminescence of lanthanide cations upon excitation on the sensitizer. On enzymatic cleavage, the intensity of the luminescence signal will increase due to the loss of a quenching agent located at a close proximity of the lanthanide. This can be monitored at the cellular level using microscopy or in small animals using macroscope equipment.
The proposed agents will be validated in infectious diseases. Methods used so far to visualise infectious processes in small animals rely on the detection of an unspecific probe or on the use of genetically modified organisms. We propose to go beyond the state of the art using a fundamentally different approach by detecting with MRI or OI pathology-related enzymatic activities that are specific to cell types.
The present proposal is the evolution of the ANR project “Enzyme-Activated Contrast Agents for Magnetic Resonance Imaging” (ENZYMRI) which ends in 2010. This project allowed us to demonstrate the feasibility of enzymatic activation of magnetic resonance imaging probes via a self-immolative approach (published in Angewandte Chemie Int. Ed.). The present project distinguishes itself from ENZYMRI by (i) including new chemical structures that afford greater transient stability of the self-immolative compounds thus more facile synthetic pathways to obtain the agents, (ii) by extending the detection mode to optical detection and (iii) by establishing the first steps towards in vivo application.