The TheraGuIma project aims at designing multifunctional nanoparticles for image-guided radiotherapy which is expected to significantly improve the brain cancer therapy. There is indeed a real need to propose innovative strategy since the survival of patients suffering from glioblastoma (the most aggressive and the most common brain tumors) does unfortunately not increase despite the tremendous efforts devoted to the improvements of cancer therapy. Although radiotherapy is presented as the most efficient treatment against brain tumors, it suffers from a lack of selectivity in the killing effect leading to numerous adverse effects in normal healthy tissues surrounding the lesion. A better selectivity is therefore required for eradicating the tumor while sparing the surrounding healthy tissues. In addition to the modulation of irradiation parameters, the accumulation of drugs containing heavy elements (high Z) in the tumor has been proposed for improving the selectivity of the radiotherapy since heavy elements generate a dose-enhancement of ionizing radiation (X- or gamma-ray). TheraGuIma project gathers eight skilled and complementary partners (from university and industry) for improving the selectivity of the radiotherapy. This innovative strategy rests on the development of nanoparticles combining medical imaging and radiosensitizing effect. These multifunctional nanoparticles will be obtained from high Z elements (Gd, Lu and Au). These elements will confer to the nanoparticles the ability to enhance both the dose effect of ionizing radiation applied for radiotherapy (radiosensitization) and the contrast of X-ray images. The composition of the nanoparticles will be tuned in order to follow up the nanoparticles by other medical imaging (magnetic resonance imaging (MRI), Single photon emission computed tomography (SPECT) or positron emission tomography (PET) and eventually fluorescence imaging). Moreover the control of the size (ideally <5 nm) and the chemical composition of the surface will receive a peculiar attention because they exert a preponderant influence on the accumulation in tumor and on the renal clearance. The optimal tumor-to-normal tissue high Z element ratio which must be as high as possible for eradicating the tumor while sparing the healthy tissue requires the functionalization of the nanoparticles by peptides (cRGD, ATWLPPR) for an active targeting of the zone to be treated. The possibility to monitor the biodistribution and the clearance of the nanoparticles by various medical imaging constitutes a great asset because the cytotoxic effect induced by the absorption of the ionizing radiation by the nanoparticles can be initiated at the most opportune moment (i.e. when the ratio is the highest). The potential of the various multifunctional nanoparticles developed in the framework of TheraGuIma for the image-guided radiotherapy will be evaluated by in vitro and in vivo irradiation experiments.

The evolution of Magnetic Resonance Imaging into one of the most prominent clinical diagnostic modalities has been largely assisted by the use of paramagnetic contrast agents. Nowadays, imaging of molecular events at the cellular level starts to become accessible. Molecular Imaging requires probes that are specific to a particular molecular event, which places chemistry into a vital position in the development of this revolutionary technology. Despite several attempts, the in vivo MRI detection of enzymes remains a challenge. This proposal offers a general approach to the rational conception of enzyme responsive MRI probes. We will synthesize GdIII complexes that change their proton relaxivity, thus MRI efficiency in the presence of an enzyme of choice. These agents are designed to function on the basis of the 'self-immolative' mechanism, a concept applied in 'Antibody Directed Enzyme Prodrug Therapy' or 'Gene Directed Enzyme Prodrug Therapy'. The contrast agent is activated or deactivated by attack of a specific enzyme which initiates a self-immolation process in a defined part of the molecule. This cascade reaction results in structural changes of the complex which in turn affects its relaxivity and result in an enzymatic MRI response. In order to target a large variety of enzyme activity, the probes have modulable structure consisting of three moieties: (i) the coordinating unit around the Gd, (ii) the chain that undergoes self-immolation on enzyme activation and (iii) the substrate for a specific enzyme which temporarily protects the self-immolative chain. For GdIII complexation, we will explore both macrocyclic (DOTA-type) and acyclic (DTPA-type) chelators, with amino glycine or an aminal for coupling to the self-immolative linker. The self-immolative mechanism is based on the intrinsic instability of benzyloxy carbamates possessing an electron donor substituent in ortho or para position. P-hydroxy mandelic acid derivatives will also be used as self-immolative linkers, with the advantage of easy coupling to macromolecules via the acid function. The substrate can be any moiety that transitionally reduces the electron donor capabilities of the substituent. This general approach allows to target a large variety of enzymes (esterase, lipase, glycosidase, …). Among the different parameters determining relaxivity of a GdIII complex, rotational dynamics and hydration number are the most accessible to modification in an enzymatically activated self-immolative process. One class of the GdIII complexes is designed to give a relaxivity response to enzymes which is related to a change in the size, hence in the rotational motion of the molecule. The size change is realized via enzymatique cleavage of the chelates from medium-sized or macromolecular (dendrimeric, protein bound, etc.) entities. Another approach is represented by compounds which undergo a hydration number change on enzyme activation. The new GdIII complexes will be characterized with regard to MRI contrast agent applications. Proton relaxivities will be measured as a function of the magnetic field. 17O NMR will be used to assess hydration number and water exchange. The project specifically aims at the understanding of the functioning of these potential enzymatic contrast agents at the molecular level. We will assess all parameters that play a role in the enzyme-promoted relaxivity change. We believe that this understanding will allow us to improve the systems on a rational basis. The most promising compounds will be tested in cell lines and small animal MRI studies. Lanthanide complexes other than GdIII (EuIII, DyIII, YbIII, TmIII) containing slow-exchanging amide protons have been widely investigated as PARACEST agents. By replacing the GdIII with one of these lanthanide ions in our enzyme-responsive systems, we might obtain potential, enzyme-responsive agents for PARACEST imaging. These aspects will be also addressed. In addition to an in vivo application, the proposed systems are prospective probes for High or Medium Throughput Screening of enzyme inhibitors by MRI. MRI represents a viable alternative to currently used high throughput screening modalities. The detection by MRI is much less prone to interferences as compared to spectrophotometric techniques, and is more convenient in terms of safety than radioactive probes. An important aspect of the project is to combine expertise from various fields, such as bioorganic chemistry and enzyme mechanisms, synthesis of prodrugs, physico-chemical characterization of MRI contrast agents and MRI technology. Through these interactions and in synergy with the industrial partner, we will develop novel, highly efficient imaging probes based on innovative coordination and bioconjugation chemistry. This project, submitted in 2006 for the first time in the programme 'ANR blanc', has been improved according to the remarks and suggestions of the referees.

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.