Project: Development of a novel diagnostic agent for the functional molecular imaging of cell death in vivo

Apoptosis, or programmed cell death, covers a set of signalisation processes inside a cell that lead to a silent death from an immunological and inflammatory stand point. The dying cells and the resultant apoptotic bodies are then eliminated by the professional phagocyte cells. In the absence of any pathology or cell aggression, apoptosis rate is low and named physiological apoptosis as it merely participates to tissue homeostasis._x000D__x000D_Apoptosis is a hallmark of numerous pathologies: _x000D__x000D_1) In oncology, apoptosis occurrence is a reliable readout of therapeutic efficacy for most types of anticancer therapy. It has now largely been observed that early assessment of therapeutic response is correlated to the patient’s outcome and is considered as a prognostic factor in a number of cancer types. In addition, the evaluation of early treatment response may avoid inefficient chemotherapies and their associated toxicities, improving quality of patient’s life. This evaluation will also allow a better adaptation of therapy to the characteristics of the patient’s cancer. To end, early evaluation may contribute to a reduction of the cost in the case of expensive therapies, which is also an important challenge nowadays. Therefore, to characterize and define early response to anticancer therapy constitute an important challenge in modern practice of oncology. _x000D__x000D_2) In cardiovascular diseases such as myocardium infarction, brain hemorrhage or ischemic accident, vulnerable atheromatous plaques, intervention time is crucial and the early and accurate evaluation of the tissue damages may be important for patient’s outcome. _x000D__x000D_3) Graft rejection and deep inflammation processes involve also cell apoptosis._x000D__x000D_The numerous pathologies involving apoptosis justify the effort made to develop tracers for its detection using various imaging modalities. _x000D__x000D_Among other cellular signals, apoptosis is characterised by a loss of membrane phospholipids asymmetry, resulting in the exposure by translocation of phosphatidylserine (PS) lipids at the cell surface where it is absent in normal cell. _x000D__x000D_Therefore the cell surface PS has become the CO target for in vivo apoptosis detection and imaging. _x000D_The CEA participant to the project has engineered and developed molecules which bind to the PS exposing membranes. These molecules are based on a mini-protein derived from a particular protein, annexin V, an endogenous protein considered as a reference molecule for apoptosis detection. They are currently evaluated in vivo using near infrared (NIR) fluorescence imaging in oncology with mouse models._x000D__x000D_The first aim of the present project is to develop new molecules with enhanced physicochemical and biological properties that can be obtained by direct chemical synthesis. These molecules will be precursors of tracers for human imaging of apoptosis using imaging modalities such as PET, SPECT, IRM, NIR fluorescence and Ultrasounds. The advantage of having a chemically synthesized tracer instead of a bacteria-produced tracer is the much lower cost for good manufacture practice (GMP) production and the much higher simplicity and rapidity of the regulatory process for human applications. _x000D__x000D_The second aim of the present project is to develop a new tracer for a more recently developed PET imaging technique using a positron emitting radionuclide, gallium-68, and to get its preclinical proof of concept. The advantage of this radionuclide is its on site hospital availability by use of a simple generator having a ~300 day half-life. This advantage is combined to the fact that our molecules could be conserved in a freeze-dried form at the hospital as a cold kit ready to use. Labeling of our molecules with other radionuclide and especially fluorine-18 will be developed to increase the offer in imaging facilities._x000D__x000D_Market launch will be done in two steps: _x000D_1) at the end of the project, a batch of the tracers, bacteria produced under good laboratory practice (GLP) conditions, will be proposed to the market by Iason for animal preclinical studiesd only; _x000D_ 2) soon after the end of this project, after GMP production, the tracers will enter into a phase I/II trial and if successful, in phase III. _x000D__x000D_The consortium is based on highly complementary Austrian and French Ps selected on their expertise. It bridges the successive steps of translational research and development involving academia, industry and the clinics: _x000D_• the French Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA) in charge of the development of the molecules,_x000D_• the Austrian ARGOS Zyklotron GmbH, focusing on the production of radioactive PET-radiopharmaceuticals(subsidiary of IASON Holding and fellow subsidiary of IASON GmbH)_x000D_• the LIMP (at UPMC), the only French authorized laboratory for gallium-68 PET imaging in animals and humans, in charge of the preclinical evaluation of the tracer._x000D_

Acronym	ApoPET (Reference Number: 6397)
Duration	01/07/2011 - 31/12/2014
Project Topic	In the presented project „ApoPET“ a new PET- radiodiagnostic for the highly sensitive detection of apoptosis in major pathologies is developed applying highly innovative technologies in the field protein engineering, bioconjugate chemistry, radiolabelling and preclinical molecular imaging.
Project Results (after finalisation)	First workpackage and aim of the project has been to develop new molecules (trimers of tetramers of AFIM) with enhanced physicochemical and biological properties . These molecules could be precursors for tracers for cell death imaging and are built by grafting a protein module to a chemical scaffold. Due to the problems with the development of suitable scaffolds, the evaluation of a Gallium-68 precursor monomer AFIM derivative has been prioritized._x000D_The second workpackage and aim has been to develop PET tracers, based on these proteins, for use in radiolabelling either with Gallium-68 or Fluorine-18. The radiometalation with Gallium-68 has been optimized using the pre-purification method. Radiolabelling with Gallium-68 was already successful. _x000D_Third workpackage has been the study of toxicity in rats. This study has not been performed as the final oligomer molecule, which was not available during project duration._x000D_Fourth workpackage has been the evaluation of the biologically evaluation of the monomer PET-tracer 68Ga-AFIM (clearance, biodistribution, uotake and comparison to known tracer)_x000D__x000D_Argos has been working on the workpackages 2 and 3:_x000D_WP2: Radioactive labelling of AFIM derivatives_x000D_ The CO goal of WP2 was the radiolabelling of various AFIM multimers which have been developed in WP1. In general two approaches should be developed which are based on the radiometalation of a corresponding NOTA-chelator with gallium-68 as well as the synthesis of fluorine-18 derivatives. The WP also includes the design of a suitable automated synthesiser module containing a pre-purification unit._x000D_M.2.1. Design of a suitable fully automated synthesis module, suitable for protein labelling_x000D_The fully automated 68Ga-synthesis module has been designed and evaluated containing a prepurification unit within the system. The 68Ga-generator eluate is collected on a SCX-cation exchange cartridge and subsequently, the 68Ga-activity is released from the cartridge using a concentrated NaCl solution. The eluate is suitable for a direct labelling of peptides, e.g. TAF-NOTA or DOTANOC – a standard peptide already used in routine applications. _x000D_M. 2.2 Development and evaluation of a GMP-compliant 68Ga and 64Cu-labelling process which can easily be implemented into an automated production under pharmaceutical conditions as well as the establishment of a suitable quality control procedure._x000D_The AFIM derivative TAF-NOTA-68Ga could be obtained in a yield of 56%. The conditions which gave the best labelling results are 37°C during 10 minutes with about 40nmol of the peptide derivative suitable for further investigations. Radiolabelling of AFIM-68Ga has also been successfully implemented at UMPC laboratory. _x000D_M. 2.3.- Development of 18F-fluorine labelled derivatives._x000D_The labelling with a 18F-prostetic groups using the so-called “Click-chemistry”has been evaluated using a 1,3-dipolar cycloaddition of azides and alkynes with FADIBO-prostetic group. The radiosynthesis of FADIBO has been optimized in a synthesizer via nucleophilic route with fluorine-18 for subsequent copper-free click reaction to the AFIM-derivative. _x000D_M 2.4.- Labelling of NOTA-derivatives with Cu-64 corresponding to task 2.1_x000D_The module has been designed and evaluated. It will allow the radio-metalation for Gallium-68 and copper-64, since the radiochemical synthesis are very similar._x000D_WP3: Toxicological evaluation_x000D_None of the tested molecules revealed very favourable characteristic in all stages of the project. Therefore,toxicology has not been tested in order to reduce the animal tests to a minimum._x000D_
Network	Eurostars
Call	Eurostars Cut-Off 6

Project partner

Number	Name	Role	Country
3	ARGOS Zyklotron GmbH	Coordinator	Austria
3	Commisariat à l'Energie Atomique et aux Energies Alternatives-Institut de Biologie et technologies de Saclay	Partner	France
3	Université Pierre et Marie Curie (UPMC) - Institut fédératif de recherche 65 (IFR65) - Laboratoire d'Imagerie Moléculaire Positronique (LIMP)	Partner	France