Water-soluble aza-BODIPY platform for multimodal imaging and trackable therapeutics – WazaBY

In order to obtain WAZABYS platforms, we worked on the functionalization of the boron atom. Indeed, this approach induces steric hindrance, which prevents aggregation of the probes. In addition, our strategy consisted in substituting the boron atom with water-soluble solubilizing groups.
From these water-soluble platforms, we considered functionalizing them, and thus introducing: 1) a bioconjugation function allowing bioconjugation and thus, the formation of an optical imaging agent; 2) a chelating agent and a bioconjugation function, allowing the formation of bimodal probes; 3) a therapeutic complex, for the formation of theranostics.
After the synthesis and development of the target compounds, we wanted to demonstrate the added value of our systems, through a pilot study in imaging and therapy. For this aim, we considered a preclinical study targeting PD-L1, using an anti-PD-L1 antibody labeled with the bimodal probe as an imaging agent (making it possible to evaluate the expression of PD-L1 in targeted tumors) , but also diagnostic companion, associated with the same antibody labeled with a theranostic, allowing the formation of a trackable ADC (ADC: antibody drug conjugate, the antibody thus having an immunotherapy type modality specific to the anti-PD antibody -L1 and a chemotherapeutic type by the added therapeutic agent).

During this year and a half, we were able to synthesize different multifunctional WAZABYs platforms. We were able to prove that these platforms were stable, water-soluble, and emitted n the near infrared region. One of them was bioconjugated with an anti-PD-L1 antibody, and a preclinical study, carried out by optical imaging, highlighted the potential of this probe. We then worked on the functionalization of the WAZABYs probes. We first introduced one or two chelating agents, which allowed us to develop bimodal probes (MRI / optical, or SPECT optical). In the latter case, the bimodal probe was bioconjugated on Trastuzumab antibody, and a complete bimodal (by SPECT and optical imaging) study, in vitro and in vivo was then carried out, associated with fluorescence-assisted surgery. Secondly, we started to work on the bioconjugation (random but also «site-specific«) of the bimodal probe on the anti-PD-L1 antibody. The preliminar in vitro studies (stability of the bioconjugate) were carried out.

In parallel, we have introduced various therapeutic complexes based on gold phosphine onto the WAZABYs probes. The first in vitro studies carried out with the resulting compounds highlighted their therapeutic potential on various cancer lines.

Finally, we discovered that certain WAZABYs compounds were able to emit in the second near infrared window NIR-II (between 1,000-1,700 nm). This optical range allows a significant gain in sensitivity and has real clinical potential, especially for cancer surgery. We studied one of these WAZABY compounds in vitro and in vivo, and demonstrated that the probe enabled to identify and visualize the small deep vessels of tumors. In addition, without vectorization, the fluorescent compound is able to accumulate strongly in the tumor, up to 7 days after injection.

Many perspectives are envisaged, after these last 18 months.
Regarding theranostic compounds, in the coming months, we now have to work on the optimization of these theranostic compounds. More particularly, we aim at increasing their water-solubilization, and to introduce a grafting function, to be able to vectorize them on PD-L1, and to assess their potential in vivo.

A complete study in imaging and therapy will then be carried out. Still in a theranostic approach, we also plan, in collaboration with the team of Dr Sancey at the Institute for Advance Biosciences, Grenoble, to introduce boron complexes on aza-BODIPYs, and in particular 10B-BSH, to perform borotherapy by neutron capture, on tumors. BSH will be introduced, in a similar way than for the organometallic complexes, on the boron atoms of the aza-BODIPYs.

Finally, during this project, we will explore the potential of WAZABY-emitting probes in NIR II, by varying the structures on the aza-BODIPYs core, in order to optimize the photophysical properties of these fluorophores. We will also build bimodal probes and theranostics from this new class of fluorophores.

Publications:
1. Investigation of B-F substitution on BODIPY and aza-BODIPY dyes: Development of B-O and B-C BODIPYs
Bodio, E.*, Goze, C.*, Dyes and Pigments, 2019, 160, 700-710. doi.org/10.1016/j.dyepig.2018.08.062.

2. A Promising Family of Fluorescent Water-Soluble aza-BODIPY Dyes for in Vivo Molecular Imaging; Jacques Pliquett, Adrien Dubois, Cindy Racoeur, Nesrine Mabrouk, Souheila Amor, Robin Lescure, Ali Bettaïeb, Bertrand Collin, Claire Bernhard, Franck Denat, Pierre Simon Bellaye, Catherine Paul, Ewen Bodio*, Christine Goze*, Bioconjugate Chem., 2019, 30 (4), 1061-1066. DOI: 10.1021/acs.bioconjchem.8b00795.
Couverture du Journal.

3. Aza-BODIPY Platform: Toward an Efficient Water-Soluble Bimodal Imaging Probe for MRI and Near-Infrared Fluorescence; Océane Florès, Jacques Pliquett, Laura Abad Galan, Robin Lescure, Franck Denat, Olivier Maury, Agnès Pallier, Pierre-Simon Bellaye, Bertrand Collin, Sandra Même, Célia S. Bonnet*, Ewen Bodio*, Christine Goze*, Inorg. Chem., 2020, 59 (2), 1306-1314.
DOI: 10.1021/acs.inorgchem.9b03017.

4. BODIPYS and aza-BODIPY derivatives as promising fluorophores for in vivo molecular imaging and theranostic application; Franck Denat, Ewen Bodio, Christine Goze*, J. Porph. Phtalo., 2019, 11(12), 1159-1183.
doi.org/10.1142/S1088424619501268.

5. Water-Soluble Aza-BODIPYs: Biocompatible Organic Dyes for High Contrast In Vivo NIR-II Imaging; Amélie Godard, Ghadir Kalot, Jacques Pliquett, Benoit Busser, Xavier Le Guével, K. David Wegner, Ute Resch-Genger, Yoann Rousselin, Jean-Luc Coll, Franck Denat, Ewen Bodio*, Christine Goze*, Lucie Sancey*, Bioconjugate Chem., 2020, 31 (4), 1088-1092.
DOI: 10.1021/acs.bioconjchem.0c00175.

Patent:

1. Utilisation de composes fluorophores de type aza-BODIPY comme agents de contraste dans l’infrarouge très lointain. Sancey L, Goze C, Bodio E, Busser B, Pliquett J, Godard A, Kalot G, Josserand V, Le Guével X, Coll J-L, Denat F ; EP19315089.3, 2019.

Among the different disciplines in medical imaging, molecular imaging is an emerging powerful tool, which enables to detect pathophysiological changes in living subject at the cellular and molecular level. More particularly, it enables to perform a fast and accurate diagnosis, or to monitor the success of a treatment. Even if it plays a central role in oncology, the use of molecular imaging opens up an incredible number of possibilities for other medical applications as for cardiovascular diseases, infections, bone disorders, thyroid disorders, brain disorders, gastrointestinal diseases… Among the different molecular imaging methods, optical imaging (OI) is the method of choice for in vitro and ex vivo studies, and is used more and more for small animal imaging studies. It is a non-ionizing technique, which presents the high resolution and the high sensitivity needed for molecular imaging investigations. It is more restricted for clinical applications, due to its tissue limited penetration, but it is of major interest for endoscopy investigations, as well as for fluorescence imaging guided surgery. The increasing interest in OI is also due to two emerging fields: bimodal imaging and trackable therapeutics. Indeed, OI is a modality of choice for bimodal imaging: its high sensitivity is suitable for a coupling to a PET/SPECT probe, which will take over the fluorophore for in vivo imaging. Concerning trackable therapeutics, grafting a fluorescent probe to a therapeutic moiety enables to track in real time the resulting therapeutic in vitro and in vivo, giving crucial information on its biodistribution and its cellular targets.
In the aim of performing in vivo studies, the use of near infrared (NIR) light offers several advantages, such as low absorption coefficient of most of the biomolecules, lower scattering, minimization of autofluorescence signals and risks to disturb the normal metabolism of the biological system to study. It therefore requires the use of called NIR fluorescent probes, which absorb and emit in the "diagnostic window" (between 650 and 900 nm). However, the choice of the fluorescent dye is almost always limited to commonly used cyanines. Despite some advantages, these fluorophores suffer from poor fluorescence efficiency, but above all from a chemical instability, rapid photobleaching, and today, there is no ideal NIR-fluorescent probe, which is suitable for biological applications (water-soluble, stable, low toxic…), which enable a large scope of functionalizations, and which can be synthesize in large scale.
We propose to solve this problematic, and to develop a NIR-fluorescent platform displaying the properties above-mentioned, and we are targeting an original and promising family of fluorophores, the aza-BODIPYs, because their synthesis is straightforward, they absorb and emit in the NIR region, they present outstanding chemical and photochemical stability, and can be produced easily in the gram scale. Therefore, the aza-BODIPYs compounds presents all the advantages for biomedical applications, except their high lipophilic character and their very poor solubility. That is why, we will develop a simple strategy to solubilize them, and we aim at elaborating a highly funtionalizable water-soluble aza-BODIPY platform, and to design, starting from this platform, new Monomolecular Mutimodal Imaging Probes (MOMIP) and optical metal-based trackable therapeutics. In the scope of the project, we will propose simple synthetic routes to get access to the water soluble aza-BODIPY platforms, the resulting optical imaging probes, MOMIP and theranostic constructs. The most promising systems will be bioconjugated to different biovectors, and, in the last part, we will demonstrate, through an imaging pilot study, the potential of the new systems.