Breast cancer remains the most commonly diagnosed cancer among women over the age of 20, as recently reported by the Canadian Cancer Society. Studies have shown a strong correlation between early detection and increased survival rates thus it is important to have a means to adequately screen and detect breast cancer. Currently, tests are limited to traditional imaging methods such as ultrasound (US), magnetic resonance imaging (MRI) and standard mammography. There remains a need for a molecular imaging probe that is capable of providing further prognostic information particularly with respect to assessing tumour aggressiveness and the likelihood of a cancer to metastasize. Overexpression of the insulin receptor (IR) has been detailed in patients with breast cancer but there is currently no means of non-invasive and quantifiable detection of the receptor. The goal of the work described here was to prepare an insulin derived nuclear imaging probe via direct coupling of a prosthetic group bearing a radionuclide to the B29 lysine (B29-Lys) residue of the hormone. Benzoic acid bearing halogens were chosen as model compounds. The lead candidate N-[4-fluorobenzoyl]-(B29-Lys) insulin (4) was prepared in 60% overall yield and showed affinity for the IR similar to that of native insulin (IC50=3.6 nM). The 18F analogue (9) was successfully synthesized and showed high stability (up to 4 hours) post formulation in both saline and phosphate buffered solution (PBS). The product represents a promising new probe for assessing the role of the IR in cancer growth and metastasis. A complementary strategy for imaging markers of tumour aggressiveness was investigated through the development of a novel ultrasound probe. A pretargeting approach involving urokinase plasminogen activator receptor (uPAR), which is known to play a role in cancer metastasis, was used to develop the agent of interest. Here an in vivo reaction between tetrazine tagged microbubbles (MBs) and anti-uPAR antibodies conjugated to trans-cyclooctene (TCO) was employed. Following preparation of the antibody conjugate and tetrazine functionalized MBs, preliminary in vitro testing in a flow cell system was conducted. Results showed the ability of the uPAR expressing cells to exclusively capture the tetrazine MBs after they have been previously incubated with the TCO anti-uPAR antibody. No capture was observed when the target and/or the antibody were absent. The contrast agent developed represents the first MB targeted against uPAR and has the potential to impact current diagnostic paradigms particularly given the widespread use of ultrasound in cancer patient management. / Thesis / Master of Science (MSc)

L’imagerie in vivo du petit animal est de plus en plus utilisée en pharmacologie pour identifier et caractériser l’activité de nouveaux agents anticancéreux.La première partie de ma thèse a consisté à développer des outils pour améliorer la quantification enbioluminescence. Une méthode, basée sur les caractéristiques spectrales des photons émis, a été établie pour corriger l’absorption tissulaire. La seconde, faisant appel aux méthodes de restauration d’images, avait pour but de corriger la diffusion pour augmenter la résolution. Dans un second temps, j’ai mis en place des modèles in vivo de tumeurs expérimentales bioluminescentes (un glioblastome intracérébral, un lymphome anaplasique à grandes cellules et un neuroblastome métastatique) en utilisant les méthodes d’imagerie décrites précédemment. Ces études ont permis d’étendre la caractérisation de l’activité préclinique d’un nouvel agent anticancéreux. L’objectif de la dernière partie de mon travail était de développer des sondes d’imagerie. La première sonde, un anticorps monoclonal anti-CD45 marqué avec un fluorochrome a permis la détection de cellules leucémiques humaines implantées chez la souris en utilisant l’imagerie de fluorescence. La seconde a été développée pour prédire l’entrée d’un agent anticancéreux, un conjugué spermine-podophyllotoxin, dans les cellules tumorales via les transporteurs des polyamines. La sonde synthétisée est une spermine à laquelle un groupement HYNIC a été ajouté afin de pouvoir lier un radioisotope : le Technétium 99m et ainsi réaliser un examen scintigraphique. Les résultats ont démontré la faisabilité d’une application préclinique de cette sonde. Ainsi à l’issu de cette thèse, les méthodes de traitement des signaux de bioluminescence développées sont disponibles pour améliorer l’application de l’imagerie optique en pharmacologie. Bien sûr des études supplémentaires sont encore nécessaires pour définir précisément dans quel contexte ces corrections seront les plus appropriées. / Small animal imaging is more and more used in pharmacology to identify and to characterize the activities of new antitumor agents. The first part of my thesis consisted in the development of new tools to improve the quantitation in bioluminescence. A method, based on spectral characteristics of emitted photons, has been established to correct tissue absorption. The second, using methods of image restoration had for objective to correct tissue scattering to increase the resolution. In a second part, I developed in vivo models of bioluminescent tumors (intracranial glioblastoma, a large cell anaplastic lymphoma and a metastatic neuroblastoma) using the imaging methods described previously. These studies allowed the characterization of the activity of a new antitumor agent. The aim of the last part was to develop imaging probes. The first, a monoclonal antibody antiCD45 labeled with a fluorochrome allowed the detection of human leukemic cells implanted in the mice using fluorescence imaging. The second was developed to predict the uptake of a antitumor agent, a spermine-podophyllotoxin conjugate, in tumor cells via the polyamine transport system. The synthesized probe is a spermine conjugated to a HYNIC group to bind a radioisotope: the Technetium 99m and to realize a scintigraphic examination. The results showed the feasibility of a preclinical use of this probe. So, at this end of this thesis, the developed methods of bioluminescent signal processing are available to improve the use of optical imaging in pharmacology. Of course, supplementary studies are necessary to define precisely in which context these corrections will be the most appropriate.

Urokinase-type plasminogen activator (uPA) protein is a serine protease of the trypsin family that is overexpressed by tumors cells seeking to metastasize. Molecular imaging methods using molecular imaging probe designed to target uPA could provide a method for the detection of aggressive cancers and monitoring response to treatment. Four classes of high affinity uPA inhibitors, three which were reversible and one irreversible, were used as platforms to develop radiolabeled probes for uPA. Based on structure-activity relationships, lead compounds were modified to allow for the introduction of a radiohalogen (radioiodine) at different sites in the corresponding molecules. Suitable synthetic strategies were developed to create libraries of iodinated phenyl guanidine, peptide, naphtamidine and phosphonate derivatives. For the phenylguanidines colorimetric assays showed the product had micromolar affinity while for the peptide derivatives low nanomolar affinity for the iodinated analogue was observed (1.4 nM to 2.53 nM). Unfortunately quantitative biodistribution studies showed low tumour uptake (<0.5% ID/g). More promising results were obtained for the irreversible iodinated phosphonated derivative which had an affinity of 2.1 nM. This reagent showed 1.95% ID/g tumour uptake and lower blood uptake in vivo which demonstrates advantageous properties over existing uPA probes in terms of tumour-to-blood ratios. A complementary development was also achieved in that the first example of a 125I-labelled tetrazine was prepared. This new reagent can be used in pre-targeted strategies that utilize bioorthogonal coupling between stained trans-cyclooctene (TCO) and tetrazines. The product was prepared using a concomitant oxidation iodo-destannylation reaction and the product isolated in 80% radiochemical yield. The reaction with transcycloctene proceeded rapidly to produce various isomers which were fully characterized through NMR analysis of the non-radioactive analogues. / Thesis / Doctor of Philosophy (PhD)

L’Imagerie par Résonance Magnétique (IRM) offre une excellente résolution à l’échelle macroscopique alors que l’imagerie optique lui est parfaitement complémentaire car elle dispose d’une haute résolution à l’échelle microscopique ainsi que d’une forte sensibilité. Les complexes de gadolinium(III) ont déjà prouvé leur efficacité en tant qu’agents de contraste IRM et d’autres lanthanides luminescents émettant dans le proche infrarouge conviennent pour l’imagerie optique. Des complexes de lanthanides bishydratés à motif pyridine montrant des résultats très prometteurs pour les deux types d’imagerie ont été développés précédemment au laboratoire. Afin d’améliorer leurs propriétés optiques, des ligands à motifs isoquinoléines ont été synthétisés. Leurs complexes de lanthanides montrent des propriétés magnétiques prometteuses et des constantes thermodynamiques qui démontrent leur faible toxicité in vitro. La longueur d’onde d’excitation et les rendements quantiques des complexes d’Yb3+ et de Nd3+ ont été augmentés pour permettre d’obtenir des images de luminescence et d’envisager des applications in vivo.Afin d’améliorer davantage ces propriétés optiques, la synthèse de ligands à motif 2-azaanthraquinone a été entreprise mais l’instabilité chimique de ces molécules n’a pas permis d’isoler les ligands désirés.Cette approche bimodale a été appliquée à la conception de sondes sensibles au zinc. La synthèse de complexes de Gd3+ à motifs pyridines adaptés permet une detection relaxométrique qualitative et sélective de ce cation. Cependant, l’absence de variation du nombre d’hydratation ne permet pas la détection par luminescence. De légères modifications chimiques sur les ligands devraient permettre d’atteindre cet objectif. / Among the state-of-the-art imaging techniques, Magnetic Resonance Imaging (MRI) offers an excellent macroscopic scale resolution whereas optical imaging shows high microscopic scale resolution and great sensitivity. Gadolinium complexes have already proved their efficiency as MRI contrast agents whereas other lanthanide cations emitting in the near infrared may suit for optical imaging purposes. Pyridine-based lanthanide complexes which showed promising results as bimodal probes have previously been developed. In the objective of improving their optical properties, isoquinoline-based ligands have been synthesized. The lanthanide complexes show promising magnetic properties and their thermodynamic stability presumes in vitro low toxicity. Excitation wavelengths and quantum yields of both Nd3+ and Yb3+ complexes have been improved to obtain luminescence images and to foresee in vivo applications. In order to further improve the optical properties, attempts to synthesize 2-azaanthraquinone-based ligands have been made.The desired ligands could not have been isolated due to their chemical instability. This bimodal approach has been applied to the design of smart probes sensitive to zinc. The qualitative and selective detection of the latter has been realized thanks to Gd3+ complexes with adapted pyridine-based ligands. However, the detection by luminescence could not be achieved. Slight chemical modifications on the ligands should allow reaching this goal.

Cancer is a leading cause of morbidity and mortality in the developed world. While much has been learned about these diseases in the last few decades, one of the main barriers to widespread advancement is the heterogeneity of cancer biology. A growing body of evidence supports the idea that certain protein receptors are overexpressed on the surface of tumor cells as compared to normal tissues. These extracellular biomarkers provide a unique opportunity to selectively target the tumor with both imaging and therapeutic modalities. The research in this dissertation focuses on targeting proteins on the tumor cell surface with peptidomimetic ligands. Following a description of various extracellular receptors, chapter one discusses targeting ligands designed to specifically and selectively bind these receptors. It reviews recent literature on targeted alpha-particle therapy and ends with an explanation of the advantages of peptide ligands. Three distinct approaches to imaging and therapeutic modalities are then discussed in subsequent chapters. First, a peptide ligand was designed to target radionuclides to malignant melanoma cells in an effort to develop companion radiotherapeutics and diagnostic imaging agents. The second research project describes the synthesis of a novel antagonist peptide ligand with conjugated near infrared dye, and its utility for real-time intraoperative guidance during pancreatic adenocarcinoma resection. Finally, the last chapter describes how the relatively new field of immunomodulatory effectors may be enhanced by their derivatization with peptide targeting ligands.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21713号 / 薬科博第104号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 小野 正博, 教授 山下 富義, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM