A radiomarcação de biomoléculas específicas com o tecnécio-99m 99mTc utilizando agentes quelantes bifuncionais é um campo em crescimento na Medicina Nuclear. Em especial, a classe de peptídeos regulatórios, como a Neurotensina, participa de processos fisiológicos essenciais no organismo, como o crescimento tumoral. O objetivo do presente trabalho foi o estudo comparativo da influência dos agentes quelantes bifuncionais 6-hidrazinonicotinamida (HYNIC) e S-acetil-mercaptoacetiltriglicina (MAG3), no comportamento in vitro e in vivo do análogo duplamente estabilizado da Neurotensina(8-13) radiomarcado com 99mTc, em células tumorais de mama da linhagem MDA-MB-231. Um elevado rendimento radioquímico (> 97%) e estabilidade frente aos agentes transquelantes foi observado para ambos análogos radiomarcados. Foram também obtidos comportamentos similares in vitro, no que diz respeito à porcentagem de ligação às proteinas plasmáticas (aproximadamente 22%), estabilidade metabólica, ligação aos receptores celulares (intervalo nM) e taxas de internalização/externalização para ambos radiocomplexos. A maior lipofilicidade encontrada para o análogo radiomarcado via MAG3 refletiu nas principais diferenças nos estudos de biodistribuição. A degradação do análogo radiomarcado via HYNIC nos estudos de estabilidade metabólica in vivo aos 90 min levou a menor retenção tumoral (0,44±0,02% DI/g), e consequentemente, às menores razões tumor/órgãos não-alvos (< 5%). Embora a superioridade do traçador marcado via MAG3 tenha sido comprovada no presente estudo, um redesenho estrutural objetivando contornar a alta captação no trato gastrointestinal deve ser realizada a fim de que sua potencial aplicabilidade não seja comprometida. / The radiolabeling of receptor specific biomolecules with 99mTc using bifunctional chelator agents represents a growing field in Nuclear Medicine, specially, regarding regulatory peptides, such as Neurotensin, which are important in several essential physiological functions, particularly in tumor growth. The aim of the study was the comparative radiolabeling evaluation of the double-stabilized NT(8-13) analog with 99mTc, via the bifunctional chelating agents 6- hydrazinonicotinamide (HYNIC) and S-acetyl-mercaptoacetyltriglycine (MAG3) in MDA-MB-231 breast cancer cell line. High radiochemical yields (> 97%) and stability toward transchelant agents was observed for both radiolabeled analogs. Also, comparable in vitro behaviour regarding the percentage of plasma protein binding (nearby 22%), metabolic stability, receptor binding affinity (nM range), and internalization/externalization rates were obtained. The greater lipophilicity found for the analog radiolabeled via MAG3, reflected in the major differences in biodistribution studies. The in vivo metabolic stability studies suggested that the degradation observed in the later time point (90 min) for the conjugate radiolabeled via HYNIC, leads not only to lower tumor uptake accumulation (0,44±0,02% ID/g), but also to lower tumor-to-non-tumor ratios (< 5%). Although the superiority of the tracer radiolabeled via MAG3 had been confirmed in the present study, a strucutural re-design aiming the reduction of the high gastrointestinal uptake must be done in order to guarantee the potential applicability of MAG3-radiocomplex.

A radiomarcação de biomoléculas específicas com o tecnécio-99m 99mTc utilizando agentes quelantes bifuncionais é um campo em crescimento na Medicina Nuclear. Em especial, a classe de peptídeos regulatórios, como a Neurotensina, participa de processos fisiológicos essenciais no organismo, como o crescimento tumoral. O objetivo do presente trabalho foi o estudo comparativo da influência dos agentes quelantes bifuncionais 6-hidrazinonicotinamida (HYNIC) e S-acetil-mercaptoacetiltriglicina (MAG3), no comportamento in vitro e in vivo do análogo duplamente estabilizado da Neurotensina(8-13) radiomarcado com 99mTc, em células tumorais de mama da linhagem MDA-MB-231. Um elevado rendimento radioquímico (> 97%) e estabilidade frente aos agentes transquelantes foi observado para ambos análogos radiomarcados. Foram também obtidos comportamentos similares in vitro, no que diz respeito à porcentagem de ligação às proteinas plasmáticas (aproximadamente 22%), estabilidade metabólica, ligação aos receptores celulares (intervalo nM) e taxas de internalização/externalização para ambos radiocomplexos. A maior lipofilicidade encontrada para o análogo radiomarcado via MAG3 refletiu nas principais diferenças nos estudos de biodistribuição. A degradação do análogo radiomarcado via HYNIC nos estudos de estabilidade metabólica in vivo aos 90 min levou a menor retenção tumoral (0,44±0,02% DI/g), e consequentemente, às menores razões tumor/órgãos não-alvos (< 5%). Embora a superioridade do traçador marcado via MAG3 tenha sido comprovada no presente estudo, um redesenho estrutural objetivando contornar a alta captação no trato gastrointestinal deve ser realizada a fim de que sua potencial aplicabilidade não seja comprometida. / The radiolabeling of receptor specific biomolecules with 99mTc using bifunctional chelator agents represents a growing field in Nuclear Medicine, specially, regarding regulatory peptides, such as Neurotensin, which are important in several essential physiological functions, particularly in tumor growth. The aim of the study was the comparative radiolabeling evaluation of the double-stabilized NT(8-13) analog with 99mTc, via the bifunctional chelating agents 6- hydrazinonicotinamide (HYNIC) and S-acetyl-mercaptoacetyltriglycine (MAG3) in MDA-MB-231 breast cancer cell line. High radiochemical yields (> 97%) and stability toward transchelant agents was observed for both radiolabeled analogs. Also, comparable in vitro behaviour regarding the percentage of plasma protein binding (nearby 22%), metabolic stability, receptor binding affinity (nM range), and internalization/externalization rates were obtained. The greater lipophilicity found for the analog radiolabeled via MAG3, reflected in the major differences in biodistribution studies. The in vivo metabolic stability studies suggested that the degradation observed in the later time point (90 min) for the conjugate radiolabeled via HYNIC, leads not only to lower tumor uptake accumulation (0,44±0,02% ID/g), but also to lower tumor-to-non-tumor ratios (< 5%). Although the superiority of the tracer radiolabeled via MAG3 had been confirmed in the present study, a strucutural re-design aiming the reduction of the high gastrointestinal uptake must be done in order to guarantee the potential applicability of MAG3-radiocomplex.

Résumé : Grâce à des caractéristiques physiques particulières, le [indice supérieur 64]Cu (T[indice inférieur 1/2]= 12.7 h; β[indice supérieur+], 0.65 MeV [17.8 %]; β[indice supérieur −], 0.58 MeV [38.4 %]) est un candidat idéal pour l’imagerie TEP et la radiothérapie ciblée du cancer. Son utilisation est actuellement limitée par la disponibilité de chélateurs bi-fonctionnels (CBFs) offrant une résistance élevée aux réactions de transmétallation in vivo. Récemment nous avons développés deux nouveaux CBFs cycliques, DOTHA[indice inférieur 2] et NOTHA[indice inférieur 2], portant des ligands hydroxamates pour la complexation au [indice supérieur 64]Cu. Ces CBFs possèdent une cinétique de marquage rapide dans des conditions très douces, une stabilité élevée in vivo et un profil de biodistribution favorable avec une clairance rapide. Nous proposons maintenant d’étendre notre approche à la préparation de CBFs acycliques plus flexibles et compacts afin de moduler les propriétés biologiques et la pharmacocinétique des traceurs peptidiques. Le but de mon projet de maîtrise est de développer une série de chélateurs acycliques dérivés de l'histidine et de l'acide glutamique et fonctionalisés avec des groupements hydroxamates pour identifier un CBF offrant un complexe stable in vivo avec le [indice supérieur 64]Cu(II). Les CBFs ont été préparés en solution pour faciliter l’optimisation de chaque étape réactionnelle. Les groupements chélatants hydroxamates ont été sélectionnés pour leur habilité à former des complexes stables avec différents métaux et ils ont été liés en position N-terminale et sur la chaîne latérale des acides aminés grâce à des réactions de substitution nucléophile. Les groupements para-methoxy-benzyles ont été judicieusement sélectionnés pour la protection des groupements hydroxamates afin de faciliter, au besoin, une déprotection sélective sous des conditions très douces. L’optimisation du marquage a été effectuée avec l’isotope stable du cuivre et ensuite avec le [indice supérieur 64]Cu en faisant varier le contre ion métallique, le pH, la concentration, et la température. Le CBF offrant la plus grande stabilité, soit celui dérivé de l’histidine, a été conjugué à un peptide, le H[indice inférieur 2]N-PEG-[D-Tyr[indice supérieur 6],βAla[indice supérieur 11],Thi[indice supérieur 13],Nle[indice supérieur 14]]bombesin(6-14) (BBN), se liant fortement aux récepteurs de la relâche de la gastrine surexprimés dans les cancers du sein et de la prostate. La stabilité et l’activité spécifique du CBF-histidine et du radiotraceur marqués au [indice supérieur 64]Cu s’est avérée faible in vitro. Il est connu que l’activité antibactérienne de ligands hydroxamates est associée à leur capacité à complexer le fer. En perspective, comme nos chélateurs complexent très fortement le Fe(III), une alternative pour ces composés serait d’évaluer leur capacité à inhiber la croissance et la prolifération des bactéries. || Abstract : Thanks to its particular physical characteristics, [superscript 64]Cu (T[subscript ½= 12.7 h; β[superscript +], 0.65MeV [17.8 %]; β[superscript −], 0.58MeV [38.4 %]) is an ideal candidate for PET imaging and targeted cancer radiotherapy. Currently, its use is limited by the availability of bi-functional chelators (BFCs) which give high resistance to in vivo transmetallation reactions. Recently, we developed two new cyclic BFCs, DOTHA[subscript 2] and NOTHA[subscript 2], bearing hydroxamate pendant arms for the complexation with [superscript 64]Cu. Those BFCs have fast labeling kinetics under very mild conditions, a high in vivo stability and a biodistribution profile which is favorable with a fast clearance. Now, we propose to expand our approach to the preparation of acyclic BFCs, which are more flexible and compact, in order to better modulate biological properties and the pharmacokinetics of the peptidic tracers. The goal of my Master’s degree project is to develop a series of acyclic chelators derived from histidine and glutamic acid and functionalized with hydroxamate pendant arms to identify a BFC that shows highly stable in vivo complexes with [superscript 64]Cu(II). BFCs have been prepared in solution to facilitate the optimization of each reactive step. Hydroxamate chelating groups have been selected for their ability to form stable complexes with different metals and they have been conjugated in N-terminal position and on the lateral chain of amino acids via nucleophilic substitution reactions. Para-methoxy-benzyl groups have been judiciously selected for the protection of the hydroxamate groups to facilitate, if needed, a selective deprotection under mild conditions. The labeling optimization has been performed with a stable copper isotope, and then with [superscript 64]Cu varying the metallic counter-ion, pH, concentration and temperature. The BFC having the highest stability, the one derived from histidine, was conjugated to a peptide, H[subscript 2]N-PEG-[D-Tyr[superscript 6],βAla[superscript 11],Thi[superscript 13],Nle[superscript 14]]bombesin(6-14) (BBN), strongly bounding the gastrin releasing peptide receptor, which is overexpressed in breast and prostate cancers. Both the stability and specific activity of BFC-histidine of the radiotracer labeled with [superscript 64]Cu were low in vitro. It is known that the antibacterial activity of hydroxamate ligands is associated with their ability to complex iron. In perspective, because our hydroxamate ligands strongly complex Fe(III), an alternative for these compounds would be to assess their ability to inhibit the growth and proliferation of bacteria.