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.

Η παρούσα διδακτορική διατριβή επιχειρεί να διερευνήσει την επίδραση της πειραματικής ηπατεκτομής στην δομή του εντερικού βλεννογόνου μελετώντας την απόπτωση και τον πολλαπλασιασμό των επιθηλιακών κυττάρων στις κρύπτες, τα επίπεδα ενδοτοξίνης στην πυλαία φλέβα και την αορτή, καθώς επίσης και τα επίπεδα του οξει-δωτικού stress. Μελετά επίσης τα επίπεδα του οξειδωτικού stress στο εναπομείναν ήπαρ. Επιπλέον, σε μια προσπάθεια θεραπευτικής παρέμ-βασης, διερευνήθηκε ο ρόλος των ρυθμιστικών εντερικών πεπτιδίων BBS και NT στις ανωτέρω παραμέτρους . Προηγούμενες μελέτες έχουν δείξει ότι τα πεπτίδια αυτά εξα-σκούν ένα ευρύ φάσμα δράσεων στον εντεροηπατικό άξονα και βελτι-ώνουν την ακεραιότητα του γαστρεντερικού βλεννογόνου έπειτα από την επίδραση διαφόρων βλαπτικών παραγόντων. Η απουσία χολής ενδοαυλικά, αποστερεί τον εντερικό βλεννο-γόνο, από τις βακτηριοστατικές, αντιενδοτοξινικές και τροφικές της ιδιότητες, οδηγώντας σε αύξηση των βακτηριδίων και της ενδοτοξίνης ενδοαυλικά και σε εντερική ατροφία. Οι μεταβολές αυτές προάγουν τη μετακίνηση βακτηρίων και ενδοτοξινών στην πυλαία φλέβα και ακο-λούθως, μέσω μιας κατασταλμένης εκκαθαριστικής ικανότητας των κυττάρων Kupffer εξαιτίας της μειωμένης μάζας τους, στη συστη-ματική κυκλοφορία. Η συστηματική ενδοτοξιναιμία ενεργοποιεί τη συστηματική φλεγμονώδη απάντηση, η οποία σχετίζεται με τη δυσλει-τουργία που αναπτύσσεται σε απομακρυσμένα όργανα, ενώ συνεισφέ-ρει και στην περαιτέρω επιδείνωση της λειτουργίας του εντερικού φραγμού και της ηπατικής βλάβης. Τα αποτελέσματα της παρούσας μελέτης επιβεβαίωσαν την παρουσία πυλαίας και συστηματικής ενδο-τοξιναιμίας σε πειραματική ηπατεκτομή. Η προκαλούμενη από την πειραματική ηπατεκτομή ατροφία του εντερικού βλεννογόνου τεκμη-ριώθηκε με μορφομετρική ανάλυση και με μετρήσεις του DNA και της πρωτεΐνης. ΄Ενας πιθανός μηχανισμός προαγωγής της ατροφίας του εντερικού βλεννογόνου είναι η διαταραχή της ισορροπίας μεταξύ κυτταρικού πολλαπλασιασμού και κυτταρικού θανάτου στις κρύπτες, με αύξηση της απόπτωσης και μείωση της μιτωτικής δραστηριότητας. Επιπλέον η πειραματική ηπατεκτομή οδήγησε τις εντερικές λειτουργί-ες σε μια κατάσταση χαμηλού οξειδωτικού stress όπως φαίνεται καθαρά από τη μείωση της υπεροξείδωσης των λιπιδίων και της οξει-δωμένης γλουταθειόνης (GSSG) καθώς και από την αύξηση της ανηγ-μένης γλουταθειόνης (GSG). Είναι άξιο παρατήρησης ότι το οξειδωτι-κό stress δεν φαίνεται να είναι το πρωταρχικό αίτιο της πρόκλησης κυτταρικής απόπτωσης και ατροφίας λαχνών που παρατηρείται μετά από μερική ηπατεκτομή, αν και αυτή η επέμβαση μειώνει το οξειδωτι-κό stress κάτω από το control. Από το άλλο μέρος αυτές οι επιδράσεις εξαλείφθηκαν υπό συνθήκες ακόμη πιο μειωμένου οξειδωτικού stress που προκλήθηκαν μετά την χορήγηση των ρυθμιστικών πεπτιδίων BBS και NT Τα ρυθμιστικά εντερικά πεπτίδια, BBS και NT, δρώντας είτε άμεσα, μέσω ειδικών υποδοχέων των επιθηλιακών κυττάρων του εντέ-ρου, είτε έμμεσα, βελτιώνοντας τη μικροκυκλοφορία του εντέρου, μείωσαν σημαντικά την απόπτωση και ανέστρεψαν την εντερική ατρο-φία. Η πρόληψη, από τα ρυθμιστικά πεπτίδια, των επαγόμενων από την ηπατεκτομή κυτταρικών και βιοχημικών μεταβολών του εντερικού βλεννογόνου, οδήγησε σε σημαντική μείωση της πυλαίας και συστη-ματικής ενδοτοξιναιμίας. Επιπλέον, η BBS και η NT, εξασκώντας αντιοξειδωτική δράση και στο ήπαρ, προστάτεψαν το εναπομείναν ήπαρ από δυο μείζονες παράγοντες ηπατικής βλάβης, που είναι το οξειδωτικό stress και η ενδοτοξιναιμία. Συμπερασματικά, τα αποτελέσματα της παρούσας μελέτης δείχ-νουν ότι η δυσλειτουργία του εντερικού φραγμού στην ηπατεκτομή σχετίζεται με την επαγωγή κυτταρικών και βιοχημικών μεταβολών στον εντερικό βλεννογόνο, οι οποίες χαρακτηρίζονται από πρόκληση οξειδωτικού stress και επαγωγή της απόπτωσης. Τα ρυθμιστικά εντε-ρικά πεπτίδια BBS και NT προλαμβάνοντας τις μεταβολές αυτές του εντερικού βλεννογόνου μειώνουν σημαντικά την πυλαία και συστημα-τική ενδοτοξιναιμία. Επίσης, εξασκούν προστατευτική δράση εναντίον του οξειδωτικού stress στο ήπαρ. Η συνδυασμένη ευεργετική επίδραση των ρυθμιστικών πεπτιδίων, τόσο στη δυσλειτουργία του εντερικού φραγμού και την ενδοτοξιναιμία, που ευθύνονται για την ανάπτυξη σηπτικών επιπλοκών και βλάβης απομακρυσμένων οργάνων, όσο και στο οξειδωτικό stress, εισηγούνται μια νέα θεραπευτική προσέγγιση στην ηπατεκτομή. / This doctoral thesis explores the effect of experimental hepatectomy on the structure of the intestinal mucosa by studying apoptosis and proliferation of epithelial cells in crypts, endotoxin levels in portal vein and aorta, as well as levels of oxidative stress. It also investigates the level of oxidative stress on the remaining liver tissue. In addition, in an attempt of therapeutical approach, the role of the regulatory intestinal peptides BBS and NT in the above parameters has also been evaluated. Previous studies have demonstrate that the above peptides present a wide range of actions on the intestinal-liver axis, thus improving integrity of the gastrointestinal mucosa after it has been affected by various harmful factors. Intestinal mucosa in the absence of bile in the lumen lacks its bacteriostatic, anti-endotoxin and trophic effects, leading in a raise of bacteria and endotoxin in the lumen as well as intestinal atrophy. These changes are promoting bacteria and endotoxin translocation to the portal vein and from there, to the systematic circulation, a procedure augmented by the supression of the cleaning ability of Kuppfer cells due to their reduced mass in hepatectomy. Systemic endotoxinaemia activates the systematic inflammatory response (SIR) related to the dysfunction of distal organs, and contributing to further damage to the mucosal barrier and the liver. This study confirmed the presence of portal and systemic endotoxinaimia in experimental hepatectomy. The subsequent atrophy of the intestinal mucosa has been documented with morphologic analyses as well as DNA and protein measurements. A possible explanation of the observed atrophy might be disturbance in balance between cellular proliferation and cellular death in the crypts by increased apoptosis and decreased mitotic activity. In addition, experimental hepatectomy led the intestinal functions in a state of low oxidative stress, as it is clearly shown by the decrease of lipid peroxidation and oxidized glutathione (GSSG), and the increase of glutathione (GSH). Interestingly enough, oxidative stress does not seem to be the primary cause of cellular apoptosis and decreased cell proliferation in the crypts (leading to mucosa atrophy), which are observed after partial hepatectomy, although this operation decreased oxidative stress below the control levels. On the other hand, these effects are prevented at more decreased levels of oxidative sress induced by the regulatory intestinal peptides BBS and NT. BBS and NT may act either directly, through specific receptors of the intestinal epithelial cells, or indirectly, by improving the intestinal microcirculation, leading to reversal of intestinal atrophy. Prevention, by these peptides, of the hepatectomy induced cellular and biochemical changes in the intestinal mucosa led to significantly reduced portal and systematic endotoxinaemia. In addition, BBS and NT, through antioxidative action to the liver, protected the remaining liver tissue from two major factors of liver lesions: oxidative stress and endotoxinaimia. Concluding, the results of this study show that dysfunction of the intestinal mucosa barrier in the hepatectomy is associated with the induction of cellular and biochemical changes in the intestinal mucosa, which are characterized by the promotion of oxidative stress and apoptosis. The regulatory intestinal peptides BBS and NT preventing these changes in the intestinal mucosa are significantly reducing portal and systematic endotoxinaimia. They also act by protecting against oxidative stress in the liver. This combined beneficial effect of these peptides, both in the dysfunction of the intestinal mucosa barrier and endotoxinaimia, which are responsible for the development of septic complications and distal organ lesions as well as in oxidative stress, might suggests a new therapeutical approach in hepatectomy.