Η παρούσα διατριβή αναφέρεται στην ανάπτυξη νέων πεπτιδικών αναλόγων μπομπεσίνης (ΒΝ) με απώτερο στόχο την ενδεχόμενη κλινική εφαρμογή τους στο πεδίο της διαγνωστικής ογκολογίας με SPECT (Υπολογιστική Τομογραφία Εκπομπής ενός Φωτονίου, Single-photon emission computed tomography) και PET (Τομογραφία Εκπομπής Ποζιτρονίων, Positron emission tomography). Η ΒΝ αποτελεί έναν εξειδικευμένο πεπτίδιο-προσδέτη για τους υποδοχείς του πεπτιδίου απελευθέρωσης της γαστρίνης (gastrin-releasing peptide receptors, GRPrs), οι οποίοι υπερεκφράζονται στην επιφάνεια διαφόρων τύπων καρκινικών κυττάρων. Οι υποδοχείς GRP δυνητικά θα μπορούσαν να θεωρηθούν ιδανικός στόχος για την απεικόνιση νευροενδοκρινικών όγκων μετά από χορήγηση ραδιοεπισημασμένων παραγώγων ΒΝ, με τεχνικές μοριακής διαγνωστικής, αλλά και για την στοχευμένη θεραπευτική αντιμετώπιση του καρκίνου. Με αυτό τον στόχο σχεδιάστηκαν και συντέθηκαν πεπτιδικά ανάλογα ΒΝ για τα οποία πραγματοποιήθηκε μία σειρά προκλινικών μελετών με σκοπό τη διαμόρφωση και την εφαρμογή ενός πλαισίου διάκρισης των κατάλληλων ραδιοχημικών και ραδιοφαρμακολογικών χαρακτηριστικών τα οποία θα μπορούσαν να οδηγήσουν ένα από αυτά τα υποψήφια διαγνωστικά σκευάσματα του σε κλινικές μελέτες ανίχνευσης του καρκίνου. Μέθοδος: Στα πλαίσια της παρούσας διατριβής σχεδιάστηκαν και συντέθηκαν πεπτιδικά ανάλογα BN, κατάλληλα για επισήμανση με ραδιονουκλίδια, με την παρακάτω γενική δομή: Υ-Χ-ΒΝ(2-14) όπου Υ = ο χηλικός παράγοντας για τη σύμπλεξη του ραδιομετάλλου, Χ = η συνδετική ομάδα και ΒΝ(2-14) το φαρμακοφόρο τμήμα. (a) Προκειμένου να απεικονισθούν όγκοι με SPECT όπου: Υ = [GlyGlyCys-] για τη σύμπλεξη με το 99mTc και το 185/187Re (μη ραδιενεργό), Χ = -(αργινίνη)3-, (BN-A), ή -(ορνιθίνη)3-, (BN-O). (b) Ενώ για τη μοριακή απεικόνιση όγκων με την τεχνική PET, όπου Υ = [c-carboxylic acid-cyclam] για σύμπλεξη με 64Cu, και Χ = -(ορνιθίνη)3-, (BN-C). Σε όλες τις παραπάνω περιπτώσεις χρησιμοποιήθηκε το τμήμα της φυσικής αλληλουχίας του πεπτιδίου μπομπεσίνη, από το 2o εως το 14ο αμινοξύ, σε συνδυασμό με αμινοξική, θετικά φορτισμένη, συνδετική ομάδα (spacer). Τα παραπάνω παράγωγα και τα αντίστοιχα σύμπλοκα τους με τα μέταλλα αρχικά αξιολογήθηκαν χημικά και ραδιοχημικά με διάφορες αναλυτικές μεθόδους. Ακολούθησε η ραδιοφαρμακολογική αξιολόγηση τους με τις παρακάτω in vitro δοκιμασίες: (a) μελέτη σταθερότητας σε δείγματα αίματος, (b) προσδιορισμός της συγγένειας τους με τον GRPr (υπολογισμός τιμών IC50) και (c) του ρυθμού εσωτερικοποίησης/εξωτερικοποίησης σε κύτταρα PC-3 (ανθρώπινου καρκίνου προστάτη). Στη συνέχεια πραγματοποιήθηκαν μελέτες in vivo (βιοκατανομή, απεικόνιση με γ-κάμερα), σε φυσιολογικά ζωικά πειραματικά πρότυπα (ποντίκια, αρουραίους και κουνέλια) και σε πειραματικά πρότυπα καρκίνου (ποντίκια SCID, με όγκους από κύτταρα PC-3). Επιπλέον με βάση τα in vivo αποτελέσματα διεξάχθηκε η φαρμακοκινητική αξιολόγηση τους και ο συσχετισμός μεταξύ δομικών και φαρμακοκινητικών χαρακτηριστικών. Τέλος επιχειρήθηκε η αλλομετρική κλιμάκωση των φαρμακοκινητικών αποτελεσμάτων από τα διάφορα είδη πειραματοζώων στον άνθρωπο. Αποτελέσματα: Για τα παράγωγα τα οποία συντέθηκαν για σύμπλεξη με 99mTc προκειμένου να απεικονισθούν όγκοι με SPECT, παρατηρήθηκαν τα εξής: Τα BN-A και BN-O συμπλέχθηκαν αποτελεσματικά με το 99mTc (απόδοση > 98%), αλλά και το χημικά όμοιο του ρήνιο (μη ραδιενεργό). Κατά τις μελέτες σταθερότητας το παράγωγο 99mTc-BN-O αποδείχθηκε σταθερότερο του 99mTc-BN-Α, (π.χ. πλάσμα του ποντικού σε 5 min επώασης το % άθικτο πεπτίδιο ήταν 44.28 και 42.48, ενώ στο ανθρώπινο πλάσμα σε 1 ώρα επώασης ήταν 63.05 και 60.2 αντίστοιχα). Η συγγένεια πρόσδεσης στον GRPr των παραπάνω παραγώγων, αλλά και των συμπλόκων τους με το μη ραδιενεργό Re, τα οποία προσομοιάζουν τα αντίστοιχα ραδιενεργά σύμπλοκα τους με το 99mTc (IC50, BN-Α, 0.5 ± 0.09, 185/187Re-BN-A, 1.58 ± 0.16, BN-Ο, 0.46 ± 0.04, 185/187Re-BN-Ο, 0.77 ± 0.07 nM), ήταν κοντά στο πρότυπο [Tyr4]-BN (0.45 ± 0.04 nM). Το συνολικό ποσοστό εσωτερικοποίησης των παραπάνω συμπλόκων 99mTc-BN-Α και 99mTc-BN-Ο στα κύτταρα PC-3 ήταν παρόμοιο (~25%), αλλά ο ρυθμός εσωτερικοποίησης τους διέφερε, καθώς χρειάστηκαν 120 min και 60 min για φτάσουν τη μέγιστη τιμή εσωτερικοποίησης αντίστοιχα. Ο ρυθμός εξωτερίκευσης ήταν ο ίδιος. Μετά από 90 min επώασης το ~ 70% της αρχικής ποσότητας πεπτιδίου παραμένει εγκλωβισμένο στα κύτταρα. Από τις μελέτες βιοκατανομής και φαρμακοκινητικής του 99mTc-BN–A και του 99mTc-BN–Ο σε φυσιολογικά ποντίκια φάνηκε ότι και τα δύο ραδιοπεπτίδια απομακρύνονται γρήγορα από το αίμα, καθώς μικρό ποσοστό της χορηγούμενης δόσης (ID)/g (2.61 και 2.76 % αντίστοιχα) παραμένει 30 min μετά τη χορήγηση (p.i.). Η βασική οδός απομάκρυνσης τους από τον οργανισμό ήταν το ουροποιητικό σύστημα καθώς μεγάλο ποσοστό των ραδιοπεπτιδίων εντοπίστηκε στα ούρα 1 h p.i. (64.16 και 56.33 % ID αντίστοιχα). Το γεγονός αυτό συσχετίσθηκε με την παρουσία της θετικά φορτισμένης συνδετικής ομάδας. Η νεφρική απέκκριση επιδιώκεται έναντι της ηπατοχολικής, γιατί με αυτόν τον τρόπο μειώνεται η συσσώρευση της ραδιενέργειας στην άνω κοιλιακή χώρα και διευκολύνεται η εντόπιση όγκων στην περιοχή αυτή. Το 99mTc-BN–A απομακρύνεται ταχύτερα από το αίμα από τα νεφρά και γενικότερα από τον οργανισμό από ότι το 99mTc-BN–Ο, αλλά εμφανίζει μεγαλύτερη συσσώρευση στο ήπαρ (4.43 ± 1.04 και 0.99 ± 0.20 %ID/g, 60 min p.i. αντίστοιχα). Από τις μελέτες βιοκατανομής και απεικόνισης με γ-κάμερα σε ποντίκια SCID με όγκους PC-3 το παράγωγο 99mTc-BN–A εντόπισε αποτελεσματικά τον όγκο. Μετά από πειράματα συγχορήγησης φυσικής ΒΝ παρατηρήθηκε μείωση του ραδιοεπισημασμένου παραγώγου στον όγκο και στο πάγκρεας, τα σημεία στα οποία εντοπίζονται υποδοχείς GRPrs, επιβεβαιώνοντας έτσι την ειδική δέσμευση του ραδιοεπισημασμένου παραγώγου σε αυτούς. Συγκρίνοντας τα παραπάνω αποτελέσματα με το 99mTc-BN–Ο διαπιστώθηκαν καλύτεροι λόγοι διάκρισης από το 99mTc-BN–A στους διάφορους χρόνους μελέτης (π.χ. 60 min p.i. ήταν όγκος/αίμα, 6.67 έναντι 3.62, όγκος/μύες, 14.0 έναντι 5.60, όγκος/ήπαρ, 2.9 έναντι 0.82, αντίστοιχα). Εξαιτίας όλων των παραπάνω αποτελεσμάτων το παράγωγο 99mTc-BN-Ο επιλέχθηκε για περαιτέρω μελέτη. Για το 99mTc-BN-Ο πραγματοποιήθηκαν περαιτέρω in vitro μελέτες σταθερότητας σε πλάσμα αίματος αρουραίων και κουνελιών και in vivo μελέτες βιοκατανομής (απεικόνισης σε γ-καμερα). Παράλληλα πραγματοποιήθηκε και η φαρμακοκινητική ανάλυση των δεδομένων. Επιπλέον επιχειρήθηκε να αντιμετωπισθεί το πρόβλημα της νεφρικής συσσώρευσης του 99mTc-BN-Ο στα νεφρά με τη συγχορήγηση Gelofusine (Gelo). Από τη συγχορήγηση Gelo στα φυσιολογικά (ποντίκια, αρουραίοι) και καρκινικά πειραματικά πρότυπα (ποντίκια SCID με όγκους PC-3) παρατηρήθηκαν ταχύτερη κάθαρση του αίματος και αποβολής από τα νεφρά. Τέλος πραγματοποιήθηκε πρωτότυπη μελέτη αλλομετρικής κλιμάκωση των φαρμακοκινητικών αποτελεσμάτων του 99mTc-BN-Ο με στόχο την πρόβλεψη της συμπεριφοράς του καθώς της συμπεριφοράς παρόμοιων μορίων στον άνθρωπο κατά τη διεξαγωγή κλινικών μελετών. Εξαιτίας των παραπάνω θετικών αποτελεσμάτων αναφορικά με το τμήμα Η2Ν-(ορνιθίνη)3-ΒΝ(2-14) και προκειμένου να επιτευχθεί η βελτιστοποίηση της ικανότητα σύμπλεξής του με ιόντα χαλκού (π.χ. 64Cu για διάγνωση με PET) συνδέθηκε ομοιοπολικά με αυτό ένας νέος χηλικός παράγοντα το c-carboxylic acid cyclam. Από την ομοιοπολική σύζευξη των παραπάνω προέκυψε ένας νέο ανάλογο ΒΝ, το ΒΝ-C. Με τη χρήση διαφόρων αναλυτικών τεχνικών (IR, UV-Vis, ESI-MS, ESR, κρυσταλλογραφία ακτίνων Χ) διαπιστώθηκε ότι τόσο ο c-carboxylic acid cyclam όσο και ΒΝ-C, ο τελικός πεπτιδικός προσδέτης, έχουν την ικανότητα σύμπλεξης με CuII. Το πεπτιδικό παράγωγο BN-C και το σύμπλοκο του με το χαλκό, Cu-BN-C. δοκιμάσθηκαν ως προς τη συγγένεια δέσμευσης τους με τον GRPr σε κυτταρικές καλλιέργειες PC-3 όπου και παρουσίαζαν συγγένεια (IC50 = 0.3 ± 0.03 και 0.33 ± 0.03 nM, αντίστοιχα) παρόμοια με το πρότυπο πεπτίδιο προσδέτη [Tyr4]-BN (0.45 ± 0.04 nM). Συμπέρασμα: Οι παραπάνω μελέτες οριοθετούν ένα γενικότερο πλαίσιο προκλινικού προσδιορισμού εκείνων των ραδιοχημικών και ραδιοφαρμακολογικών in vitro και in vivo ιδιοτήτων, οι οποίες θα μπορούσαν να οδηγήσουν ένα παράγωγο ΒΝ σε περαιτέρω κλινικές μελέτες. Με βάση τα παραπάνω επιλέχθηκε και αναπτύχθηκε ένα πεπτιδικό ανάλογο ΒΝ, το οποίο σε κάθε περίπτωση αποδείχθηκε ότι διαθέτει τον κατάλληλο συνδυασμό των παραπάνω χαρακτηριστικών για περαιτέρω ανάπτυξη ως ραδιοδιαγνωστικό σκεύασμα για SPECT (BN-O) και PET (ΒΝ-C) σε κλινικό επίπεδο. Επιπλέον η φαρμακοκινητική ανάλυση και η αλλομετρική κλιμάκωση των παραπάνω αποτελεσμάτων συνεισφέρουν σημαντικά στην αύξηση της πιθανότητας μελλοντικής κλινικής εφαρμογής των παραπάνω παραγώγων στον άνθρωπο. / The present Thesis refers to the development of new peptidic bombesin (BN) analogs for application in the field of tumor diagnosis with the SPECT (Single-photon emission computed tomography) and PET (Positron emission tomography) techniques. The BN peptide is a specific ligand for the gastrin-releasing peptide receptors (GRPrs), which are over-expressed on the surface of various types of tumor cells. GRPrs can be considered as an ideal target for radiolabeled BN analogues in the molecular diagnosis of neuro-endocrine tumors as well as in the targeted treatment of cancer. Taking the above into consideration, three BN analogues were designed and synthesized as potential molecular diagnostics of tumors. Thus, a series of preclinical studies were conducted in order to find out the best candidate for further development in clinical studies. Methods: All the BN analogues of the present study can be described by the following general structure: Υ-Χ-ΒΝ(2-14) Where Y = the chelator group for the radionuclide, X = a spacer group and BN(2-14) = the pharmacophore group. (a) For the diagnosis of tumors with SPECT: Υ = [GlyGlyCys-], suitable for the complexation of 99mTc and 185/187Re and X= -(arginine)3-, (BN-A), or -(ornithine)3-, (BN-O). (b) Further on, for tumor diagnosis with PET: Υ = [c-carboxylic acid-cyclam] suitable for the complexation of 64Cu, και Χ = -(ornithine)3-, (BN-C). For all the above cases the part from the 2nd to the 14th amino acid of the naturally occurring BN was used, in combination with a spacer group composed of three positively charged amino acids. The amino acids were either naturally occurring (arginine) or not (ornithine). The above BN analogues as well as their metal complexes were chemically and radiochemically analyzed and evaluated in vitro. The in vitro evaluation of the BN derivatives included assays for the determination of (a) their stability in blood samples, (b) their affinity for the GRPrs (by the calculation of their IC50 values), (c) as well as of their internalization and externalization rates in PC-3 cell (human prostate cancer cell line) cultures. Additionally a series of in vivo assays were conducted including: biodistibution studies in normal (mice, rats, rabbits) and in experimental cancer animal models (SCID mice with PC3 tumors). For the later in addition to the biodistribution tumor imaging studies were carried out using an experimental small animal gamma camera. Based on the results of the in vivo studies, a series of pharmacokinetic analyses were performed and the results were correlated with the structural characteristics of the BN analogues. Finally, the results from the pharmacokinetic analyses of the three different animal species, (mouse, rat, rabbit) were combined and scaled up for a human of 70 Kg, by using the allometric approach. Results: The BN analogues BN-A and BN-O, which were designed for application in tumor imaging with SPECT, were able to form stable complexes with 99mTc (in high yield > 98%), as well as with Re (non radioactive). The two elements 99mTc and Re belong to the same group of the Periodic Table of elements and thus they are considered to have similar chemical properties. During the in vitro stability assays 99mTc-BN-O prove to be more stable than the 99mTc-BN-Α i.e. the % percentages of intact peptide for mouse plasma (after 5 min incubation) were 44.28 and 42.48 %, while for human plasma the percentages of intact peptide (after 1h of incubation) were 63.05 and 60.2 respectively. The affinities (IC50 values) of BN-Α (0.5 ± 0.09 nM), and BN-Ο (0.46 ± 0.04 nM), as well as of their non radioactive complexes with Rhenium 185/187Re-BN-A, 1(.58 ± 0.16 nM) and 185/187Re-BN-Ο (0.77 ± 0.07 nM) were similar to the one of the control peptide [Tyr4]-BN (0.45 ± 0.04 nM). Although the total amount of internalized radiolabeled peptide was the same for both 99mTc-BN-Α and 99mTc-BN-O (~25%), their rates of internalization differed. The ornithine-spacer analogue, 99mTc-BN-O, internalized a lot faster (60 min for maximum internalization plateau) than the arginine-spacer analogue, 99mTc-BN-Α (120 min for maximum internalization plateau). The externalization rates for both analogues were the same, with ~ 70% of the initially internalized radiolabeled peptide remaining inside the cells even after 90 min of incubation. From the biodistribution and pharmacokinetic studies of 99mTc-BN–A and 99mTc-BN–Ο a fast blood clearance was observed for both peptides, with small amounts of the initial dose remaining in the blood stream 30 min p.i. (2.61 and 2.76 % ID/g, respectively). The main clearance route throughout the body in both cases was the urinary system and high percentages of the radiolabeled peptides were detected in the urine 1 h p.i (64.16 and 56.33 % ID respectively). The latter was related to the presence of the positively charged spacer group. A clearance through the urinary system is preferred instead of the hepatobiliary system since in that way the decrease of the upper abdominal region background radiation is achieved. Although the arginine-spacer analogue 99mTc-BN–A was cleared from the kidneys and the body with higher rates than 99mTc-BN–Ο, it showed a higher liver accumulation than the latter (4.43 ± 1.04 and 0.99 ± 0.20 %ID/g, 60 min p.i. respectively). Both in the biodistribution and in the gamma-camera tumor-imaging studies the BN analogue 99mTc-BN–A was able to efficiently locate the tumour. During the GRPrs blocking studies the amount of 99mTc-BN–A was reduced both in the tumour and in the pancreas, where the GRPrs are located, proving thus the specific binding of 99mTc-BN–A to those receptors. Comparing the tumor/normal tissue contrast ratios between 99mTc-BN–Ο and 99mTc-BN–A the ornithine spacer analogue proved a better choice since it presented greater values (i.e. 60 min p.i. tumor/blood, 6.67 vs 3.62, tumor/muscle, 14.0 vs 5.60, tumor/liver, 2.9 vs 0.82, respectively). According to all the above results 99mTc-BN-Ο was selected between the two SPECT tumor imaging agent candidates for further studies. For 99mTc-BN-Ο additional in vitro stability assays were conducted in rats and rabbits plasma, which were followed by in vivo biodistribution studies in normal rats and imaging studies in normal rabbits. The in vivo data were analyzed with the appropriate pharmacokinetic approaches. In an effort to achieve a faster clearance of 99mTc-BN-Ο from the kidneys a new approach was tested by its co-injection with Gelofusine (Gelo). During the Gelo co-injection studies in normal mice, in rats as well as in SCID mice with PC-3 tumors a faster renal clearance was observed. Finally by combining the in vivo results of 99mTc-BN-Ο from the three different animal species (mice, rats, rabbits) the allometric scale up of the pharmacokinetic parameters was achieved and the prediction of those parameters for a man of 70 kg. The results of latter study could probably be extended to predict the pharmacokinetic profile of other similar molecules in the preclinical stage of development. Due to promising results of obtained from the BN analogue with the ornithine spacer, Η2Ν-(ornithine)3-ΒΝ(2-14), and in order to improve its ability to form complexes with copper radionuclides i.e. 64Cu, which are suitable for PET imaging, a new chelator group was introduced into the peptide sequence. The chelator group c-carboxylic acid cyclam was covalently attached to the peptide chain Η2Ν-(ornithine)3-ΒΝ(2-14) to give a new bombesin analogue ΒΝ-C. The chelator group and the new BN analogue were able to form complexes with cold copper fast and under mild conditions. The copper complexes of the above molecules were analyzed with a variety of techniques like IR, UV-Vis, ESI-MS, ESR, X ray crystallography. Additionally both the BN analogue BN-C and its complex with copper Cu-BN-C were tested for their affinity for GRPrs in PC-3 cell lines, where it was found that their IC50 values of both were similar (IC50 = 0.3 ± 0.03 και 0.33 ± 0.03 nM, respectively) to the control peptide [Tyr4]-BN (0.45 ± 0.04 nM). Conclusion: The above studies can be considered the essential parts of a discrimination process of the ideal radiochemical and radiopharmacological characteristics, which could lead a BN analogue to clinical trials. According to the results of the above preclinical studies a suitable BN analogue was selected for further clinical development as a tumor imaging agent with SPECT (BN-O) as well as PET (ΒΝ-C). Additionally the pharmacokinetic analysis in three different animal species and allometric scale up of those parameters for the human will enhance the probability of its further application in clinical trials.

Meduloblastoma é o tumor intracranial mais comum em crianças, provavelmente derivado de células precursoras da camada granular externa do cerebelo durante seu desenvolvimento. O tratamento padrão consiste em cirurgia, radioterapia e quimioterapia, que produzem graves sequelas nos pacientes e garantem uma sobrevida baixa, o que demonstra a necessidade de novas alternativas terapêuticas para a doença. Evidências demonstram que o receptor do peptídeo liberador de gastrina (GRPR) está superexpresso em diversos tumores humanos, assim como seu agonista (GRP) pode atuar como um fator de crescimento autócrino em tumores cerebrais. No presente estudo, avaliamos a expressão de GRPR e o efeito de seus agonistas, bombesina (BB) e GRP, além do antagonista RC-3095, sobre a viabilidade celular de linhagens de meduloblastoma humano DAOY, D283 e ONS76. Mostramos que meduloblastomas, apesar de expressarem GRPR, não têm sua viabilidade celular afetada por agonistas e antagonista desse receptor. Uma vez que há evidências de que BDNF (fator neurotrófico derivado de cérebro) esteja relacionado à diferenciação celular em meduloblastomas, também avaliamos o efeito de BDNF sobre a viabilidade celular das linhagens de meduloblastoma humano. As linhagens DAOY e D283 tiveram sua viabilidade celular reduzida pela presença de BDNF. Uma vez que a via da PKA tem sido implicada na iniciação e progressão de vários tumores, também avaliamos o efeito de rolipram, um inibidor de fosfodiesterase tipo IV, sobre a viabilidade celular das linhagens de meduloblastoma humano, sendo que rolipram reduziu a viabilidade celular de todas as linhagens estudadas. Os receptores de BDNF e a via da PKA podem, portanto, ser alvos moleculares promissores para o desenvolvimento de novas terapias para meduloblastomas. / Medulloblastoma is the most common intracranial tumor in children and is believed to arise from the precursor cells of the external granule layer of the developing cerebellum. The standard treatment, consisting of surgery, craniospinal radiotherapy and chemotherapy, produces severe sequelae in patients and provides a poor overall survival, indicating the need for new therapeutic alternatives for treating this disease. Evidences show that the gastrin releasing peptide receptor (GRPR) is overexpressed in various human tumors and its agonist (GRP) can act as an autocrine growth factor in brain tumors. In the present study, we evaluated GRPR expression, as well as the effect of its agonists, bombesin (BB) and GRP, and its antagonist RC-3095, over cell viability of the human medulloblastoma cell lines DAOY, D283 and ONS76. We found that medulloblastomas, in spite of expressing GRPR, do not have its viability affected by the presence of agonists and antagonist of this receptor. Since there are evidences that BDNF (brain-derived neurotrophic factor) is related to cell differentiation in medulloblastomas, we also evaluated the effect of BDNF over the viability of medulloblastoma cell lines. The viability of the cell lines DAOY and D283 was reduced by the presence of BDNF. Since the PKA pathway has been implicated in the initiation and progression of various tumors, we also evaluated the effect of rolipram, a phosphodiesterase IV inhibitor, over the viability of the same medulloblastoma cell lines and we found that rolipram inhibited the viability of all the cell lines studied. BDNF receptors, as well as the PKA pathway, may be therefore promising molecular targets for the development of new therapies for treating medulloblastomas.

Meduloblastoma é o tumor intracranial mais comum em crianças, provavelmente derivado de células precursoras da camada granular externa do cerebelo durante seu desenvolvimento. O tratamento padrão consiste em cirurgia, radioterapia e quimioterapia, que produzem graves sequelas nos pacientes e garantem uma sobrevida baixa, o que demonstra a necessidade de novas alternativas terapêuticas para a doença. Evidências demonstram que o receptor do peptídeo liberador de gastrina (GRPR) está superexpresso em diversos tumores humanos, assim como seu agonista (GRP) pode atuar como um fator de crescimento autócrino em tumores cerebrais. No presente estudo, avaliamos a expressão de GRPR e o efeito de seus agonistas, bombesina (BB) e GRP, além do antagonista RC-3095, sobre a viabilidade celular de linhagens de meduloblastoma humano DAOY, D283 e ONS76. Mostramos que meduloblastomas, apesar de expressarem GRPR, não têm sua viabilidade celular afetada por agonistas e antagonista desse receptor. Uma vez que há evidências de que BDNF (fator neurotrófico derivado de cérebro) esteja relacionado à diferenciação celular em meduloblastomas, também avaliamos o efeito de BDNF sobre a viabilidade celular das linhagens de meduloblastoma humano. As linhagens DAOY e D283 tiveram sua viabilidade celular reduzida pela presença de BDNF. Uma vez que a via da PKA tem sido implicada na iniciação e progressão de vários tumores, também avaliamos o efeito de rolipram, um inibidor de fosfodiesterase tipo IV, sobre a viabilidade celular das linhagens de meduloblastoma humano, sendo que rolipram reduziu a viabilidade celular de todas as linhagens estudadas. Os receptores de BDNF e a via da PKA podem, portanto, ser alvos moleculares promissores para o desenvolvimento de novas terapias para meduloblastomas. / Medulloblastoma is the most common intracranial tumor in children and is believed to arise from the precursor cells of the external granule layer of the developing cerebellum. The standard treatment, consisting of surgery, craniospinal radiotherapy and chemotherapy, produces severe sequelae in patients and provides a poor overall survival, indicating the need for new therapeutic alternatives for treating this disease. Evidences show that the gastrin releasing peptide receptor (GRPR) is overexpressed in various human tumors and its agonist (GRP) can act as an autocrine growth factor in brain tumors. In the present study, we evaluated GRPR expression, as well as the effect of its agonists, bombesin (BB) and GRP, and its antagonist RC-3095, over cell viability of the human medulloblastoma cell lines DAOY, D283 and ONS76. We found that medulloblastomas, in spite of expressing GRPR, do not have its viability affected by the presence of agonists and antagonist of this receptor. Since there are evidences that BDNF (brain-derived neurotrophic factor) is related to cell differentiation in medulloblastomas, we also evaluated the effect of BDNF over the viability of medulloblastoma cell lines. The viability of the cell lines DAOY and D283 was reduced by the presence of BDNF. Since the PKA pathway has been implicated in the initiation and progression of various tumors, we also evaluated the effect of rolipram, a phosphodiesterase IV inhibitor, over the viability of the same medulloblastoma cell lines and we found that rolipram inhibited the viability of all the cell lines studied. BDNF receptors, as well as the PKA pathway, may be therefore promising molecular targets for the development of new therapies for treating medulloblastomas.

Targeted radionuclide therapy is becoming a widely used cancer treatment strategy. By radiolabeling receptor-specific peptides, cancer cells overexpressing the receptor can be selectively targeted, and the cytotoxic radionuclide can be delivered to the target cell or tissue for therapeutic or diagnostic purposes. Bombesin analogues have been previously developed and utilized to target the gastrin-releasing peptide receptor (GRPR), a receptor commonly overexpressed in prostate cancer cells. The RM26 analogue derived from the native bombesin is an antagonistic ligand of GRPR and a possible candidate for targeted radiotherapy. Prolonging the half-life of the molecule is an important aspect of developing a new protein therapeutic. Using albumin binding domain (ABD) for this purpose is an emerging strategy in recent years. ABD is able to bind to serum albumin and thus remains in the blood circulation for a long period of time. It is also a scaffold for protein engineering efforts and by coupling receptor-specific ligands to ABD, the target-specific binding along with extended in vivo halflife can be achieved. In this project, an RM26 analogue with a PEG linker and ABD with a DOTA chelator for future radiolabeling were synthesized with solid phase peptide synthesis (SPPS), conjugated, purified by RP-HPLC and analyzed by mass spectrometry. The binding properties of the conjugate were evaluated by SPR-based biosensory studies, and further experiments are planned for the testing the product and its potential application in radionuclide therapy. / Riktad radioterapi är en allt vanligare metod för behandling av cancer. Genom att radioinmärka receptor-specifika peptider kan dessa selektivt levereras till tumörceller som uttrycker receptorn. Radioterapi kan användas för diagnostik eller terapi, beroende på kopplad radionuklid. Bombesinanaloger har utvecklats och använts för att selektivt binda gastrinfrisättande peptidreceptor (gastrin-releasing peptide receptor, GRPR), en receptor som ofta är överuttryckt i prostatacancer. Bombesinanalogen RM26, som har sitt ursprung från nativt bombesin, är en antagonist till GRPR och kan möjligen användas för riktad radioterapi av prostatacancer. Vid utvecklingen av nya proteinläkemedel är halveringstiden i serum en viktig aspekt. En nyligen utvecklad strategi för att förlänga halveringstiden i serum är fusion av det  tumörspecifika proteinet till en albumin-bindande domän (ABD). ABD binder till albumin, ochsåledes kan fusionsproteinet bevaras i blodcirkulationen under en längre tid. I detta projekt, har både RM26 med en PEG-linker, och ABD med en DOTA kelator syntetiserats med fastfaspeptidsyntes (solid phase peptide synthesis, SPPS). RM26-PEG och DOTA-ABD har därefter konjugerats, renats med RP-HPLC och analyserats med massspektrometri. Bindning till albumin har utvärderats med ytplasmonresonans (surface plasmon resonance, SPR). Vidare studier planeras för att utvärdera peptid-proteinkonjugatet och dess potential för riktad radioterapi.

Coordination of transition metals to peptides, either through the incorporation of unnatural chelating groups or amino acid ligating side-chains, expands the utility of peptides for biological studies. The first part of this project describes induction of α-helical secondary structure in pentapeptides upon side-chain coordination of inert transition metal ions. The second part of this project describes the syntheses of biologically active peptide species that contain a macrobicyclic hexaamine ligand that can complex radioactive metal ions for diagnostic imaging purposes. / Short peptide sequences do not form thermodynamically stable α-helices in water. The capacity of two metal clips, cis-[Ru(NH3)4(solvent)2]2+ and cis [Pd(en)(solvent)2]2+ to induce α-helicity in peptides that are five amino acids long, Ac HARAH NH2 and Ac MARAM-NH2 has been explored. In all cases at pH < 5, the metal ions bind to the side-chains of amino acid residues at positions i, i+4 of the pentapeptides resulting in formation of bidentate macrocyclic species. Circular dichroism and 1H nuclear magnetic resonance data indicate that the metal complexes of Ac-MARAM-NH2 are highly α helical in water, and in the most spectacular case, coordination of Ac-MARAM-NH2 to cis-[Ru(NH3)4(solvent)2]2+ results in up to 80% α-helicity. In contrast, metal complexes of Ac-HARAH-NH2 exhibit significantly less α-helicity in water. / 64Cu-radiolabelled peptides have been investigated for their ability to target specific tissue or cell types. These peptides require a chelating group that binds copper ions strongly. Macrobicyclic hexaamine ligands, based on the compound commonly referred to as “sarcophagine”, have demonstrated extremely high stability under biological conditions. Here we describe the synthesis of diaminosarcophagine chelators with carboxylate groups for conjugation to peptides. These new chelators have been attached to the N-terminus or lysine side-chain of biologically-active peptides, including Tyr3 octreotate, Lys3-bombesin and an integrin targeting peptide. Spectroscopic and voltammetric studies of these species suggest that the conjugated sarcophagine group retains the high metal binding affinity and structural properties of the parent species, diaminosarcophagine. These are among the first sarcophagine-peptide compounds that have been properly characterised. The new sarcophagine-peptide conjugates can be easily radiolabelled with 64Cu2+ over a wide pH range at ambient temperature.

Muitas evidências demonstram que o peptídeo liberador de gastrina (GRP) é um fator de crescimento que afeta funções neuroendócrinas, incluindo proliferação e diferenciação celular, comportamento alimentar, formação de memória, respostas a estresses, desenvolvimento de neoplasias, desordens neurológicas e psiquiátricas. Porém, os eventos moleculares pelos quais isso ocorre ainda não são totalmente compreendidos. No presente estudo, nós avaliamos as interações entre o receptor do peptídeo liberador de gastrina (GRPR) e a via de sinalização celular da PKA, tanto na proliferação celular de glioblastoma humano (in vitro) quanto na consolidação da memória no hipocampo de ratos Wistar (in vivo). Mostramos que o GRP age em sinergismo com agentes que estimulam a via do cAMP/PKA, promovendo a proliferação de células de glioblastoma humano, pois o tratamento com GRP combinado com um ativador de adenilil ciclase (AC), forskolin, ou um análogo de cAMP, 8-Br-cAMP, ou um inibidor do tipo IV de fosfodiesterase, rolipram, aumentaram a proliferação das células de U- 138MG, quando avaliadas pelo método de MTT. Nenhum destes compostos teve efeito sozinho. O mRNA de GRPR e a expressão protéica em U-138MG foram detectados pelas técnicas de RT-PCR e imuno-histoquímica. No estudo in vivo a bombesina em baixas doses induziu um aumento na consolidação da memória. O resultado foi potencializado na combinação com um ativador do receptor de dopamina D1/D5 (D1R), além de ser prevenido quando combinado com um inibidor da via da PKA. Os resultados sugerem que GRP e GRPR interagem com a via de sinalização cAMP/PKA tanto na estimulação da proliferação celular em linhagem de câncer humano quanto na modulação da memória no hipocampo de ratos. / Increasing evidence indicates that gastrin-releasing peptide (GRP) acts as an autocrine growth factor for brain tumors as well as been implicated in memory formation, however, underlying molecular events are poorly understood. In the present study, we examined interactions between the GRPR and cellular signaling pathways in influencing memory consolidation in the hippocampus and on proliferation of glioblastoma cell in vitro. We show here that GRP acts synergistically with agents that stimulate the cAMP/PKA pathway to promote proliferation of human gliobastoma cells. Treatment with GRP combined with the adenylyl cyclase (AC) activator forskolin, the cAMP analog 8-Br-cAMP, or the phosphodiesterase type IV (PDE4) inhibitor rolipram increased proliferation of U138-MG cells in vitro measured by MTT assay. None of the compounds had an effect when given alone. GRP receptor (GRPR) mRNA and protein expression in U138-MG cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. We investigated the interactions between the GRPR and the PKA pathway in male Wistar rats. BB-induced enhancement of consolidation was potentiated by co infusion of activators of the dopamine D1/D5 receptor (D1R) pathway and prevented by a PKA inhibitor. The results suggest that GRP and the GRPR interact with the cAMP/PKA signaling pathway in stimulating a cancer cell line proliferation and in memory modulation by hippocampal.

Introdução: Os neuropeptídeos regulam uma variedade de aspectos da função nervosa e neuroendócrina, atuando através da ativação de receptores específicos da membrana celular. No sistemana nervoso central (SNC) os receptores do pepetídeo liberador de gastrina (GRPR) são amplamente expressos, e numerosos efeitos centrais têm sido descritos com a sua ativação, incluindo efeitos sobre a saciedade, regulação do ritmo circadiano, termorregulação, modulação do stress, resposta ao medo, ansiedade e memória. Pesquisas mostram que o bloqueio farmacológico do GRPR em modelos animais leva ao aparecimento de deficits na interação social, padrões restritivos de comportamento e estereotipias motoras, sintomas semelhantes ao comportamento autista em humanos, sugerindo a possibilidade de que o complexo GRP/GRPR possa ter um papel na patogênese do transtorno do espectro autista (TEA). Recentemente, dois estudos não controlados com administração do peptídeo liberador de gastrina (GRP) a 13 crianças com autismo sugeriram que ele é seguro e que possa melhorar alguns sintomas do transtorno, principalmente interação social e sintomas associados à irritabilidade. Objetivos: Comparar a eficácia, segurança, tolerabilidade do GRP em relação ao placebo em sintomas do TEA. Metodologia: Ensaio clínico crossover, randomizado, duplo-cego, controlado por placebo, com uso de GRP 160 picomol/kg por 4 dias consecutivos, em 10 crianças com autismo. Os desfechos foram medidos através da escala Aberrant Behavior Checklist (ABC). Resultados: Todos os participantes eram do sexo masculino, com idade entre 4 e 9 anos. Houve uma redução nos escores da escala ABC e suas subescalas após o uso de GRP e de placebo. Apesar dessa redução ser mais proeminente com o GRP, principalmente nas subescalas Irritabilidade, Comportamento estereotipado e Hiperatividade, não houve diferença estatística entre os resultados (p 0,334). Após uma semana da infusão, 5 crianças apresentavam melhora maior que 25% no escore total da escala ABC com uso de GRP e 2 com uso de placebo, não apresentando diferença estatística (p 0,375). Não houve efeitos adversos, alterações dos sinais vitais ou variações laboratoriais associados ao uso de GRP em nenhum paciente. Conclusões: Os resultados deste estudo, apesar do tamanho reduzido da amostra, reforçam os dados anteriores sobre a segurança do GRP no uso a curto prazo. Apesar de ter ocorrido redução dos escores da escala ABC após uso de GRP, não houve diferença estatística em relação ao placebo. Devido ao desenho crossover e tamanho pequeno da amostra do estudo atual, não foi possível esclarecer a real eficácia do GRP na redução dos sintomas do TEA na infância. Existe a necessidade de novas pesquisas com outros delineamentos e tamanho amostral maior para confirmar a eficácia e segurança do GRP em crianças com autismo. / Introduction: The neuropeptides regulate a variety of aspects of the nervous and neuroendocrine function, acting through activation of specific receptors of the cellular membrane. In system central nervous (CNS) the gastrin-releasing peptide recptors (GRPR) are widely expressed, and numerous central effects have been reported with their activation, including effects on satiety, regulating the circadian rhythm, thermoregulation, stress modulation, response to fear, anxiety and memory. Research has shown that pharmacological blockade of GRPR in animal models leads to the deficits in social interaction, restrictive patterns of behavior and motor stereotypies, autistic symptoms similar to human behavior, suggesting the possibility that the complex GRP/GRPR may have a role in the pathogenesis of autism spectrum disorder (ASD). Recently, two studies are not controlled with the administration of gastrin releasing peptide (GRP) to 13 children with autism suggest that it is safe and can improve some symptoms of the disorder, especially social interaction and symptoms associated with irritability. Objectives: To compare the efficacy, safety, tolerability GRP compared to placebo in ASD symptoms. Methodology: crossover clinical trial, randomized, double-blind, placebo-controlled, using GRP 160 picomol/kg for 4 consecutive days in 10 children with autism. Outcomes were measured by the Aberrant Behavior Checklist scale (ABC). Results: All participants were male, aged between 4 and 9 years. There was a reduction in the scores of the ABC range and its subscales after use GRP and placebo. Despite this reduction be more prominent with the GRP, particularly in subscales Irritability, Stereotypic behavior and Hyperactivity and noncompliance, there was no statistical difference between the results (p 0.334). After a week of infusion, 5 children showed improvement greater than 25% in the total score of the ABC scale in GRP use and 2 with placebo use, however there was no statistical difference (p 0.375). No adverse effects, changes in vital signs or laboratory abnormalities associated with use of GRP in any patient. Conclusions: The results of this study, despite the small sample size, reinforce previous data on the safety of the GRP in the short-term use. Although there was a reduction in ABC scale scores after use of GRP, there was no statistical difference from placebo. Due to the small sample size and design of the current study, it was not possible to clarify the real effectiveness of GRP in reducing the symptoms of ASD in childhood. There is a need for further research with other designs and larger sample size to confirm the efficacy and safety of GRP in children with autism.

Muitas evidências demonstram que o peptídeo liberador de gastrina (GRP) é um fator de crescimento que afeta funções neuroendócrinas, incluindo proliferação e diferenciação celular, comportamento alimentar, formação de memória, respostas a estresses, desenvolvimento de neoplasias, desordens neurológicas e psiquiátricas. Porém, os eventos moleculares pelos quais isso ocorre ainda não são totalmente compreendidos. No presente estudo, nós avaliamos as interações entre o receptor do peptídeo liberador de gastrina (GRPR) e a via de sinalização celular da PKA, tanto na proliferação celular de glioblastoma humano (in vitro) quanto na consolidação da memória no hipocampo de ratos Wistar (in vivo). Mostramos que o GRP age em sinergismo com agentes que estimulam a via do cAMP/PKA, promovendo a proliferação de células de glioblastoma humano, pois o tratamento com GRP combinado com um ativador de adenilil ciclase (AC), forskolin, ou um análogo de cAMP, 8-Br-cAMP, ou um inibidor do tipo IV de fosfodiesterase, rolipram, aumentaram a proliferação das células de U- 138MG, quando avaliadas pelo método de MTT. Nenhum destes compostos teve efeito sozinho. O mRNA de GRPR e a expressão protéica em U-138MG foram detectados pelas técnicas de RT-PCR e imuno-histoquímica. No estudo in vivo a bombesina em baixas doses induziu um aumento na consolidação da memória. O resultado foi potencializado na combinação com um ativador do receptor de dopamina D1/D5 (D1R), além de ser prevenido quando combinado com um inibidor da via da PKA. Os resultados sugerem que GRP e GRPR interagem com a via de sinalização cAMP/PKA tanto na estimulação da proliferação celular em linhagem de câncer humano quanto na modulação da memória no hipocampo de ratos. / Increasing evidence indicates that gastrin-releasing peptide (GRP) acts as an autocrine growth factor for brain tumors as well as been implicated in memory formation, however, underlying molecular events are poorly understood. In the present study, we examined interactions between the GRPR and cellular signaling pathways in influencing memory consolidation in the hippocampus and on proliferation of glioblastoma cell in vitro. We show here that GRP acts synergistically with agents that stimulate the cAMP/PKA pathway to promote proliferation of human gliobastoma cells. Treatment with GRP combined with the adenylyl cyclase (AC) activator forskolin, the cAMP analog 8-Br-cAMP, or the phosphodiesterase type IV (PDE4) inhibitor rolipram increased proliferation of U138-MG cells in vitro measured by MTT assay. None of the compounds had an effect when given alone. GRP receptor (GRPR) mRNA and protein expression in U138-MG cells was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. We investigated the interactions between the GRPR and the PKA pathway in male Wistar rats. BB-induced enhancement of consolidation was potentiated by co infusion of activators of the dopamine D1/D5 receptor (D1R) pathway and prevented by a PKA inhibitor. The results suggest that GRP and the GRPR interact with the cAMP/PKA signaling pathway in stimulating a cancer cell line proliferation and in memory modulation by hippocampal.

Introdução: Os neuropeptídeos regulam uma variedade de aspectos da função nervosa e neuroendócrina, atuando através da ativação de receptores específicos da membrana celular. No sistemana nervoso central (SNC) os receptores do pepetídeo liberador de gastrina (GRPR) são amplamente expressos, e numerosos efeitos centrais têm sido descritos com a sua ativação, incluindo efeitos sobre a saciedade, regulação do ritmo circadiano, termorregulação, modulação do stress, resposta ao medo, ansiedade e memória. Pesquisas mostram que o bloqueio farmacológico do GRPR em modelos animais leva ao aparecimento de deficits na interação social, padrões restritivos de comportamento e estereotipias motoras, sintomas semelhantes ao comportamento autista em humanos, sugerindo a possibilidade de que o complexo GRP/GRPR possa ter um papel na patogênese do transtorno do espectro autista (TEA). Recentemente, dois estudos não controlados com administração do peptídeo liberador de gastrina (GRP) a 13 crianças com autismo sugeriram que ele é seguro e que possa melhorar alguns sintomas do transtorno, principalmente interação social e sintomas associados à irritabilidade. Objetivos: Comparar a eficácia, segurança, tolerabilidade do GRP em relação ao placebo em sintomas do TEA. Metodologia: Ensaio clínico crossover, randomizado, duplo-cego, controlado por placebo, com uso de GRP 160 picomol/kg por 4 dias consecutivos, em 10 crianças com autismo. Os desfechos foram medidos através da escala Aberrant Behavior Checklist (ABC). Resultados: Todos os participantes eram do sexo masculino, com idade entre 4 e 9 anos. Houve uma redução nos escores da escala ABC e suas subescalas após o uso de GRP e de placebo. Apesar dessa redução ser mais proeminente com o GRP, principalmente nas subescalas Irritabilidade, Comportamento estereotipado e Hiperatividade, não houve diferença estatística entre os resultados (p 0,334). Após uma semana da infusão, 5 crianças apresentavam melhora maior que 25% no escore total da escala ABC com uso de GRP e 2 com uso de placebo, não apresentando diferença estatística (p 0,375). Não houve efeitos adversos, alterações dos sinais vitais ou variações laboratoriais associados ao uso de GRP em nenhum paciente. Conclusões: Os resultados deste estudo, apesar do tamanho reduzido da amostra, reforçam os dados anteriores sobre a segurança do GRP no uso a curto prazo. Apesar de ter ocorrido redução dos escores da escala ABC após uso de GRP, não houve diferença estatística em relação ao placebo. Devido ao desenho crossover e tamanho pequeno da amostra do estudo atual, não foi possível esclarecer a real eficácia do GRP na redução dos sintomas do TEA na infância. Existe a necessidade de novas pesquisas com outros delineamentos e tamanho amostral maior para confirmar a eficácia e segurança do GRP em crianças com autismo. / Introduction: The neuropeptides regulate a variety of aspects of the nervous and neuroendocrine function, acting through activation of specific receptors of the cellular membrane. In system central nervous (CNS) the gastrin-releasing peptide recptors (GRPR) are widely expressed, and numerous central effects have been reported with their activation, including effects on satiety, regulating the circadian rhythm, thermoregulation, stress modulation, response to fear, anxiety and memory. Research has shown that pharmacological blockade of GRPR in animal models leads to the deficits in social interaction, restrictive patterns of behavior and motor stereotypies, autistic symptoms similar to human behavior, suggesting the possibility that the complex GRP/GRPR may have a role in the pathogenesis of autism spectrum disorder (ASD). Recently, two studies are not controlled with the administration of gastrin releasing peptide (GRP) to 13 children with autism suggest that it is safe and can improve some symptoms of the disorder, especially social interaction and symptoms associated with irritability. Objectives: To compare the efficacy, safety, tolerability GRP compared to placebo in ASD symptoms. Methodology: crossover clinical trial, randomized, double-blind, placebo-controlled, using GRP 160 picomol/kg for 4 consecutive days in 10 children with autism. Outcomes were measured by the Aberrant Behavior Checklist scale (ABC). Results: All participants were male, aged between 4 and 9 years. There was a reduction in the scores of the ABC range and its subscales after use GRP and placebo. Despite this reduction be more prominent with the GRP, particularly in subscales Irritability, Stereotypic behavior and Hyperactivity and noncompliance, there was no statistical difference between the results (p 0.334). After a week of infusion, 5 children showed improvement greater than 25% in the total score of the ABC scale in GRP use and 2 with placebo use, however there was no statistical difference (p 0.375). No adverse effects, changes in vital signs or laboratory abnormalities associated with use of GRP in any patient. Conclusions: The results of this study, despite the small sample size, reinforce previous data on the safety of the GRP in the short-term use. Although there was a reduction in ABC scale scores after use of GRP, there was no statistical difference from placebo. Due to the small sample size and design of the current study, it was not possible to clarify the real effectiveness of GRP in reducing the symptoms of ASD in childhood. There is a need for further research with other designs and larger sample size to confirm the efficacy and safety of GRP in children with autism.

Introdução: Os neuropeptídeos regulam uma variedade de aspectos da função nervosa e neuroendócrina, atuando através da ativação de receptores específicos da membrana celular. No sistemana nervoso central (SNC) os receptores do pepetídeo liberador de gastrina (GRPR) são amplamente expressos, e numerosos efeitos centrais têm sido descritos com a sua ativação, incluindo efeitos sobre a saciedade, regulação do ritmo circadiano, termorregulação, modulação do stress, resposta ao medo, ansiedade e memória. Pesquisas mostram que o bloqueio farmacológico do GRPR em modelos animais leva ao aparecimento de deficits na interação social, padrões restritivos de comportamento e estereotipias motoras, sintomas semelhantes ao comportamento autista em humanos, sugerindo a possibilidade de que o complexo GRP/GRPR possa ter um papel na patogênese do transtorno do espectro autista (TEA). Recentemente, dois estudos não controlados com administração do peptídeo liberador de gastrina (GRP) a 13 crianças com autismo sugeriram que ele é seguro e que possa melhorar alguns sintomas do transtorno, principalmente interação social e sintomas associados à irritabilidade. Objetivos: Comparar a eficácia, segurança, tolerabilidade do GRP em relação ao placebo em sintomas do TEA. Metodologia: Ensaio clínico crossover, randomizado, duplo-cego, controlado por placebo, com uso de GRP 160 picomol/kg por 4 dias consecutivos, em 10 crianças com autismo. Os desfechos foram medidos através da escala Aberrant Behavior Checklist (ABC). Resultados: Todos os participantes eram do sexo masculino, com idade entre 4 e 9 anos. Houve uma redução nos escores da escala ABC e suas subescalas após o uso de GRP e de placebo. Apesar dessa redução ser mais proeminente com o GRP, principalmente nas subescalas Irritabilidade, Comportamento estereotipado e Hiperatividade, não houve diferença estatística entre os resultados (p 0,334). Após uma semana da infusão, 5 crianças apresentavam melhora maior que 25% no escore total da escala ABC com uso de GRP e 2 com uso de placebo, não apresentando diferença estatística (p 0,375). Não houve efeitos adversos, alterações dos sinais vitais ou variações laboratoriais associados ao uso de GRP em nenhum paciente. Conclusões: Os resultados deste estudo, apesar do tamanho reduzido da amostra, reforçam os dados anteriores sobre a segurança do GRP no uso a curto prazo. Apesar de ter ocorrido redução dos escores da escala ABC após uso de GRP, não houve diferença estatística em relação ao placebo. Devido ao desenho crossover e tamanho pequeno da amostra do estudo atual, não foi possível esclarecer a real eficácia do GRP na redução dos sintomas do TEA na infância. Existe a necessidade de novas pesquisas com outros delineamentos e tamanho amostral maior para confirmar a eficácia e segurança do GRP em crianças com autismo. / Introduction: The neuropeptides regulate a variety of aspects of the nervous and neuroendocrine function, acting through activation of specific receptors of the cellular membrane. In system central nervous (CNS) the gastrin-releasing peptide recptors (GRPR) are widely expressed, and numerous central effects have been reported with their activation, including effects on satiety, regulating the circadian rhythm, thermoregulation, stress modulation, response to fear, anxiety and memory. Research has shown that pharmacological blockade of GRPR in animal models leads to the deficits in social interaction, restrictive patterns of behavior and motor stereotypies, autistic symptoms similar to human behavior, suggesting the possibility that the complex GRP/GRPR may have a role in the pathogenesis of autism spectrum disorder (ASD). Recently, two studies are not controlled with the administration of gastrin releasing peptide (GRP) to 13 children with autism suggest that it is safe and can improve some symptoms of the disorder, especially social interaction and symptoms associated with irritability. Objectives: To compare the efficacy, safety, tolerability GRP compared to placebo in ASD symptoms. Methodology: crossover clinical trial, randomized, double-blind, placebo-controlled, using GRP 160 picomol/kg for 4 consecutive days in 10 children with autism. Outcomes were measured by the Aberrant Behavior Checklist scale (ABC). Results: All participants were male, aged between 4 and 9 years. There was a reduction in the scores of the ABC range and its subscales after use GRP and placebo. Despite this reduction be more prominent with the GRP, particularly in subscales Irritability, Stereotypic behavior and Hyperactivity and noncompliance, there was no statistical difference between the results (p 0.334). After a week of infusion, 5 children showed improvement greater than 25% in the total score of the ABC scale in GRP use and 2 with placebo use, however there was no statistical difference (p 0.375). No adverse effects, changes in vital signs or laboratory abnormalities associated with use of GRP in any patient. Conclusions: The results of this study, despite the small sample size, reinforce previous data on the safety of the GRP in the short-term use. Although there was a reduction in ABC scale scores after use of GRP, there was no statistical difference from placebo. Due to the small sample size and design of the current study, it was not possible to clarify the real effectiveness of GRP in reducing the symptoms of ASD in childhood. There is a need for further research with other designs and larger sample size to confirm the efficacy and safety of GRP in children with autism.