Dissertation committee:
Ανδρέας Τσοτίνης, Καθηγητής, Τομέας Φαρμακευτική Χημείας, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Ιωάννης Πιρμεττής, Ερευνητής Α΄, ΙΠΡΕΤΕΑ, ΕΚΕΦΕ “Δημόκριτος”
Μηνάς Παπαδόπουλος, Ερευνητής Α΄, ΙΠΡΕΤΕΑ, ΕΚΕΦΕ “Δημόκριτος”
Μαρία Πελεκάνου, Ερευνήτρια Α', ΙΒ-Ε, ΕΚΕΦΕ "Δημόκριτος"
Νικολαΐς Πουλή, Καθηγήτρια, Τομέας Φαρμακευτικής Χημείας, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Παναγιώτης Μαράκος, Καθηγητής, Τομέας Φαρμακευτικής Χημείας, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Νικόλαος Λουγιάκης, Επικ. Καθηγητής, Τομέας Φαρμακευτικής Χημείας, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Summary:
The current project aims at the design, synthesis, and biological evaluation of a series of mannosylated dextran derivatives labeled with 99mTc and 68Ga, bearing dextrans of different molecular weight, 10-500 kDa, for potential use in sentinel lymph node detection.
In this context, 2-imino-2-methoxy-ethyl-1-thio-β-D-mannopyranoside, a mannose derivative, was initially synthesized according to the literature procedure, to act as a substrate for binding to the mannose receptor CD206 in the macrophages of the lymph nodes.
DCM derivatives, 19-24, were then synthesized in three steps. In the first step, by adding allyl bromide to a dextran of different molecular weight each time, the intermediate allyl dextrans D10Α-D500Α, 7-12, were obtained. Cysteine was added to the allyl-dextrans to give the D10C-D500C derivatives, 13-18. Finally, freshly prepared 2-imino-2-methoxy-ethyl-1-thio-β-D-mannopyranoside solution was added to the D10CM-D500CM derivatives, affording the mannosylated DCM derivatives, 19-24. NODAGA chelating system was added to D20CM, 20, to give NODAGA-D20CM, 25, followed by fluorescein isothiocyanate (FITC), a fluorochrome for potential use in optical imaging, to give NODAGA-D20CM-FITC, 26. All derivatives were characterized by NMR spectroscopy.
Derivatives 19-24 were labeled with the fac-[99mTc(CO)3(H2O)3]+ core, to give complexes fac-[99mTc(CO)3(DCM)], 19'-24', where DCM derivatives, 19-24, act as efficient SNO tridentate ligands. Derivatives 25 and 26 were labeled with 68GaCl3, to yield complexes 68Ga-NODAGA-D20CM, 25 ', and 68Ga-NODAGA-D20CM -FITC, 26', where 68Ga is coordinated by the three nitrogen atoms and the three carboxylic groups of the NODAGA chelating system.
At tracer level, all complexes were prepared in high yield. 99mTc complexes, 19'-24', were stable overtime, in the labeling solution, and during histidine/cysteine challenge experiments up to 24 h and 6 h, accordingly. 68Ga complexes, 25' and 26', showed also high stability in the labeling solution at room temperature up to 4 h.
Biodistribution studies with 99mTc complexes, 19'-24', at 15, 60 and 180 min after injection showed rapid injection site clearance at 15 min, with a slower clearance up to 60 min, which then remained quite stable. Sentinel lymph node uptake was high for all complexes at 15 min (2-3%). At 60 and 180 min after injection, uptake was further increased or remained stable (5-15%). The highest values were presented for complex fac-[99mΤc(D75CM)(CO)3], 22’, which at 180 min showed the highest 1st/2nd lymph node ratio. The uptake in the second lymph node was low throughout the studies (0.49-1.84% at 15 min, 1.81-2.37% at 180 min). For comparison purposes, the biodistribution of the non-mannosylated fac-[99mΤc(D75C)(CO)3], 16', was studied to confirm the uptake of complex 22’, due to the interaction of mannose with the mannose receptor CD206 in the macrophages of the lymph nodes. Biodistribution results, for complexes 22’ and 16’, were also confirmed by imaging studies with SPECT/CT camera.
68Ga complexes, 25' and 26', were studied in vivo at 15, 30 and 60 min. An initially rapid injection site clearance was observed, which increased further overtime. The data for the sentinel lymph node showed remarkable and stable uptake at all time points. The common characteristics of both biodistribution studies indicate that binding FITC to complex 26’ does not significantly alter the biodistribution of the molecule.
Complex fac-[99mΤc(D75CM)(CO)3], 22’, showed the best biological characteristics, making it a candidate for further evaluation in larger animals and clinical trials as a new radiopharmaceutical for sentinel lymph node detection.
