Dissertation committee:
Σταμπάκη-Χατζηπαναγιώτη Δέσποινα Αναπλ. Καθηγήτρια ΕΚΠΑ (Επιβλέπουσα), Μερτής Κωνσταντίνος Ομότ. Καθηγητής ΕΚΠΑ, Μάινα - Nock Θεοδοσία Ερευνήτρια Α (Ε.Κ.Ε.Φ.Ε. Δημόκριτος)
Summary:
In recent years important progress has been made in diagnostic oncology by
applying specialized site-specific radiolabeled compounds. Research is mainly
focusing on the development of radiolabeled peptides because they offer several
advantages such as rapid biokinetics, high penetration capacity into cancer
tissue due to their low molecular weight and low antigenicity. Radiopeptides
interact selectively with target-biomolecules (peptide receptors) which are
overexpressed on the surface of cancer cells. Therefore, they act as
transporters of diagnostic or therapeutic radionuclides to the target
(malignant lesions), providing the possibility of high contrast imaging or cell
damage.
Bombesin is a 14-amino acid peptide with high affinity for gastrin releasing
peptide receptors (GRP-Rs) which are overxpressed in a variety of human tumors
including prostate and breast cancers.
In the present thesis, the synthesis and preclinical evaluation of novel
bombesin antagonists radiolabeled with 99mTc for potential application in
targeted diagnostic imaging of GRP-R+ tumors, is described.
Synthesis of the new peptides was based on the bombesin antagonist peptide
[D-Phe6,Leu-NHEt13]BBN(6-13) and modifications occurred at the N-terminus,
C-terminus and/or at the peptide chain of this antagonist.
The radionuclide of choice was 99mTc because of its ideal nuclear properties. A
bifunctional tetraamine precursor H2NCH2CH2NH(CH2)3NHCH2CH2NH2 (2,3,2-tet) was
used for the stable binding of 99mTc. Incorporation of 99mTc by the tetraamine
unit was almost quantitative as it was confirmed by chromatographic analysis.
Binding affinity of the new analogs for the GRP-R was studied with competition
binding experiments in PC-3 cell membranes and it was high, even higher than
the peptide of reference [Tyr4]BBN. These results show the favorable effect of
the tetraamine complex at the N-terminus of the new peptides. Also the binding
affinity of the radiolabeled peptide was studied for [99gTc/99mTc]Demobesin 8
by saturation binding experiments in whole PC-3 cells and it was significantly
high (Kd = 0.24±0.03 nM) indicating that the presence of the [TcV(O)2N4)]+
complex at the N-terminus of the peptide affects positively the binding
affinity to GRP-R.
Metabolic studies for [99mTc]Demobesin 8 were conducted in mouse plasma, kidney
homogenates and urine. [99mTc]Demobesin 8 survived long enough in mouse
plasma, whereas in kidney homogenates it rapidly degraded in hydrophilic
metabolites and it is finally excreted into urine. HPLC analysis in urine that
were collected 30 min after the intravenous injection of the radiopeptide
showed full degradation in the kidneys.
Biodistribution studies in healthy and pathological animal models after
intravenous injection of the radiolabeled peptides confirmed the in vitro
results. The radioactivity rapidly cleared from the blood and background
tissues and it was excreted mainly via kidneys and the urinary system.
As it was mentioned before the main route of excretion of [99mTc]Demobesin 8 is
via the kidneys and the urinary system and not through the hepatobiliary system
due to high hydrophilicity. This is expected to lead in rapid excretion from
background tissues and hence a possible better imaging of GRP-R positive
tumors. Also higher uptake in organs that expressing GRP-R such as pancreas was
observed for [99mTc]Demobesin 8. The process was GRP-R mediated, as shown by
in vivo receptor blockade by co-injection of excess of [Tyr4]BBN with each
radiopeptide. In SCID mice bearing human PC-3 xenografts expressing the human
GRP-R, [99mTc]Demobesin 8 achieved the higher uptake in tumor.
In comparison with other radiolabeled bombesin antagonists, which are known in
the literature, [99mTc]Demobesin 8 showed the highest binding affinity for
GRP-R and also in vivo the highest tumor uptake. Moreover compared with agonist
radiopeptides such as [99mTc]Demobesin 3-6 showed the more effective targeting
of GRP-R by antagonist peptides.
In conclusion, this thesis successfully led in the development of new
99mTc-radiolabeled bombesin antagonists for diagnostic imaging of GRP-R
positive tumors in human. This preclinical evaluation showed as the
radiopeptide of choice [99mTc]Demobesin 8 because satisfied important criteria
and combined a variety of favorable properties for successful application in
the targeted diagnostic imaging of GRP-R positive tumors such as favorable
pharmacokinetics, metabolic stability as well as rapid and high in vivo GRP-R
targeting which can led to satisfactory tumor images
Keywords:
Technetium, Biodistribution studies , Diagnostic oncology, Gastrin releasing peptide receptors
