99mTc-EDDA/HYNIC-TOC and 99mTc-EDDA/HYNIC-TATE - from CRP to extended clinical application

Full text: Somatostatin is 14-amino acid peptide naturally occurring which controls the secretion of many hormones. A high density of somatostatin receptors have been found on tumours mainly of neuroendocrine origin. Somatostatin has a very short biological half-life of several minutes, hampering sufficient receptor binding to allow adequate imaging. Derivatives of somatostatin represent the most important peptides for receptor targeting in oncological applications. In this study two promising radiopharmaceuticals 99mTc-HYNIC-TOC and 99mTc-HYNIC-TATE with the potential to replace 111In-DTPA-D-Phe1-Octreotide in somatostatin receptor scintigraphy are presented HYNIC - Tyr3-octreotide (HYNIC - TOC) and HYNIC - Tyr3 - Octreotate (HYNIC - TATE) were synthesized in our laboratory on solid phase using the Fmoc strategy. Then for the HYNIC chelators coupling, the peptides were cleaved from the resin, cyclization and coupling was done after that in solution. The overall yields after deprotection with preparative HPLC were between 14% and 20 %. Wet 99mTc-labelling of HYNIC-TOC and HYNIC-TATE were performed to optimize the amount and concentration of reagents, temperature and reaction time which was then transferred to HYNIC-TOC and HYNIC-TATE dry kit formulation. The kit contains two vials: the first vial: 20 μg HYNIC-TOC or HYNIC-TATE, 40 μg SnCl2, 50 mg tricine, 10 mg mannitol, the second vial: 10 mg EDDA (ethylenediamine- N,N'-diacetic acid). Radiolabelling was carried out by the addition 1 ml of generator eluate (20-40mCi radioactivity) to kit and 0,5 ml EDDA followed by 30 min incubation at 80 deg. C. Radiochemical purity of 99mTc-HYNIC-TOC and 99mTc-HYNIC-TATE, controlled by TLC and HPLC methods showed over 90% radiochemical yield and percentage of unbound free 99mTc-pertechnetate as well as colloidal forms of 99mTc was in the range of 2 - 3%. The stability of the freeze-dried kits during storage at 2-8 deg. C was performed once a month by radiolabelling and determination of the radiochemical purity by HPLC. The stability of these kits was determined for 1 year. Stability of the obtained kits allowed extensive clinical studies of 99mTc-HYNIC-Tyr3-octreotide and 99mTc-HYNIC-Tyr3-Octreotate. Biodistribution studies in normal mice showed rapid urine excretion of unbound radioactivity. Accumulation in kidneys was at the level of 5% at 3.5h p.i. Activity accumulated in pancreas, organ reach in somatostatin receptors, was 2.8 times higher than in lungs in normal animals and 2.2 times lower than in lungs in blocked animals, respectively. Similar accumulation in adrenals was observed. The internalization experiments were tested using AR42J rat pancreatic tumour cell membranes. The 99mTc-labelled peptides showed the following order in the rate of internalization: 99mTc-HYNIC-TOC - 10.9%, 99mTc-HYNIC-TATE - 18.7%. Hynic-Tyr3-octreotide and Hynic-Tyr3-octreotate conjugates obtained in our laboratory were successfully labelled with technetium-99m with the yields over 90% and tested in vitro for human serum stability and internalization. To make labelling procedure easier, dry kits were produced with Tricine and EDDA as co-ligands. The in vitro and in vivo features of the 99mTc- Hynic-Tyr3-octreotide and 99mTc-Hynic-Tyr3-octreotate confirmed their diagnostic potential. (author)