Investigation Of ～(188)Re, ～(99)Tc～m Directly Labeling Octreotide And Receptor Binding Analysis In Vitro

Somatostatin analogues such as octreotide has numerous functions including inhibition of hormone release, immuno- modulation and neurotransmission and are endogenous inhibitors of cell prolixferation and angiogenesis.1. Investigation of 188Re, 99Tcm directly labeling octreotideObjective To establish a useful and stable method for directly labeling octreotide with 188Re,99TcmMethods (1) 99Tcm directly labeling octreotide: Two-step reduction method was adopted. The peptide was reduced by using the ascorbic acid method, and 99mTc was reduced with a fresh solution of Na2S2O4. And the labeling conditions were changed. 1) the study were carried out under the temperature of 22, 60, 100℃ respectively. 2) the pH value of ascorbic acid were 3.0,4.5,5.5,6.5 respectively. 3) Sodium gluconate or acetate buffer were selected as buffer.(2) 188Re directly labeling octreotide: The 'pretinning' procedurewas adopted. Both the peptide and ReO4- were reduced by SnCl2.Results (1) In 99Tcm directly labeling octreotide, 99Tcm- octreotide was prepared in 53%-60% radiochemical yield. After purification, radiochemical purity was > 95%. As to the labeling condition: 1) when the study carried out under the temperature of 22℃ ,the highest radiochemical purity of (57.8±2.3)% was obtained. 2) the pH value of ascorbic acid was 4.5, the radiochemical purity of 99Tcm-octreotide was 57.8±2.3%. 3) we got plenty of radiocolloid when sodium gluconate actas buffer.(2) 188Re-octreotide was prepared with yield varying between 92%-95%.Conclusions (1)Preparation of technetium-99m-labeled octreotide:The mixture of 0.025ml ascorbic acid(40mg/ml) and octreotide 5μg(1mg/ml) was incubated for 30 min. 0.1ml 99mTc reduced with a freshsolution of 0.6mg Na2S2O4 in acetate acid for 1 min, and added to themixture of octreotide and ascorbic acid for further incubation for 15 min(2) Preparation of rhenium-188-octreotide: rhenium-188- octreotide was prepared by reacting 0.1ml sodium gluconate (0.3M),0.03ml stannous chloride (20mg/ml), 0.01ml octreotide (1mg/ml) in 0.04mlacetate buffer(pH 5.0) for 1 hour, then 0.2ml Re perrhenate was added, incubating for 3 hour.2. Comparison of 188Re-octreotide, 99mTc-octreotide of receptor binding analysis in vitroObjective: To determine the in vitro receptor binding properties of 99Tcm-octreotide,188Re-octreotideMethods: The binding assay for radiolabeled peptides was carried out using SD rats brain cortex cell suspension as SSTR(somatostatin receptor), 99Tcm-octreotide and 188Re- octreotide as ligands. The data were analyzed using the method of Scatchard, giving the dissociation constant (Kd) and the number of binding sites (Bmax) for the SD rat brain cortex cell preparation.Results: 99Tcm-octreotide was shown to have a dissociation constant (Kd) of 11.25±1.26nmol/L, and a receptor concentration (Bmax) of 35.06±1.87 fmol/ug; 188Re-octreotide has a Kd=18.28±2.13nM, Bmax= 34.70±2.01fmol/ug.The binding affinities of 188Re-octreotide was significantly (t=5.39,P < 0.05) lower than that of 99Tcm-octreotide.Conclusions: Re-octreotide was shown to have lower affinity for SD rats brain cortex cell than that 99Tcm-octreotide do. Both they maintain relatively high binding properties and were likely to be the radio-pharmaceutical for detection and radiotherapy of SSTR-expressing tumor and other tissues.