Ccomplished by 30 min incubation on the activity of 30 MBq [99mTc]Tc/nmol DB15 with Tc-99m for preclinical testing (molecular radiolabel99m Tc]Tc/nmol peptide) was 200 MBq [mixture, containing DB15, [99maccomplished by 30 citrate anions, atof the radiing reaction Tc]TcO4-, SnCl2 and min incubation pH 11 and olabeling reaction mixture, containing DB15, [99m Tc]TcO4 – , SnCl2 and citrate to reachat at area temperature. For clinical testing, the labeling reaction was modified anions, a pH 11 molecular activity (higher than 50 MBq [99mTc]Tc/nmol peptide). was modified to labeling reaction higher and at space temperature. For clinical testing, the 99m attain a both circumstances, good quality manage of the radiolabeled product involved ITLC and HPLC higher molecular activity (greater than 50 MBq [ Tc]Tc/nmol peptide). In In both situations, high-quality handle on the radiolabeled solution involved ITLC and HPLC procedures (Bcr-Abl| Supplementary File). Almost quantitative radiochemical yields (98 ) were esmethods (Supplementary File). Nearly quantitative radiochemical yields (98 ) were 99m tablished with much less than two of total radiochemical impurities ([99mTc]TcO4-, [- Tc]Tc-citestablished with significantly less than 2 of total radiochemical impurities ([99m Tc]TcO4 , [99m Tc]Tc99mTc]TcO2 nH2O) present, whereas HPLC revealed the formation of a single rate and [ 99m citrate and [ Tc]TcO nH2 O) present, whereas HPLC revealed the formation of a single 99m radiochemical species2 (Figures S1 and S2; Supplementary File). Therefore, [99mTc]Tc-D15 radiochemical species (Figures S1 and S2; Supplementary File). As a result, [ Tc]Tc-D15 was used without further radiochemical purification [35]. was utilised without the need of further radiochemical purification [35]. 3.two. SID 7969543 Cancer binding Affinity of DB15 for the Human GRPR 3.two. Binding Affinity of DB15 for the Human GRPR 125 four As shown in Figure 2, DB15 displaced the [125 I]I-[Tyr4]BBN radioligand from PC-3 As shown in Figure two, DB15 displaced the [ I]I-[Tyr ]BBN radioligand from PC-3 cell membranes in a monophasic and dose-dependent way, displaying a larger binding cell membranes within a monophasic and dose-dependent way, displaying a greater binding four affinity for the human GRPR (IC 50 = 0.37 0.03 nM, n = 3) compared with the [Tyr4 ]BBN affinity for the human GRPR (IC = 0.37 0.03 nM, n = 3) compared using the [Tyr ]BBN 50 reference (IC50 = 1.33 0.09 nM, n = three). reference (IC = 1.33 0.09 nM, n = 3).distinct binding75 50 25 0 ten -13 10 -12 ten -11 10 -10 10 -9 ten -8 ten -7 ten -[peptide] (M)Figure 2. Displacement of [125of [125 I]I-[Tyrby increasing concentrations of DB15 (, ICDB15 ( two. Displacement I]I-[Tyr4]BBN four ]BBN by growing concentrations of 50 = 0.37 0.03 nM) or0.03 nM) ( reference, 1.33 reference, 1.33 0.09cell membrane homogenates; final results [Tyr4]BBN or [Tyr4 ]BBN ( 0.09 nM) from PC-3 nM) from PC-3 cell membrane hoIC50 = 0.37 represent typical values SD of 3 experiments performed in triplicate. mogenates; final results represent average values SD of three experiments performed in triplicate.three.3. GRPR-Specific Uptake of [99m Tc]Tc- DB15 by PC-3 and T-47D Cells Time-dependent cell association curves of [99m Tc]Tc-DB15 in PC-3 and T-47D cells are shown in Figure three. High and specific uptake was observed throughout 30 min incubation of [99m Tc]Tc-DB15 in PC-3 (13.1 0.1 ) and T-47D cells (24.two 0.7 ) at 37 C. The majority of radioactivity was associated with the cell-membrane having a smaller portion (six ) discovered inside the cells, as expected for a GRPR-radioantagonist [20,35,41]. Cel.
