Ccomplished by 30 min incubation in 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 larger and at room temperature. For clinical testing, the 99m reach a each situations, high quality handle of the radiolabeled solution involved ITLC and HPLC larger molecular activity (greater than 50 MBq [ Tc]Tc/nmol peptide). In In each circumstances, good quality manage from the radiolabeled product involved ITLC and HPLC techniques (Supplementary File). Pretty much quantitative Tenofovir diphosphate triethylamine radiochemical yields (98 ) were esmethods (Supplementary File). Almost quantitative radiochemical yields (98 ) had been 99m tablished with much less than two of total radiochemical impurities ([99mTc]TcO4-, [- Tc]Tc-citestablished with much less than two 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). Thus, [ Tc]Tc-D15 was employed without having additional radiochemical purification [35]. was employed without having additional radiochemical purification [35]. three.2. Binding Affinity of DB15 for the Human GRPR three.2. Binding Affinity of DB15 for the Human GRPR 125 4 As shown in Figure two, DB15 displaced the [125 I]I-[Tyr4]BBN radioligand from PC-3 As shown in Figure 2, DB15 displaced the [ I]I-[Tyr ]BBN radioligand from PC-3 cell membranes in a monophasic and dose-dependent way, displaying a greater binding cell membranes in a monophasic and dose-dependent way, displaying a greater binding 4 affinity for the human GRPR (IC 50 = 0.37 0.03 nM, n = 3) compared together with the [Tyr4 ]BBN affinity for the human GRPR (IC = 0.37 0.03 nM, n = 3) compared with all the [Tyr ]BBN 50 Mefenpyr-diethyl Autophagy reference (IC50 = 1.33 0.09 nM, n = 3). reference (IC = 1.33 0.09 nM, n = three).distinct binding75 50 25 0 10 -13 ten -12 10 -11 ten -10 ten -9 10 -8 10 -7 ten -[peptide] (M)Figure 2. Displacement of [125of [125 I]I-[Tyrby rising concentrations of DB15 (, ICDB15 ( 2. Displacement I]I-[Tyr4]BBN 4 ]BBN by growing concentrations of 50 = 0.37 0.03 nM) or0.03 nM) ( reference, 1.33 reference, 1.33 0.09cell membrane homogenates; benefits [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; results represent average values SD of three experiments performed in triplicate.3.three. 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. Higher and precise uptake was observed through 30 min incubation of [99m Tc]Tc-DB15 in PC-3 (13.1 0.1 ) and T-47D cells (24.2 0.7 ) at 37 C. The majority of radioactivity was connected with all the cell-membrane with a smaller portion (6 ) located inside the cells, as anticipated for any GRPR-radioantagonist [20,35,41]. Cel.
