A preclinical evaluation of Ga-68 labeled VPAC receptor specific peptide using different chelating agents, NODAGA and N2S2. (2016)

1171 Objectives Our laboratory has successfully designed and characterized a peptide which has high affinity for VPAC1 receptors expressed in high density on breast (BC) and prostate cancers (PC). The purpose was to conjugate the peptide with two different chelating agents- NODAGA (1,4,7-triazacyclononane-1-glutamic acid-4,7-diacetic acid) and N2S2 [bis(thiosemicarbazone)], to label the conjugates with gallium-68 (Ga-68) and evaluate them for stability, ability to micro-PET image human breast cancer in a mouse model and to determine their biodistribution. Methods The NODAGA-peptide (TP-3718) was synthesized with C-terminal NODAGA chelator on a Wang resin using a peptide synthesizer (Protein Technologies, Tucson, Arizona, USA) while N2S2-peptide (TP-3805) was purchased from American Peptide Co, Sunniville, CA. Both peptide-conjugates were characterized by electrospray mass spectroscopy. Ga-68 was eluted from [Ge-68/Ga-68] generator using 0.1 N HCl (5.0 mL). The eluate was then passed through a cation exchange column and the column was washed with 80% acetone (in 0.1 N HCl). Ga-68 retained on column was eluted with 98% acetone (in 0.05 N HCl) and the excess of acetone was evaporated by heating at 70° C for 5 min. For labeling TP-3718, 20 µg of peptide-conjugate in 200 µL of deionized water, and 20 µL of Ga-68-Cl3 were incubated at 90°C for 30 min. The final pH was adjusted to 7.2 ± 0.2 with 0.1 N NaOH. For radiolabeling TP-3805, 200 µL of glycine buffer (pH=9.2), 20 µL SnCl2 (10 mg/mL in 50 mg/mL glucoheptonate), 20 µg TP-3805 and 20 µL of Ga-68-Cl3 were mixed (pH- 7.2 ± 0.2) and incubated at 90°C for 30 min. The radiochemical purity and in vitro stability of the radiolabeled peptides were determined by Radio-HPLC. Ga-68-labeled peptides (~ 100 µCi in 200 µL) each was injected intravenously to a separate group of immunocompromised female nude mice (n = 5) bearing BT474 human BC. Micro-PET/CT imaging was performed at 1 hr post-injection and data were analyzed by calculating regional standardized uptake values. After imaging, the animals were sacrificed and % injected dose per gram was calculated for each organ. Results As determined by mass spectrometry, the purity of the peptide was 99.9%. Radio-HPLC showed retention time for Ga-68-TP-3718 at 9.9 ± 0.3 min and Ga-68-TP-3805 at 4.9 ± 0.4 min as compare to free Ga-68-Cl3 at 3.4 ± 0.4 min. The radiolabeling efficiency for each peptide conjugate was > 95.0 % and their stability was greater than 95.0% at 4 h. Micro-PET images showed tumor to muscle ratio of 3.2 ± 0.2 for Ga-68-TP-3718 and 3.4 ± 0.2 for Ga-68-TP-3805 at 1 hr post injection. The biodistribution data at 1 hr post injection showed that Ga-68-TP-3718 had maximum uptake in the kidneys (30.4 ± 5.0 %ID/g) associated with high renal clearance whereas Ga-68-TP-3805 had lesser uptake in kidneys (4.1 ± 2.5 %ID/g). Conclusions The post processing of Ga-68 ensured removal of cationic impurities and yielded higher specific activity. Data demonstrated that either Ga-68-TP-3718 or Ga-68-TP-3805 could be used as a potential agent for PET imaging for breast cancer.
Research Support: The research, in part, was supported by NIH/NCI RO1CA157372 (MLT), NIH/NCI 1S10OD012406 (MLT) and NIH/NCI S10RR23709 (MLT).