Development of a hydroxamamide-based bifunctional chelating agent to prepare technetium-99m-labeled bivalent ligand probes

Hydroxamamide (Ham) is a thiol-free chelating agent that forms technetium-99m (99mTc)-complexes with a metal-to-ligand ratio of 1:2 under moderate reaction conditions. Therefore, Ham-based chelating agents will produce 99mTc-labeled compounds with a bivalent targeting scaffold. For their universal usage, we developed a novel Ham-based bifunctional chelating agent, “Ham-Mal”, with a maleimide group that can easily conjugate with a thiol group, for to preparing 99mTc-labeled bivalent ligand probes. Ham-Mal was synthesized by a four-step reaction, and then reacted with cysteine or c(RGDfC) to produce Ham-Cys or Ham-RGD. These precursors were reacted with 99mTcO4- for 10 min under room temperature to obtain 99mTc-(Ham-Cys)2 and 99mTc -(Ham-RGD)2. The cellular uptake level of 99mTc-(Ham-RGD)2 by U87MG (high Integrin ɑvβ3 expression) cells was significantly higher than that by PC3 (low Integrin ɑvβ3 expression) cells at 60 min after the incubation, and the uptake was significantly suppressed by pre-treatment for 15 min with excess c(RGDfK) peptide. In the in vivo study with U87MG/PC3 dual xenografted BALB/c-nu mice, the radioactivity of U87MG tumor tissue was significantly higher than that of PC3 tumor tissue at 360 min after the administration of 99mTc-(Ham-RGD)2. These results suggest Ham-Mal may have potential as a bifunctional chelating agent for 99mTc-labeled bivalent ligand probes.

Development of a Hydroxamamide-Based Bifunctional Chelating Agent to Prepare Technetium-99m-Labeled Bivalent Ligand Probes

Hydroxamamide (Ham) is a thiol-free chelating agent that forms technetium-99m (99mTc)-complexes with a metal-to-ligand ratio of 1:2 under moderate reaction conditions. Therefore, Ham-based chelating agents will produce 99mTc-labeled compounds with a bivalent targeting scaffold. For their universal usage, we developed a novel Ham-based bifunctional chelating agent, “Ham-Mal”, with a maleimide group that can easily conjugate with a thiol group, for to preparing 99mTc-labeled bivalent ligand probes. Ham-Mal was synthesized by a four-step reaction, and then reacted with cysteine or c(RGDfC) to produce Ham-Cys or Ham-RGD. These precursors were reacted with [99mTc]TcO4− for 10 min under room temperature to obtain [99mTc]Tc-(Ham-Cys)2 and [99mTc]Tc-(Ham-RGD)2. The cellular uptake level of [99mTc]Tc-(Ham-RGD)2 by U87MG (high Integrin ɑvβ3 expression) cells was significantly higher than that by PC3 (low Integrin ɑvβ3 expression) cells at 60 min after the incubation, and the uptake was significantly suppressed by pre-treatment for 15 min with excess c(RGDfK) peptide. In the in vivo study with U87MG/PC3 dual xenografted Balb/c-nu mice, the radioactivity of U87MG tumor tissue was significantly higher than that of PC3 tumor tissue at 360 min after the administration of [99mTc]Tc-(Ham-RGD)2. These results suggest Ham-Mal may have potential as a bifunctional chelating agent for 99mTc-labeled bivalent ligand probes.

68Ga-labeled HBED-CC Variant of uPAR Targeting Peptide AE105 Compared with 68Ga-NODAGA-AE105

Aims: The urokinase Plasminogen Activator Receptors (uPAR) over-expressed on tumor cells and their invasive microenvironment are clinically significant molecular targets for cancer research. uPARexpressing cancerous lesions can be suitably identified and their progression can be monitored with radiolabeled uPAR targeted imaging probes. Hence this study aimed at preparing and evaluating two 68Ga-labeled AE105 peptide conjugates, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 as uPAR PET-probes. Method: The peptide conjugates, HBED-CC-AE105-NH2 and NODAGA-AE105-NH2 were manually synthesized by standard Fmoc solid phase strategy and subsequently radiolabeled with 68Ga eluted from a commercial 68Ge/68Ga generator. In vitro cell studies for the two radiotracers were performed with uPAR positive U87MG cells. Biodistribution studies were carried out in mouse xenografts with the subcutaneously induced U87MG tumor. Results: The two radiotracers, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 that were prepared in >95% radiochemical yield and >96% radiochemical purity, exhibited excellent in vitro stability. In vivo evaluation studies revealed higher uptake of 68Ga-HBED-CC-AE105 in U87MG tumor as compared to 68Ga-NODAGAAE105; however, increased lipophilicity of 68Ga-HBED-CC-AE105 resulted in slower clearance from blood and other non-target organs. The uPAR specificity of the two radiotracers was ascertained by significant (p<0.05) reduction in the tumor uptake with a co-injected blocking dose of unlabeled AE-105 peptide. Conclusion: Amongst the two radiotracers studied, the neutral 68Ga-NODAGA-AE105 with more hydrophilic chelator exhibited faster clearance from non-target organs. The conjugation of HBED-CC chelator (less hydrophilic) resulted in negatively charged 68Ga-HBED-CC-AE105 which was observed to have high retention in blood that decreased target to non-target ratios.

Early Monitoring Antiangiogenesis Treatment Response of Sunitinib in U87MG Tumor Xenograft by18F-FLT MicroPET/CT Imaging

Aim. It was aimed to monitor early treatment response of Sunitinib in U87MG models mimicking glioblastoma multiforme by longitudinal18F-FLT microPET/CT imaging in this study.Methods. U87MG tumor mice were intragastrically injected with Sunitinib at a dose of 80 mg/kg for consecutive 7 days.18F-FLT microPET/CT scans were acquired on days 0, 1, 3, 7, and 13 after therapy. Tumor sizes and body weight were measured. Tumor samples were collected for immunohistochemical analysis of proliferation and microvessel density (MVD) with anti-Ki67 and anti-CD31, respectively.Results. The uptake ratios of tumor to the contralateral muscle (T/M) of18F-FLT in the Sunitinib group decreased from baseline to day 3 (T/M0= 2.98 ± 0.33; T/M3= 2.23 ± 0.36;P<0.001), reached the bottom on day 7 (T/M7= 1.96 ± 0.35;P<0.001), and then recovered on day 13. The T/M of18F-FLT uptake in the control group remained around 3.0. There was no difference for the tumor size between both groups until day 11.18F-FLT uptakes of tumor were correlated with Ki67 staining index and MVD.Conclusion. Early therapy response to Sunitinib could be predicted via18F-FLT PET, which will contribute to monitoring antiangiogenesis treatment.