Preparation of a Promising Angiogenesis PET Imaging Agent: ⁶⁸Ga-Labeled c(RGDyK)–Isothiocyanatobenzyl-1,4,7-Triazacyclononane-1,4,7-Triacetic Acid and Feasibility Studies in Mice

¹ Department of Nuclear Medicine, Cancer Research Institute College of Medicine, Seoul National University, Seoul, Korea
² Department of Nuclear Medicine, Korea Institute of Radiological and Medical Sciences, Seoul, Korea

^* To whom correspondence should be addressed. E-mail: jmjng{at}snu.ac.kr.

	Abstract

Arg-Gly-Asp (RGD) derivatives have been labeled with various radioisotopes for the imaging of angiogenesis in ischemic tissue, in which _v3 integrin plays an important role. In this study, cyclic Arg-Gly-Asp-D-Tyr-Lys [c(RGDyK)] was conjugated with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA) and then labeled with ⁶⁸Ga. The labeled RGD so produced was subjected to an in vitro binding assay and in vivo biodistribution and PET studies. Methods: A mixture of SCN-Bz-NOTA (660 nmol) and c(RGDyK) (600 nmol) in 0.1 M sodium carbonate buffer (pH 9.5) was allowed to react for 20 h at room temperature in the dark for thiourea bond formation. The conjugate obtained was purified by semipreparative high-performance liquid chromatography (HPLC). The purified c(RGDyK)–SCN-Bz-NOTA (NOTA-RGD) was then labeled with ⁶⁸Ga from a ⁶⁸Ge/⁶⁸Ga generator and purified by semipreparative HPLC. A competitive binding assay for c(RGDyK) and NOTA-RGD was performed with ¹²⁵I-c(RGDyK) as a radioligand and _v3 integrin–coated plates as a solid phase. ⁶⁸Ga-NOTA-RGD (0.222 MBq/100 µL) was injected, through a tail vein, into mice with hind limb ischemia and into mice bearing human colon cancer SNU-C4 xenografts. Biodistribution and imaging studies were performed at 1 and 2 h after injection. Results: The labeling of NOTA-RGD with ⁶⁸Ga was straightforward. The K_i values of c(RGDyK) and NOTA-RGD were 1.3 and 1.9 nM, respectively. In the biodistribution study, the mean ± SD uptake of ⁶⁸Ga-NOTA-RGD by ischemic muscles was 1.6 ± 0.2 percentage injected dose per gram (%ID/g); this uptake was significantly blocked by cold c(RGDyK) to 0.6 ± 0.3 %ID/g (P < 0.01). Tumor uptake was 5.1 ± 1.0 %ID/g, and the tumor-to-blood ratio was 10.3 ± 4.8. Small-animal PET revealed rapid excretion through the urine and high levels of tumor and kidney uptake. Conclusion: Stable ⁶⁸Ga-NOTA-RGD was obtained in a straightforward manner at a high yield and showed a high affinity for _v3 integrin, specific uptake by angiogenic muscles, a high level of uptake by tumors, and rapid renal excretion. ⁶⁸Ga-NOTA-RGD was found to be a promising radioligand for the imaging of angiogenesis.

Key Words: integrin, NOTA, gallium, peptide, ischemia, DOTA

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