Indocyanine Green Derived Dual-modality Agent for Imaging-guided Therapy of Hepatocellular Carcinomas

1171 Objectives: Indocyanine green (ICG), a near-infrared (NIR) and US-FDA-approved agent, has been reported for high local spatial and temporal resolution imaging of hepatocellular carcinomas (HCC)1. However, the limited penetration depth of NIR fluorescence imaging technology hampers the clinical application of ICG for noninvasive HCC assessment. To overcome this limitation, we designed and synthesized an ICG analogue to enable positron emission tomography (PET) imaging when labeled with 64Cu, which was chosen based on the fact that its half-life matches the hepatic clearance of ICG. Such a dual-modality imaging probe may find potential applications in the initial whole-body screening of cancer patients with PET and subsequent intraoperative excision of HCC. Methods: The ICG analogue (ICG-PEG3-CB-TE1A-GA) is composed of a heptamethine cyanine chromophore core, a “cross-bridged” cyclam (1,4,8,11-tetraazabicyclo[6.6.2]-hexadecane-4,11-diyl-8-acetic acid-1-glutaric acid, CB-TE1A-GA) for 64Cu labeling, and a tetra(ethylene glycol) diamine (NH2-PEG3-NH2) spacer in between. To install a carboxy-functionalized aryl moiety onto the heptamethine cyanine chromophore via the meso-chlorine atom, we applied the Suzuki-Miyaura cross-coupling method to generate a robust C-C linkage rather than photochemical labile aryl-ether linkage to enhance the chemical stability. This intermediate was further conjugated with NH2-PEG3-NH2 and CB-TE1A-GA. The CB-TE1A-GA ligand was chosen because it forms an inert complex with 64Cu(II), which is essential to our dual-modality imaging probe design as the transchelation of 64Cu must be avoided in the liver.2 Radiolabeling of ICG-PEG3-CB-TE1A-GA with 64Cu was performed in 0.1M NH4OAc/MeCN (v/v 10/1)at 70oC for 30 min. The radiolabeled complex was purified by a light C18 cartridge and eluted with ethanol. The elution was concentrated by gently blowing positive flow of nitrogen (~20 min) and reconstructed in 5% ethanol, 0.9% NaCl solution to 9.0 MBq/150 µL for mouse iv injection. The reaction mixture and final product were analyzed by analytical radio-HPLC (gradient: 45%-60% MeCN + 0.1% TFA 0-5 min, 60% MeCN +0.1% TFA 5-10 min, Luna C18 column, 1mL/min). For in vitro stability, the radiolabeling product was reconstructed in 5% ethanol, 1x PBS buffer, 0.01 mg/mL ascorbic acid to 18.5 MBq/500 µL, incubated at 37oC, and analyzed by radio-HPLC at 4h, 8h and 24h. For the PET/CT study, healthy Balb/c mice were scanned at 1h, 6h, 24h and 48h post injection to evaluate the in vivo distribution and clearance profile of the agent. Results: The radiolabeling with 64Cu was accomplished with 95% radiochemical yield. The stability test indicated that ICG-PEG3-CB-TE1A-GA-64Cu remained greater than 99%, 93%, 87% and 70% intact at 0h, 4h, 8h and 24h after incubation in PBS buffer at 37oC, respectively, likely due to the optical instability of the chromophore but not dissociation of 64Cufrom ligand. As revealed by PET imaging, the radioactivity remained nearly constant (18-21%ID/g) in the liver along with a gradual decrease in the kidney (8.8 - 4.7%ID/g) out to 48 h post-injection. The clearance of the agent was through urine and feces. Low (< 1.4%ID/g) uptake was observed in other organs and tissues, such as brain, bone, joint, and muscle. Conclusions: We designed and successfully synthesized a 64Cu-labeled PET/NIR dual-modality agent derived from ICG. Preliminary imaging evaluation of the agent showed an in vivo uptake and clearance profile similar to ICG in mice. Studies using orthotopic HCC tumor models are ongoing. Research Support: This project was partially supported by a grant from the Cancer Prevention and Research Institute of Texas (RP110771) and the Dr. Jack Krohmer Professorship Funds. Reference:1. Ishizawa, T., et. al., Cancer 2009,115 (11), 2491-2504. 2. Sun, X., et. al., Journal of Medicinal Chemistry 2002,45 (2), 469-477.