A dual-labeled octreotide analog for fluorescence-guided surgery

Objectives Dual-labeled agents possessing radioactive and fluorescent reporters have the potential to provide preoperative nuclear imaging for surgical planning and real-time fluorescence-guided surgery with a single agent. Ideally, converting a clinical radiotracer into its hybrid counterpart could extend PET findings into the operating room and improve surgical outcomes. However, prior attempts with proven PET tracers (e.g., glucose and octreotide analogs) have yet to produce an agent with translational utility primarily due to technical challenges in agent design that occur from the attachment of large molecular weight fluorescent dyes to targeting agents of similar or even smaller size. To overcome this, we have developed a customized multimodality chelator (MMC) scaffold that permits dual labeling of Tyr3-octreotide (TOC) with IRDye800 and 64Cu for multimodal imaging of somatostatin receptor (SSTR)-expressing tumors. Here, we describe the synthesis and preliminary evaluation of the hybrid SSTR-targeted agent, 64Cu-MMC(NIR)-TOC, and demonstrate the importance of the MMC in maintaining bioactivity.

Methods To synthesize the MMC, a DOTA mimetic was functionalized with two pendant arms containing different reactive groups. MMC was then conjugated to TOC on solid-phase, and the resulting peptide conjugate was fluorescently labeled with IRDye800 via copper-free click chemistry in solution to afford MMC(NIR)-TOC. For comparison, a second peptide conjugate was produced without the MMC scaffold by sequential conjugation of an azide-containing amino acid and DOTA to the N-terminus of TOC, and then labeled with IRDye to yield DA(NIR)-TOC. 64Cu labeling methods were optimized and used to prepare cold-labeled agents. Potency assays in SSTR2-transfected human embryonic kidney (HEK)-293 cells were performed to measure the ability of the agents to inhibit cyclic adenosine monophosphate (cAMP). Uptake and competition studies with 64Cu-MMC(NIR)-TOC were conducted in IMR-32 neuroblastoma cells and compared to 68Ga-DOTA-TOC and 64Cu-DA(NIR)-TOC. PET/CT imaging of 64Cu-MMC(NIR)-TOC was performed in normal mice to examine biodistribution and clearance.

Results MMC-TOC was produced using standard solid-phase techniques and dual labeling was confirmed by HPLC identification of the corresponding UV, fluorescent, and radiometric peaks. cAMP inhibition studies revealed an EC50 value of 0.125±0.034 nM (mean ±SEM) for natCu-MMC(NIR)-TOC, which was only slightly higher than that for octreotide (EC50 0.027±0.005) nM) and significantly lower than for natCu-DA(NIR)-TOC (EC50 >1 nM), confirming retention of agonistic properties with the MMC approach (Figure). These findings were consistent with the radioactive uptake studies which showed nearly 6-fold higher uptake for the MMC agent. Competitive binding in the presence octreotide caused a dose-dependent reduction in 64Cu-MMC(NIR)-TOC and 68Ga-DOTA-TOC binding, while 64Cu-DA(NIR)-TOC binding was not affected. This suggests a loss of SSTR2 specificity for 64Cu-DA(NIR)-TOC which may be attributed to steric effects of IRDye800 on the pharmacophore of the peptide. PET/CT imaging showed that 64Cu-MMC(NIR)-TOC was cleared primarily by the liver, along with notable renal uptake and low signal in other tissues.

Conclusions Agent design is critical in the development of a dual-labeled octreotide analog. These findings show that customized dual labeling with the MMC produces a compound that retains the binding and agonistic properties of the original peptide, and may offer significant advantages over conventional methods for dual labeling. Further characterization of 64Cu-MMC(NIR)-TOC in tumor models is warranted to examine its utility for multimodal imaging. Research Support: This work was supported by NIH/NIBIB R01EB017279 (Azhdarinia).