Preclinical Melanoma Imaging with ⁶⁸Ga-Labeled α-Melanocyte-Stimulating Hormone Derivatives Using PET

Abstract

It is estimated that melanoma accounted for 76,380 new cases and 10,130 deaths in the United States in 2016. The melanocortin 1 receptor (MC1R) is highly expressed in the vast majority of melanomas, which makes it an attractive target for molecular imaging and radionuclide therapy. Lactam bridge-cyclized α-melanocyte-stimulating hormone (Ac-Nle4-cyclo[Asp5-His-D-Phe7-Arg-Trp-Lys10]-NH2, or Nle-CycMSHhex) analogues have been successfully developed and studied for MC1R-targeted imaging, predominantly with single-photon emission computed tomography (SPECT). The goal of this study was to design and evaluate novel peptides for melanoma imaging with positron emission tomography (PET). We designed and synthesized three peptides, DOTA-PEG2-Nle-CycMSHhex (CCZ01047), DOTA-4-amino-(1-carboxymethyl) piperidine (Pip)-Nle-CycMSHhex (CCZ01048), and DOTA-Pip-Pip-Nle-CycMSHhex (CCZ01056). All three peptides exhibited high binding affinity to MC1R with sub-nanomolar Ki values, rapid internalization into B16F10 melanoma cells and high in vivo stability with more than 93% remaining intact at 15 min post-injection (p.i.) in blood plasma. All three 68Ga-labeled tracers produced high contrast PET images in C57BL/6J mice bearing B16F10 tumors, and their respective tumor uptakes were 8.0 ± 3.0, 12.3 ± 3.3, and 6.5 ± 1.4 %ID/g at 1 h p.i. Minimal normal organ activity was observed at 1 h p.i., except for kidneys (5.1 ± 1.4, 4.7 ± 0.5, and 6.2 ± 2.0 %ID/g, respectively), and thyroid (4.1 ± 0.6 %ID/g for CCZ01047 and 2.4 ± 0.6 %ID/g for CCZ01048). Due to high accumulation at tumor sites and rapid background clearance of 68Ga-CCZ01048, we further evaluated it at 2 h p.i., and a tumor uptake of 21.9 ± 4.6 %ID/g was observed, with background activity further decreased. Exceptional image contrast was also achieved, i.e. tumor-to-blood, tumor-to-muscle, tumor-to-bone and tumor-to-kidney ratios were 96.4 ± 13.9, 210.9 ± 20.9, 39.6 ± 11.9 and 4.0 ± 0.9, respectively. A blocking study was also performed by co-injection of excess amount of non-radioactive Ga-coupled of CCZ01048, which confirmed that the tumor uptake was MC1R mediated. In conclusion, the introduction of a cationic Pip linker to Nle-CycMSHhex, CCZ01048, not only improved tumor uptake, but also generated high tumor-to-normal tissue contrast with PET imaging in a preclinical melanoma model. Therefore, CCZ01048 is a promising candidate for PET imaging of melanoma, and potentially as a theranostic agent for radionuclide therapy of melanoma when labeled with α or β emitters.

Synthesis and Evaluation of a Macrocyclic Actinium‐225 Chelator, Quality Control and In Vivo Evaluation of 225Ac‐crown‐αMSH Peptide

“…Ideally, Ac‐radiopharmaceuticals should have low normal tissue uptake and fast tumor internalization to help mitigate any cytotoxicity induced by irradiation of healthy tissue, and ensure alpha‐emitting daughter radionuclides released from the targeting vector are contained inside the tumors. An α‐melanocyte‐stimulating hormone (αMSH) derivative, CCZ01048, designed for MC1R‐targeted melanoma imaging and treatment is a highly promising candidate that has been shown to exhibit rapid tumor uptake and internalization and was chosen for subsequent functionalization and in vivo uptake studies in tumor bearing mice . Late‐stage metastatic melanoma is a deadly disease with low long‐term survival rate even with immunotherapy agents .…”

“…MC1R is specifically expressed in primary and metastatic melanoma with low normal tissue expression . We have extensive experience in developing αMSH‐based radiopharmaceuticals targeting MC1R with [ 68 Ga]Ga‐CCZ01048 and [ 18 F]CCZ01064 for positron emission tomography (PET) imaging in a preclinical model of mouse B16F10 melanoma. We have also evaluated a novel αMSH‐based [ 18 F]CCZ01096 radiotracer in a preclinical model of human melanoma with the SK‐MEL‐1 cell line .…”

Synthesis and Evaluation of [18F]FEtLos and [18F]AMBF3Los as Novel 18F-Labelled Losartan Derivatives for Molecular Imaging of Angiotensin II Type 1 Receptors

“…The in vivo AT 1 R binding specificity of [ 18 F]AMBF 3 Los was evaluated in healthy mice by a dynamic PET scan. Healthy NSG mice were injected in the tail vein with 6 ± 1 MBq (170 ± 38 µCi) of [ 18 F]AMBF 3 Los and a sixty-minute dynamic PET scans was carried out on a µPET/CT scanner (Inveon, Siemens) following procedures reported previously [67]. Alternatively, [ 18 F]AMBF 3 Los was concomitantly injected with the AT 1 R blocker losartan (18 mg/kg) before the dynamic acquisition.…”

“…Kidneys harvested from healthy NSG mice (after the biodistribution studies), were rinsed in PBS and frozen in isopentane/dry-ice bath. Ten µm-thick slides of the kidneys were obtained using a cryostat (Leica, Wetzlar, Germany), and thaw-mounted onto Superfrost Plus microscope slides (Fischerbrand from Thermo Fisher Scientific, Toronto, ON, Canada), following general procedures of the lab [67]. Afterwards, kidney slides were exposed to a phosphor screen overnight and imaged on a Typhoon FLA 9500 scanner (GE Healthcare, Chicago, IL, USA).…”

Synthesis of DOTA-Pyridine Chelates for 64Cu Coordination and Radiolabeling of αMSH Peptide

“…We have extensively studied analogues of α-melanocyte-stimulating hormone (αMSH) for MC1R-targeted imaging and therapy. We have developed 68 Ga or 18 F labeled cyclic αMSH (CCZ01048, CCZ01064, CCZ01096) for PET imaging in preclinical models of mouse or human melanoma (Zhang et al 2020;Zhang et al 2019;Zhang et al 2018;Zhang et al 2017). In all cases, tracers achieved good tumor visualization in PET images with excellent tumor-to-normal tissue ratios.…”