Design and preclinical evaluation of a novel apelin-based PET radiotracer targeting APJ receptor for molecular imaging of angiogenesis

Abstract

APJ has been extensively described in the pathophysiology of angiogenesis and cell proliferation. The prognostic value of APJ overexpression in many diseases is now established. This study aimed to design a PET radiotracer that specifically binds to APJ. Apelin-F13A-NODAGA (AP747) was synthesized and radiolabeled with gallium-68 ([⁶⁸Ga]Ga-AP747). Radiolabeling purity was excellent (> 95%) and stable up to 2 h. Affinity constant of [⁶⁷Ga]Ga-AP747 was measured on APJ-overexpressing colon adenocarcinoma cells and was in nanomolar range. Specificity of [⁶⁸Ga]Ga-AP747 for APJ was evaluated in vitro by autoradiography and in vivo by small animal PET/CT in both colon adenocarcinoma mouse model and Matrigel plug mouse model. Dynamic of [⁶⁸Ga]Ga-AP747 PET/CT biodistributions was realized on healthy mice and pigs for two hours, and quantification of signal in organs showed a suitable pharmacokinetic profile for PET imaging, largely excreted by urinary route. Matrigel mice and hindlimb ischemic mice were submitted to a 21-day longitudinal follow-up with [⁶⁸Ga]Ga-AP747 and [⁶⁸Ga]Ga-RGD₂ small animal PET/CT. [⁶⁸Ga]Ga-AP747 PET signal in Matrigel was significantly more intense than that of [⁶⁸Ga]Ga-RGD₂. Revascularization of the ischemic hind limb was followed by LASER Doppler. In the hindlimb, [⁶⁸Ga]Ga-AP747 PET signal was more than twice higher than that of [⁶⁸Ga]Ga-RGD₂ on day 7, and significantly superior over the 21-day follow-up. A significant, positive correlation was found between the [⁶⁸Ga]Ga-AP747 PET signal on day 7 and late hindlimb perfusion on day 21. We developed a new PET radiotracer that specifically binds to APJ, [⁶⁸Ga]Ga-AP747 that showed more efficient imaging properties than the most clinically advanced tracer of angiogenesis, [⁶⁸Ga]Ga-RGD₂.

Keywords: APJ; Angiogenesis; Apelin; PET imaging; Theranostics.

Fig. 1

In vivo longitudinal study using [⁶⁸Ga]Ga-AP747 PET compared with [⁶⁸Ga]Ga-RGD₂ PET on Matrigel and HLI mouse models

Fig. 2

ESI (+) MS spectrum showing the different doubly charged adducts of AP747 (insert: calculated isotopic pattern of [M + 2H]2 + species) (a). [⁶⁸ Ga]Ga-AP747 radiochemical purity and stability in physiological serum (b) and in human serum (c) for two hours

Fig. 3

Semi-quantifications of APJ-to-GAPDH expression ratios by Western blot on human adenocarcinoma T84 cells and HUVECs with or without TNF-alpha activation (a). Saturation binding curve of [⁶⁷Ga]Ga-AP747 towards APJ receptor on T84 cells (b). Representative maximum intensity projections (MIP) images of small animal [⁶⁸Ga]Ga-AP747 PET/CT in an ectopic mice model of human colon adenocarcinoma, in baseline or APJ-blocking conditions (c). Representative MIP of small animal [⁶⁸Ga]Ga-AP747 PET/CT images of in a Matrigel plug mice model, in baseline or APJ-blocking conditions (d). [⁶⁸Ga]Ga-AP747 PET signal quantifications in a mouse model of ectopic human colon adenocarcinoma in baseline or APJ-blocking conditions (n = 3) (e). [⁶⁸Ga]Ga-AP747 PET signal quantifications in a Matrigel plug mouse model in baseline or APJ-blocking conditions (n = 5) (f). c/b standing for cell-to-background; t/b standing for tumor-to-background, mean. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 4

Representative MIP PET images of [⁶⁸Ga]Ga-AP747 biodistribution (a) and associated organ quantifications (b) in healthy mice (n = 3). Blood kinetics of [⁶⁸Ga]Ga-AP747 during 120 min (c) and ex vivo gamma counting at 120 min post-injection (d) in healthy mice (n = 3). Representative [⁶⁸Ga]Ga-AP747 biodistribution MIP PET images (e) and associated organ PET quantifications (f) in healthy swine (n = 3)

Fig. 5

Representative [⁶⁸Ga]Ga-RGD₂ PET and [⁶⁸Ga]Ga-AP747 MIP PET images (a) and related quantifications (b) in the Matrigel mouse model over 21 days (n = 7). m/m standing for Matrigel-to-muscle. *P ≤ 0.05; **P ≤ 0.01

Fig. 6

[⁶⁸Ga]Ga-RGD₂ and [⁶⁸Ga]Ga-AP747 in a hindlimb ischemia model representative MIP PET images a [⁶⁸Ga]Ga-RGD₂ small animal PET/CT and [⁶⁸ Ga]Ga-AP747 small animal PET/CT quantifications (n = 8) b. LASER Doppler quantifications of hindlimb blood perfusion c and correlation studies (d, e). Representative images of anti-APJ immunofluorescence on day 7 in contralateral hindlimb and ischemic hindlimb of an HLI mouse (f). i/c standing for ipsi-to-contralateral. D standing for day. *P < 0.05