Summary                         

APJ, a G-protein coupled receptor and apelin, its endogenous ligand is involved in angiogenezis. It 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 specifcally 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. Specifcity 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 quantifcation of signal in organs showed a suitable pharmacokinetic profle 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 signifcantly 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 signifcantly superior over the 21-day follow-up. A signifcant, positive correlation was found between the [⁶⁸Ga]Ga-AP747 PET signal on day 7 and late hindlimb perfusion on day 21. In this study, a new PET radiotracer that specifcally binds to APJ, [⁶⁸Ga]Ga-AP747 was developed. It showed more efficient imaging properties than the most clinically advanced tracer of angiogenesis, [⁶⁸Ga]Ga-RGD₂.

Results from nanoScan® PET/CT

[⁶⁸Ga]Ga‑AP747 biodistribution in healthy mice:

Nine-week-old male Swiss mice (n = 3) were injected in the lateral caudal vein with [⁶⁸Ga]Ga-AP747 (4.45 ± 0.32 MBq/70 μL), and small animal PET images were continuously acquired right after, up to 2 h post-injection. The quantified PET signal in organs was presented as mean ± SD percentage of the decay-corrected injected dose (%ID). Acquisition of small animal dynamic PET/CT was performed for 120 min on a NanoScan PET/CT camera (Mediso, Budapest, Hungary) under 2% isoflurane in medical air anesthesia [PET parameters: numbers of iterations: 4, coincidence: 1–3, field of view (FOV): 10 cm]. CT parameters were fixed at 35 kV voltage, 300 ms exposure at medium zoom, acquired by semi-circular method on the same FOV as PET. CT attenuation-corrected reconstruction was performed using Nucline software (Mediso, Budapest, Hungary) on the following time frames: 0–5 min, 6–10 min, 11–15 min, 16–20 min, 21–25 min, 26–30 min, 31–45 min, 46–60 min, 61–75 min, 76–90 min, 91–105 min, and 106–120 min. Quantitative volume-of-interest (VOI) analysis of the small animal PET images was CT-based manually performed on attenuation- and decay-corrected PET images using VivoQuant software (v.3.5, InVicro, Boston, USA).

Three 9-week-old Swiss male mice were injected in the lateral caudal vein with [⁶⁸Ga]Ga-AP747 (4.02 ± 0.16 MBq/70 μL) and maintained under isoflurane anesthesia (2%) for two hours. Blood was collected at 2, 5, 10, 15, 20, 30, 45, 60, 75, 90, 105, and 120 min post-injection and gamma counted with decay correction. Plasmatic half-life (t1/2) was estimated by nonlinear regression. At two hours post-injection, mice were euthanatized and the main organs (heart, liver, lungs, muscle, brain, spleen, intestines, bone, pancreas, and kidneys) were collected, washed in PBS, weighted, and gamma counted. Results were decay corrected and expressed as percentage of injected dose corrected by organ weight (%ID/g).

In vivo specificity of [⁶⁸Ga]Ga‑AP747 PET signal:

Mice bearing ectopic colon adenocarcinoma xenograft (n = 3, 1370±167.3 mm³) or Matrigel plug (n = 5, 635 ± 146 mm³) were injected in the lateral caudal vein with [⁶⁸Ga] Ga-AP747 (5.14 ± 0.60 MBq/80 μL) and underwent small animal PET imaging acquired 1 h p.i. followed by a CT scan. Small animal PET imaging acquisition lasted 20 min (number of iterations: 4, coincidence: 1–3) using a field of view (FOV) of 10 cm. CT parameters were fixed at 35 kV voltage, 300 ms exposure at medium zoom, acquired by semi-circular method on the same FOV as PET. CT attenuation-corrected reconstruction was performed using Nucline software (Mediso, Budapest, Hungary). The day after, the mice received an intravenous injection of a 100X excess of unconjugated peptide (apelin-F13A, 100 μg/100 μL) 30 min before the intravenous injection of 5.5 ± 0.20 MBq/80 μL [⁶⁸Ga]Ga-AP747. PET images were acquired 1 h after [⁶⁸Ga] Ga-AP747 injection. Tissue uptake values were expressed as a mean target-to-background PET signal ratio (TBRmean) with background represented by the left gastrocnemius muscle.

In vivo longitudinal study using [⁶⁸Ga]Ga‑AP747 PET compared with [⁶⁸Ga]Ga‑RGD PET₂:

Matrigel plug mouse model (n = 7) and HLI mice (n = 8) were injected in the lateral caudal vein with [⁶⁸Ga] Ga-AP747 (5.66 ± 0.57 MBq) and 11 h later with [⁶⁸Ga] Ga-RGD₂ (5.96 ± 0.95 MBq) on days 1, 3, 7, 10, 13, and 21 after ischemia (Fig. 1). Static PET images were acquired 1 h after intravenous injection of [⁶⁸Ga]Ga-AP747 or [⁶⁸Ga] Ga-RGD₂ and followed by a CT. Small animal PET imaging acquisition lasted 20 min (numbers of iterations: 4, coincidence: 1–3) using a field of view (FOV) of 10 cm. CT parameters were fixed at 35 kV voltage, 300 ms exposure at medium zoom, acquired by semi-circular method on the same FOV as PET. CT attenuation-corrected reconstruction was performed using Nucline software (Mediso, Budapest, Hungary). Tissue uptake values were expressed as a targetto-background (Matrigel-to-muscle) or as an ischemic-tocontralateral hindlimb PET signal ratio.

Results show:

• In healthy mice, the highest [⁶⁸Ga]Ga-AP747 dynamic small animal PET signal was quantified at 120 min in the bladder and in the kidneys without noticeable accumulation in the liver, lungs, or brain (Fig. 4a, b). Ex vivo gamma counting confirmed these results (Fig. 4d). Plasmatic half-life of [⁶⁸Ga]Ga-AP747 was estimated at 13.3 min (Fig. 4c).

• In Matrigel mice from day 1 to day 21, the growing target-to-background [⁶⁸Ga]Ga-AP747 small animal PET signal was significantly higher than that of [⁶⁸Ga]Ga-RGD₂ (Fig. 5a, b).

• In HLI mice from day 1 to day 21, the i/c [⁶⁸Ga]Ga-AP747 small animal PET signal ratio was significantly higher than that of [⁶⁸Ga]Ga-RGD₂, especially on day 7 (Fig. 6a, b).

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