Synthesis, gallium labelling and characterization of P04087, a functionalized phosphatidylserine-binding peptide

Abstract

Background: Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis and thrombus. The hexapeptide PGDLSR was described as a selective and high affinity ligand for PS. In this work, we synthesized and evaluated a gallium labelled-PGDLSR peptide as a potential and selective radiopharmaceutical for nuclear imaging. PGDLSR-β-alanine-NODAGA (P04087) was prepared using Fmoc-based synthesis and then chelated with cold gallium, ⁶⁸Ga and ⁶⁷Ga. The affinity of Ga-P04087 for PS was evaluated by a competitive binding assay using biotinylated AnnexinV. The in vitro stability of the radiotracer was checked at room temperature and after incubation in human serum at 37 °C with and without a metalloprotease inhibitor. The in vivo binding of ⁶⁷Ga-P04087 to phosphatidylserine was evaluated in a rat model of infective endocarditis.

Results: PGDLSR was successfully prepared with a yield of 31%. P04087 was obtained with a yield of 28% and in high chemical purity (>95%). The radiochemical purities of ⁶⁷Ga-P04087 and ⁶⁸Ga-P04087 exceeded 98% in all cases. IC50 of P04087 and Ga-P04087 were in the same order of magnitude (10^-7M). The radiolabelled product was stable for 24 h at room temperature, but was very rapidly degraded in human serum in the absence of a protease inhibitor, which had a stabilizing effect. No focal uptake could be detected visually in the cardiac area on SPECT images. On autoradiography however, a focal uptake of ⁶⁷Ga-P04087 in the valve area was present and histological slices demonstrated localization of peptide binding at the peripheral layer of vegetations.

Conclusion: In spite of the preservation of the peptide affinity to the PS after its conjugation to the NODAGA chelator, and of the presence of ⁶⁷Ga-P04087 uptake on autoradiography, the absence of detectable foci in vivo in the valve area may be attributed to both the low intensity of the signal and the presence of background activity originating from blood pool and surrounding tissues in the living animals. Further modifications are necessary to design a radiolabeled peptide with higher binding potencies to PS while possessing enhanced metabolic stability in vivo.

Keywords: Gallium; Infective endocarditis; NODAGA; PGDLSR; Peptide; Phosphatidylserine.

Fig. 1

The coupling of R824-β-Alanine and NODAGA

Fig. 2

Labelling of P04087 with natural gallium

Fig. 3

HPLC chromatograms of (1) P04087 (RT = 12.7), (2) Ga-P04087 (RT = 11.7) and (3) a mixture (P04087/Ga-P04087)

Fig. 4

Phosphatidylserine saturation by (a) R824 peptide, (b) the Ga-P04087 tracer. When Annexin V is present without competitor, the signal is considered as reference and is equal 100%

Fig. 5

Radio-ITLC-SG of the ⁶⁷Ga-citrate solution purchased from iba molecular. Mobile phase: 0.4 M sodium acetate pH 4. Rf (67Ga-citrate) = 1

Fig. 6

ITLC-SG of the ⁶⁷Ga-chloride obtained by the elution of ⁶⁷Ga from Sep-Pak SI cartridge using 0.1 M HCl. Mobile phase: 0.4 M sodium acetate pH 4. Rf (⁶⁷GaCl3) = 0, Rf (⁶⁷Ga-citrate) = 1 The same retention profile was obtained when ⁶⁸GaCl³ eluate was analyzed using the same ITLC-SC conditions

Fig. 7

Typical HPLC chromatogram of (1) ^NatGa-P04087 and radio-chromatograms of (2) ⁶⁷Ga-P04087 and (3) ⁶⁸Ga-P04087. (Analytic conditions: column : ACE® 100 Å C18; 150 mm × 4.6 mm, 3 μm particle size. Gradient elution: A) H2O, 0.1% TFA; B) acetonitrile, 0.1% TFA; UV detection at 220 nm; flow: 1 mL/min; 0-2 min isocratic 5% B, 5-35% B linear gradient 20 min, 35-5% B linear gradient 2 min. Retention time (tR) for signal was 11.7 ± 0.07 min

Fig. 8

Degradation kinetics of [Ga]-P04087 in human serum. Gallium radiolabelled P04087 peptide was incubated in human serum at 37 °C in presence and in absence of a metalloprotease inhibitor

Fig. 9

Infective endocarditis in rat. Whole body SPECT/CT acquired 10 minutes after intravenous injection of 67Ga-P04087 showing intense signal in the bladder due to rapid renal elimination. There is no detectable uptake in the heart area

Fig. 10

Infective endocarditis in rat. Comparative analysis of autoradiography (a) and histology (hematoxylin-eosin staining, ×10) of adjacent slices showing localization of 67Ga-P04087 uptake matching with vegetations on aorta root (b) and aorta valve (c), and the absence of uptake in remote myocardium (d)