Synthesis of DOTA conjugated GF-17 and RAWVAWR-NH2 and radiolabeling with 68Ga as a potential PET tracer for infection imaging

1115 Objectives GF-17 is a peptide fragment of human cathelicidin LL-37 which has antimicrobial activity against gram negative organism E.coli. RAWVAWR-NH2 is the peptide sequence of human lysozyme and active against E.coli and S.aureus. The objective of the present study was to synthesize antimicrobial peptide fragments GF-17and RAWVAWR-NH2, their conjugation with bifunctional chelator DOTA and radiolabeling of the newly synthesised peptide conjugates with gallium-68 (68Ga) and its preclinical evaluation as a potential PET tracer for infection imaging. Methods The peptide fragments were GF-17 and RAWVAWR-NH2 and were synthesised in-house by solid phase peptide synthesis. DOTA was coupled to GF-17 in solution phase and conjugated to RAWVAWR-NH2 by solid phase conjugation. The peptide conjugates were purified by HPLC and characterized by mass spectroscopy. Radiolabeling with 68Ga was standardized by varying peptide conjugate concentration, buffer pH, reaction time. DOTA-peptide conjugates (5-30nmol) were radiolabeled with 68Ga in 800.0 µL of sodium acetate:acetic acid buffer (pH=3.5-5) by heating at 95°C for different time periods. The radiolabeled compounds were purified by using C8 SPE cartridge. Biodistribution of radiolabeled peptide conjugates was done in female Wistar rats. A dose of 100µCi of radiolabeled peptide conjugate was injected in tail veil of each animal. Animals (n=5 for each time point) were sacrificed at each time interval of 15 min, 30 min, 45 min, 60 min, 120 min post injection. Results The mass spectroscopy analysis demonstrated that the molecular masses of DOTA-GF-17, NOTA-GF-17 and DOTA-RAWVAWR-NH2 were 2489.2 (calculated mass 2487.44), 2388.2 (calculated mass 2386.40) and 1329.9 (calculated mass 1328.71) respectively. The radiolabeling efficiencies for 68Ga-DOTA-GF-17 and 68Ga-DOTA-RAWVAWR-NH2 were evaluated and were found to be 95.0% and 96.0 % respectively at peptide conjugate concentration of 20nmol, buffer pH 4 and reaction time of 25 min. The radiolabeled products remained stable for up to 3h. Rf value for both the compounds was 0.1 (Stationary phase: TLC SG, mobile phase: 0.1M sodium citrate). The plasma protein binding and lipophilicity for 68Ga-DOTA-GF-17 were 80.98% and -3.12; and that for 68Ga-DOTA-RAWVAWR-NH2 were 40.98% and -0.81 respectively. Normal biodistribution studies showed an initial uptake 68Ga-DOTA-GF-17 in blood, heart, thyroid, lung, liver and small intestine which decreased by 120 min. Data showed maximum uptake in kidneys at 15 and 120 min that indicates the mode of excretion is renal. Likewise maximum renal uptake of 68Ga-DOTA-RAWVAWR-NH2 was at 60 min and minimum at 120 min suggesting renal mode of excretion. Conclusions A successful radiolabeling of 68Ga with GF-17 & RAWVAWR-NH2 was achieved. The normal biodistribution pattern of 68Ga labeled peptides in bone, muscles and fast clearance from liver may be useful for targeting of bacterial infections in these areas. However, the potential of these radiopharmaceuticals for infection imaging requires further pre-clinical validation before translation to clinical use.