Synthesis and characterization of antimicrobial peptide fragment derived from human lysozyme, its radiolabeling with 68Ga and evaluation as an infection imaging PET probe.

1081 Objectives: Antimicrobial peptide RAWVAWR-NH2, a human lysozyme derivative is known to have antimicrobial activity against gram positive and gram negative bacteria. In this study, RAWVAWR-NH2 was synthesised, characterized and radiolabeled with 68Ga using DOTA conjugation. Further, a detailed evaluation of 68Ga-DOTA-RAWVAWR-NH2 was done through a battery of in vitro quality control tests and in vivo animal studies for its suitability as a human infection imaging agent. Methods: DOTA-RAWVAWR-NH2 was synthesised by solid phase peptide synthesis. Purification and characterization was done using HPLC and ESI-MS. For radiolabelling, 25.0µL (stock solution: 1.0 mM/mL) of DOTA-RAWVAWR-NH2 was added to 800.0µL of sodium acetate buffer (pH 4.0) followed by addition of 1.00 mL (185-200 MBq) of freshly eluted 68Ga activity. The solution was heated for 30 min at 95oC. 68Ga-DOTA-RAWVAWR-NH2 was purified using C-8 SPE light cartridge and subjected to quality control tests. Biodistribution studies were performed in female Wistar rats (n=15) by injecting 3.7 MBq (i.v.) of radiotracer. Infection models were developed by injecting 2×106CFUs of S.aureus into the right thigh muscle of male Balb/c mice (n=15). Sterile inflammation was developed in left thigh muscle of male Balb/c mice by injecting 50.0µL of turpentine oil. Animals were sacrificed after injecting (i.v.) 3.7 MBq of radiotracer at different time points. PET imaging (Inveon, Siemens, Animal PET scanner) was performed in normal Balb/c mice following i.v. injection of 20.0-25.0 MBq of radiotracer. Results: The HPLC data demonstrated retention time tr= 6.4 min for DOTA-RAWVAWR-NH2 (40%-60% gradient in 20 min). The ESI-MS analysis demonstrated the m/z ratios of 665.9 ([M+2H]2+) and 1329.9 ([M+H]+) corresponding to molecular mass of 1328 Da. The radiolabeling efficiency of 68Ga-DOTA-RAWVAWR-NH2 was found to be 91.75% which remained stable up to 3 h. RCP was found to be 96.48%. Rf values for radiotracer were found to be 0.6, 0.1 and 0.9 using 50.0% CH3CN/TLC-SG, 0.1 M sodium citrate/TLC-SG and 1.0 M ammonium acetate and CH3OH (1:1) solvent systems respectively. The plasma protein binding and lipophilicity of radiotracer were 40.98% and -0.81 respectively. Normal biodistribution showed an initial uptake in blood, heart, lung, liver and small intestine that decreased by 120 min. Maximum uptake of the radiotracer was seen in kidneys at 60 and 120 indicating renal mode of excretion. The initial organ uptake in infection/inflammation models was similar to that in normal animals. The uptake was highest in infection site at 45 min. The initial uptake of radiotracer in inflammation site was more as compared to that in infection site but there was a rapid clearance of radiotracer from the inflammation site with time. PET imaging demonstrated intense uptake in kidneys with excretion into the urinary bladder, moderate grade uptake in the liver, salivary glands, blood pool and bone marrow and minimal uptake in the muscles. Conclusions: A successful radiolabeling of 68Ga with RAWVAWR-NH2 was achieved. The normal biodistribution pattern of 68Ga labeled peptide was in agreement with PET imaging data. The biodistribution studies and PET imaging indicated renal mode of excretion for the radiotracer. A significant uptake of the radiotracer at infection site and its slow clearance is viewed as having good potential as an infection imaging agent. However, the potential of this radiotracer for infection imaging needs further preclinical validation before proving its translational relevance.