Development and characterization of Ga-68 labelled antimicrobial peptides as potential PET probes for the detection of human bacterial infections - a pre-clinical study

Antimicrobial peptides (AMPs) have a unique property to kill microorganisms and are seen as potential substitute for the conventional antibiotics. As a part of the innate host immunity and unlike antibiotics, bacteria do not develop any resistance against them. Therefore, radiolabelling of AMPs with positron emitters (⁶⁸Ga) may provide specific molecular probes for the accurate detection of active bacterial human infections. The aim of the present study was to synthesize and characterize DOTA-AMP conjugates, standardization of ⁶⁸Ga radiolabelling conditions and pre-clinical evaluation by using small animal PET imaging. The peptide fragments GF-17 and RAWVAWR-NH₂ (active against both gram positive and negative bacteria) were synthesised by solid phase peptide synthesis. DOTA coupling for GF-17 and RAWVAWR-NH₂ was done in solution phase and solid phase respectively. Purification and characterization were done by HPLC and mass spectroscopy. DOTA-peptide conjugates (20.0 μL; 1 mmol/mL) were radiolabelled with ⁶⁸Ga in 800.0 μL of sodium acetate:acetic acid buffer (pH 4) by heating at temperature of 95°C for approximately 25.0 min. The radiolabelled compounds were purified by using C8 SPE cartridge. Quality control tests were performed for⁶⁸Ga-DOTA-GF-17 and ⁶⁸Ga-DOTA-RAWVAWR-NH₂. PET imaging (Inveon, Siemens, Animal PET scanner) was performed in normal Balb/c mice following i.v. administration of 20.0-25.0 MBq of each of the ⁶⁸Ga-labeled peptides. Results: The mass spectroscopy analysis demonstrated that the molecular masses of DOTA-GF-17, and DOTA-RAWVAWR-NH₂ were 2487.2 (calculated mass 2487.44) and 1328.9 (calculated mass 1328.71) respectively. The radiolabelling efficiencies for ⁶⁸Ga-DOTA-GF-17 and ⁶⁸Ga-DOTA-RAWVAWR-NH₂ were found to be 95.0% and 96.0 % respectively and remained stable for up to 3h. 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. Rf value for both the compounds was 0.1 ( Stationary phase: TLC SG, mobile phase: 0.1M sodium citrate). ⁶⁸Ga-DOTA-GF-17 PET dynamic images (90 min) showed initial uptake in heart, liver, kidneys and low grade uptake in bone marrow and muscles. There was significant clearance of tracer from the heart and liver. However, the clearance from the kidneys was minimal. ⁶⁸Ga- DOTA-RAWVAWR-NH₂ PET static image (90 min) demonstrated intense uptake of the tracer 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. A successful radiolabelling of ⁶⁸Ga with GF-17 and RAWVAWR-NH2 was achieved. The normal bio-distribution pattern of ⁶⁸Ga labelled 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. (author)