Developing pet infection imaging agent for visualizing bacterial peptidoglycan assembly

Abstract

Diagnosis of bacterial infections remains a serious medical challenge making them one of the main causes of mortality and morbidity worldwide. A nuclear imaging modality such as Positron Emission Tomography – Computed Tomography (PET/CT) scanning has been considered one of the better diagnostic alternatives due to its ability to assess anatomical and physiological abnormalities in real-time. Despite excellent pre-clinical studies, specific radiotracers capable of discriminating infection from sterile inflammation or detecting infective foci, especially in the early phases or in deeply seated tissue are lacking in clinical practice. For this reason, the uniqueness of the peptidoglycan cell wall presents an attractive target to improve the specificity and selectivity of PET radiotracers with the potential to identify the causative pathogen (gram-negative vs gram-positive). Many studies have demonstrated the inventiveness of peptidoglycan targeting and imaging using amino acids and glycan core-based radiotracers, however, their prospects in clinical settings are still under development. As part of the study objectives, diagnostic accuracy, and clinical impact of currently available peptidoglycan-based radiotracers for imaging of bacterial infections were evaluated using QUADAS. The findings revealed that most radiotracers demonstrated high non-target background signals, which might limit their specificity and sensitivity for diagnostic imaging of bacterial infections. Therefore, this warrants further research and development of target-specific radiotracers for infection imaging. The AeK tripeptide is considered an essential building block of peptidoglycan required by bacteria to proliferate as previously shown by fluorescence imaging in vitro using AeK-NBD. In this study, we developed AeK tripeptide radiotracer by replacing the fluorescent tag (NBD) with DOTA chelator to warrant 68Ga-complexation without altering the peptide chain. Given the structural similarity to Aek tripeptide, bacterial uptake of [68Ga]Ga-DOTA-AeK was investigated in vitro and in vivo for potential infection imaging using PET/CT. An easy-to-implement radiolabeling procedure and SPE purification method were successfully developed for [68Ga]Ga-DOTA-AeK with RCP≥95 %. The radiotracer showed proteolytic stability with limited protein binding. Unlike AeK-NBD, [68Ga]Ga-DOTA-AeK was not successfully taken up by both E .coli (gram-negative) and S.aureus (gram-positive) bacteria in vitro and in vivo, ultimately hindering metabolic integration into the peptidoglycan cell wall. Interestingly, the biodistribution data further revealed inflammatory localization of radiotracer, however, whether this may or may not limit the specificity of AeK tripeptide as a potential radiotracer, needs further investigation. Based on these findings, it was concluded that the addition of the DOTA hindered the active intracellular transportation of [68Ga]Ga-DOTA-AeK necessary for ultimate metabolic processing and integration into the peptidoglycan. Therefore, alternative radiolabeling strategies without the need for radioisotope chelation, eg. 11C and 18F are recommended to maintain the targeting moiety of AeK tripeptide.