Synthesis, radiolabelling and preliminary in vitro assessment of novel radiopharmaceuticals as potential bacterial-specific radiotracers using positron emission tomography imaging

Background: The current positron emission tomography (PET)-radiopharmaceuticals used for diagnosis of bacterial infections rely on secondary host-mediated inflammatory responses, leading to false-positive findings because of misinterpreted, sterile inflammation. To discriminate between infection and inflammation, a more direct targeting mechanism for bacteria is desirable. Therefore, the development of bacteria- selective PET-radiotracers is a valid approach to improve differential diagnosis of bacterial infections and monitoring the performance of treatment interventions using non-invasive nuclear imaging. In this study three candidate molecules with known bacterial-specific interaction were deemed appropriate radiotracer vector scaffolds when functionalised with 1,4,7-triazacyclononane-1-succinic acid- 4,7-diacetic acid (NODASA) to subsequently allow for gallium-68 complexation: (1) NODASA-D-lysine (NDL) and two Mycobacterium tuberculosis (MTB)-specific phage display generated peptides also known as (2) NODASA-PH1 and (3) NODASA-H8. Methods: Solid phase peptide synthesis (SPPS) was used to synthesise these compounds, including NODASA functionalised L-lysine (Nell; negative control), followed by purification using reverse-phase preparative high-performance liquid chromatography (HPLC), and characterisation using Harms/L CMS. Compounds were radiolabelled with generator eluted 68Ga-activity, the radiosynthesis was optimised by adjusting reaction conditions such as pH, temperature and NODASA-compound concentration. Radiolabelling efficiency, radiochemical yield and radiochemical purity was assessed with radio-HPLC or TLC. The radio-metal complex stability was assessed using the EDTA trans-chelation challenge. In vitro studies included quantification of plasma stability, blood cell association, and plasma-protein binding assays to characterise the compound stability, radioisotope integrity and estimated bioavailability following intravenous administration into human blood as a potential PET radiotracer. Results: NDL, NLL, H8 and PH1 were successfully synthesised and conjugated to NODASA, purified (> 95%) and characterised. Compounds were subsequently radiolabelled with gallium-68, optimal labelling conditions were explored to produce radiochemically pure products (> 99%) including suitable radiochemical yields and activity concentrations for future preclinical applications. All radiometal complexations demonstrated good integrity when challenged by 1000-times excess of EDTA (> 90%). [68Ga]Ga-PH1, [68Ga]Ga-NDL and [68Ga]Ga-NLL were stable for up to 2 h (> 90%) in human plasma whereas 68Ga-H8 degraded rapidly (< 40% remained intact after 30 min). All compounds showed minimal RBC interaction (< 10%) and plasma protein binding ([68Ga]Ga- NDL/-NLL < 14%; [68Ga]Ga-PH1/-H8 < 9%), thereby mimicking a favourable expected bioavailability. Conclusion: Following successful radiolabelling and in vitro characterisation, [68Ga]Ga-NDL and [68Ga]Ga-PH1 are deemed promising candidate PET-radiotracers. Upcoming bacterial cell uptake assays and micro positron emission tomography/computed tomography imaging studies in mice will further assess the potential of these compounds as bacterial-specific ([68Ga]Ga-NDL), or even MTB-specific ([68Ga]Ga-PH1) radiotracers. (authors)