The diagnosis of invasive pulmonary aspergillosis (IPA) is difficult and lacks specificity and sensitivity. In the pathophysiology of Aspergillus fumigatus, iron plays an essential role as a nutrient during infection. A. fumigatus uses a specific and highly efficient iron uptake mechanism based on iron-complexing ferric ion Fe(III) siderophores, which are a requirement for A. fumigatus virulence. We aimed to evaluate the potential of siderophores radiolabeled with ⁶⁸Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA. Methods: ⁶⁸Ga radiolabeling of the A. fumigatus siderophores desferri-triacetylfusarinine C (TAFC) and desferri-ferricrocin (FC) was performed at high specific activity. Stability, protein binding, and log P values were determined. In vitro uptake in A. fumigatus cultures was tested under varying conditions. Biodistribution was studied in healthy noninfected BALB/c mice, and uptake was studied in a model of A. fumigatus infection using immunosuppressed Lewis rats. Results: High-specific-activity ⁶⁸Ga labeling could be achieved, and resulting complexes were stable in serum, toward diethylenetriaminepentaacetic acid and Fe(III) challenge. Both siderophores showed hydrophilic properties (⁶⁸Ga-TAFC, log P = −2.59; ⁶⁸Ga-FC, log P = −3.17) with low values of protein binding for ⁶⁸Ga-TAFC (<2%). Uptake of both siderophores was highly dependent on the mycelial iron load and could be blocked with an excess (10 μM) of siderophore or NaN₃, indicating specific, energy-dependent uptake. In noninfected mice, ⁶⁸Ga-TAFC showed rapid renal excretion and low blood values (1.6 ± 0.37 percentage injected dose per gram [%ID/g] at 30 min); in urine only intact ⁶⁸Ga-TAFC was detected. In contrast, ⁶⁸Ga-FC revealed high retention in blood (16.1 ± 1.07 %ID/g at 90 min) and rapid metabolism. In the rat IPA model, lung uptake of ⁶⁸Ga-TAFC was dependent on the severity of infection, with less than 0.04 %ID/g in control rats (n = 5) and 0.29 ± 0.11 %ID/g in mildly infected (n = 3) and 0.95 ± 0.37 %ID/g in severely infected (n = 4) rats. PET showed focal accumulation in infected lung tissue. Conclusion: Both siderophores bound ⁶⁸Ga with high affinity, and ⁶⁸Ga-TAFC, especially, showed high stability. ⁶⁸Ga-TAFC displayed highly selective accumulation by A. fumigatus subspecies in vitro and in vivo. The high and specific uptake by A. fumigatus proves the potential of ⁶⁸Ga-labeled siderophores for the specific detection of A. fumigatus during infection. They hold promise as new PET agents for IPA.

This review covers publications on siderophores applied for molecular imaging applications, mainly for radionuclide-based imaging. Siderophores are low molecular weight chelators produced by bacteria and fungi to scavenge essential iron. Research on these molecules has a continuing history over the past 50 years. Many biomedical applications have been developed, most prominently the use of the siderophore desferrioxamine (DFO) to tackle iron overload related diseases. Recent research described the upregulation of siderophore production and transport systems during infection. Replacing iron in siderophores by radionuclides, the most prominent Ga-68 for PET, opens approaches for targeted imaging of infection; the proof of principle has been reported for fungal infections using ⁶⁸Ga-triacetylfusarinine C (TAFC). Additionally, fluorescent siderophores and therapeutic conjugates have been described and may be translated to optical imaging and theranostic applications. Siderophores have also been applied as bifunctional chelators, initially DFO as chelator for Ga-67 and more recently for Zr-89 where it has become the standard chelator in Immuno-PET. Improved DFO constructs and bifunctional chelators based on cyclic siderophores have recently been developed for Ga-68 and Zr-89 and show promising properties for radiopharmaceutical development in PET. A huge potential from basic biomedical research on siderophores still awaits to be utilized for clinical and translational imaging.

Purpose Invasive pulmonary aspergillosis is mainly caused by Aspergillus fumigatus, and is one of the major causes of morbidity and mortality in immunocompromised patients. The mortality associated with invasive pulmonary aspergillosis remains high, mainly due to the difficulties and limitations in diagnosis. We have shown that siderophores can be labelled with ⁶⁸Ga and can be used for PET imaging of A. fumigatus infection in rats. Here we report on the further evaluation of the most promising ⁶⁸Ga-siderophore candidates, triacetylfusarinine (TAFC) and ferrioxamine E (FOXE). Methods Siderophores were labelled with ⁶⁸Ga using acetate buffer. Log P, protein binding and stability values were determined. Uptake by A. fumigatus was studied in vitro in cultures with high and low iron loads. In vivo biodistribution was determined in normal mice and an infection model was established using neutropenic rats inoculated with A. fumigatus. Static and dynamic μPET imaging was performed and correlated with CT images, and lung infection was evaluated ex vivo. Results ⁶⁸Ga-siderophores were labelled with high radiochemical purity and specific activity. ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed high uptake by A. fumigatus in iron-deficient cultures. In normal mice, ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed rapid renal excretion with high metabolic stability. In the rat infection model focal lung uptake was detected by μPET with both compounds and increased with severity of the infection, correlating with abnormal CT images. Conclusion ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE displayed excellent in vitro stability and high uptake by A. fumigatus. Both compounds showed excellent pharmacokinetics, highly selective accumulation in infected lung tissue and good correlation with severity of disease in a rat infection model, which makes them promising agents for A. fumigatus infection imaging.

Purpose: With the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with⁶⁸Ga for imaging of bacterial infections.Methods: In vitro characterization of [⁶⁸Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [⁶⁸Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens.Results: DFO-B was labelled with⁶⁸Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [⁶⁸Ga]Ga-DFO-B in selected strains ofPseudomonas aeruginosa,Staphylococcus aureusandStreptococcus agalactiaecould be blocked with an excess of iron-DFO-B. [⁶⁸Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [⁶⁸Ga]Ga-DFO-B in bothP. aeruginosaandS. aureusinfections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivatedP. aeruginosaorS. aureusandEscherichia colilacking DFO-B transporters.Conclusion: DFO-B can be easily radiolabelled with⁶⁸Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [⁶⁸Ga]Ga-DFO-B byP. aeruginosaandS. aureuswas confirmed both in vitro and in vivo, proving the potential of [⁶⁸Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other⁶⁸Ga-labelled radiopharmaceuticals, we believe that [⁶⁸Ga]Ga-DFO-B has a great potential for clinical translation.

Purpose ⁶⁸Ga-triacetylfusarinine C (⁶⁸Ga-TAFC) and ⁶⁸Ga-ferrioxamine E (⁶⁸Ga-FOXE) showed excellent targeting properties in Aspergillus fumigatus rat infection model. Here, we report on the comparison of specificity towards different microorganisms and human lung cancer cells (H1299). Procedures The in vitro uptake of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE was studied in various fungal, bacterial and yeast cultures as well as in H1299 cells. The in vivo imaging was studied in fungal and bacterial rat infection and inflammation models. Results ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed rapid uptake in A. fumigatus cultures, significantly lower in other fungal species and almost no uptake in other microorganisms and H1299 cells, except for ⁶⁸Ga-FOXE in Staphylococcus aureus. ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE revealed rapid uptake in the lungs of A. fumigatus-infected rats, low accumulation in sterile inflammation and no uptake in bacterial abscess. Conclusions We have shown that ⁶⁸Ga-FOXE and ⁶⁸Ga-TAFC have high uptake in A. fumigatus both in vitro and in vivo. ⁶⁸Ga-TAFC showed higher specificity, while ⁶⁸Ga-FOXE showed higher sensitivity.