⁶⁸Ga-Siderophores for PET Imaging of Invasive Pulmonary Aspergillosis: Proof of Principle

Abstract

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 68 Ga, a positron emitter with complexing properties comparable to those of Fe(III), as a radiopharmaceutical for imaging IPA. Methods: 68 Ga radiolabeling of the A. fumigatus siderophores desferri-triacetylfusarinine C (TAFC) and desferriferricrocin (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 68 Ga labeling could be achieved, and resulting complexes were stable in serum, toward diethylenetriaminepentaacetic acid and Fe(III) challenge. Both siderophores showed hydrophilic properties ( 68 Ga-TAFC, log P 5 22.59; 68 Ga-FC, log P 5 23.17) with low values of protein binding for 68 Ga-TAFC (,2%). Uptake of both siderophores was highly dependent on the mycelial iron load and could be blocked with an excess (10 mM) of siderophore or NaN 3 , indicating specific, energy-dependent uptake. In noninfected mice, 68 Ga-TAFC showed rapid renal excretion and low blood values (1.6 6 0.37 percentage injected dose per gram [%ID/g] at 30 min); in urine only intact 68 Ga-TAFC was detected. In contrast, 68 Ga-FC revealed high retention in blood (16.1 6 1.07 %ID/g at 90 min) and rapid metabolism. In the rat IPA model, lung uptake of 68 Ga-TAFC was dependent on the severity of infection, with less than 0.04 %ID/g in control rats (n 5 5) and 0.29 6 0.11 %ID/g in mildly infected (n 5 3) and 0.95 6 0.37 %ID/g in severely infected (n 5 4) rats. PET showed focal accumulation in infected lung tissue. Conclusion: Both siderophores bound 68 Ga with high affinity, and 68 Ga-TAFC, especially, showed high stability. 68 Ga-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 68 Ga-labeled siderophores for the specific detection of A. fumigatus during infection. They hold promise as new PET agents for IPA.

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo

“…Especially in the field of preclinical imaging, an increasing number of biologicals including small molecules, antibodies, their fragments and peptides have been developed and evaluated for imaging infectious diseases (24)(25)(26)(27)(28). Recently, attempts have been made to visualize IPA in A. fumigatus-infected animals with microPET/CT using 68 Ga radiolabeled siderophores (29,30). These small high-affinity chelating compounds are produced by fungi and bacteria to scavenge iron from the host, and by Gram-negative bacterial pathogens as virulence factors (31 TAFC has been shown to occur under conditions of iron depletion, patients who acquire fungal infections typically suffer from iron overload thereby potentially reducing tracer uptake and sensitivity of this method.…”

Mevalonate governs interdependency of ergosterol and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus

“…Several lines of evidence indicate that A. fumigatus faces iron limitation during mammalian infection: (i) Defects impairing adaptation to iron limitation, such as inactivation of the siderophore system or the iron regulator HapX, attenuate virulence in murine models of pulmonary aspergillosis (3, 5, 21); (ii) RIA and the siderophore system are transcriptionally up-regulated during initiation of murine infection (22); and (iii) TAFC-chelated 68 Ga, the uptake of which is induced by iron starvation, allows in vivo PET imaging of invasive pulmonary aspergillosis in rats (23). Consistent with these findings, human protection against A. fumigatus includes growth inhibition by polymorphonuclear leukocytes via lactoferrin-mediated iron depletion and possibly siderocalin-mediated scavenging of siderophores (24,25).…”

Siderophores for molecular imaging applications

“…More than 30 years after the first attempts of labelling siderophores with 67 Ga [36-38, 40, 41], we evaluated the use of 68 Ga labelled siderophores for PET imaging of fungal infections [47]. In proof of concept studies, which should confirm or refute the possibility of PET imaging of infections caused by Aspergillus fumigatus (A. f.) using 68 Ga-siderophores [48], it was demonstrated that desferrisiderophores, particularly triacetylfusarinine C (TAFC), can be easily radiolabelled with 68 Ga using a few micrograms of the siderophore and exhibit high chemical stability. Uptake of 68 Ga-TAFC by A. f. was upregulated under iron starvation conditions and could be blocked with an excess of siderophore or NaN 3 , indicating specific and energy-dependent uptake.…”