PurposeInvasive 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).MethodsSiderophores 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.Results68Ga-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.Conclusion68Ga-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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-012-2110-3) contains supplementary material, which is available to authorized users.

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.

Development of CCK2 receptor ligands especially for therapeutic purposes in patients with MTC or small cell lung cancer (SCLC) is still ongoing in different laboratories. This comparative study provided valuable insight into the importance of biological stability especially in the context of other results of this comparative trial within the COST Action BM0607.

IntroductionSiderophores are low-molecular-mass iron chelators serving as iron transporters for almost all bacteria, fungi and some plants. Iron is an essential element for majority of organisms and plays an important role in virulence of pathogenic organisms. 68Ga is a positron emitter with complexing properties comparable to those of Fe(III) and readily available from a generator. Initial studies with 68Ga-triacetylfusarinine C (TAFC) showed excellent targeting properties in a rat infection model. We report here on the in vitro and in vivo evaluation of other siderophores radiolabelled with 68Ga as potential radiopharmaceuticals for infection imaging.Methods68Ga labelling was performed using acetate buffer. Stability, log P and protein binding values were determined. In vitro uptake was tested using iron-deficient and iron-sufficient Aspergillus fumigatus (A.f.) cultures. Biodistribution of 68Ga-siderophores was studied in Balb/c mice.ResultsSignificant differences among studied siderophores were observed in labelling efficiency, stability and protein binding. Uptake in A.f. cultures was highly dependent on iron load and type of the siderophore. In mice, 68Ga-TAFC and 68Ga-ferrioxamine E (FOXE) showed rapid renal excretion and low blood values even at a short period after injection; in contrast, 68Ga-ferricrocin and 68Ga-ferrichrome revealed high retention in blood and 68Ga-fusarinine C showed very high kidney retention.ConclusionsSome of the studied siderophores bind 68Ga with high affinity and stability, especially 68Ga-TAFC and 68Ga-FOXE. Low values of protein binding, high and specific uptake in A.f., and excellent in vivo biodistribution make them favourable agents for Aspergillus infection imaging.

Radiolabeling of nanoparticles (NPs) has been performed for a variety of reasons, such as for studying pharmacokinetics, for imaging, or for therapy. Here, we describe the in vitro and in vivo evaluation of DTPA-derivatized lipid-based NP (DTPA-NP) radiolabeled with different radiometals, including (111)In and (99m)Tc, for single-photon emission computed tomography (SPECT), (68)Ga for positron emission tomography (PET), and (177)Lu for therapeutic applications. PEGylated DTPA-NP with varying DTPA amounts, different composition, and size were radiolabeled with (111)In, (177)Lu, and (68)Ga, using various buffers. (99m)Tc-labeling was performed directly and by using the carbonyl aquaion, [(99m)Tc(H(2)O)(3)(CO)(3)](+). Stability was tested and biodistribution evaluated. High labeling yields (>90%) were achieved for all radionuclides and different liposomal formulations. Specific activities (SAs) were highest for (111)In (>4 MBq/mug liposome), followed by (68)Ga and (177)Lu; for (99m)Tc, high labeling yields and SA were only achieved by using [(99m)Tc(H(2)O)(3)(CO)(3)](+). Stability toward DTPA/histidine and in serum was high (>80 % RCP, 24 hours postpreparation).). Biodistribution in Lewis rats revealed no significant differences between NP in terms of DTPA loading and particle composition; however, different uptake patterns were found between the radionuclides used. We observed lower retention in blood (<3.3 %ID/g) and lower liver uptake (< 2.7 %ID/g) for (99m)Tc- and (68)Ga, compared to (111)In-NP (blood, <4 %ID/g; liver, <3.6 %ID/g). Imaging potential was shown by both PET magnetic resonance imaging fusion imaging and SPECT imaging. Overall, our study shows that PEGylated DTPA-NP are suitable for radiolabeling studies with a variety of radiometals, thereby achieving high SA suitable for targeting applications.