Milos Petrik

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

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.

Preclinical evaluation of two 68Ga-siderophores as potential radiopharmaceuticals for Aspergillus fumigatus infection imaging

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.

In vitro and in vivo evaluation of selected 68Ga-siderophores for infection imaging

Introduction Siderophores 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. Methods 68Ga 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. Results Significant 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. Conclusions Some 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.

Full automation of 68Ga labelling of DOTA-peptides including cation exchange prepurification

Here we describe a fully automated approach for the synthesis of (68)Ga-labelled DOTA-peptides based on pre-concentration and purification of the generator eluate by using a cation exchange-cartridge and its comparison with fully automated direct labelling applying fractionated elution. Pre-concentration of the eluate on a cation exchange cartridge both using a resin-based and a disposable cation-exchange cartridge efficiently removed (68)Ge as well as major metal contaminations with Fe and Zn. This resulted in a high labelling efficiency of DOTA-peptides at high specific activity (SA) with short synthesis times.

Novel Bifunctional Cyclic Chelator for (89)Zr Labeling-Radiolabeling and Targeting Properties of RGD Conjugates.

Within the last years 89Zr has attracted considerable attention as long-lived radionuclide for positron emission tomography (PET) applications. So far desferrioxamine B (DFO) has been mainly used as bifunctional chelating system. Fusarinine C (FSC), having complexing properties comparable to DFO, was expected to be an alternative with potentially higher stability due to its cyclic structure. In this study, as proof of principle, various FSC-RGD conjugates targeting αvß3 integrins were synthesized using different conjugation strategies and labeled with 89Zr. In vitro stability, biodistribution, and microPET/CT imaging were evaluated using [89Zr]FSC-RGD conjugates or [89Zr]triacetylfusarinine C (TAFC). Quantitative 89Zr labeling was achieved within 90 min at room temperature. The distribution coefficients of the different radioligands indicate hydrophilic character. Compared to [89Zr]DFO, [89Zr]FSC derivatives showed excellent in vitro stability and resistance against transchelation in phosphate buffered saline (PBS), ethylenediaminetetraacetic acid solution (EDTA), and human serum for up to 7 days. Cell binding studies and biodistribution as well as microPET/CT imaging experiments showed efficient receptor-specific targeting of [89Zr]FSC-RGD conjugates. No bone uptake was observed analyzing PET images indicating high in vivo stability. These findings indicate that FSC is a highly promising chelator for the development of 89Zr-based PET imaging agents.

68Ga-Triacetylfusarinine C and 68Ga-Ferrioxamine E for Aspergillus Infection Imaging: Uptake Specificity in Various Microorganisms

Purpose68Ga-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).ProceduresThe 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.Results68Ga-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.ConclusionsWe 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.

An Iron-Mimicking, Trojan Horse-Entering Fungi—Has the Time Come for Molecular Imaging of Fungal Infections?

Despite recent advancements in the diagnosis and management of fungal infections [1], invasive fungal diseases remain a major cause of morbidity and mortality in immunocompromised patients and are major drivers of elevated healthcare costs [2]. In this context, early diagnosis is a key factor. However, current diagnostic approaches, including laboratory tests and computer tomography, have limitations, especially in terms of sensitivity and specificity [3]. Therefore, empirical therapy has often evolved as the standard of care, irrespective of the immediate and long-term consequences in terms of cost, development of drug resistance, or toxicity [4]. An exceptional challenge is the development of imaging modalities providing not only high specificity and sensitivity but also localization of the infection site. In particular, nuclear medicine imaging techniques using radiolabelled probes (radiotracers) have the potential to specifically target the underlying pathophysiological mechanisms of the pathogen leading to molecular localization of the infection site in patients. Traditionally applied in the context of planar scintigraphy and single photon emission tomography (SPECT) in the past decade, Positron-Emission Tomography (PET) has evolved as a major clinical imaging technique, particularly in oncology [5]. This technology provides improved sensitivity and resolution based on the coincidence detection of photons emitted from radionuclei resulting from annihilation of positrons. Its tremendous success in oncology is mainly based on 2-[18F] fluorodeoxyglucose specifically accumulating in cells in dependence of their glucose consumption [6]. In this process, termed “molecular trapping,” the radiolabelled glucose molecule is actively transported into the cell, followed by its phosphorylation. The incorporated Fluor blocks further metabolic processing and traps the radionuclide 18F inside the cell, leading to an intense radioactive signal in affected cells. Other clinically used PET probes, such as [18F]-3′-fluoro-3′-deoxy-L-thymidine, [18F]-choline derivatives, or radiolabelled peptides, such as 68Ga-DOTATOC, show a similar trapping mechanism based on initial active transport in, or receptor-specific recognition by, diseased cells with promising clinical applications in oncology [7]. Besides accumulation in the target, favorable pharmacokinetics of radiotracers, such as rapid transport to and low retention in non-target sites/cells as well as efficient elimination from the body, ideally via renal excretion, are required. These features allow early imaging with short-lived radionuclides such as 18F with a half-life of 110 minutes, resulting in a low radiation burden for the patient. Therefore, a radiotracer for specific imaging of fungal infections should ideally fulfill similar criteria: specific accumulation in the pathogen combined with favorable pharmacokinetics, including rapid elimination from healthy tissue. Ideally, the pathogen should recognize the radiotracer as an apparent molecule of interest, boosting its active uptake and accumulation.

Radiolabelling of peptides for PET, SPECT and therapeutic applications using a fully automated disposable cassette system.

ObjectivesRadiolabelled somatostatin analogues have found wide clinical use in nuclear medicine for both diagnostic and therapeutic applications. Here, we describe the development of a fully automated synthesis system allowing radiolabelling of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-derivatized peptides with 68Ga/111In/177Lu and 90Y, meeting radiation safety and pharmaceutical requirements. Materials and methodsThe system consists of a syringe pump, a holder for insertion of a single use multivalve cassette, a heater and a removable radiation shielding. 68Ga labelling was performed in acetate buffer and 177Lu, 90Y and 111In labelling in ascorbate buffer, respectively, followed by purification on a C18 cartridge and final sterile filtration. Cross-contamination was prevented by using disposable cassettes and also by ensuring pharmaceutical standards. Radiochemical purity (RCP) was determined by instant thin-layer chromatography on silica gel impregnated glass fibres and reversed-phase high performance liquid chromatography. Results68Ga-DOTA-peptides were prepared with high RCP (>91%) and radiochemical yields (RCY>80% decay corrected) and 68Ge content was less than 0.0001% in all cases. Synthesis time did not exceed 30 min. 111In, 177Lu and 90Y labelling of DOTA-peptides resulted again in high yields (approximately 90%) and RCP (approximately 95%) and total synthesis time of less than 45 min. Radiation dose to fingers was considerably reduced when compared with manual labelling procedures. ConclusionThe described system allows fully automated, aseptic preparation of DOTA-peptides radiolabelled with different radionuclides in high radiochemical yields and pharmaceutical quality suitable for clinical application.

[68Ga]NS3-RGD and [68Ga] Oxo-DO3A-RGD for imaging αvβ3 integrin expression: synthesis, evaluation, and comparison

INTRODUCTION ⁶⁸Ga-labeled RGD peptides in combination with PET allow non-invasive determination of α(v)β₃ integrin expression which is highly increased during tumor-induced angiogenesis. The aim of this study was to synthesize and evaluate two RGD peptides containing alternative chelating systems, namely [⁶⁸Ga]NS₃-RGD-RGD and [⁶⁸Ga]Oxo-DO3A-RGD and to compare their in vitro and in vivo properties with [⁶⁸Ga]DOTA- and [⁶⁸Ga]NODAGA-RGD. METHODS Syntheses of both radiotracers followed standard SPPS protocols. For in vitro characterization distribution coefficients, protein binding abilities, serum stabilities, and α(v)β₃ integrin binding affinities were determined. For in vitro tests as well as for the biodistribution assay α(v)β₃ positive human melanoma M21 and α(v)β₃ negative M21-L cells were used. RESULTS ⁶⁸Ga-labeling of NS₃-RGD resulted in good radiochemical purity, whereas HPLC analysis showed two peaks with a ratio of 1:6 for [⁶⁸Ga]Oxo-DO3A-RGD. Distribution coefficients were -3.4 for [⁶⁸Ga]Oxo-DO3A-RGD and -2.9 for [⁶⁸Ga]NS₃-RGD. Both radiotracers were stable in PBS solution at 37°C for 2 h but lack stability in human serum. Protein binding was approximately 40% of the total activity for [⁶⁸Ga]NS₃-RGD and 70% for [⁶⁸Ga]Oxo-DO3A-RGD, respectively, resulting in high blood pool activities. Biodistribution assays confirmed these findings and showed an additional high uptake in liver and kidneys, especially for [⁶⁸Ga]NS₃-RGD. Furthermore, [⁶⁸Ga]Oxo-DO3A-RGD showed nearly the same activity concentrations in α(v)β₃ positive and α(v)β₃ negative tumors. CONCLUSIONS [⁶⁸Ga]Oxo-DO3A-RGD and [⁶⁸Ga]NS₃-RGD have inferior characteristics compared to already existing ⁶⁸Ga-labeled RGD peptides and thus, both are not suited to image α(v)β₃ integrin expression. Of all our tested RGD peptides, [⁶⁸Ga]NODAGA-RGD still possesses the most favorable imaging properties. Moreover this study shows that the use of appropriate chelators to achieve good targeting properties of ⁶⁸Ga-labeled biomolecules and careful in vitro and in vivo evaluation including comparative studies of different strategies are essential components in designing an effective imaging agent for PET.

In Vitro and In Vivo Comparison of Selected Ga-68 and Zr-89 Labelled Siderophores

PurposeSome [68Ga]siderophores show promise in specific and sensitive imaging of infection. Here, we compare the in vitro and in vivo behaviour of selected Ga-68 and Zr-89 labelled siderophores.ProceduresRadiolabelling was performed in HEPES or sodium acetate buffer systems. Radiochemical purity of labelled siderophores was determined using chromatography. Partition coefficients, in vitro stability and protein binding affinities were determined. Ex vivo biodistribution and animal imaging was studied in mice.ResultsCertain differences among studied siderophores were observed in labelling efficiency. Protein binding and stability tests showed highest stabilities and lowest protein binding affinities for Ga-68 and [89Zr]triacetylfusarinine C (TAFC). All studied Ga-68 and [89Zr]siderophores exhibited a similar biodistribution and pharmacokinetics in mice with the exception of [89Zr]ferrioxamine E (FOXE).ConclusionsZr-89 and [68Ga]siderophores showed analogous in vitro and in vivo behaviour. Tested [89Zr]siderophores could be applied for longitudinal positron emission tomography (PET) studies of fungal infections and especially TAFC for the development of novel bioconjugates.