Preclinical characterisation of gallium-68 labeled ferrichrome siderophore stereoisomers for PET imaging applications.

BACKGROUND: Siderophores are small iron-binding molecules produced by microorganisms to facilitate iron acquisition from the environment. Radiolabelled siderophores offer a promising solution for infection imaging, as they can specifically target the pathophysiological mechanisms of pathogens. Gallium-68 can replace the iron in siderophores, enabling molecular imaging with positron emission tomography (PET). Stereospecific interactions play a crucial role in the recognition of receptors, transporters, and iron utilisation. Furthermore, these interactions have an impact on the host environment, affecting pharmacokinetics and biodistribution. This study examines the influence of siderophore stereoisomerism on imaging properties, with a focus on ferrirubin (FR) and ferrirhodin (FRH), two cis-trans isomeric siderophores of the ferrichrome type. RESULTS: Tested siderophores were labelled with gallium-68 with high radiochemical purity. The resulting complexes differed in their in vitro characteristics. [68Ga]Ga-FRH showed less hydrophilic properties and higher protein binding values than [68Ga]Ga-FR. The stability studies confirmed the high radiochemical stability of both [68Ga]Ga-siderophores in all examined media. Both siderophores were found to be taken up by S. aureus, K. pneumoniae and P. aeruginosa with similar efficacy. The biodistribution tested in normal mice showed rapid renal clearance with low blood pool retention and fast clearance from examined organs for [68Ga]Ga-FR, whereas [68Ga]Ga-FRH showed moderate retention in blood, resulting in slower pharmacokinetics. PET/CT imaging of mice injected with [68Ga]Ga-FR and [68Ga]Ga-FRH confirmed findings from ex vivo biodistribution studies. In a mouse model of S. aureus myositis, both radiolabeled siderophores showed radiotracer accumulation at the site of infection. CONCLUSIONS: The 68Ga-complexes of stereoisomers ferrirubin and ferrirhodin revealed different pharmacokinetic profiles. In vitro uptake was not affected by isomerism. Both compounds had uptake with the same bacterial culture with similar efficacy. PET/CT imaging showed that the [68Ga]Ga-complexes accumulate at the site of S. aureus infection, highlighting the potential of [68Ga]Ga-FR as a promising tool for infection imaging. In contrast, retention of the radioactivity in the blood was observed for [68Ga]Ga-FRH. In conclusion, the stereoisomerism of potential radiotracers should be considered, as even minor structural differences can influence their pharmacokinetics and, consequently, the results of PET imaging.

[68Ga]Ga-Ornibactin for Burkholderia cepacia complex Infection Imaging Using Positron Emission Tomography.

Bacteria from the Burkholderia cepacia complex are generally considered to be non-pathogenic to the healthy population. However, some of these species may cause serious nosocomial infections in immunocompromised patients; as such, it is essential to diagnose these infections rapidly so that adequate treatment can be initiated. We report here the use of a radiolabeled siderophore, ornibactin (ORNB), for positron emission tomography imaging. We successfully radiolabeled ORNB with gallium-68 with high radiochemical purity and proved that the resulting complex has optimal in vitro characteristics. In mice, the complex did not show excessive accumulation in organs and was excreted in the urine. We demonstrated that the [68Ga]Ga-ORNB complex accumulates at the site of Burkholderia multivorans infection, including pneumonia, in two animal infection models. These results suggest that [68Ga]Ga-ORNB is a promising tool for the diagnosis, monitoring, and evaluation of the therapeutic response to B. cepacia complex infection.

Modulating the pharmacokinetic profile of Actinium-225-labeled macropa-derived radioconjugates by dual targeting of PSMA and albumin.

Rationale: Small 225Ac-labeled prostate-specific membrane antigen (PSMA)-targeted radioconjugates have been described for targeted alpha therapy of metastatic castration-resistant prostate cancer. Transient binding to serum albumin as a highly abundant, inherent transport protein represents a commonly applied strategy to modulate the tissue distribution profile of such low-molecular-weight radiotherapeutics and to enhance radioactivity uptake into tumor lesions with the ultimate objective of improved therapeutic outcome. Methods: Two ligands mcp-M-alb-PSMA and mcp-D-alb-PSMA were synthesized by combining a macropa-derived chelator with either one or two lysine-ureido-glutamate-based PSMA- and 4-(p-iodophenyl)butyrate albumin-binding entities using multistep peptide-coupling chemistry. Both compounds were labeled with [225Ac]Ac3+ under mild conditions and their reversible binding to serum albumin was analyzed by an ultrafiltration assay as well as microscale thermophoresis measurements. Saturation binding studies and clonogenic survival assays using PSMA-expressing LNCaP cells were performed to evaluate PSMA-mediated cell binding and to assess the cytotoxic potency of the novel radioconjugates [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Biodistributions of both 225Ac-radioconjugates were investigated using LNCaP tumor-bearing SCID mice. Histological examinations of selected organs were performed to analyze the occurrence of necrosis using H&E staining, DNA damage via γH2AX staining and proliferation via Ki67 expression in the tissue samples. Results: Enhanced binding to serum components in general and to human serum albumin in particular was revealed for [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Moreover, the novel derivatives are highly potent PSMA ligands as their KD values in the nanomolar range (23.38 and 11.56 nM) are comparable to the reference radioconjugates [225Ac]Ac-mcp-M-PSMA (30.83 nM) and [225Ac]Ac-mcp-D-PSMA (10.20 nM) without albumin binders. The clonogenic activity of LNCaP cells after treatment with the 225Ac-labeled ligands was affected in a dose- and time-dependent manner, whereas the bivalent radioconjugate [225Ac]Ac-mcp-D-alb-PSMA has a stronger impact on the clonogenic cell survival than its monovalent counterpart [225Ac]Ac-mcp-M-alb-PSMA. Biodistribution studies performed in LNCaP tumor xenografts showed prolonged blood circulation times for both albumin-binding radioconjugates and a substantially increased tumor uptake (46.04 ± 7.77 %ID/g for [225Ac]Ac-mcp-M-alb-PSMA at 128 h p.i. and 153.48 ± 37.76 %ID/g at 168 h p.i. for [225Ac]Ac-mcp-D-alb-PSMA) with favorable tumor-to-background ratios. Consequently, a clear histological indication of DNA damage was discovered in the tumor tissues, whereas DNA double-strand break formation in kidney and liver sections was less pronounced. Conclusion: The modification of the PSMA-based 225Ac-radioconjugates with one or two albumin-binding entities resulted in an improved radiopharmacological behavior including a greatly enhanced tumor accumulation combined with a rather low uptake in most non-targeted organs combined with a high excretion via the kidneys.

Preparation, In Vitro Affinity, and In Vivo Biodistribution of Receptor-Specific 68Ga-Labeled Peptides Targeting Vascular Endothelial Growth Factor Receptors.

As angiogenesis plays a key role in tumor growth and metastasis, the angiogenic process has attracted scientific interest as a target for diagnostic and therapeutic agents. Factors influencing angiogenesis include the vascular endothelial growth factor (VEGF) family and the two associated receptor types (VEGFR-1 and VEGFR-2). VEGFR-1/-2 detection and quantification in cancer lesions are essential for tumor process management. As a result of the advantageous pharmacokinetics and image contrast, peptides radiolabeled with PET emitters have become interesting tools for the visualization of VEGFR-1/-2-positive tumors. In this study, we prepared 68Ga-labeled peptides containing 15 (peptide 1) and 23 (peptide 2) amino acids as new PET tracers for tumor angiogenic process imaging. METHODS: The peptides were conjugated with NODAGA-tris(t-Bu ester) and subsequently radiolabeled with [68Ga]Ga-chloride. The prepared [68Ga]Ga-NODAGA-peptide 1 and [68Ga]Ga-NODAGA-peptide 2 were tested for radiochemical purity and saline/plasma stability. Consequently, the binding affinity toward VEGFRs was assessed in vitro on human glioblastoma and kidney carcinoma cells. The found peptide receptor affinity was compared with the calculated values in the PROtein binDIng enerGY prediction (PRODIGY) server. Finally, the biodistribution study was performed on BALB/c female mice to reveal the basic pharmacokinetic behavior of radiopeptides. RESULTS: The in vitro affinity testing of [68Ga]Ga-NODAGA-peptides 1 and 2 showed retained receptor binding as characterized by equilibrium dissociation constant (KD) values in the range of 0.5-1.2 µM and inhibitory concentration 50% (IC50) values in the range of 3.0-5.6 µM. Better binding properties of peptide 2 to VEGFR-1/-2 were found in the PRODIGY server. The biodistribution study on mice showed remarkable accumulation of both peptides in the kidneys and urinary bladder with a short half-life after intravenous application. The in vitro plasma stability of [68Ga]Ga-NODAGA-peptide 2 was superior to that of [68Ga]Ga-NODAGA-peptide 1. CONCLUSIONS: The obtained results demonstrated a high radiolabeling yield with no need for purification and preserved binding potency of 68Ga-labeled peptides 1 and 2 toward VEGFRs in cancer cells. The peptide-receptor protein interaction assessed in protein-peptide docking determined the strongest interaction of peptide 2 with domain 2 of VEGFR-2 in addition to a more acceptable plasma stability (t1/2 = 120 min) than that for peptide 1. We found both radiolabeled peptides very potent in their receptor binding, which makes them suitable imaging agents. The rapid transition of the radiopeptides into the urinary tract indicates suitable pharmacokinetic characteristics.

[68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice.

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvß3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [68Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [68Ga]Ga-DFO-c(RGDyK) can be used for αvß3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.

Towards Targeted Alpha Therapy with Actinium-225: Chelators for Mild Condition Radiolabeling and Targeting PSMA-A Proof of Concept Study.

Currently, targeted alpha therapy is one of the most investigated topics in radiopharmaceutical cancer management. Especially, the alpha emitter 225Ac has excellent nuclear properties and is gaining increasing popularity for the treatment of various tumor entities. We herein report on the synthesis of two universal 225Ac-chelators for mild condition radiolabeling and binding to conjugate molecules of pharmacological interest via the copper-mediated click chemistry. A convenient radiolabeling procedure was investigated as well as the complex stability proved for both chelators and two PSMA (prostate-specific membrane antigen)-targeting model radioconjugates. Studies regarding affinity and cell survival were performed on LNCaP cells followed by biodistribution studies, which were performed using LNCaP tumor-bearing mice. High efficiency radiolabeling for all conjugates was demonstrated. Cell binding studies revealed a fourfold lower cell affinity for the PSMA radioconjugate with one targeting motif compared to the radioconjugate owing two targeting motifs. Additionally, these differences were verified by in vitro cell survival evaluation and biodistribution studies, both showing a higher cell killing efficiency for the same dose, a higher tumor uptake (15%ID/g) and a rapid whole body clearance after 24 h. The synthesized chelators will overcome obstacles of lacking stability and worse labeling needs regarding 225Ac complexation using the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid) chelator. Moreover, the universal functionalization expands the coverage of these chelators in combination with any sensitive bio(macro)molecule, thus improving treatment of any addressable tumor target.

Preclinical evaluation of anti-VEGFR2 monoclonal antibody ramucirumab labelled with zirconium-89 for tumour imaging.

The key factors participating in angiogenesis include vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), particularly VEGFR2. Angiogenesis suppression comprises the blocking of the VEGFR2 binding site by the monoclonal antibody ramucirumab (RAM). Our study focused on RAM radiolabelling with zirconium-89 along with subsequent in vitro and in vivo biological evaluation. RAM was conjugated with the bifunctional chelator p-SCN-Bn-deferoxamine (DFO) and subsequently radiolabelled with [89 Zr]Zr-oxalate. The binding affinity of [89 Zr]Zr-DFO-RAM to VEGFR2 was tested in vitro on prostate (PC-3) and ovary adenocarcinoma (SK-OV-3) cell lines. The positron emission tomography/computed tomography (PET/CT) imaging and ex vivo biodistribution experiments were performed in PC-3 and SK-OV-3 xenografted mice. The in vitro experiments revealed the preserved binding affinity of [89 Zr]Zr-DFO-RAM to VEGFR2. The obtained ex vivo biodistribution data showed the uptake in PC-3 and SK-OV-3 tumours at about 8.7 ± 0.2 and 12.1 ± 1.6%ID/g, respectively. The tumour-to-muscle ratio for 1, 3 and 6 days post injection was 3.9, 5.5 and 5.12 for PC-3 and 6.0, 8.0 and 8.82 for SK-OV-3 tumours, respectively. PET/CT images showed high radioactivity accumulation in the tumours starting already on the first day after tracer administration. The obtained results proved the potency of [89 Zr]Zr-DFO-RAM to target and image VEGFR2-positive tumours in vivo.

68Ga-labelled desferrioxamine-B for bacterial 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 with 68Ga 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 with 68Ga 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 of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could 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 both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters. CONCLUSION: DFO-B can be easily radiolabelled with 68Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [68Ga]Ga-DFO-B by P. aeruginosa and S. aureus was 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 other 68Ga-labelled radiopharmaceuticals, we believe that [68Ga]Ga-DFO-B has a great potential for clinical translation.

Iodinated Choline Transport-Targeted Tracers.

We present a novel series of radioiodinated tracers and potential theranostics for diseases accompanied by pathological function of proteins involved in choline transport. Unlike choline analogues labeled with 11C or 18F that are currently used in the clinic, the iodinated compounds described herein are applicable in positron emission tomography, single-photon emission computed tomography, and potentially in therapy, depending on the iodine isotope selection. Moreover, favorable half-lives of iodine isotopes result in much less challenging synthesis by isotope exchange reaction. Six of the described compounds were nanomolar ligands, and the best compound possessed an affinity 100-fold greater than that of choline. Biodistribution data of 125I-labeled ligands in human prostate carcinoma bearing (PC-3) mice revealed two compounds with a biodistribution profile superior to that of [18F]fluorocholine.

Head-To-Head Comparison of Biological Behavior of Biocompatible Polymers Poly(Ethylene Oxide), Poly(2-Ethyl-2-Oxazoline) and Poly[N-(2-Hydroxypropyl)Methacrylamide] as Coating Materials for Hydroxyapatite Nanoparticles in Animal Solid Tumor Model.

Nanoparticles (NPs) represent an emerging platform for diagnosis and treatment of various diseases such as cancer, where they can take advantage of enhanced permeability and retention (EPR) effect for solid tumor accumulation. To improve their colloidal stability, prolong their blood circulation time and avoid premature entrapment into reticuloendothelial system, coating with hydrophilic biocompatible polymers is often essential. Most studies, however, employ just one type of coating polymer. The main purpose of this study is to head-to-head compare biological behavior of three leading polymers commonly used as "stealth" coating materials for biocompatibilization of NPs poly(ethylene oxide), poly(2-ethyl-2-oxazoline) and poly[N-(2-hydroxypropyl)methacrylamide] in an in vivo animal solid tumor model. We used radiolabeled biodegradable hydroxyapatite NPs as a model nanoparticle core within this study and we anchored the polymers to the NPs core by hydroxybisphosphonate end groups. The general suitability of polymers for coating of NPs intended for solid tumor accumulation is that poly(2-ethyl-2-oxazoline) and poly(ethylene oxide) gave comparably similar very good results, while poly[N-(2-hydroxypropyl)methacrylamide] was significantly worse. We did not observe a strong effect of molecular weight of the coating polymers on tumor and organ accumulation, blood circulation time, biodistribution and biodegradation of the NPs.

Preclinical Evaluation of Radiolabeled Peptides for PET Imaging of Glioblastoma Multiforme.

In this study, we have compared four 68Ga-labeled peptides (three Arg-Gly-Asp (RGD) peptides and substance-P) with two 18F-tracers clinically approved for tumor imaging. We have studied in vitro and in vivo characteristics of selected radiolabeled tracers in a glioblastoma multiforme tumor model. The in vitro part of the study was mainly focused on the evaluation of radiotracers stability under various conditions. We have also determined in vivo stability of studied 68Ga-radiotracers by analysis of murine urine collected at various time points after injection. The in vivo behavior of tested 68Ga-peptides was evaluated through ex vivo biodistribution studies and PET/CT imaging. The obtained data were compared with clinically used 18F-tracers. 68Ga-RGD peptides showed better imaging properties compared to 18F-tracers, i.e., higher tumor/background ratios and no accumulation in non-target organs except for excretory organs.

In Situ In Vivo radiolabeling of polymer-coated hydroxyapatite nanoparticles to track their biodistribution in mice.

The imaging of healthy tissues and solid tumors benefits from the application of nanoparticle probes with altered pharmacokinetics, not available to low molecular weight compounds. However, the distribution and accumulation of nanoprobes in vivo typically take at least tens of hours to be efficient. For nanoprobes bearing a radioactive label, this is contradictory to the requirement of minimizing the radiation dose for patients by using as-short-as-feasible half-life radionuclides in diagnostics. Thus, we developed a two-stage diagnostic concept for monitoring long-lasting targeting effects with short-lived radioactive labels using bone-mimicking biocompatible polymer-coated and colloidally fully stabilized hydroxyapatite nanoparticles (HAP NPs) and bone-seeking radiopharmaceuticals. Within the pretargeting stage, the nonlabeled nanoparticles are allowed to circulate in the blood. Afterward, 99mTc-1-hydroxyethylidene-1.1-diphosphonate (99mTc-HEDP) is administered intravenously for in situ labeling of the nanoparticles and subsequent single-photon emission computed tomography/computed tomography (SPECT/CT) visualization. The HAP NPs, stabilized with tailored hydrophilic polymers, are not cytotoxic in vitro, as shown by several cell lines. The polymer coating prolongs the circulation of HAP NPs in the blood. The nanoparticles were successfully labeled in vivo with 99mTc-HEDP, 1 and 24 h after injection, and they were visualized by SPECT/CT over time in healthy mice.

Modifying the Siderophore Triacetylfusarinine C for Molecular Imaging of Fungal Infection.

PURPOSE: Aspergillus fumigatus produces the siderophore triacetylfusarinine C (TAFC) for iron acquisition which is essential for its virulence. Therefore, TAFC is a specific marker for invasive aspergillosis. We have shown previously that positron emission tomography (PET) imaging with [68Ga]TAFC exhibited excellent targeting properties in an A. fumigatus rat infection model. In this study, we aimed to prepare TAFC analogs modifying fusarinine C (FSC) by acylation with different carbon chain lengths as well as with charged substituents and investigated the influence of introduced substituents on preservation of TAFC characteristics in vitro and in vivo. PROCEDURES: Fifteen TAFC derivatives were prepared and labeled with gallium-68. In vitro uptake assays were carried out in A. fumigatus under iron-replete as well as iron-depleted conditions and distribution coefficient was determined. Based on these assays, three compounds, [68Ga]tripropanoyl(FSC) ([68Ga]TPFC), [68Ga]diacetylbutanoyl(FSC) ([68Ga]DABuFC), and [68Ga]trisuccinyl(FSC) ([68Ga]FSC(suc)3), with high, medium, and low in vitro uptake in fungal cultures, were selected for further evaluation. Stability and protein binding were evaluated and in vivo imaging performed in the A. fumigatus rat infection model. RESULTS: In vitro uptake studies using A. fumigatus revealed specific uptake of mono- and trisubstituted TAFC derivatives at RT. Lipophilicities as expressed by logD were 0.34 to - 3.80. The selected compounds displayed low protein binding and were stable in PBS and serum. Biodistribution and image contrast in PET/X-ray computed tomography of [68Ga]TPFC and [68Ga]DABuFC were comparable to [68Ga]TAFC, whereas no uptake in the infected region was observed with [68Ga]FSC(suc)3. CONCLUSIONS: Our studies show the possibility to modify TAFC without losing its properties and specific recognition by A. fumigatus. This opens also new ways for multimodality imaging or theranostics of fungal infection by introducing functionalities such as fluorescent dyes or antifungal moieties.

Antiangiogenic Human Monoclonal Antibody Ramucirumab Radiolabelling: In Vitro Evaluation on VEGFR2-positive Cell Lines.

Background/Aim: Radiolabelling of monoclonal antibodies (mAbs) could be beneficial in cancer diagnosis and therapy, however it may cause structural changes and consequently deteriorate their immunoreactivity. Materials and Methods: The therapeutic mAb ramucirumab (RAM) was technetium-99m labelled using either a direct or an indirect method with the use of two bifunctional chelating agents (HYNIC, DTPA). The radiochemical purity was assessed using instant thin-layer chromatography (ITLC) and high-performance liquid chromatography (HPLC) technique. The affinity of radiolabelled RAM was tested on human cancer cell lines. Results: The radiolabelling provided the following stable compounds: [ 99m Tc]RAM, [ 99m Tc]HYNIC-RAM and [ 99m Tc]DTPA-RAM. Their radiochemical purity was over 95%. All prepared radiopharmaceuticals showed moderate affinity to the targeted receptor, in vitro. However, their affinity was one order lower compared to that of the natural mAb. Moreover, directly and DTPA-radiolabelled RAM demonstrated less favourable binding kinetics. Conclusion: Radiolabelling negatively affected the affinity of RAM to its targeted receptor.

Imaging of Pseudomonas aeruginosa infection with Ga-68 labelled pyoverdine for positron emission tomography.

Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen that causes a variety of life-threatening nosocomial infections. Novel strategies for the development of new antibacterial treatments as well as diagnostic tools are needed. One of the novel diagnostic strategies for the detection of infection could be the utilization of siderophores. Siderophores are low-molecular-weight chelators produced by microbes to scavenge essential iron. Replacing iron in siderophores by suitable radiometals, such as Ga-68 for positron emission tomography (PET) imaging, opens approaches for targeted imaging of infection. Here we report on pyoverdine PAO1 (PVD-PAO1), a siderophore produced by P. aeruginosa, labelled with Ga-68 for specific imaging of Pseudomonas infections. PVD-PAO1 was labelled with Ga-68 with high radiochemical purity. The resulting complex showed hydrophilic properties, low protein binding and high stability in human serum. In vitro uptake of 68Ga-PVD-PAO1 was highly dependent on the type of microbial culture. In normal mice 68Ga-PVD-PAO1 showed rapid pharmacokinetics with urinary excretion. PET imaging in infected animals displayed specific accumulation of 68Ga-PVD-PAO1 in infected tissues and better distribution than clinically used 18F-fluorodeoxyglucose (18F-FDG) and 68Ga-citrate. Ga-68 labelled pyoverdine PAO1 seems to be a promising agent for imaging of P. aeruginosa infections by means of PET.

Early and Non-invasive Diagnosis of Aspergillosis Revealed by Infection Kinetics Monitored in a Rat Model.

Background: Aspergillus fumigatus is a ubiquitous saprophytic airborne fungus responsible for more than one million deaths every year. The siderophores of A. fumigatus represent important virulence factors that contribute to the microbiome-metabolome dialog in a host. From a diagnostic point of view, the monitoring of Aspergillus secondary metabolites in urine of a host is promising due to the non-invasiveness, rapidity, sensitivity, and potential for standardization. Methods: Using a model of experimental aspergillosis in immunocompromised Lewis rats, the fungal siderophores ferricrocin (FC) and triacetylfusarinine C (TAFC) were monitored in rat urine before and after lung inoculation with A. fumigatus conidia. Molecular biomarkers in high-dose (HD) and low-dose (LD) infection models were separated using high performance liquid chromatography (HPLC) and were detected by mass spectrometry (MS). In the current work, we corroborated the in vivo MS infection kinetics data with micro-positron emission tomography/computed tomography (µPET/CT) kinetics utilizing 68Ga-labeled TAFC. Results: In the HD model, the initial FC signal reflecting aspergillosis appeared as early as 4 h post-infection. The results from seven biological replicates showed exponentially increasing metabolite profiles over time. In A. fumigatus, TAFC was found to be a less produced biomarker that exhibited a kinetic profile identical to that of FC. The amount of siderophores contributed by the inoculating conidia was negligible and undetectable in the HD and LD models, respectively. In the µPET/CT scans, the first detectable signal in HD model was recorded 48 h post-infection. Regarding the MS assay, among nine biological replicates in the LD model, three animals did not develop any infection, while one animal experienced an exponential increase of metabolites and died on day 6 post-infection. All remaining animals had constant or random FC levels and exhibited few or no symptoms to the experiment termination. In the LD model, the TAFC concentration was not statistically significant, while the µPET/CT scan was positive as early as 6 days post-infection. Conclusion: Siderophore detection in rat urine by MS represents an early and non-invasive tool for diagnosing aspergillosis caused by A. fumigatus. µPET/CT imaging further determines the infection location in vivo and allows the visualization of the infection progression over time.

Lasioglossins LLIII affect the morphogenesis of Candida albicans and reduces the duration of experimental vaginal candidiasis in mice.

Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D-isomeric variant LLIII-D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII-D at concentrations of 11.5 µM and 21 µM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida-inhibitory effect of daily intravaginal administration of 0.7 or 1.4 µg of LLIII was assessed in mice with experimentally-induced vaginal candidiasis. LLIII and LLIII-D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature-induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.

Developing Targeted Hybrid Imaging Probes by Chelator Scaffolding.

Positron emission tomography (PET) as well as optical imaging (OI) with peptide receptor targeting probes have proven their value for oncological applications but also show restrictions depending on the clinical field of interest. Therefore, the combination of both methods, particularly in a single molecule, could improve versatility in clinical routine. This proof of principle study aims to show that a chelator, Fusarinine C (FSC), can be utilized as scaffold for novel dimeric dual-modality imaging agents. Two targeting vectors (a minigastrin analogue (MG11) targeting cholecystokinin-2 receptor overexpression (CCK2R) or integrin αVß3 targeting cyclic pentapeptides (RGD)) and a near-infrared fluorophore (Sulfo-Cyanine7) were conjugated to FSC. The probes were efficiently labeled with gallium-68 and in vitro experiments including determination of logD, stability, protein binding, cell binding, internalization, and biodistribution studies as well as in vivo micro-PET/CT and optical imaging in U-87MG αVß3- and A431-CCK2R expressing tumor xenografted mice were carried out. Novel bioconjugates showed high receptor affinity and highly specific targeting properties at both receptors. Ex vivo biodistribution and micro-PET/CT imaging studies revealed specific tumor uptake accompanied by slow blood clearance and retention in nontargeted tissues (spleen, liver, and kidneys) leading to visualization of tumors at early (30 to 120 min p.i.). Excellent contrast in corresponding optical imaging studies was achieved especially at delayed time points (24 to 72 h p.i.). Our findings show the proof of principle of chelator scaffolding for hybrid imaging agents and demonstrate FSC being a suitable bifunctional chelator for this approach. Improvements to fine-tune pharmacokinetics are needed to translate this into a clinical setting.

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

PURPOSE: Some [(68)Ga]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. PROCEDURES: Radiolabelling 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. RESULTS: Certain 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 [(89)Zr]triacetylfusarinine C (TAFC). All studied Ga-68 and [(89)Zr]siderophores exhibited a similar biodistribution and pharmacokinetics in mice with the exception of [(89)Zr]ferrioxamine E (FOXE). CONCLUSIONS: Zr-89 and [(68)Ga]siderophores showed analogous in vitro and in vivo behaviour. Tested [(89)Zr]siderophores could be applied for longitudinal positron emission tomography (PET) studies of fungal infections and especially TAFC for the development of novel bioconjugates.

Selected 68Ga-siderophores versus 68Ga-colloid and 68Ga-citrate: biodistribution and small animal imaging in mice.

BACKGROUND: (68)Ga-triacetylfusarinine C (TAFC) and (68)Ga-ferrioxamine E (FOXE) show great potential to be used as highly sensitive and selective tracers for Aspergillus infection imaging. Here we report on a comparison of the ex vivo biodistribution and small animal imaging of (68)Ga-TAFC and (68)Ga-FOXE versus (68)Ga-colloid and (68)Ga-citrate as unspecific control in mice. METHODS: The radiochemical purity of tested (68)Ga labelled tracers was determined by RP-HPLC or ITLC-SG. Ex vivo biodistribution was studied in normal DBA/2 mice 30 min and 90 min p.i. Static and dynamic imaging were performed using µPET/CT. RESULTS: (68)Ga-TAFC and (68)Ga-FOXE showed rapid renal excretion and low blood values even 90 min p.i. (68)Ga-TAFC showed almost no retention in other organs while (68)Ga-FOXE displayed some uptake in gastrointestinal tract. (68)Ga-colloid and (68)Ga-citrate revealed significantly different ex vivo biodistribution. (68)Ga-colloid showed pronounced radioactivity retention in the liver, while (68)Ga-citrate displayed high blood values and significant retention of radioactivity in highly perfused organs. CONCLUSIONS: From the results, both (68)Ga-TAFC and (68)Ga-FOXE have excellent and significantly different in vivo behaviour compared to (68)Ga-colloid and (68)Ga-citrate. (68)Ga-TAFC in particular confirmed its great potential use as a specific tracer for Aspergillus infection imaging.