68Ga-NOTA-Functionalized Ubiquicidin: Cytotoxicity, Biodistribution, Radiation Dosimetry, and First-in-Human PET/CT Imaging of Infections.

Ubiquicidin is an antimicrobial peptide with great potential for nuclear imaging of infectious diseases, as its cationic-rich fragment TGRAKRRMQYNRR (UBI) has been functionalized with NOTA to allow complexation to 68Ga (68Ga-NOTA-UBI). We herein assess the cytotoxicity and radiation dosimetry for 68Ga-NOTA-UBI and a first-in-human evaluation to diagnose infectious processes. Methods: Cytotoxicity was evaluated in green monkey kidney epithelial (Vero) cells and MT-4 leukocytes. Tracer susceptibility was studied in vitro using different bacterial and fungal strains. PET/CT-based biodistribution, pharmacokinetics, and radiation dosimetry were performed on nonhuman primates. Two healthy volunteers and 3 patients with suspected infection underwent 68Ga-NOTA-UBI PET/CT imaging. Results: Negligible cytotoxicity was determined for NOTA-UBI. 68Ga-NOTA-UBI showed moderate blood clearance (29-min half-life) and predominant renal clearance in nonhuman primates. Human radiation dose estimates indicated the bladder wall as the dose-critical tissue (185 µSv/MBq), followed by the kidneys (23 µSv/MBq). The total absorbed body dose was low (<7 µSv/MBq); the effective dose was estimated at 17 µSv/MBq. 68Ga-NOTA-UBI could diagnose bone- and soft-tissue infection in 3 of 3 patients. Conclusion:68Ga-NOTA-UBI is considered a nontoxic, safe-to-administer radiopharmaceutical unlikely to cause adverse effects in humans. The favorable tracer biodistribution and the first-in-human results will make 68Ga-NOTA-UBI PET/CT an encouraging future diagnostic technique with auxiliary clinical relevance.

Detection of sites of infection in mice using 99mTc-labeled PN(2)S-PEG conjugated to UBI and 99mTc-UBI: a comparative biodistribution study.

UNLABELLED: The antimicrobial peptide ubiquicidin (UBI) directly labeled with technetium-99m ((99m)Tc) has recently been shown to be specifically taken up at sites of infection; however, its chemical structure is not well defined. To address this problem, the aim of the present study was to label UBI using poly(ethyleneglycol)-N-(N-(3-diphenylphosphinopropionyl)glycyl)-S-tritylcysteine ligand (PEG-PN(2)S) in order to compare its ability to detect infection sites with that of (99m)Tc-UBI. METHODS: The PN(2)S-PEG-UBI conjugate was prepared and labeled with (99m)Tc, and its radiochemical purity was subsequently assessed. The stability of the conjugate to cysteine challenge and dilution with both saline solution and phosphate buffer was determined and serum stability and protein binding were also assessed. In vivo studies were carried out in healthy mice to study the biodistribution of (99m)Tc-PN(2)S-PEG-UBI and its precursor (99m)Tc-PN(2)S-PEG and in infected mice to compare the uptakes of (99m)Tc-UBI and (99m)Tc-PN(2)S-PEG-UBI at the site of infection using scintigraphic imaging and ex vivo tissue counting. RESULTS: (99m)Tc-PN(2)S-PEG-UBI was obtained with high radiochemical purity (98+/-1%) and high stability. The amphiphilic nature of the conjugate leads to a tendency to form micellar aggregates that explain the high protein binding values obtained. Biodistribution studies in mice showed low renal clearance followed by a predominant reticuloendothelial system clearance that limits its application in the abdominal area. Statistical analysis revealed no significant difference between (99m)Tc-UBI and (99m)Tc-PN(2)S-PEG-UBI uptake in infected mouse thigh, and the site of infection was clearly visualized using scintigraphic imaging. CONCLUSIONS: (99m)Tc-PN(2)S-PEG-UBI proved to be as effective as (99m)Tc-UBI in detecting sites of infection; however, the well-defined chemical structure of (99m)Tc-PN(2)S-PEG-UBI makes it a better candidate for clinical imaging of infection.

The pharmacology of radiolabeled cationic antimicrobial peptides.

Cationic antimicrobial peptides are good candidates for new diagnostics and antimicrobial agents. They can rapidly kill a broad range of microbes and have additional activities that have impact on the quality and effectiveness of innate responses and inflammation. Furthermore, the challenge of bacterial resistance to conventional antibiotics and the unique mode of action of antimicrobial peptides have made such peptides promising candidates for the development of a new class of antibiotics. This review focuses on antimicrobial peptides as a topic for molecular imaging, infection detection, treatment monitoring and additionally, displaying microbicidal activities. A scintigraphic approach to studying the pharmacokinetics of antimicrobial peptides in laboratory animals has been developed. The peptides were labeled with technetium-99m and, after intravenous injection into laboratory animals, scintigraphy allowed real-time, whole body imaging and quantitative biodistribution studies of delivery of the peptides to the various body compartments. Antimicrobial peptides rapidly accumulated at sites of infection but not at sites of sterile inflammation, indicating that radiolabeled cationic antimicrobial peptides could be used for the detection of infected sites. As the number of viable micro-organisms determines the rate of accumulation of these peptides, radiolabeled antimicrobial peptides enabled to determine the efficacy of antibacterial therapy in animals to be monitored as well to quantify the delivery of antimicrobial peptides to the site of infection. The scintigraphic approach provides to be a reliable method for investigating the pharmacokinetics of small cationic antimicrobial peptides in animals and offers perspective for diagnosis of infections, monitoring antimicrobial therapy, and most important, alternative antimicrobial treatment infections with multi-drug resistant micro-organisms in humans.

[Imaging of inflammatory foci with 99m technecium labeled antimicrobial oligopeptide].

A direct method using 99mtechnecium to label the synthetic antimicrobial oligopeptide ubiquicidine with a molecular mass of 1.7 kDa has been developed. The radiochemical purity of the resultant labeled compound was more than 98%. A combination of ubiquicidine and 99mtechnecium showed stability in blood plasma. The biological study indicated that the complex was renally excreted from blood flow rapidly (the half-life is 30 min). It accumulated in the experimental inflammatory focus of laboratory animals, peaking 1-2 hours after intravenous injection when the inflammatory focus/intact tissue ratio amounted up to 2.6-3.4, which could yield satisfactory abscess scintigrams. The findings make it possible to consider ubiquicidine as a promising compound for the design of radiopharmaceutical to image inflammatory foci.

Biokinetics of (99m)Tc-UBI 29-41 in humans.

Antimicrobial peptides have been proposed as new agents to distinguish between bacterial infections and sterile inflammatory processes. (99m)Tc-UBI labeled by a direct method has shown high in vitro and in vivo stability, specific uptake at the site of infection, rapid background clearance, minimal accumulation in non-target tissues and rapid detection of infection sites in mice. The aim of this study was to establish a (99m)Tc-UBI biokinetic model and evaluate its feasibility as an infection imaging agent in humans. Whole-body images from 6 children with suspected bone infection were acquired at 1, 30, 120, 240 min and 24 h after (99m)Tc-UBI administration. Regions of interest (ROIs) were drawn around source organs (heart, liver, kidneys and bladder) on each time frame. The same set of ROIs was used for all 6 scans and the cpm of each ROI were converted to activity using the conjugate view counting method. Counts were corrected by physical decay and by the background correction factor derived from preclinical phantom studies. The image sequence was used to extrapolate (99m)Tc-UBI time-activity curves in each organ and calculate the cumulated activity (A). Urine samples were used to obtain the cumulative percent of injected activity (% I.A.) versus time renal elimination. The absorbed dose in organs was evaluated according to the general equation described in the MIRD formalism. In addition, (67)Ga-citrate images were obtained from all the patients and used as a control. Biokinetic data showed a fast blood clearance with a mean residence time of 0.52 h. Approximately 85% of the injected activity was eliminated by renal clearance 24 h after (99m)Tc-UBI administration. Images showed minimal accumulation in non-target tissues with an average target/non-target ratio of 2.18 +/- 0.74 in positive lesions at 2 h. All infection positive(99m)Tc-UBI images were in agreement with those obtained with (67)Ga-citrate. The mean radiation absorbed dose calculated was 0.13 mGy/MBq for kidneys and the effective dose was 4.34 x 10(-3)mSv/MBq.

Lys and Arg in UBI: a specific site for a stable Tc-99m complex?

The aim of this study was to help establish if ubiquicidin peptide 29-41 fragment (UBI) contains a specific site for 99mTc labeling by a new direct method under alkaline conditions. Since this peptide does not have cysteine residues, it is possible that neighboring arginine and lysine in the peptide amino acid sequence (Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) could be a specific coordination site to form a stable 99mTc-UBI complex. Following direct labeling, the in vitro stability of 99mTc-UBI was compared to UBI radiolabeled by one indirect method using HYNIC/tricine and HYNIC/tricine/EDDA. Radiochemical purity of 99mTc-UBI averaged 97% compared to 88% for 99mTc-HYNIC-UBI/tricine and 98% for 99mTc-HYNIC-UBI/tricine/EDDA. Both 99mTc-HYNIC-UBI (tricine or EDDA) and 99mTc-UBI showed stability in human serum and solutions of cysteine. 99mTc-UBI radiochemical purity 24 h after dilution in 0.9% NaCl was greater than 90% at pH 9 and greater than 95% at pH 6.5. Under one set of experimental conditions, in vitro binding to bacteria of 99mTc-UBI was 35% and identical to that of 99mTc-HYNIC-UBI/tricine and 99mTc-HYNIC-UBI/tricine/EDDA at 32% and 31% respectively. The biodistribution of 99mTc-UBI in mice showed a rapid renal clearance. To help identify the site(s) of 99mTc binding following direct labeling, molecular mechanics and quantum-mechanical calculations were performed which showed that the amine groups of Arg(7) and Lys are the most probable site. The calculations show that these groups can form a square pyramid with two water molecules for the Tc cation (dxysp(3)). It will be necessary to isolate and characterize the 99Tc(V)(O)-UBI.(H2O)n complex to confirm these results.

99mTc-antimicrobial peptides: promising candidates for infection imaging.

This review presents the state of the art of imaging of bacterial and fungal infections in laboratory animals using antimicrobial peptides labelled with technetium-99m ((99m)Tc). The mechanistic basis of this approach is that these peptides accumulate at sites of infection, but not in sterile inflammatory lesions, because of their preferential binding to bacteria and fungi over mammalian cells. For practical reasons, such as production of large amounts of peptides under good laboratory practice conditions and favourable pharmacokinetics, synthetic peptides representing such binding domains of natural antimicrobial peptides are preferred. On the basis of their preferential in vitro and in vivo binding to microorganisms over human cells, fast and easy penetration into the target area, and rapid clearance from the circulation via the urinary tract, various (99m)Tc-antimicrobial peptides were identified. Next, it was determined whether these radiopharmaceuticals distinguish infectious foci from sites of sterile inflammation. Further experiments with (99m)Tc-ubiquicidin-derived peptides in infected laboratory animals have revealed that the radioactivity at the infectious site correlated well with the number of viable bacteria present, indicating that these (99m)Tc-labelled peptides may enable the monitoring of the efficacy of antimicrobial therapy. Together, (99m)Tc-labelled synthetic peptides derived from human ubiquicidin are promising candidates for imaging of bacterial and fungal infections in nuclear medicine.

Radiochemical and biological characteristics of 99mTc-UBI 29-41 for imaging of bacterial infections.

A technetium-99m-labeled peptide derived from ubiquicidine, further referred to as 99mTc-UBI 29-41, targets bacterial and fungal infections, but not sterile inflammatory processes, in experimental animals. This paper reports on the radiochemical and biological features of this radioactive agent and the importance of the amino acid sequence of UBI 29-41 for imaging of infections. Radiochemical analyses of 99mTc-UBI 29-41 and a radiolabeled scrambled version of this peptide, i.e. 99mTc-Sc-UBI 29-41, revealed that both peptides were labeled rapidly (within 10 min) and effectively with little colloid formation (less than 5% of the total radioactivity) and very little free pertechnetate (or radioactive intermediates) in the preparations containing radiolabeled peptide. Furthermore, association of the peptides with bacteria could be competed with excess unlabeled peptide and this association proved to be temperature-dependent. Based on this in vitro data we concluded that labeling of peptides with 99mTc by this direct method is rapid, efficient, and safe. Scintigraphy demonstrated that radioactivity is rapidly removed from the circulation (half-lifes of UBI 29-41 and Sc-UBI 29-41 were 16 and 21 min, respectively) mainly by renal clearance. Analysis of murine blood revealed that only a small proportion of the intravenously injected 99mTc-peptides is associated with blood cells. Although both radiolabeled peptides accumulated rapidly at sites of infection, the values for 99mTc-UBI 29-41 were higher (P < 0.05) than for 99mTc-Sc-UBI 29-41. Moreover, injection of excess unlabeled UBI 29-41, but not Sc-UBI 29-41, into Staphylococcus aureus-infected mice prior to injection of 99mTc-UBI 29-41 significantly (P < 0.05) reduced the accumulation of this radiopharmaceutical at the site of infection. In addition, we observed significantly (P < 0.01) higher amounts of 99mTc-UBI 29-41 at the site of infection in mice using a carrier-free radiolabeled UBI 29-41 as compared with unpurified preparations containing radiolabeled UBI 29-41. This in vivo data indicates that the amino acid sequence of 99mTc-UBI 29-41 contributes to its accumulation at the site of infection.

Technetium-99m labelled fluconazole and antimicrobial peptides for imaging of Candida albicans and Aspergillus fumigatus infections.

The aim of this study was to investigate whether technetium-99m labelled fluconazole can distinguish fungal from bacterial infections. Fluconazole was labelled with (99m)Tc and radiochemical analysis showed less than 5% impurities. The labelling solution was injected into animals with experimental infections. For comparison, we used two peptides for infection detection, i.e. UBI 29-41 and hLF 1-11, and human IgG, all labelled with (99m)Tc. Mice were infected with Candida albicans or injected with heat-killed C. albicans or lipopolysaccharides to induce sterile inflammation. Also, mice were infected with Staphylococcus aureus or Klebsiella pneumoniae. Next, accumulation of (99m)Tc-fluconazole and (99m)Tc-labelled peptides/IgG at affected sites was determined scintigraphically. (99m)Tc-fluconazole detected C. albicans infections (T/NT ratio=3.6+/-0.47) without visualising bacterial infections (T/NT ratio=1.3+/-0.04) or sterile inflammatory processes (heat-killed C. albicans: T/NT ratio=1.3+/-0.2; lipopolysaccharide: T/NT ratio=1.4+/-0.1). C. albicans infections were already seen within the first hour after injection of (99m)Tc-fluconazole (T/NT ratio=3.1+/-0.2). A good correlation (R(2)=0.864; P<0.05) between T/NT ratios for this tracer and the number of viable C. albicans was found. Although (99m)Tc-UBI 29-41 and (99m)Tc-hLF 1-11 were able to distinguish C. albicans infections from sterile inflammatory processes in mice, these (99m)Tc-labelled peptides did not distinguish these fungal infections from bacterial infections. It is concluded that (99m)Tc-fluconazole distinguishes infections with C. albicans from bacterial infections and sterile inflammations.