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Abstract Background The urgent demand for innovative theranostic strategies to combat bacterial resistance to antibiotics is evident, with substantial implications for global health. Rapid diagnosis of life-threatening infections can expedite treatment, improving patient outcomes. Leveraging diagnostic modalities i.e., positron emission tomography (PET) and single photon emission computed tomography (SPECT) for detecting focal infections has yielded promising results. Augmenting the sensitivity of current PET and SPECT tracers could enable effective imaging of pathogenic bacteria, including drug-resistant strains.UBI (29–41), an antimicrobial peptide (AMP) fragment recognizes the S. aureus membrane through electrostatic binding. Radiolabeled UBI (29–41) is a promising SPECT and PET-based tracer for detecting focal infections. 2-APBA (2-acetyl-phenyl-boronic acid), a non-natural amino acid, specifically targets lysyl-phosphatidyl-glycerol (lysyl-PG) on the S. aureus membranes, particularly in AMP-resistant strains. We propose that combining UBI with 2-APBA could enhance the diagnostic potential of radiolabeled UBI. Results Present work aimed to compare the diagnostic potential of two radiolabeled peptides, namely UBI (29–41) and 2-APBA modified UBI (29–41), referred to as UBI and UBI-APBA. APBA modification imparted antibacterial activity to the initially non-bactericidal UBI against S. aureus by inducing a loss of membrane potential. The antibacterial activity demonstrated by UBI-APBA can be ascribed to the synergistic interaction of both UBI and UBI-APBA on the bacterial membrane. To enable PET imaging, we attached the chelator 1,4,7-triazacyclononane 1-glutaric acid 4,7-acetic acid (NODAGA) to the peptides for complexation with the positron emitter Gallium-68 (68Ga). Both NODAGA conjugates were radiolabeled with 68Ga with high radiochemical purity. The resultant 68Ga complexes were stable in phosphate-buffered saline and human serum. Uptake of these complexes was observed in S. aureus but not in mice splenocytes, indicating the selective nature of their interaction. Additionally, the APBA conjugate exhibited superior uptake in S. aureus while preserving the selectivity of the parent peptide. Furthermore, [68Ga]Ga-UBI-APBA demonstrated accumulation at the site of infection in rats, with an improved target-to-non-target ratio, as evidenced by ex-vivo biodistribution and PET imaging. Conclusions Our findings suggest that linking UBI, as well as AMPs in general, with APBA shows promise as a strategy to augment the theranostic potential of these molecules.

Background: The urgent demand for innovative theranostic strategies to combat bacterial resistance to antibiotics is evident, with substantial implications for global health. Rapid diagnosis of life-threatening infections can expedite treatment, improving patient outcomes. Leveraging diagnostic modalities i.e., positron emission tomography (PET) and single photon emission computed tomography (SPECT) for detecting focal infections has yielded promising results. Augmenting the sensitivity of current PET and SPECT tracers could enable effective imaging of pathogenic bacteria, including drug-resistant strains.UBI (29–41), an antimicrobial peptide (AMP) fragment recognizes the S. aureus membrane through electrostatic binding. Radiolabeled UBI (29–41) is a promising SPECT and PET-based tracer for detecting focal infections. 2-APBA (2-acetyl-phenyl-boronic acid), a non-natural amino acid, specifically targets lysyl-phosphatidyl-glycerol (lysyl-PG) on the S. aureus membranes, particularly in AMP-resistant strains. We propose that combining UBI with 2-APBA could enhance the diagnostic potential of radiolabeled UBI. Results: Present work aimed to compare the diagnostic potential of two radiolabeled peptides, namely UBI (29–41) and 2-APBA modified UBI (29–41), referred to as UBI and UBI-APBA. APBA modification imparted antibacterial activity to the initially non-bactericidal UBI against S. aureus by inducing a loss of membrane potential. The antibacterial activity demonstrated by UBI-APBA can be ascribed to the synergistic interaction of both UBI and UBI-APBA on the bacterial membrane. To enable PET imaging, we attached the chelator 1,4,7-triazacyclononane 1-glutaric acid 4,7-acetic acid (NODAGA) to the peptides for complexation with the positron emitter Gallium-68 (68Ga). Both NODAGA conjugates were radiolabeled with 68Ga with high radiochemical purity. The resultant 68Ga complexes were stable in phosphate-buffered saline and human serum. Uptake of these complexes was observed in S. aureus but not in mice splenocytes, indicating the selective nature of their interaction. Additionally, the APBA conjugate exhibited superior uptake in S. aureus while preserving the selectivity of the parent peptide. Furthermore, [68Ga]Ga-UBI-APBA demonstrated accumulation at the site of infection in rats, with an improved target-to-non-target ratio, as evidenced by ex-vivo biodistribution and PET imaging. Conclusions: Our findings suggest that linking UBI, as well as AMPs in general, with APBA shows promise as a strategy to augment the theranostic potential of these molecules. [ABSTRACT FROM AUTHOR]

Introduction/Justification: Infectious diseases are the second leading cause of mortality worldwide. In this context, considerable efforts are being made to develop radiopharmaceuticals that enable the accurate diagnosis of bacterial infections. A new field of research is focusing on antimicrobial peptides, such as Ubiquicidin. The 29-41 fragment (Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) UBI(29-41) labeled with radionuclides has proved to be an important tool for the specific diagnosis of infectious processes. Objectives: To present the radiochemical and ''in vitro'' studies of UBI(29-41) with the chelating agents 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for labeling with gallium-68 (68Ga) and to validate with a clinical application. Materials and Methods: After 68GaCl3 elution, the NOTA-UBI and DOTA-UBI reactions were performed at 85oC for 5 min and 95oC for 15 min, respectively. In both cases, the purification was carried out using a Sep-Pak C18 filter and radiochemical control by Thin-Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). The partition coefficient (Log P) and serum protein binding were determined, as well as the stability in saline and serum up to 90 min. After these studies, assays were carried out to determine the binding of the radiopharmaceuticals to ''Staphylococcus aureus'' bacteria. For the clinical study, the patient selected had a diagnosis of chronic osteomyelitis in the distal tibia, a positive blood culture for ''Staphylococcus aureus'' and a possible infectious/inflammatory process at the site identified by magnetic resonance imaging. The patient was injected intravenously with 281 MBq of 68Ga-DOTA-UBI to confirm the presence of an infectious process by PET-CT and the images were obtained 1 h post-administration. Resultados: The radiochemical purity (RP) of the radiopharmaceuticals after purification was 99.28±0.28% and 99.78±0.06% for 68Ga-NOTA-UBI and 68Ga-DOTA-UBI, respectively. Log P was -3.57±0.20 for 68Ga-NOTA-UBI and -3.63±0.17 for 68Ga-DOTA-UBI. Stability studies up to 90 min showed RP of 98.13±0.78% and 99.75±0.08% in saline; and 79.94±5.10% and 94.69±1.14% in serum, for 68Ga-NOTA-UBI and 68Ga-DOTA-UBI, respectively. The percentage of serum protein binding, evaluated at 30 and 60 min, was 59.90±1.21% and 53.45±2.16% for 68Ga-NOTA-UBI and 60.22±2.96% and 44.06±1.88% for 68Ga-DOTA-UBI, respectively. The binding of radiopharmaceuticals to ''Staphylococcus aureus'' revealed a direct relation to the amount of bacteria in the culture. Clinical images showed intense uptake of the radiopharmaceutical in the entire remnant of the talus and in the adjacent soft tissues of the left ankle. After scan, the secretion collected from the surgical site was cultured and the presence of ''Staphylococcus aureus'' was confirmed. Conclusion: The radiochemical and ''in vitro'' assays showed that the radiopharmaceuticals studied presented similar characteristics with the potential to be implemented in clinical practice. The clinical study showed that the UBI(29-41) fragment radiolabeled with 68Ga can be used as a potential biomarker for infectious processes, according to the availability of the chelating agent.

Background: Postsurgical spinal infections are a severe complication and a challenge to the neurosurgeon due to their complex management. Revision surgeries and the removal of hardware are usually necessary. Recently, advances in nuclear medicine have made it possible to employ radiotracers to identify infections. The radiolabeled antimicrobial peptide technetium-99m-ubiquicidin (99mTc-UBI) (29-41) has been demonstrated to detect bacterial infections. UBI 29-41 is a peptide sequence with selective binding to the anionic cell membrane of bacteria, which has recently been used to differentiate between infection and inflammation. Here, we describe the clinical utility of 99mTc-UBI 29-41 single-photon emission computed tomography-computed tomography (SPECT-CT) in a patient suspected of a postoperative infection. Case description: A 54-year-old male who presented with conus medullaris syndrome secondary to T12 spondylodiscitis and multiple abscess collections was initially managed with debridement, corpectomy, and minimally invasive lateral instrumentation. The patient developed postsurgical empyema near the surgical site. The image study avoided the need for a second surgery and hardware removal. Conclusion: The use of 99mTc-UBI 29-41 SPECT-CT served as a tool to avoid a second invasive procedure; instead, conservative management with antibiotics was performed with an effective outcome after two weeks. This radiotracer has utility in cases in which infection is suspected, but the location is not entirely clear, and information is needed to guide the therapeutic approach. [ABSTRACT FROM AUTHOR]

We studied an original radiolabeled complex of antimicrobial peptides UBI29-41 and UBI18-35, ubiquicidin derivatives, for distinguishing between bacterial and aseptic inflammation. For radiolabeling of the peptides with technetium-99m, a bifunctional chelating agent succinimide-1-yl 6-(bis(pyridin-2-ylmethyl)amino)hexanoate was used. The obtained complexes 99mТс-DPAH-UBI29-41 and 99mТс-DPAH-UBI18-35 had radiolabeling yield >80% and radiochemical purity >96%. Accumulation of the complexes in the focus of bacterial inflammation in bone structures and the absence of this complex in the site of aseptic inflammation was confirmed in a rat model of traumatic osteomyelitis by single-photon emission computed tomography. [ABSTRACT FROM AUTHOR]

Published methods for radiolabelling of 1,4,7-triazacyclononane-1,4,7-triacetic acid ubiquicidin (NOTA-UBI) 29–41 to date describe manual or kit-based procedures. The purpose of this study was to develop an automated synthesis method for the synthesis of [68Ga]Ga-NOTA-UBI. NOTA-UBI was successfully labelled with gallium-68 using three different automated procedures. The use of radical scavengers to improve radiochemical purity is also discussed. The automated procedures showed a high degree of robustness and repeatability. The validated automated synthesis protocols using a Scintomics GRP Module will contribute to provide GMP-compliant [68Ga]Ga-NOTA-UBI for clinical infection imaging. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Human antimicrobial peptides are of interest for the development of positron emission tomography (PET) tracers as they exhibit desirable characteristics that make them good candidates for targeting vectors. Due to their natural role in the innate immune system they selectively bind to pathogenic bacteria and yeast, whilst remaining minimally immunogenic and cytotoxic to humans. Research into ubiquicidin (UBI)-based tracers has focused on 99mTc as a radionuclide, however, the use of bi-functional chelators such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), in combination with 68Ga as a radionuclide, allows for a simple radiolabeling procedure which is preferable in a clinical setting using PET/CT. Methods: The peptides fragments UBI29-41, UBI30-41 were synthesized by standard microwave Fmoc/tert-butyl (tBu)-solid phase synthetic protocols. Characterizations were performed using analytical HPLC and LC/MS. Both NOTA-conjugated peptides were exposed to natGa3+; their complexed form was quantified by direct LC/MS injection. This complexation was utilized to testify bacterial and mammalian cell binding potential of fluorophore-linked NOTA-UBI29-41/30-41. 68Ga labeled NOTA-UBI fragments were also tested for competitive interaction to Staphylococcus aureus to proof the binding target. 68Ga was eluted from SnO2- and TiO2-based 68Ge/68Ga generators using fractionated elution and anion exchanged-based post-procession. NOTA-peptide radiolabeling was carried out including optimization of buffer molarity, NOTA-peptide concentration(s), incubation temperature and –duration as well as considering various SPE purification cartridges. Results: Pure UBI29-41, UBI30-41 and NOTA-UBI30-41 were successfully characterized. Both, NOTA-UBI fragments exhibited complexation rates to natGa3+ ≥99%. The percentage binding was significantly higher to Staphylococcus aureus bacilli over Mt4 human leucocytes (P>0.05) for NOTA-UBI29-41[Lys(Abz)]0.03) after pre-incubation with excess unlabeled NOTA-UBI. Reproducible 68Ga radiolabeling ranged for 51–85% and 46–78% for NOTA-UBI29-41 and NOTA-UBI30-41, respectively. Conclusion: Aside from successful peptide syntheses the first ever 68Ga-radiolabeling method is reported for NOTA-UBI fragments. The NOTA-conjugation didn’t compromise the selective and specific interaction with bacterial cells in vitro. Both tracers are warranting prospective imaging of infection with PET/CT. [Copyright &y& Elsevier]

Purpose: Detection of osteomyelitis beneath a diabetic foot ulcer is imperative for proper management; however, accurate and noninvasive diagnosis of osteomyelitis remains a challenge. Ubiquicidin 29-41 (UBI 29-41) is a synthetic antimicrobial peptide fragment reported to be highly infection-specific. Tc-UBI 29-41 has recently been reported to be a promising radiotracer for infection imaging. The aim of this prospective study was to evaluate the utility of Tc-UBI 29-41 scintigraphy in diabetic patients with suspected osteomyelitis of the foot. Methods: Included in the study were 65 patients with type 2 diabetes mellitus and foot ulcer and with clinical suspicion of osteomyelitis . Each patient had a three-phase bone scan and a Tc-UBI scan at 30 and 60 min after injection. The scan was considered to be consistent with osteomyelitis when the Tc-UBI 29-41 uptake was concordant with the Tc-MDP uptake. It was considered negative for osteomyelitis if there was no uptake of Tc-UBI 29-41 or if Tc-UBI 29-41 accumulated in an area not concordant with the abnormal uptake of Tc-MDP on the bone scan. In the latter case a diagnosis of soft-tissue infection was made. Bone infection was confirmed by bone biopsy/culture and by clinical and radiological follow-up. Results: Final analysis was done in 55 patients. Osteomyelitis was confirmed in 37 patients, and 18 patients were free of bone infection. Tc-UBI 29-41 was positive in all 37 patients and with the bone scan as the reference for the bone identified all osteomyelitic foci (68 in total). Tc-UBI 29-41 was negative for osteomyelitis in all 18 patients, and 17 of these patients were diagnosed with soft-tissue infection (Tc-UBI 29-41 accumulation without concordant abnormal uptake on bone scintigraphy). The sensitivity, specificity and accuracy of Tc-UBI 29-41 scan in combination with three-phase bone scan for the diagnosis of osteomyelitis in diabetic foot was 100 %. Accuracy for soft-tissue infection was also 100 %. Maximum accumulation of the Tc-UBI 29-41 with maximum target to background activity was observed in the infectious foci at 30 min after injection. Conclusion: Tc-UBI 29-41 may be a useful agent for the accurate diagnosis of bone infection in diabetic foot because of the high accuracy demonstrated in this pilot study. It was able to differentiate between bone and soft-tissue involvement effectively in combination with a bone scan. [ABSTRACT FROM AUTHOR]

Introduction: Ubiquicidin (UBI) 29-41 is a synthetic antimicrobial peptide that binds with the microbial cell membrane at the location of infection. This study was conducted to evaluate its probable efficacy as an infection- imaging agent with potential to differentiate bacterial infection from sterile inflammation in humans. Methods: Fifteen diabetic foot patients (10 males and 5 females) with suspected bacterial infection, prior to starting antibiotic treatment, were selected for this study. First a routine three phase bone scan and later a 99mTc-UBI scan was performed for all the patients. 555-740 MBq of 99mTc-UBI was injected intravenously. A 10 minute dynamic study was followed by spot views of the suspected region of infection and corresponding normal areas (liver and kidneys) at 60 and 120 mm. Whole-body anterior and posterior images were also acquired. To interpret the studies as positive or negative, visual score (0-3) was used, with scores of 0 (minimal or no uptake; equivalent to soft tissue) and 1 (mild; less uptake than in liver) being considered negative and scores of 2 (moderate; uptake equal to or greater than that in liver) and 3 (intense uptaike equal to or greater than that in kidneys) being considered positive. Results: Of 15 studies performed with 99mTc-UBI, all had positive bacterial cultures. The result of bone scan was positive for osteomyelitis in 12 patients (80%). 99mTc-UBI Scintigraphy was positive in 6 patients, but negative in nine. The sensitivity of 99mTc-UBI for detection of infection was therefore 40%. From 12 patients who had positive bone scans, only 6 had a positive 99mTc-UBI (50%) indicating the sensitivity of 50% for 99~Tc-UBI in osteomyelitis cases. 99mTc-UBI was not positive in any patient who had evidence of soft tissue infection in the bone scan. Conclusion: Although 99mTc-UBl 29-41 was well tolerated by all the patients without any side effects, considering low sensitivity of this agent, this radiopharmaceutical is not of great value for diabetic foot infection diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Ubiquicidin (UBI) 29–41 is a cationic synthetic antimicrobial peptide fragment that binds preferentially with the anionic microbial cell membrane at the site of infection. This study was conducted to evaluate the potentiality of [99mTc/Tricine/HYNIC0]UBI 29–41 prepared from lyophilized kits as an infection imaging agent in humans. Methods: Seven patients (5 males and 2 females; mean age=55 years; age range=35–75 years) with suspected bone or soft-tissue infections participated in this study. [99mTc/Tricine/HYNIC]UBI 29–41, corresponding to activity in the range 555–740 MBq added to 40 μg of peptide obtained from instant freeze-dried kit formulations with radiochemical purities >95%, was injected intravenously. A 45-min dynamic study was followed by spot views of the suspected region of infection (target) and a corresponding normal area (nontarget). Whole-body anterior and posterior images were also acquired at 30, 60 and 120 min after injection. True- or false-positive or true- or false-negative images were interpreted upon bacterial culture, radiography, clinical tests and bone scanning. Results: The biodistribution of [99mTc/Tricine/HYNIC]UBI 29–41 in patients showed rapid accumulation of activity in the kidneys in the first 30 min after injection that gradually declined and accumulated in the urinary bladder. There were positive findings in five studies and negative findings in two. Findings were subsequently confirmed to be true positive or negative. Images showed minimal accumulation in nontarget tissues, with an average target/nontarget ratio of 2.10±0.33 in positive lesions at 30 min. Conclusion: Given its favorable clinical characteristics, [99mTc/Tricine/HYNIC]UBI 29–41 shows promise as a tracer for infection imaging that allows early diagnosis (30 min) of infection. [Copyright &y& Elsevier]

Abstract: The antimicrobial peptide ubiquicidin (UBI) directly labeled with technetium-99m (99mTc) 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-PN2S) in order to compare its ability to detect infection sites with that of 99mTc-UBI. Methods: The PN2S-PEG-UBI conjugate was prepared and labeled with 99mTc, 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 99mTc-PN2S-PEG-UBI and its precursor 99mTc-PN2S-PEG and in infected mice to compare the uptakes of 99mTc-UBI and 99mTc-PN2S-PEG-UBI at the site of infection using scintigraphic imaging and ex vivo tissue counting. Results: 99mTc-PN2S-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 99mTc-UBI and 99mTc-PN2S-PEG-UBI uptake in infected mouse thigh, and the site of infection was clearly visualized using scintigraphic imaging. Conclusions: 99mTc-PN2S-PEG-UBI proved to be as effective as 99mTc-UBI in detecting sites of infection; however, the well-defined chemical structure of 99mTc-PN2S-PEG-UBI makes it a better candidate for clinical imaging of infection. [Copyright &y& Elsevier]

Background: Previous studies demonstrated that 99mTc-labeled-ubiquicidin 29–41 (99mTc-UBI 29–41) imaging is an accurate method for detection of bacterial infections. This study was conducted to evaluate the clinical use of 99mTc-UBI 29–41 for detection of mediastinitis after cardiac surgery. Methods: Thirteen patients with suspected mediastinitis after cardiac surgery were included. Qualitative and semiquantitative analyses of 99mTc-UBI 29–41 images were performed. Mediastinitis was confirmed by bacterial culture. Results: Qualitative analysis correctly identified the infection in 5/6 patients with mediastinitis. For observer 1, there were five true-positive results, six true-negative results, one false-positive result and one false-negative result (sensitivity: 83%, specificity: 85%, positive predictive value: 83%, negative predictive value: 85%, and overall diagnostic accuracy: 84%). For observer 2, there were five true-positive results, five true-negative results, two false-positive results and one false-negative result (sensitivity: 83%, specificity: 71%, positive predictive value: 71%, negative predictive value: 83%, and overall diagnostic accuracy: 76%). Agreement between observers was 0.847 (SE=0.145, p =0.002). Semiquantitative analysis showed a higher mediastinum uptake of the 99mTc-UBI 29–41 in patients with mediastinitis than that derived from patients without mediastinitis. Mean uptake of 99mTc-UBI 29–41 was 60.4±10.3 counts/pixel and 47.4±5.5 counts/pixel, respectively (p =0.01). At the threshold value of ≥57 counts/pixel using ROC analysis, the sensitivity, specificity, positive predictive value, negative predictive value and overall diagnostic accuracy for detecting patients with mediastinitis were 83, 100, 100, 87, and 92%, respectively (p =0.02; 95% CI: 0.65–1.10%). Conclusions: 99mTc-UBI 29–41 imaging yielded fast and promising first results for patients with suspected mediastinitis after cardiac surgery and, as such, deserves further investigation. [Copyright &y& Elsevier]

Introduction: Ubiquicidin 29-41 (UBI) is a fragment of the cationic antimicrobial peptide that is present in various species including humans. The purpose of this study was to investigate radiochemical and biological characteristics of [6-hydrazinopyridine-3-carboxylic acid (HYNIC)]-UBI 29-41 designed for the labeling with 99mTc using tricine as coligand. Methods: Synthesis was preformed on a solid phase using a standard Fmoc strategy and HYNIC precursor coupled at the N-terminus. Purified peptide conjugate was labeled with 99mTc at 100°C for 10 min, Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate stability and affinity to human serum was challenged for 24 hours and its in vitro binding to bacteria was assessed. Biodistribution and accumulation of radiopeptide in staphylococcus aureus infected mice were studied using scintigraphy and ca- vivo counting. Results: Radiolabeling was performed at high specific activities, and radiochemical purity was >95%. The stability of radiolabeled peptide in human serum was excellent. In vitro studies showed 70% of radioactivity was bound to bacteria. After injection into mice with a bacterial infection, removing from the circulation occurred mainly by renal clearance and site of infection was rapidly detected within 30 rain. Target to nontarget muscle ratio was 2.099 ± 0.05% at 30 rain post injection. Conclusion: [99mTc-HYNIC]-UBI 29-41 showed favorable radiochemical and biological characteristics which permitted detection of the infection with optimal visualization within 30 min. [ABSTRACT FROM AUTHOR]

The uptake of 99mTc-UBI (29–41) was evaluated at sites of bacterial infections in rabbits before and after treatment with ciprofloxacin. Staphylococcus aureus susceptible to ciprofloxacin was used to induce a focal infection in each rabbit of group 1 (G1), group 2 (G2), and group 3 (G3) with 2 × 104, 2 × 106, and 2 × 108 colony forming units (CFU), respectively. After 24 h, images of infected thighs (target: T) and contralateral thighs (nontarget: NT) were acquired. Animals then received ciprofloxacin intramuscularly for 5 days followed by imaging on the third and fifth days. The control group 4 (G4) was imaged at days 1, 3, and 5 under the same acquisition parameters. Group 5 (G5) was employed to study biodistribution of the peptide. Increases in (T/NT) ratios in G1, G2, and G3 were observed from 5 min onwards with maximum values at 60 min. G3 revealed the highest accumulation of the peptide. Growth of the same strain of S. aureus on blood agar medium was visualized after fine needle aspiration. After ciprofloxacin treatment, the images for G1–G3 resulted in significantly decreased ( P < 0.05) T/NT values on the third and fifth days that correlated with reduction in number of viable bacteria. No significant difference ( P < 0.05) in left to right thigh ratios in the control group (G4) was observed. Biodistribution of the peptide showed rapid removal of tracer from circulation through the kidneys. 99m Tc-UBI (29–41) accumulation directly correlates with the number of viable bacteria. This infection localization agent can be utilized for monitoring efficacy and duration of antibiotic treatment. [ABSTRACT FROM AUTHOR]

99mTc-UBI 29–41 is an antimicrobial peptide fragment that directly radiolabeled with 99mTc shows high in vitro and in vivo stability, rapid background clearance, minimal accumulation in non-target tissues and rapid detection of infection sites. Molecular mechanics (MM) calculation has been an essential tool in explaining experimental results associated with molecular recognition and stability. This work is an attempt to explain the 99mTc-UBI 29–41 specificity for bacteria and to understand from a structural point of view, the experimental results indicative of a molecular recognition and stability not well favored for two other cationic peptides (99mTc-Tat-1-Scr and 99mTc-Tat-2-Scr ) used as control. Structures of 99mTc-UBI, 99mTc-Tat-1-Scr, 99mTc-Tat-2-Scr and of the corresponding free cationic peptides were built and the optimized structures, in the best stable configurations, were calculated by a MM procedure. In order to correlate the calculated and experimental results, in vitro stability tests with cysteine challenge and stability to dilution in human serum and in saline solution, were performed for the three labeled cationic peptides. The three complexes can be represented by the general formula [Tc(V)(O)(H2O)2(Lysn=1,2-Argn=0,1-peptide)]10+,11+. The potential energies were 104.5, 95.6 and 90.8 kcal/mol for 99mTc-Tat-1-Scr, 99mTc-Tat-2-Scr and 99mTc-UBI 29–41, respectively. Experimental and calculated results were in good agreement. It is thus possible to predict and explain that in similar solution media 99mTc-Tat-2-Scr would be more stable than 99mTc-Tat-1-Scr and why 99mTc-UBI shows the highest stability. In conclusion, the in vitro specific binding to bacteria and the accumulation at infection sites in humans of 99mTc-labeled UBI could be the result of its high thermodynamic stability, selectivity and stereospecificity. [Copyright &y& Elsevier]

The lumen of the human colon is heavily colonized with microbes, but infections across its epithelial surface are infrequent. To address the hypothesis that antimicrobial polypeptides contribute to the barrier function of colonic epithelial cells, we examined cellular extracts from non-inflamed colonic mucosa using an antimicrobial assay. This approach yielded five polypeptides: three antimicrobials were previously identified as ribosomal polypeptides (L30, S19 and ubiquicidin), and two were members of the histone family (H1.5 and H2B). All exhibited bactericidal activity against Escherichia coli, and with the exception of S19, had been isolated by others based on their potent antimicrobial activity in other cells and tissues. These polypeptides normally reside inside cells and are proposed to contribute to the formation of the functional antimicrobial barrier of the colonic epithelium. [Copyright &y& Elsevier]

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 Arg7 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 (dxysp3). It will be necessary to isolate and characterize the 99Tc(V)(O)-UBI.(H2O)n complex to confirm these results. [Copyright &y& Elsevier]

Antimicrobial peptides and proteins are effector molecules in the protection of epithelial surfaces. We have evaluated the presence of antimicrobial peptides/proteins that can participate in human colonic defence against microbes. A peptide/protein extract of normal human colon mucosa was found to be active against Gram-positive bacteria, Gram-negative bacteria, and fungi. Four polypeptides with antimicrobial activity were isolated from this material and they were identified by N-terminal amino acid sequence analysis as ubiquicidin, histone H2B, eosinophil cationic protein, and phospholipase A2 (PLA2). Using immunodetection and mass spectrometry, LL-37, HNP1–3, and HBD-1 were also identified. Combined, these results indicate that the colon mucosa is protected by a complex mixture of polypeptides, able to kill invading microbes and working in synergy as a barrier against bacterial invasion. [Copyright &y& Elsevier]

Introduction: Radiotracers are widely used in medical imaging, using techniques of gamma-camera imaging (scintigraphy and SPECT) or positron emission tomography (PET). In bone marrow infection, there is no single routine test available that can detect infection with sufficiently high diagnostic accuracy. Here, we review radiotracers used for imaging of bone marrow infection, also known as osteomyelitis, with a focus on why these molecules are relevant for the task, based on their physiological uptake mechanisms. The review comprises [67Ga]Ga-citrate, radiolabelled leukocytes, radiolabelled nanocolloids (bone marrow) and radiolabelled phosphonates (bone structure), and [18F]FDG as established radiotracers for bone marrow infection imaging. Tracers that are under development or testing for this purpose include [68Ga]Ga-citrate, [18F]FDG, [18F]FDS and other non-glucose sugar analogues, [15O]water, [11C]methionine, [11C]donepezil, [99mTc]Tc-IL-8, [68Ga]Ga-Siglec-9, phage-display selected peptides, and the antimicrobial peptide [99mTc]Tc-UBI29-41 or [68Ga]Ga-NOTA-UBI29-41. Conclusion: Molecular radiotracers allow studies of physiological processes such as infection. None of the reviewed molecules are ideal for the imaging of infections, whether bone marrow or otherwise, but each can give information about a separate aspect such as physiology or biochemistry. Knowledge of uptake mechanisms, pitfalls, and challenges is useful in both the use and development of medically relevant radioactive tracers. [ABSTRACT FROM AUTHOR]

Unlabelled: Antimicrobial peptides such as ubiquicidin (UBI) are believed to differentiate between mammalian and bacterial or fungal cells. (99m)Tc-UBI29-41 was previously tested for detecting infection in humans using SPECT. For the present study, the UBI fragment UBI29-41 (TGRAKRRMQYNRR) was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA), radiolabeled with (68)Ga, and investigated in a rabbit infection model., Methods: (68)Ga was obtained from a 1.85-GBq (68)Ge/(68)Ga generator. New Zealand White rabbits were anesthetized with ketamine/medetomidine before tracer administration and placed in a clinical PET/CT scanner. (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic-acid-ubiquicidin29-41 ((68)Ga-NOTA-UBI29-41) was formulated in saline solution, and 101 ± 41 MBq were administered intravenously. The tracer distribution was studied by PET/CT imaging in animals (a) that were healthy, (b) bearing muscular Staphylococcus aureus infections and turpentine oil-induced muscular inflammations, and (c) bearing ovalbumin-induced lung inflammations. Static PET/CT imaging was performed at different time intervals up to 120 min after injection. For calculation of target-to-nontarget ratios, standardized uptake values were normalized against healthy thigh muscle, representing nontargeted tissue., Results: PET/CT images of healthy animals showed predominant distribution in the kidneys, liver, and bladder; heart and spleen showed moderate, declining uptake, only. The biologic half-life in blood was 29 min. Urinary accumulation of (68)Ga-NOTA-UBI29-41 peaked at 3.8 ± 0.91 percentage injected dose per gram (%ID) at 120 min, and 88 ± 5.2 %ID was recovered in total urine. (68)Ga-NOTA-UBI29-41 imaging in (b) selectively visualized the muscular infection site and was differentiated from sterile inflammatory processes. Standardized uptake value ratios for muscles (infected/inflamed) were 2.9 ± 0.93, 2.9 ± 0.50, 3.5 ± 0.86, and 3.8 ± 0.90 at 5, 30, 60, and 90 min after injection, respectively. Rabbit lungs with asthma showed insignificant uptake., Conclusion: (68)Ga-NOTA-UBI29-41 was strongly localized in bacteria-infected areas and minimally detected in a sterile inflammation area in rabbit muscles. The findings propose this compound to be an excellent first-line PET/CT tracer to allow the distinguishing of infection from inflammation.