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Purpose Sentinel lymph node biopsy is a routine procedure for nodal staging in penile cancer. Most commonly, this procedure is guided by radioactive tracers, providing various forms of preoperative and intraoperative guidance. This is further extended with fluorescence imaging using hybrid radioactive–fluorescence tracers. Alternatively, a magnetic-based approach has become available using superparamagnetic iron-oxide nanoparticles (SPIONs). This study investigates a novel freehand magnetic particle imaging and navigation modality (fhMPI) for intraoperative localization, along with a hybrid approach, combining magnetic and fluorescence guidance. Materials and methods The fhMPI set-up was built with a surgical navigation device, optical tracking system and magnetometer probe. A dedicated reconstruction software based on a look-up-table method was used to reconstruct a superficial 3D volume of the SPION distribution in tissue. For fluorescence guidance, indocyanine green (ICG) was added to the SPIONs. The fhMPI modality was characterized in phantoms, ex vivo human skin and in vivo porcine surgery. Results Phantom and human skin explants illustrated that the current fhMPI modality had a sensitivity of 2.2 × 10–2 mg/mL SPIONs, a resolving power of at least 7 mm and a depth penetration up to 1.5 cm. Evaluation during porcine surgery showed that fhMPI allowed for an augmented reality image overlay of the tracer distribution in tissue, as well as 3D virtual navigation. Besides, using the hybrid approach, fluorescence imaging provided a visual confirmation of localized nodes. Conclusion fhMPI is feasible in vivo, providing 3D imaging and navigation for magnetic nanoparticles in the operating room, expanding the guidance possibilities during magnetic sentinel lymph node procedures. Furthermore, the integration of ICG provides the ability to visually refine and confirm correct localization. Further clinical evaluation should verify these findings in human patients as well.

BackgroundWith the rise of prostate-specific membrane antigen (PSMA) radioguided surgery, which is performed using a microdosing regime, demand for visual target confirmation via fluorescence guidance is growing. While proven very effective for radiotracers, microdosing approaches the detection limit for fluorescence imaging. Thus, utility will be highly dependent on the tracer performance, the sensitivity of the fluorescence camera used, and the degree of background signal. Using a porcine model the ability to perform robot-assisted radical prostatectomy under fluorescence guidance using the bimodal or rather hybrid PSMA tracer (99mTc-EuK-(SO3)Cy5-mas3) was studied, while employing the tracer in a microdosing regime. This was followed by ex vivo evaluation in surgical specimens obtained from prostate cancer patients.ResultsT50% bloodand T50% urinewere reached at 85 min and 390 min, in, respectively, blood and urine. Surgical fluorescence imaging allowed visualization of the prostate gland based on the basal PSMA-expression in porcine prostate. Together, in vivo visualization of the prostate and urinary excretion suggests at least an interval of > 7 h between tracer administration and surgery. Confocal microscopy of excised tissues confirmed tracer uptake in kidney and prostate, which was confirmed with PSMA IHC. No fluorescence was detected in other excised tissues. Tumor identification based on ex vivo fluorescence imaging of human prostate cancer specimens correlated with PSMA IHC.ConclusionIntraoperative PSMA-mediated fluorescence imaging with a microdosing approach was shown to be feasible. Furthermore, EuK‐(SO3)Cy5‐mas3allowed tumor identification in human prostate samples, underlining the translational potential of this novel tracer.Trial registrationApproval for use of biological material for research purposes was provided by the Translational Research Board of the Netherlands Cancer Institute-Antoni van Leeuwenhoek hospital (NKI-AvL) under reference IRBm19-273 (22/10/2019).

Prostate cancer surgery is currently being revolutionized by the use of prostate-specific membrane antigen (PSMA)-targeted radiotracers, for example, (99)mTc-labeled PSMA tracer analogs for radioguided surgery. The purpose of this study was to develop a second-generation (99)mTc-labeled PSMA-targeted tracer incorporating a fluorescent dye. Methods: Several PSMA-targeted hybrid tracers were synthesized: glutamic acid- urea-lysine (EuK)-Cy5-mas(3), EuK-(SO3)Cy5-mas(3), EuK-Cy5(SO3)-mas(3), EuK-(Ar)Cy5-mas(3), and EuK-Cy5(Ar)-mas(3); the Cy5 dye acts as a functional backbone between the EuK targeting vector and the 2-mercaptoacetyl-seryl-seryl-seryl (mas(3)) chelate to study the dye's interaction with PSMA's amphipathic entrance funnel. The compounds were evaluated for their photophysical and chemical properties and PSMA affinity. After radiolabeling with (99)mTc, we performed in vivo SPECT imaging, biodistribution, and fluorescence imaging on BALB/c nude mice with orthotopically transplanted PC346C tumors. Results: The dye composition influenced the photophysical properties (brightness range 0.3-1.5 x 10(4) M-1 x cm(-1)), plasma protein interactions (range 85.0% +/- 2.3%-90.7%+/- 1.3% bound to serum, range 76%+/- 0%-89%+/- 6% stability in serum), PSMA affinity (half-maximal inhibitory concentration [IC50] range 19.2 +/- 5.8-175.3 +/- 61.1 nM) and in vivo characteristics (tumorto-prostate and tumor-to-muscle ratios range 0.02 +/- 0.00-154.73 +/- 28.48 and 0.46 +/- 0.28-5,157.50 +/- 949.17, respectively; renal, splenic, and salivary retention). Even though all tracer analogs allowed tumor identification with SPECT and fluorescence imaging, (99)mTc-EuK(SO3)Cy5-mas(3) had the most promising properties (e.g., half-maximal inhibitory concentration, 19.2 +/- 5.8, tumor-to-muscle ratio, 5,157.50 +/- 949.17). Conclusion: Our findings demonstrate the intrinsic integration of a fluorophore in the pharmacophore in PSMA-targeted smallmolecule tracers. In this design, having 1 sulfonate on the indole moiety adjacent to EuK ((99)mTc-EuK-(SO3)Cy5-mas(3)) yielded the most promising tracer candidate for imaging of PSMA.

Background: Today, both radioactive SPECT and PET imaging radiopharmaceuticals are used for clinical diagnosis of bacterial infections. Due to the possible applications in image-guided surgery, fluorescent imaging of infections has gained interest. We here present the highlights and recent developments in the use of fluorescence imaging for bacterial infections. In this overview, we also include the latest developments in multimodal bacterial imaging strategies that combine radioactive and fluorescent imaging. Based on this literature, we present our future perspectives for the field including the translational potential. Methods: In the current review, we complement earlier reports with the most recent fluorescent and multimodal radiopharmaceuticals for bacterial infection imaging. Where possible, in this review, the chemical structure of the compounds and clinical images were shown. Results: A total of 35 out of 77 original articles on pre-clinical and clinical imaging of bacterial infections with fluorescent tracers and multimodality radiopharmaceuticals were included for reviewing. Conclusion: In our view, the highest translational potential lies with compounds that are based on targeting vectors that are specific for bacteria: e.g., fluorescently labelled UBI 29–41 , polymyxin B, vancomycin, ZnDPA and a M. tuberculosis-specific β-lactamase-cleavable linker CNIP800. Multimodal concepts using dually labelled UBI 29–41 , vancomycin, and ZnDPA help connect optical imaging to the more traditional use of radiopharmaceuticals in infectious diseases. Multimodal bacterial imaging is a promising strategy not only to diagnose bacterial infections but also to evaluate the effectivity of surgical treatment for infections.

The potential of receptor-mediated fluorescence-based image-guided surgery tracers is generally linked to the near-infrared emission profile and good-manufacturing-production availability of fluorescent dyes. Surprisingly, little is known about the critical interaction between the structural composition of the dyes and the pharmacokinetics of the tracers. In this study, a dual-modality tracer design was used to systematically and quantitatively evaluate the influence of elongation of the polymethine chain in a fluorescent cyanine dye on the imaging potential of a targeted tracer. Methods: As a model system, the integrin marker αvβ3 was targeted using arginylglycylaspartisc acid [RGD]-based vectors functionalized with a 111In-diethylenetriaminepentaacetic acid (DTPA) chelate and a fluorescent dye: (Cy3-(SO3)methyl-COOH [emission wavelength (λem), 580 nm], Cy5-(SO3)methyl-COOH [λem, 680 nm], or Cy7-(SO3)methyl-COOH [λem, 780 nm]). Tracers were analyzed for differences in photophysical properties, serum protein binding, chemical or optical stability, and signal penetration through tissue. Receptor affinities were evaluated using saturation and competition experiments. In vivo biodistribution (SPECT imaging and percentage injected dose per gram of tissue) was assessed in tumor-bearing mice and complemented with in vivo and ex vivo fluorescence images obtained using a clinical-grade multispectral fluorescence laparoscope. Results: Two carbon-atom-step variations in the polymethine chain of the fluorescent cyanine dyes were shown to significantly influence the chemical and photophysical characteristics (e.g., stability, brightness, and tissue penetration) of the hybrid RGD tracers. DTPA-Cy5-(SO3)methyl-COOH-c[RGDyK] structurally outperformed its Cy3 and Cy7 derivatives. Radioactivity-based evaluation of in vivo tracer pharmacokinetics yielded the lowest nonspecific uptake and highest tumor-to-background ratio for DTPA-Cy5-(SO3)methyl-COOH-c[RGDyK] (13.2 ± 1.7), with the Cy3 and Cy7 analogs trailing at respective tumor-to-background ratios of 5.7 ± 0.7 and 4.7 ± 0.7. Fluorescence-based assessment of tumor visibility revealed a similar trend. Conclusion: These findings underline that variations in the polymethine chain lengths of cyanine dyes have a profound influence on the photophysical properties, stability, and in vivo targeting capabilities of fluorescent imaging tracers. In a direct comparison, the intermediate-length dye (Cy5) yielded a superior c[RGDyK] tracer, compared with the shorter (Cy3) and longer (Cy7) analogs.

Timely and specific diagnosis of infectious diseases can be clinically challenging but essential for the patient's outcome. Laboratory tests, such as a blood culture or urine specimen, can detect the responsible micro-organism but cannot discriminate between sterile inflammatory disease and truly infectious disease. Imaging tests, like scintigraphic techniques, can pinpoint the infection in the body. There are a number of clinical scintigraphic tests from which to choose, and no single test is optimal for the various presentations of clinical infectious disease. The currently available radiopharmaceuticals often are not capable of distinguishing between sterile inflammation, and bacterial or fungal infections. Neutrophil-mediated processes, characteristic for both inflammatory and infectious processes, can be targeted in situ by radiolabeled leukocytes, antibodies or fragments, or even by cytokines and (18)F-fluorodeoxyglucose. Unfortunately those techniques are not infection-specific markers, and ongoing research is in progress to tackle this problem. The most promising option in this respect is directly targeting bacteria or fungi with radiolabeled antibiotics or antimicrobial peptides. These theoretically highly infection-specific radiopharmaceuticals could be used for monitoring the success of antimicrobial therapy of infectious disease. Although results from preclinical experiments and pilot studies in patients are promising, radiolabeled anti-infective agents are not currently in routine clinical use and studies are continuing to prove their effectiveness for diagnostic imaging of infections in the future.

(99m)Tc-UBI 29-41 (UBI), an antimicrobial peptide, specifically targets bacteria. We tested the ability of UBI to discriminate between infected and uninfected prosthetic joints using a rabbit model previously validated.Left knee arthroplasty was performed on 20 New Zealand rabbits, then 10(7) cfu of S. aureus (n = 12) or sterile saline (n = 8) was injected into the joint. On days 9 and 20 after surgery, planar UBI scintigraphy was performed in six infected and four uninfected rabbits, 1 h and 4 h p.i. (150 MBq), on a gamma camera. Operated-to-normal knee activity ratio (ONKR) was calculated on each scintigram. Then, after sacrifice, tissue samples of both knees were counted in a gamma counter.One rabbit injected with sterile saline had cutaneous infection at sacrifice and was excluded from analysis. ONKR was higher in infected than in uninfected animals 4 h p.i. 20 days after surgery: 1.75 +/- 0.48 vs 1.13 +/- 0.11, p = 0.04. From 1 h to 4 h p.i., ONKR increased in 9/12 infected and 0/7 uninfected animals. According to UBI uptake intensity and kinetics, scintigraphy was truly positive in all infected cases on day 9 and in four of six infected cases on day 20. It was truly negative in two of three sterile inflamed prosthetic knees on day 9, and in all cases on day 20. Biodistribution studies revealed increased UBI uptake in periprosthetic tissues in all animals 9 days after surgery, and only in infected animals on day 20.In this experimental study, (99m)Tc-UBI 29-41 scintigraphy permitted the early detection of acute prosthetic joint infection, and exclusion of infection in chronic sterile prosthetic joint inflammation.

Introduction Llama single domain antibody fragments (VHH), which can pass endothelial barriers, are being investigated for targeting amyloid plaque load in Alzheimer's disease (AD). Contrary to conventional human or murine antibodies consisting of IgG or F(ab′)2 antibody fragments, VHH are able to effectively pass the blood brain barrier (BBB) in vitro . However, in earlier in vivo studies, anti-amyloid VHH showed poor BBB passage due to their short serum half-lives. It would be of interest to develop a VHH based protein with elongated serum half-life to enhance BBB passage, allowing the VHH to more easily reach the cerebral amyloid deposits. Methods To increase serum persistence, the Fc portion of the human IgG1 antibody (hinge plus CH2 and CH3 domains) was fused to the C-terminus of the VHH (VHH-pa2H-Fc). To determine the pharmacokinetics and biodistribution profile of the fusion protein, the chelator p-SCN-Bz-DTPA was linked to the protein and thereafter labeled with radioactive indium-111 ( 111 In). Double transgenic APPswe/PS1dE9 and wild type littermates were injected with 20μg VHH-pa2H-Fc-DTPA- 111 In (10-20MBq). Pharmacokinetics of the tracer was determined in blood samples at 10 intervals after injection and imaging using microSPECT was performed. The biodistribution of the radioactivity in various excised tissues was measured at 48h after injection. Results We succeeded in the expression of the fusion protein VHH-pa2H-Fc in HEK293T cells with a yield of 50mg/L growth medium. The fusion protein showed homodimerization – necessary for successful Fc neonatal receptor recycling. Compared to VHH-pa2H, the Fc tailed protein retained high affinity for amyloid beta on human AD patient brain tissue sections, and significantly improved serum retention of the VHH. However, at 48h after systemic injection of the non-fused VHH-DTPA- 111 In and the VHH-Fc-DTPA- 111 In fusion protein in transgenic mice, the specific brain uptake of VHH-Fc-DTPA- 111 In was not improved compared to non-fused VHH-DTPA- 111 In. Conclusion Using VHH-Fc conjugates increases the blood half-life of the protein. However, purely extending the time window for brain uptake does not increase BBB passage. Nevertheless, VHH-Fc holds promise for therapeutic applications where a sustained systemic circulation of VHH is advantageous.

The purpose of this study was to investigate radiochemical and biological characteristics of an instant kit for the preparation of 99mTc-labelled UBI 29-41 for specific detection of infections. The kit is based on 99mTc-labelling via HYNIC conjugated to the terminal amine of the peptide, producing a well-understood labelled compound. One hour after the addition of fresh 99mTcO to the kit ITLC and HPLC reverse-phase analysis was performed. Stability of the labelled complex was challenged and the binding to bacterial pellets was assessed. Finally, the biodistribution and accumulation in MRSA-infected tissues were studied using scintigraphy and ex vivo countings. Data were compared to a non-kit control method. Radiochemical analysis indicated >96% labelling, stability for 24 h and the preparation was used without purification. In vitro studies showed 41% of radioactivity was bound to bacteria. After injection into mice with a bacterial infection the site of infection was visualized within 30 min. Kit prepared 99mTc-HYNIC-UBI 29-41 was rapidly (half-life 113 min) cleared via the kidneys and urinary bladder, essentially slower than control peptide (half-life 74 min). This slower clearance results in higher activities in blood and other tissues. Nevertheless, 99mTc-HYNIC-UBI 29-41 shows favourable radiochemical characteristics and deserves further evaluation in a clinical setting. Copyright © 2005 John Wiley & Sons, Ltd.

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 (P0.05) 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 (P0.05) reduced the accumulation of this radiopharmaceutical at the site of infection. In addition, we observed significantly (P0.01) 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.

The aim of this study was to select technetium-99m labelled peptides that can discriminate between bacterial infections and sterile inflammations. For this purpose, we first assessed the binding of various 99mTc-labelled natural or synthetic peptides, which are based on the sequence of the human antimicrobial peptide ubiquicidin (UBI) or human lactoferrin (hLF), to bacteria and to leucocytes in vitro. In order to select peptides that preferentially bind to bacteria over host cells, radiolabelled peptides were injected into mice intraperitoneally infected with Klebsiella pneumoniae (K. pneumoniae) and the amount of radioactivity associated with the bacteria and with the leucocytes was quantitated. The next phase focussed on discrimination between bacterial infections and sterile inflammatory processes using 99mTc-labelled peptides in mice intramuscularly infected with various bacteria (e.g. multi-drug-resistant Staphylococcus aureus) and in animals that had been injected with lipopolysaccharides (LPS) of bacterial origin to create a sterile inflammatory process. Also, we studied the distribution of 99mTc-labelled UBI 29-41 and UBI 18-35 in rabbits having an experimental thigh muscle infection with K. pneumoniae and in rabbits injected with LPS. Based on the results of our in vitro and in vivo binding assays, two peptides, i.e. UBI 29-41 and UBI 18-35, were selected as possible candidates for infection imaging. The radiolabelled peptides can detect infections with both gram-positive and gram-negative bacteria in mice as early as 5–30 min after injection, with a target-to-non-target (T/NT) ratio between 2 and 3; maximum T/NT ratios were seen within 1 h after injection. In rabbits, high T/NT ratios (>5) for 99mTc-labelled UBI 29-41 were observed from 1 h after injection. No accumulation of the selected 99mTc-labelled UBI-derived peptides was observed in thighs of mice and rabbits previously injected with LPS. Scintigraphic investigation into the biodistribution of 99mTc-labelled UBI peptides revealed that these peptides were rapidly removed from the circulation by renal excretion. Similar data were observed for 99mTc-labelled defensin 1-3. Our data for 99mTc-labelled hLF and related peptides indicate that these compounds are less favourable for infection detection. Taken together, 99mTc-labelled UBI 18-35 and UBI 29-41 enable discrimination between bacterial infections and sterile inflammatory processes in both mice and rabbits. Based on their characteristics, we consider these peptides the candidates of preference for detection of bacterial infections in man.

Integration of optical imaging technologies can further strengthen the field of radioguided surgery. Rather than using two separate chemical entities to achieve this extension, hybrid imaging agents can be used that contain both radionuclear and optical properties. Two types of such hybrid imaging agents are available: (1) hybrid imaging agents generated by Cerenkov luminescence imaging (CLI) of β-emitters and (2) hybrid imaging agents that contain both a radioactive moiety and a fluorescent dye. One major challenge clinicians are now facing is to determine the potential value of these approaches. With this tutorial review we intend to clarify the differences between the two approaches and highlight the clinical potential of hybrid imaging during image-guided surgery applications.

The purpose of this study was to assess the contribution of phagocytic cells and bacteria to the accumulation of technetium-99m labelled polyclonal human immunoglobulin (HIG) at sites of inflammation. Mice were intraperitoneally injected withStaphylococcus aureus (SA animals), with heat-inactivated newborn calf serum (NBCS, to mimic a non-bacterial inflammation) or with physiological saline (controls); 1 h thereafter they received HIG. At various intervals after the administration of HIG the mice were killed, and the percentages of radioactivity in the peritoneal effluent and attached to the cellular and bacterial fraction thereof were established. Furthermore, the total number of cells and that of bacteria in the fluid were quantitated. The percentage of activity in the effluent in the SA animals was (P

Prosthetic joint replacement surgery is performed with increasing frequency. Overall the incidence of prosthetic joint infection (PJI) and subsequently prosthesis revision failure is estimated to be between 1 and 3%. Differentiating infection from aseptic mechanical loosening, which is the most common cause of prosthetic failure, is especially important because of different types of therapeutic management. Despite a thorough patient history, physical examination, multiple diagnostic tests and complex algorithms, differentiating PJI from aseptic loosening remains challenging. Among imaging modalities, radiographs are neither sensitive nor specific and cross-sectional imaging techniques, such as computed tomography and magnetic resonance imaging, are limited by hardware-induced artefacts. Radionuclide imaging reflects functional rather than anatomical changes and is not hampered by the presence of a metallic joint prosthesis. As a result scintigraphy is currently the modality of choice in the investigation of suspected PJI. Unfortunately, there is no true consensus about the gold standard technique since there are several drawbacks and limitations inherent to each modality. Bone scintigraphy (BS) is sensitive for identifying the failed joint replacement, but cannot differentiate between infection and aseptic loosening. Combined bone/gallium scintigraphy (BS/GS) offers modest improvement over BS alone for diagnosing PJI. However, due to a number of drawbacks, BS/GS has generally been superseded by other techniques but it still may have a role in neutropenic patients. Radiolabelled leucocyte scintigraphy remains the gold standard technique for diagnosing neutrophil-mediated processes. It seems to be that combined in vitro labelled leucocyte/bone marrow scintigraphy (LS/BMS), with an accuracy of about 90%, is currently the imaging modality of choice for diagnosing PJI. There are, however, significant limitations using in vitro labelled leucocytes and considerable effort has been devoted to developing alternative radiotracers, such as radiolabelled HIGs, liposomes, antigranulocyte antibodies and fragments, as well as more investigational tracers such as radiolabelled antibiotics, antimicrobial peptides, bacteriophages and thymidine kinase. On the other hand, positron emission tomography (PET) is still growing in the field of PJI imaging with radiotracers such as (18)F-fluorodeoxyglucose (FDG), (18)F-FDG white blood cells and (18)F-fluoride. But unfortunately this superb tomographic technique will only receive full acceptance when specific PET uptake patterns can be successfully developed. The emergence of hybrid modality imaging using integrated single photon emission computed tomography (SPECT) and PET with computed tomography (SPECT/CT and PET/CT) may also have a contributing role for more accurate assessment of joint replacement complications, especially combined with new radiotracers such as (68)Ga and (64)Cu. Finally, in searching for infection-specific tracers, currently there is no such diagnostic agent available.

A new direct labeling technique for proteins with technetium-99m (99mTc) has been developed and makes use of borohydride and stannous chloride. The method is simple and reproducible and gives a high labeling efficiency and high retention of biological activity for proteins, including polyclonal immunoglobulin (Ig), antifibrin monoclonal antibody, tissue type plasminogen activator, fibrinogen and low density lipoprotein (LDL). This method can be used in kit-format and takes about 20 min preparation time at room temperature. Both in vitro and in vivo studies indicate good stability of the label. In vivo, mice and rabbit images show significant accumulation of 99mTc-Ig at an inflammation area, 99mTc-antifibrin at a thrombus site and 99mTc-LDL in atherosclerotic lesions. The method is attractive for routine research and clinical purposes.

Objective The aim of this study was to investigate the differences in biological performance between a technetium-99m (Tc-99m)-labelled glucose derivative, the Tc-99m-labelled 1-thio-beta-D-glucose 2,3,4,6-tetra-acetate analogue (Tc-99m-TG) with F-18-fluoro-2-deoxy-glucose ([F-18]FDG). Methods Binding of both tracers was performed in vitro to viable tumour cells and bacteria. Both tracers were injected into mice for targeting Staphylococcus aureus thigh muscle infections and subcutaneous rat lymphoma (RMA) tumours by using scintigraphy, or by radioactivity counts in excised tissues to determine the biodistribution. Results In-vitro binding studies revealed that both tracers bind more effectively to tumour cells expressing the glucose transporter 1 rather than the glucose transporter 2, and this binding was specific for [F-18]FDG. (99)mTc-TG shows the highest binding to bacteria and, in addition, gives the highest rate of accumulation in infected thigh muscles in mice. Both tracers were rapidly removed from the circulatory system through the kidneys, and the majority of the injected radioactivity accumulated in the urinary bladder. Two hours after the injection radioactivity accumulation in two high-energy-dependent organs, heart, and liver, increased. Within 15 min of the injections, Tc-99m-TG visualized the site of S. aureus infection or the tumour. Conclusion We conclude that the new tracer Tc-99m-TG may have potential use as a SPECT agent for infection and tumour imaging. Nucl Med Commun 31: 239-248 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Dear Sirs, With great interest, we read the short comment of Giovanni Lucignani about infection imaging [1]. Here, commentaries are given, derived from a literature search including summaries of recently published articles. An important part of the publication was dedicated to the labelling of living bacteria with antimicrobial agents, mainly to the application of radiolabelled antibiotic ciprofloxacin for the specific detection of bacteria. Dr. Lucignani stated; “that the approach with technetium-labelled ciprofloxacin showed considerable promise in the preliminary studies but clinical trials have shown limitations”. On the contrary, most pre-clinical studies have shown poor specificity of technetium-labelled ciprofloxacin for bacterial infections [2–7] and vivid discussions related to this topic took place [8–10, 17]. One of the most tested radiolabelled ciprofloxacin is Infecton® (Draximage, Quebec, Canada). This compound was evaluated in the International Atomic Energy Agency (IAEA, Austria, Vienna) sponsored study of more than 500 patients and specificity was claimed for bacterial infections [11]. However, related to its specificity, other studies showed contradicting results in pre-clinical [2–7] and clinical settings as well [12]. Although it was suggested that this was due to sub-optimal labeling conditions and sample preparations, such statements could be easily refuted by routine radiochemical analysis and biodistribution studies in laboratory animals. More recently, claims for Infecton® in its specificity for detecting of bacterial infections in humans (depending on the discrepancy between early and late imaging) was refuted as well by Palestro et al., as his group conducted a Phase II trial sponsored by Draximage, the manufacturer of Infecton®, and they experienced that this tracer disappeared from sites of infection as well as from inflammation with equal rapidity [13]. As Infecton® shows detection of bacterial infections with poor specificity and accuracy [14], this group seriously considered that it is unlikely that radiolabelled antibiotics will ever be a viable method for imaging infection. Moreover, the recent press release from Draximage clearly stated that formulation development of Infecton® targeting orthopaedic indications has, to date, not been successful and Draximage will allocate the resources devoted to this product to other projects (http://www.draxishealth.com/pdf/Draxis_Q3_PR_US_GAAP.pdf). These findings are supported by two recent studies in which for synthetic 18F-ciprofloxacin, no specific binding to bacteria was observed [15] and poor retention of this tracer in bacteria-infected tissues in patients [16]. These findings raise concerns about the specificity of radiolabelled antibiotics, especially for Infecton®, for the detection of bacterial infections. The issue of whether the uptake is not more than a blood pool effect or the non-specific penetration of radiolabelled ciprofloxacin through membranes of bacterial and mammalian cells as well may explain the conflicting findings [17, 18]. The rapid introduction of Infecton® in clinical settings may pointing out the necessity of the support of pre-clinical data and the analysis of radiochemical structure for any new radiochemical tracer dedicated to the detection of bacterial infections in clinical practice.

Drug resistance remains a significant impediment to successful chemotherapy and constitutes a major prognostic factor in osteosarcoma (OS) patients. This study was designed to identify the role and prognostic significance of multidrug-resistance (MDR)-related transporters, such as multidrug resistance protein 1 (MDR1), multidrug-resistance-associated protein (MRP1) and breast-cancer-related protein (BCRP), in OS using cationic lipophilic radiotracers. We evaluated the chemosensitivity of four OS cell lines (Saos-2, 143B, MNNG/HOS and U-2OS) to doxorubicin (DOX), cisplatin (CIS) and methotrexate. The expression of MDR-related transporters was analyzed at mRNA level by quantitative polymerase chain reaction and at functional level by 99mTc sestamibi and 99mTc tetrofosmin. The effectiveness of MDR modulators [cyclosporin A (CsA) and imatinib] on transporter inhibition and on the reversal of resistance was also assessed. MNNG/HOS and U-2OS cells expressing high levels of MDR1 were highly resistant to DOX and showed reduced accumulation and higher efflux for radiotracers. Although MRP1 was uniformly expressed in all cells, only U-2OS was resistant to CIS. CsA restored sensitivity to DOX and CIS, and enhanced the accumulation and efflux half-life of radiotracers in MDR1-expressing cell lines. The chemosensitivity of OS cells to DOX was strongly dependent on mRNA MDR1 expression and could be circumvented by adding CsA. The kinetic parameters of radiotracers correlated with MDR1 expression levels, hence predicting DOX resistance. We concluded that sensitivity to chemotherapy is strongly dependent on the expression of MDR1 transporter and that radiotracer studies could prove clinically useful in predicting chemotherapy response and in evaluating the efficacy of MDR-reversing agents.

Earlier we reported that UBI 29-41, a synthetic peptide corresponding to residues 29-41 of human ubiquicidin, directly labeled with technetium-99m ( 99m Tc-UBI 29-41) distinguishes bacterial and fungal infections from sterile inflammations in animals. This study was undertaken to evaluate the radiochemical and biological characteristics of 99m Tc-UBI 29-41 labeled through the intermediacy of a HYNIC or N 2 S 2 moiety, which were introduced at the N-terminus of UBI 29-41 during solid phase synthesis, with 99m Tc-UBI 29-41. Methods were as follows: UBI 29-41 and HYNIC- or N 2 S 2 -conjugated peptide were labeled with technetium-99m. Preparations of these radiolabeled UBI 29-41 were purified by HPLC and Sep-Pak. Next, the stability of these tracers in human serum was challenged for 24 hours and their in vitro binding to bacteria assessed. Using scintigraphy up to 2 hours after injection of the tracer and ex vivo countings at the last interval we evaluated the ability of the three tracers to detect bacterial infections in mice inoculated with 2 × 10 7 viable Staphylococcus aureus or Klebsiella pneumoniae as well as their biodistribution. We observed the following results: HPLC analysis of purified 99m Tc-HYNIC-UBI 29-41, 99m Tc-UBI 29-41 and 99m Tc-N 2 S 2 -UBI 29-41 revealed that within 60 minutes >90% of the radioactivity was associated with the peptide. In addition, the stability of these radiolabeled UBI 29-41 peptides in human serum was excellent. All three tracers bound equally well to bacteria in vitro. After intravenous injection into mice with an experimental bacterial infection 99m Tc-HYNIC-UBI 29-41 and 99m Tc-UBI 29-41 were rapidly removed from the circulation mainly by renal clearance (at t = 120 minutes approximately 60% of the injected dose/gram tissue; % ID/g). In contrast, 99m Tc-N 2 S 2 -UBI 29-41 was removed mainly by the liver ( t = 120 minutes; 52% ID/g) showing deposits in the intestines ( t = 120 minutes; 31% ID/g) and to a lesser extent by renal clearance (19% ID/g). All three tracers rapidly detected bacterial infections in mice and highest accumulation (target-to-nontarget ratios between 3.2 and 3.6 and between 2.9 and 4.4 for infections with S. aureus and K. pneumoniae, respectively) was found at 2 hours after injection of the tracer. In conclusion, purified 99m Tc-HYNIC-UBI 29-41 and 99m Tc-N 2 S 2 -UBI 29-41 were as effective as 99m Tc-UBI 29-41 in detecting infections in mice injected intramuscularly with bacteria. However, 99m Tc-N 2 S 2 -UBI 29-41 should not be advised for the imaging of abdominal infections as this tracer, in contrast to the other tracers, is cleared via the liver and intestines.

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; P0.05) 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.

To improve standardization in analytical reagents we investigated Chloramine-T radioiodination (125I) of several biomolecules based on the use of a single amount of the oxidizing agent Chloramine-T as the limiting reagent being exhausted during the course of the reaction. Whenever the labeling yield resulted in less than one atom 125I/molecule, a second amount of the oxidizing agent was added. Thereafter, the integrity of the various biomolecules was assessed using radioimmunoassays, radioreceptor binding assays, or radioimmunometric assays. Purification yields were done by gel permeation (56% +/- 19%, n=230) or by precipitation with trichloroacetic acid (59% +/- 19%, n=230). Specific activity (117 +/- 61 MBq/nmol) and the degree of iodine incorporation (1.4 +/- 0.8 atoms of 125I/molecule) were achieved after 300 sec of incubation. A second addition of Chloramine-T resulted in an increased labeling yield of all biomolecules tested by a mean factor of 1.8 +/- 0.9. After the second addition of Chloramine-T, we observed for some biomolecules a significant (p

Epidermal growth factor (EGF) has been detected by radioimmunoassay (RIA) in different body fluids such as serum, amniotic fluid, and urine. Human tumor tissues with EGF receptors (EGF-Rc) may be saturated with EGF, which may be of prognostic value. An RIA was envisaged to measure human epidermal growth factor (hEGF) levels using EGF-Rc as capture agent and a monoclonal antibody anti-hEGF (MAb anti-hEGF) labeled with 125Iodine as a marker for this binding. The purpose of this work was to study the feasibility of MAb anti-hEGF to detect the receptor binding sites and to investigate the interaction between MAb anti-hEGF and the EGF-Rc. Various binding experiments were performed to study possible interference and interactions in the complex MAb anti-hEGF and the receptor. Affinity constants were determined by means of Scatchard plot analysis to interpret the complex stability challenged with other compounds for a better understanding of the interaction process. Binding constants were of the same order for all the ligands tested separately involving the EGF-Rc, but were significantly higher (t = 15.7, p0.05) for hEGF in its binding to MAb anti-hEGF. It was possible with equilibrium studies and competition experiments to evaluate the interaction of EGF and MAb anti-hEGF with the EGF receptor. This observation makes the MAb anti-hEGF a potential tracer for the quantitation of receptors in vitro, and possibly for the detection of membrane receptors on tumor cells in vivo.

SUMMARY At present, it is difficult to distinguish between bacterial infections and sterile inflammatory processes using radiopharmaceuticals. This is so for a variety of reasons, including binding to bacteria with low affinity (e.g. infection) and binding to a specific micro-organism (e.g. radiolabelled monoclonal antibodies or F(ab)2 fragments thereof against micro-organisms). In this review, we propose that radiolabelled antimicrobial peptides should be the first choice in the development of new radiopharmaceuticals for imaging of bacterial infections. Antimicrobial peptides are a recently discovered component of the innate defence system of plants, animals and humans. These peptides, which now number more than 100, with proven microbicidal activity against a variety of micro-organisms, share certain properties, such as their small size and cationic charge. The latter allows them to bind preferentially to a broad spectrum of microorganisms. We have recently demonstrated that radiolabelled human defensins allow the rapid visualization of bacterial infections in mice. Furthermore, binding of this antimicrobial peptide to bacteria is the major factor contributing to the accumulation of this tracer in bacterial infections. Based on these considerations, we believe that radiolabelled antimicrobial peptides will be an important asset in the imaging of infections in patients.

The aim of this study was to determine the contribution of various IgG subclasses to the scintigraphic detection of a staphylococcal infection. An experimental thigh infection in mice was used to determine the accumulation of the various 99 Tc m -labelled IgG preparations with enriched IgG1, IgG2 or IgG4 subclass. Multiple-regression analysis was used to investigate a relationship between the IgG subclasses and the time-dependent accumulation in infected sites. Eighteen hours after infection with Staphylococcus aureus bacteria, 20 μg of 99 Tc m -labelled IgG preparations enriched with one of the IgG1, IgG2 or IgG4 subclasses by thiophilic absorption were administered intravenously and target-to-nontarget (T/NT) ratios were determined at 15 min, 1 h, 4 h and 24 h after injection of the tracer. Moreover, the binding of these preparations to S. aureus was assessed using an in vitro bacterial pellet model as an indication for the potency of detecting infections. As a control agent, 99 Tc m -labelled polyclonal IgG (HIG) was used. In vivo, the T/NT ratios were significantly (P < 0.05) higher for the IgG1-enriched preparation at all time points, and for the IgG2-enriched preparation at 4 h and 24 h after injection, compared with HIG. In contrast, IgG4 did not yield higher T/NT ratios at any time. Using multiple-regression analysis, it became evident that IgG3 at all time intervals, IgG1 for early scans (up to 4 h) and IgG2 for late scans (24 h) contribute significantly (P < 0.05) to the accumulation. The abundance of IgG subclasses in the various preparations appeared to influence the accumulation of tracer at infected sites. The percentage of binding to S. aureus in vitro was significantly (P < 0.05) higher for enriched IgG subclass preparations than for HIG. We conclude that specific subclass enrichment of 99 Tc m -labelled IgG preparations improves the scintigraphic detection of staphylococcal infections at various time intervals post-injection.

This study was designed to assess monoclonal antibodies (MAbs) directed against tumor necrosis factor-alpha (TNF-alpha) (anti-TNF) or interleukin-8 (anti-IL-8) as radioactive agents for the detection of Staphylococcus aureus-or Klebsiella pneumoniae-infected thighs in mice. At 5 min (acute infection) or 20 h (established) post-infection, 20 micrograms of the 99mTc-labeled MAbs were injected. At various time intervals, the accumulation of the radiotracer in the infected thighs was assessed and expressed as a target-to-nontarget (T/NT) ratio. The binding of 99mTc-labeled MAbs to circulating mononuclear cells and granulocytes was quantitated 20 h after injection. The pharmacokinetics of the MAbs, in relation to the control agents 99mTc-labeled polyclonal human immunoglobulin (IgG) and a 99mTc-labeled nonspecific IgG1 MAb, were also studied. In acute infections, 99mTc-anti-TNF accumulated to a higher extent (p0.05) in S. aureus-infected thighs in mice until 4 h after the injection than 99mTc-IgG and was higher at 0.25 h in K. pneumoniae-infected mice (p0.03) compared with 99mTc-IgG. In established S. aureus and K. pneumoniae infections, 99mTc-anti-IL-8 detected the infection more intensely than 99mTc-IgG until 1 h after injection. In both S. aureus and K. pneumoniae infections, localization of sites of infection correlates (p0.05) with increased binding of the 99mTc-labeled MAbs to granulocytes and mononuclear cells in both acute and established infections. It was concluded that 99mTc-labeled MAbs, directed against TNF-alpha and IL-8, accumulate in bacterial infections in mice to a higher extent than does 99mTc-IgG after infection and is related to the binding of the antibodies to blood leukocytes. With these 99mTc-labeled MAbs, information might be gained about the development of an infection.

Our objective was to investigate the mechanism of accumulation of 99 Tc m -labelled non-specific polyclonal human immunoglobulin ( 99 Tc m -HIG) in inflamed synovial tissue (ST) in an experimental animal model of arthritis. Following 99 Tc m -HIG scintigraphy, the in vivo localization of 99 Tc m -HIG in the ST of knee joints of rats with adjuvant arthritis was studied using immunohistochemical techniques. In addition, the in vitro binding of 99 Tc m -HIG to extracellular matrix proteins was analysed by means of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). After 99 Tc m -HIG scintigraphy, 99 Tc m -HIG was detected in the ST of rats with adjuvant arthritis. 99 Tc m -HIG was diffusely distributed and not bound to cells. In vitro incubation of 99 Tc m -HIG on the ST of rats with adjuvant arthritis revealed binding of 99 Tc m -HIG to inflamed, but not to non-inflamed, ST. In addition, specific binding of 99 Tc m -HIG to fibronectin, fibrin, collagen type I and III was demonstrated by ELISA. We conclude that the accumulation of 99 Tc m -HIG in inflamed ST can be explained by the binding of 99 Tc m -HIG to extracellular matrix proteins.

To improve the scintigraphic detection of bacterial infections a protein charge-purified fraction of polyclonal human immunoglobulin was applied as a radiopharmaceutical. This purification was achieved by attaching the immunoglobulin to an anion-exchanger column and by obtaining the column-bound fraction with buffer. The binding to bacteria in vitro and the target to non-target ratios of an experimental thigh infection with Staphylococcus aureus or Klebsiella pneumoniae in mice were evaluated to compare the purified and the unpurified immunoglobulin. The percentage of binding to all gram-positive and gramnegative bacteria used in this study was significantly (P

The purpose of this work was the development of an orthotopic model of osteosarcoma based on luciferase-expressing tumour cells for the in vivo imaging of multidrug resistance (MDR) with (99m)Tc-sestamibi.Doxorubicin-sensitive (143B-luc(+)) and resistant (MNNG/HOS-luc(+)) osteosarcoma cell lines expressing different levels of P-glycoprotein and carrying a luciferase reporter gene were inoculated into the tibia of nude mice. Local tumour growth was monitored weekly by bioluminescence imaging and X-ray. After tumour growth, a (99m)Tc-sestamibi dynamic study was performed. A subset of animals was pre-treated with an MDR inhibitor (PSC833). Images were analysed for calculation of (99m)Tc-sestamibi washout half-life (t (1/2)), percentage washout rate (%WR) and tumour/non-tumour (T/NT) ratio.A progressively increasing bioluminescent signal was detected in the proximal tibia after 2 weeks. The t (1/2) of (99m)Tc-sestamibi was significantly shorter (p0.05) in drug-resistant MNNG/HOS-luc(+) tumours (t (1/2) = 87.3 +/- 15.7 min) than in drug-sensitive 143B-luc(+) tumours (t (1/2) = 161.0 +/- 47.4 min) and decreased significantly with PSC833 (t (1/2) = 173.0 +/- 24.5 min, p0.05). No significant effects of PSC833 were observed in 143B-luc(+) tumours. The T/NT ratio was significantly lower (p0.05) in MNNG/HOS-luc(+) tumours than in 143B-luc(+) tumours at early (1.55 +/- 0.22 vs 2.14 +/- 0.36) and delayed times (1.12 +/- 0.11 vs 1.62 +/- 0.33). PSC833 had no significant effects on the T/NT ratios of either tumour.The orthotopic injection of tumour cells provides an animal model suitable for functional imaging of MDR. In vivo bioluminescence imaging allows the non-invasive monitoring of tumour growth. The kinetic analysis of (99m)Tc-sestamibi washout provides information on the functional activity of MDR related to P-glycoprotein expression and its pharmacological inhibition in osteosarcoma.