Protocol for Clinical GLP-1 Receptor PET/CT Imaging with [68Ga]Ga-NODAGA-Exendin-4.

Imaging with radiolabeled exendin enables detection and characterization of glucagon-like peptide 1 receptors (GLP-1Rs) in vivo with high specificity. The novel radiotracer [68Ga]Ga-NODAGA-exendin-4 forms a stable complex after a simple and fast labeling procedure. Beta-cell mass in the islets of Langerhans can be visualized using [68Ga]Ga-NODAGA-exendin-4, which is promising for research into diabetes mellitus (DM) pathophysiology. Furthermore, this radiotracer enables very sensitive detection of insulinomas, resulting from vast overexpression of GLP-1Rs, and seems promising for the detection of focal lesions in congenital hyperinsulinism (CHI). Here, we describe the procedures involved in [68Ga]Ga-NODAGA-exendin-4 positron emission tomography (PET)/computed tomography (CT) imaging including the radiolabeling of the NODAGA-exendin conjugate with 68Ga, quality controls, and PET/CT.

New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals.

BACKGROUND: The introduction of a GMP-certified 68Ga-generator spurred the application of 68Ga-radiopharmaceuticals. Several radiosynthesis of 68Ga-radiopharmaceuticals are more efficient and robust when performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity in the quality control (QC) procedure. Thus, prior to clinical use, QC must be conducted to ensure that HEPES does not exceed the maximum dose of 200 µg/V Injected as described in European Pharmacopoeia (Ph Eur) for edotreotide. However, when applying the thin-layer chromatography (TLC) method described in the Ph Eur to quantify the HEPES amount present in the 68Ga-octreotide or in the remaining 68Ga-radiopharmaceuticals that were tested, no amount was detectable after 4 min of iodine incubation. Here we tested our modified TLC method and validate a new high-performance liquid chromatography (HPLC) method to quantify HEPES in 68Ga-radiopharmaceuticals and compare it to the TLC-method described in Ph Eur. In addition, samples collected from various institutes were tested to evaluate whether the synthesis of different 68Ga-radiopharmaceuticals or the use of different synthesis methods could affect the amounts of HEPES. RESULTS: HEPES could not be detected by the TLC method described in the Ph Eur within 4 min incubation in an iodine-saturated chamber. As for our modified TLC method, only after 2 h, spots were only visible > 1 mg/mL. The HPLC method had a limit-of-quantification (LOQ) of 3 µg/mL and a limit-of-detection (LOD) of 1 µg/mL. From the three 68Ga-radiopharmaceuticals tested, only in the [68Ga]Ga-NODAGA-Exendin samples exceeding amounts of HEPES were found and its concentration in the [68Ga]Ga-NODAGA-Exendin was significantly higher, when compared to [68Ga]Ga-DOTATOC and [68Ga]Ga-PSMA-11. CONCLUSION: The TLC method described in Ph Eur and our modified TLC method may not be sufficiently sensitive and thus unsuitable to use for QC release. The new HPLC method was sensitive, quantitative, reproducible and suitable for QC release. With this method, we were able to determine that some 68Ga-radiopharmaceuticals may exceed the HEPES limit of 200 µg/ V Injected. This new analytical system would allow correcting for the maximum injected dose in order not to exceed this amount.

PET and SPECT imaging of a radiolabeled minigastrin analogue conjugated with DOTA, NOTA, and NODAGA and labeled with (64)Cu, (68)Ga, and (111)In.

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

Monitoring hypoxia and vasculature during bevacizumab treatment in a murine colorectal cancer model.

The purpose of this study was to assess the effect of bevacizumab on vasculature and hypoxia in a colorectal tumor model. Nude mice with subcutaneous LS174T tumors were treated with bevacizumab or saline. To assess tumor properties, separate groups of mice were imaged using (18) F-Fluoromisonidazole (FMISO) and (18) F-Fluorodeoxyglucose (FDG) positron emission tomography or magnetic resonance imaging before and 2, 6 and 10 days after the start of treatment. Tumors were harvested after imaging to determine hypoxia and vascular density immunohistochemically. The T2 * time increased significantly less in the bevacizumab group. FMISO uptake increased more over time in the control group. Vessel density significantly decreased in the bevacizumab-treated group. The Carbonic anhydrase 9 (CAIX) and glucose uptake transporter 1 (GLUT1) fractions were higher in bevacizumab-treated tumors. However, the hypoxic fraction showed no significant difference. Bevacizumab led to shorter T2 * times and higher GLUT1 and CAIX expression, suggesting an increase in hypoxia and a higher glycolytic rate. This could be a mechanism of resistance to bevacizumab. The increase in hypoxia, however, could not be demonstrated by pimonidazole/FMISO, possibly because distribution of these tracers is hampered by bevacizumab-induced effects on vascular permeability and perfusion.

Anti-CEA antibody fragments labeled with [(18)F]AlF for PET imaging of CEA-expressing tumors.

A facile and rapid method to label peptides with (18)F based on chelation of [(18)F]AlF has been developed recently. Since this method requires heating to 100 °C, it cannot be used to label heat-sensitive proteins. Here, we used a two-step procedure to prepare (18)F-labeled heat-labile proteins using the [(18)F]AlF method based on hot maleimide conjugation. 1,4,7-Triazacyclononae-1,4-diacetate (NODA) containing a methyl phenylacetic acid group (MPA) functionalized with N-(2-aminoethyl)maleimide (EM) was used as a ligand which was labeled with [(18)F]AlF and then conjugated to the humanized anti-CEA antibody derivatives hMN-14-Fab', hMN-14-(scFv)2 (diabody), and a Dock-and-Lock engineered dimeric fragment hMN-14 Fab-AD2 at room temperature. The in vivo tumor targeting characteristics of the (18)F-labeled antibody derivatives were determined by PET imaging of mice with s.c. xenografts. NODA-MPAEM was radiolabeled with [(18)F]AlF at a specific activity of 29-39 MBq/nmol and a labeling efficiency of 94 ± 2%. The labeling efficiencies of the maleimide conjugation ranged from 70% to 77%, resulting in [(18)F]AlF-labeled hMN14-Fab', hMN14-Fab-AD2, or hMN14-diabody with a specific activity of 15-17 MBq/nmol. The radiolabeled conjugates were purified by gel filtration. For biodistribution and microPET imaging, antibody fragments were injected intravenously into BALB/c nude mice with s.c. CEA-expressing LS174T xenografts (right flank) and CEA-negative SK-RC-52 xenografts (left flank). All [(18)F]AlF-labeled conjugates showed specific uptake in the LS174T xenografts with a maximal tumor uptake of 4.73% ID/g at 4 h after injection. Uptake in CEA-negative SK-RC-52 xenografts was significantly lower. Tumors were clearly visualized on microPET images. Using a [(18)F]AlF-labeled maleimide functionalized chelator, antibody fragments could be radiofluorinated within 4 h at high specific activity. Here, we translated this method to preclinical PET imaging studies and showed feasibility of [(18)F]AlF-fluorinated hMN-14-Fab', [(18)F]AlF-hMN-14-Fab-AD2, and [(18)F]AlF-hMN-14-diabody for microPET imaging of CEA-expressing colonic cancer.

Intravenous technetium-99m labelled PEG-liposomes in horses: a safety and biodistribution study.

REASONS FOR PERFORMING STUDY: Liposomes are phospholipid nanoparticles that extravasate at sites of increased vascular permeability. They have potential in equine medicine for targeted drug delivery and diagnostic imaging of infectious, inflammatory and neoplastic lesions. OBJECTIVES: This study evaluates the safety and biodistribution of i.v. polyethyleneglycol(PEG) liposomes in normal horses. METHODS: PEG-liposomes were prepared by the film hydration method and labelled using (99m) Tc-hexamethyl-propylene-amine-oxime. A single dose of 0.24 µmol/kg bwt (99m) Tc-PEG-liposomes and 2.4 µmol/kg bwt unlabelled PEG-liposomes was administered to 10 conscious horses via i.v. infusion at a rate of 6 µmol/min for the first 15 min and 60 µmol/min thereafter. Clinical parameters, haematology, plasma biochemistry and serum complement activity were monitored serially. Scintigraphic imaging was performed at 1, 12 and 21 h post infusion (PI). Six horses were subjected to euthanasia at 24 h PI. The percentage injected dose per kilogram of tissue was calculated for multiple organs. Results were analysed using repeated measures ANOVA. RESULTS: Horses did not demonstrate adverse reactions during or after liposome infusion. There was a significant elevation in heart rate and respiratory rate at 20 and 25 min PI. No significant complement consumption was detected, although a trend for decreased total haemolytic complement values at 20 min PI was present. Scintigraphic studies revealed a prolonged vascular phase that lasted to 21 h PI, with a reproducible pattern of organ distribution. Biodistribution studies revealed the highest concentrations of radiopharmaceutical within the lung, kidney, liver and spleen. CONCLUSIONS: Intravenous liposome administration appears to be safe in horses. When administered in combination with PEG-liposomes, (99m) Tc-PEG-liposomes have long circulating characteristics and a reproducible pattern of organ distribution in horses. POTENTIAL RELEVANCE: Radiolabelled liposomes may be useful for detecting infection, inflammation and neoplasia in the horse. Liposomes have significant potential for targeted drug delivery in the horse. This study establishes the scintigraphic findings and tissue distribution of 99mTc-PEG-liposomes after i.v. administration in healthy horses.

Quantitative multi-pinhole small-animal SPECT: uniform versus non-uniform Chang attenuation correction.

Attenuation of photon flux on trajectories between the source and pinhole apertures affects the quantitative accuracy of reconstructed single-photon emission computed tomography (SPECT) images. We propose a Chang-based non-uniform attenuation correction (NUA-CT) for small-animal SPECT/CT with focusing pinhole collimation, and compare the quantitative accuracy with uniform Chang correction based on (i) body outlines extracted from x-ray CT (UA-CT) and (ii) on hand drawn body contours on the images obtained with three integrated optical cameras (UA-BC). Measurements in phantoms and rats containing known activities of isotopes were conducted for evaluation. In (125)I, (201)Tl, (99m)Tc and (111)In phantom experiments, average relative errors comparing to the gold standards measured in a dose calibrator were reduced to 5.5%, 6.8%, 4.9% and 2.8%, respectively, with NUA-CT. In animal studies, these errors were 2.1%, 3.3%, 2.0% and 2.0%, respectively. Differences in accuracy on average between results of NUA-CT, UA-CT and UA-BC were less than 2.3% in phantom studies and 3.1% in animal studies except for (125)I (3.6% and 5.1%, respectively). All methods tested provide reasonable attenuation correction and result in high quantitative accuracy. NUA-CT shows superior accuracy except for (125)I, where other factors may have more impact on the quantitative accuracy than the selected attenuation correction.

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies.

Pegylated liposomal doxorubicin (Doxil) and 99mTc-HYNIC PEG liposomes (HPL) were reported earlier to cause hypersensitivity reactions (HSRs) in a substantial percentage of patients treated i.v. with these formulations. Here we report that (1) Doxil, HPL, pegylated phosphatidylethanolamine (PEG-PE)-containing empty liposomes matched with Doxil and HPL in size and lipid composition, and phosphatidylglycerol (PG)-containing negatively charged vesicles were potent C activators in human serum in vitro, whereas small neutral liposomes caused no C activation. (2) Doxil and other size-matched PEG-PE and/or PG-containing liposomes also caused massive cardiopulmonary distress with anaphylactoid shock in pigs via C activation, whereas equivalent neutral liposomes caused no hemodynamic changes. (3) A clinical study showed more frequent and greater C activation in patients displaying HSR than in non-reactive patients. These data suggest that liposome-induced HSRs in susceptible individuals may be due to C activation, which, in turn, is due to the presence of negatively charged PEG-PE in these vesicles.

Microscopic localization of PEG-liposomes in a rat model of focal infection.

In the present study the microscopic localization of polyethylene glycol (PEG) liposomes in infected tissues was studied with both light microscopy (LM) and transmission electron microscopy (TEM) in rats with focal intramuscular Staphylococcus aureus infection. PEG-liposomes containing colloidal gold were prepared and injected intravenously in rats with focal S. aureus infection and tissues were dissected at 24 h post injection. Sections were cut and liposomes were visualized for microscopic evaluation using silver enhancement. Uptake of PEG-liposomes was visualized by both scintigraphy and LM in the abscess, liver and spleen. In the infected area, the liposomes were mainly found in the vicinity of blood vessels. TEM showed that the liposomes were localized in the macrophages and to a lesser extent in endothelial cells in the infectious tissue. In the liver, the liposomes appeared mainly localized in Kupffer cells. In the spleen, uptake was only seen in cells of the red pulp and in cells around the central arteries. Our microscopic observations indicate that uptake and retention of PEG-liposomes in the infectious focus is a result of enhanced extravasation due to increased vascular permeability and subsequent phagocytosis of PEG-liposomes by macrophages in the infected tissue.

Factors affecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injection.

Previously, we showed that long-circulating polyethylene glycol (PEG)-liposomes are cleared rapidly from the circulation when injected repeatedly in the same animal. In this article, we describe the effects of PEG-coating, the circulation time, the lipid dose, and the presence of encapsulated doxorubicin on the pharmacokinetics upon repeated injection in rats. Furthermore, the role of liver and splenic macrophages was investigated. Liposomes without PEG-coating also showed the so-called "enhanced clearance effect": blood levels at 4 h post injection decreased from 62.8 +/- 13.7% of injected dose (%ID) after the first injection to 0.54 +/- 0.21%ID after the second injection. This decrease was independent of the circulation time of the first dose. Decreasing the first lipid dose of PEG-liposomes to 0.05 micromol/kg still led to enhanced clearance of a second dose of 5 micromol/kg. No changes in pharmacokinetics were observed when the second dose was 50 micromol/kg. When hepatosplenic macrophages were depleted, no enhanced clearance of repeated liposome injections was observed. A dose of doxorubicin containing PEG-liposomes (Doxil), injected 1 week after injection of empty PEG-liposomes, was cleared rapidly from the circulation in rats. Our results indicate that hepatosplenic macrophages play an essential role in the enhanced clearance effect and that the change in pharmacokinetic behavior upon repeated injection is a general characteristic of liposomes, unrelated to the presence of PEG. Therefore, these findings may have a considerable impact on the clinical application of liposomal formulations that are administered repeatedly.

Recognition and clearance of methoxypoly(ethyleneglycol)2000-grafted liposomes by macrophages with enhanced phagocytic capacity. Implications in experimental and clinical oncology.

Intravenous injection of an endotoxin-free solution of poloxamine-908 to rats can enhance the phagocytic clearance capacity of tissue macrophages, particularly those of the liver and the spleen. Such stimulated cells were able to clear a significant portion of intravenously injected methoxypoly(ethyleneglycol)2000 liposomes (mean size of 87 nm), labelled with technetium-99m via the N-hydroxysuccinimidyl hydrazine nicotinate hydrochloride derivative of distearoyl phosphatidylethanolamine, within 4 h post administration. These liposomes, otherwise, exhibit long circulatory behaviour in control animals, with poor localization to the liver and spleen. We suggest that such technetium-99m-labelled engineered vesicles may be of aid for detection of the liver and spleen macrophages with enhanced phagocytic clearance capacity by gamma scintigraphy. Alterations in the phagocytic activity of liver and spleen macrophages is known to occur during cancer. Therefore, such diagnostic procedures may prove useful for patient selection or for monitoring the progress of treatment with long circulating nanoparticles carrying anti-cancer agents, thus minimizing damage to this important line of body's defence cells, and are discussed.

The effect of molecular weight on nonspecific accumulation of (99m)T-labeled proteins in inflammatory foci.

UNLABELLED: Although several proteins have been proposed and tested for scintigraphic detection of infection, the most optimal characteristics of a protein for this application have not yet been determined. Molecular weight (MW) of the protein, its charge, shape, carbohydrate content, characteristics of the radionuclide and receptor interactions are factors that could affect the in vivo behavior of the infection imaging agent. The effect of molecular weight on nonspecific accumulation of (99m)Tc-labeled proteins in inflammatory foci was studied in a rat model. METHODS: Eleven proteins whose MWs ranged from 2.5 kDa up to 800 kDa were labeled with (99m)Tc using the hydrazinonicotinamide (HYNIC) chelator. Rats with S. aureus infection were injected i.v. with 15 MBq (99m)Tc-labeled protein. Gamma camera images were acquired and biodistribution of the radiolabel was determined ex vivo. RESULTS: From biodistribution data no significant correlation was found between abscess uptake and molecular size of the (99m)Tc-labeled proteins that were studied. Fast blood clearance with predominant uptake in liver and spleen was found for the largest proteins (MW 669 kDa-800 kDA). For proteins of intermediate size (MW 66 kDa -206 kDa) we found relatively slow blood clearance with relatively moderate uptake in liver and spleen. For smaller proteins (MW 2.5 kDa -29 kDa) rapid blood clearance with predominant kidney uptake was observed. The abscess uptake of the (99m)Tc-labeled proteins (%ID/g, 24 h p.i.) was highest for serum proteins IgG and BSA. Abscess uptake correlated well with blood levels: r = 0.95 and 0.84 at 4 and 24 h respectively (P < 0.005). The abscess-to-muscle ratios varied from 2.1 to 17.8 at 24 h p.i. with highest values for alpha-2 macroglobulin (MW 725 kDa) and the intermediate sized proteins (MW 66-206 kDa). Gamma camera imaging showed localization of all radiotracers at the site of infection with abscess-to-background ratios (A/B) ranging from 1.4 to 7.0 (IgG) at 20 h p.i. The serum proteins IgG and BSA showed highest blood levels and best infection imaging characteristics. CONCLUSION: Not molecular weight but blood residence time is the principal factor that determines localization of a nonspecific tracer protein in infectious foci. The ideal nonspecific infection imaging agent is a protein with a long circulatory half-life. From the proteins tested here IgG and albumin showed the best characteristics for an infection imaging agent.

In vivo applications of PEG liposomes: unexpected observations.

Recent studies with PEG liposomes in patients have consistently shown that liposomes can induce side effects (flushing, tightness of the chest). Furthermore, the blood clearance of PEG liposomes was shown to be dose-dependent: at lipid doses lower than 1 micromol/kg, PEG liposomes do not show the long-circulation property but instead are cleared relatively rapidly from the bloodstream. Another remarkable observation was that repeated injections of PEG liposomes led to significant pharmacokinetic changes: the circulatory half-life of a second dose of radiolabeled PEG liposomes dramatically decreased when given from 5 days to up to 4 weeks after a first injection. In these three unexpected phenomena, proteins of the complement system seem to play a key role. Therefore, one has to consider that PEG liposomes are not inert drug-carrying vehicles in vivo. Pharmacological effects can occur, induced solely by using liposomal particles irrespective of the drug content.

Radiolabeled liposomes for scintigraphic imaging.

Liposomes have been investigated extensively as carriers for drugs in attempts to achieve selective deposition and/or reduced toxicity. Liposomes radiolabeled with gamma emitters such as (67)Ga, (111)In and (99m)Tc, can be used for imaging purposes. Liposomes as formulated in the past, are rapidly taken up by cells of the mononuclear phagocyte system (MPS), primarily those located in liver and spleen. The recent development of long-circulating liposomes (LCLs), yielded liposomes that oppose recognition by the MPS. The development of these LCLs with enhanced circulatory half-lives has broadened the potential of liposomes to scintigraphically visualize pathologic processes in vivo. Liposomes have been proposed for tumor imaging, infection imaging and blood pool imaging. Strategies have been developed that allow rapid, easy and efficient labeling of preformed liposomes with (111)In and (99m)Tc. There is now a vast body of preclinical evidence showing that LCLs can be used to image a wide variety of tumors as well as inflammatory lesions. The first studies in patients show that radiolabeled liposomes can image tumor and inflammatory lesions with good sensitivity and good specificity. Here, the present status of liposome-based radiopharmaceuticals for scintigraphic application is reviewed.

Preclinical and clinical evidence for disappearance of long-circulating characteristics of polyethylene glycol liposomes at low lipid dose.

This study describes the effect of the lipid dose of (99m)Tc-polyethylene glycol (PEG) liposomes in the low-dose range (0. 02-1.0 micromol/kg) on the pharmacokinetics and biodistribution in rats, rabbits, and humans. The biodistribution and pharmacokinetics of (99m)Tc-PEG liposomes at various dose levels were studied in rats and rabbits with a focal Escherichia coli infection. Scintigraphic images were recorded on a gamma camera. In addition, the role of macrophages in the biodistribution of a low-dose PEG liposome injection was studied. Finally, the pharmacokinetics of (99m)Tc-PEG liposomes at two lipid dose levels was studied in four patients. At a dose level of 0.03 micromol/kg, the blood level in rats at 4 h postinjection was significantly lower than at the highest dose level (1.1 micromol/kg). The same effect was observed in rabbits where enhanced clearance was observed at a dose level of 0.02 micromol/kg. The circulatory half-life decreased from 10.4 to 3.5 h (at 1.0 and 0. 02 micromol/kg, respectively). At the lowest dose level, liposomes were mainly taken up by the liver and to a lesser extent by the spleen. Injection of a low dose of PEG liposomes in macrophage-depleted rabbits resulted in normal pharmacokinetics, suggesting involvement of macrophages in the effectuation of the rapid elimination of the liposomes from the circulation. Most importantly, the rapid clearance of low-dose PEG liposomes was also observed in humans when relatively low lipid doses were administered. This study showed that at very low lipid doses the biodistribution of PEG liposomes is dramatically altered.

Scintigraphic evaluation of experimental chronic osteomyelitis.

UNLABELLED: Assessment of disease activity and disease extent in chronic osteomyelitis remains a difficult diagnostic problem. Radiography is not particularly sensitive. Scintigraphic techniques can be more helpful, but the routinely available agents lack specificity (99mTc-methylene diphosphonate [MDP], 67Ga-citrate) or are laborious to prepare (111In-leukocytes). We evaluated the performance of 2 new radiopharmaceuticals, 99mTc-polyethyleneglycol (PEG) liposomes and 99mTc-hydrazinonicotinamide (HYNIC)-immunoglobulin G (IgG), in an experimental model of chronic osteomyelitis. METHODS: Chronic osteomyelitis was induced in rabbits by inserting S. aureus into the right reamed and washed femoral canal. The canal was closed with cement. A sham operation was performed on the left femur. Routine radiographs were obtained immediately after surgery and before scintigraphy. Four weeks after surgery, each rabbit was injected with 37 MBq 99mTc-PEG liposomes, 99mTc-HYNIC-IgG, and 99mTc-MDP on 3 consecutive days and imaged up to 4 (MDP) or 22 (liposomes and IgG) h after injection. On day 4, rabbits received either 18 MBq 111In-granulocytes or 67Ga-citrate and were imaged up to 44 h after injection. Uptake in the infected femur was determined by drawing regions of interest. Ratios of infected-to-sham-operated femur were calculated. After the last image, the rabbits were killed, and the left and right femur were scored for microbiologic and histopathologic evidence of osteomyelitis. RESULTS: 99mTc-PEG liposomes and 99mTc-HYNIC-IgG correctly identified all 6 rabbits with osteomyelitis. 11In-granulocytes and 67Ga-citrate gave equivocal results in 1 infected rabbit. 99mTc-MDP missed 1 case of osteomyelitis. The uptake in the affected region did not differ significantly between the agents, although 99mTc-MDP tended to have higher values (MDP, 4.75 +/- 1.23 percentage injected dose per gram [%ID/g]; 67Ga, 2.05 +/- 0.54 %ID/g; granulocytes, 1.56 +/- 0.83 %ID/g; liposomes, 1.75 +/- 0.76 %ID/g, and IgG, 1.96 +/- 0.27 %ID/g). The ratios of infected-to-normal femur were also not significantly different for the respective radiopharmaceuticals. Radiography visualized only severe osteomyelitis. CONCLUSION: In this rabbit model, 99mTc-PEG liposomes and 99mTc-HYNIC-IgG performed at least as well as 111In-granulocytes and 67Ga-citrate in the localization of chronic osteomyelitis. The ease of preparation, the better image quality, and the lower radiation dose suggest that 99mTc-PEG liposomes and 99mTc-HYNIC-IgG might be suitable alternatives for 67Ga-citrate and 111In-granulocytes in the scintigraphic evaluation of osteomyelitis.

Improved imaging of infections by avidin-induced clearance of 99mTc-biotin-PEG liposomes.

UNLABELLED: This article describes the preparation and optimization of biotin-polyethyleneglycol (PEG) liposomes and their application in experimental infection models to improve the scintigraphic imaging of infection and inflammation. METHODS: Biotin was coupled to PEG-distearoylphosphatidylethanolamine (DSPE) and subsequently incorporated in the PEG liposomes. Biotinylated liposomes were radiolabeled with 99mTc-hydrazinonicotinamide. In vitro binding studies were performed to find the optimal biotin concentration in the liposomes. In rats the biodistribution of the 99mTc-biotin-PEG liposomes was compared with the biodistribution of normal (nonbiotinylated) 99mTc-PEG liposomes. Furthermore, in vivo studies in rats were performed to study both the effect of the biotin content and the optimal avidin dose for efficient clearance of the liposomes. Liposomes containing 0.5 or 1.0 mol% biotin-PEG-DSPE were compared in rats with a Staphylococcus aureus infection in the left calf muscle. Avidin was injected 4 h after injection of the liposomes. RESULTS: Biotinylation of the liposomes did not affect their in vivo behavior. All biotin-PEG liposome formulations tested showed good in vitro avidin binding with 50% inhibitory concentrations ranging from 36 to 8 micromol/L. With avidin doses higher than 100 microg, both preparations rapidly cleared from the circulation. As a result, abscess-to-blood ratios increased 5-fold. To illustrate the potential of the avidin-induced clearance of radiolabeled PEG liposomes, we also studied the 99mTc-biotin-PEG liposomes in rabbits with a subcutaneous S. aureus abscess. The infection was visualized only after injection of 100 microg avidin. CONCLUSION: This study shows that biotin-coated 99mTc-PEG liposomes in combination with the injection of avidin can lead to improved imaging of infection or inflammation localized especially in regions with high blood-pool activity.

99mTc-PEG liposomes for the scintigraphic detection of infection and inflammation: clinical evaluation.

UNLABELLED: Polyethyleneglycol (PEG) liposomes have been shown to be excellent vehicles for scintigraphic imaging of infection and inflammation in various experimental models. In this article we report on a series of patients with possible infectious and inflammatory disease in whom the performance of 99mTc-PEG liposomes was evaluated. The results of 99mTc-PEG liposome scintigraphy were directly compared with those of 111In-immunoglobulin G (IgG) scintigraphy. METHODS: Thirty-five patients (22 men, 13 women; mean age, 51 y; range, 20-76 y), suspected of having infectious or inflammatory disease, received 740 MBq 99mTc-PEG liposomes intravenously. Imaging was performed at 4 and 24 h after injection. Patients received 75 MBq 111In-IgG 24 h after administration of the liposomes. The scintigraphic results were compared and verified by culture, biopsy, surgery, and follow-up of at least 6 mo. RESULTS: Of the 16 proven infections and inflammations, 15 were detected by 99mTc-PEG liposome scintigraphy: soft-tissue infection (n = 3), septic arthritis (n = 3), autoimmune polyarthritis (n = 2), infected hip prosthesis (n = 1), infected osteosynthesis (n = 1), spondylodiscitis (n = 1), infected aortic prosthesis (n = 1), colitis (n = 1), abdominal abscess (n = 1), and pneumonia (n = 1). 99mTc-PEG liposome and 111In-IgG scintigraphy both missed 1 case of endocarditis. In addition, an 111In-IgG scan of a patient with mild soft-tissue infection was false-negative. Concordantly false-positive scans were recorded from 2 patients, both with uninfected pseudarthrosis and focal signs of sterile inflammation. During liposomal administration, 1 patient experienced flushing and chest tightness, which rapidly disappeared after lowering the infusion rate. No other adverse events were observed. CONCLUSION: This clinical evaluation of 99mTc-PEG liposomes shows that focal infection and inflammation can be adequately imaged with this new agent. The performance of 99mTc-PEG liposomes is at least as effective as that of 111In-IgG. With the simple and safe preparation and the physical and logistic advantages of a 99mTc label, 99mTc-PEG liposomes could be an attractive agent for infection or inflammation imaging.

Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes.

Sterically stabilized liposomes are considered promising carriers of therapeutic agents because they can facilitate controlled release of the drugs, thereby reducing drug-related toxicity and/or targeted delivery of drugs. Herein, we studied the pharmacokinetics and biodistribution of repeated injections of radiolabeled polyethyleneglycol (PEG) liposomes. Weekly injections of (99m)Tc-PEG liposomes dramatically influenced the circulatory half-life in rats. Biodistribution 4 h after the second dose showed a significantly reduced blood content (from 52.6 +/- 3.7 to 0.6 +/- 0.1% injected dose (ID), P <.01) accompanied by a highly increased uptake in the liver (from 8.1 +/- 0.8 to 46.2 +/- 9.8%ID, P <.01) and in the spleen (from 2.2 +/- 0.2 to 5.3 +/- 0.7%ID, P <.01). At subsequent injections the effect was less pronounced: after the fourth dose, the pharmacokinetics of the radiolabel had almost returned to normal. The same phenomenon was observed in a rhesus monkey, but not in mice. The enhanced blood clearance of the PEG liposomes also was observed in rats after transfusion of serum from rats that had received PEG liposomes 1 week earlier, indicating that the enhanced blood clearance was caused by a soluble serum factor. This serum factor was a heat-labile molecule that coeluted on a size exclusion column with a 150-kDa protein. In summary, i.v. administration of sterically stabilized PEG liposomes significantly altered the pharmacokinetic behavior of subsequently injected PEG liposomes in a time- and frequency-dependent manner. The observed phenomenon may have important implications for the repeated administration of sterically stabilized liposomes for targeted drug delivery.