Detection of Intestinal Inflammation by Vascular Adhesion Protein-1-Targeted [68Ga]Ga-DOTA-Siglec-9 Positron Emission Tomography in Murine Models of Inflammatory Bowel Disease.

PURPOSE: Inflammatory bowel disease (IBD) can be imaged with positron emission tomography (PET), but existing PET radiopharmaceuticals have limited diagnostic accuracy. Vascular adhesion protein-1 (VAP-1) is an endothelial cell surface molecule that controls leukocyte extravasation into sites of inflammation. However, the role of inflammation-induced VAP-1 expression in IBD is still unclear. Therefore, this study investigated the utility of VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 positron emission tomography/computed tomography (PET/CT) for assessing inflammation in two mouse models of IBD. PROCEDURES: Studies were performed using K8-/- mice that develop a chronic colitis-phenotype and C57Bl/6NCrl mice with acute intestinal inflammation chemically-induced using 2.5% dextran sodium sulfate (DSS) in drinking water. In both diseased and control mice, uptake of the VAP-1-targeting peptide [68Ga]Ga-DOTA-Siglec-9 was assessed in intestinal regions of interest using in vivo PET/CT, after which ex vivo gamma counting, digital autoradiography, and histopathological analyses were performed. Immunofluorescence staining was performed to determine VAP-1-expression in the intestine, including in samples from patients with ulcerative colitis. RESULTS: Intestinal inflammation could be visualized by [68Ga]Ga-DOTA-Siglec-9 PET/CT in two murine models of IBD. In both models, the in vivo PET/CT and ex vivo studies of [68Ga]Ga-DOTA-Siglec-9 uptake were significantly higher than in control mice. The in vivo uptake was increased on average 1.4-fold in the DSS model and 2.0-fold in the K8-/- model. Immunofluorescence staining revealed strong expression of VAP-1 in the inflamed intestines of both mice and patients. CONCLUSIONS: This study suggests that the VAP-1-targeting [68Ga]Ga-DOTA-Siglec-9 PET tracer is a promising tool for non-invasive imaging of intestinal inflammation. Future studies in patients with IBD and evaluation of the potential value of [68Ga]Ga-DOTA-Siglec-9 in diagnosis and monitoring of the disease are warranted.

Vascular adhesion protein-1-targeted PET imaging in autoimmune myocarditis.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and primary amine oxidase, and Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetra-acetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([68Ga]Ga-DOTA-Siglec-9) is a positron emission tomography (PET) tracer targeting VAP-1. We evaluated the feasibility of PET imaging with [68Ga]Ga-DOTA-Siglec-9 for the detection of myocardial lesions in rats with autoimmune myocarditis. METHODS: Rats (n = 9) were immunized twice with porcine cardiac myosin in complete Freund's adjuvant. Control rats (n = 6) were injected with Freund's adjuvant alone. On day 21, in vivo PET/computed tomography (CT) imaging with [68Ga]Ga-DOTA-Siglec-9 was performed, followed by ex vivo autoradiography, histology, and immunohistochemistry of tissue sections. In addition, myocardial samples from three patients with cardiac sarcoidosis were studied. RESULTS: [68Ga]Ga-DOTA-Siglec-9 PET/CT images of immunized rats showed higher uptake in myocardial lesions than in myocardium outside lesions (SUVmean, 0.5 ± 0.1 vs 0.3 ± 0.1; P = .003) or control rats (SUVmean, 0.2 ± 0.03; P < .0001), which was confirmed by ex vivo autoradiography of tissue sections. Immunohistochemistry showed VAP-1-positive staining in lesions of rats with myocarditis and in patients with cardiac sarcoidosis. CONCLUSION: VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 PET is a potential novel technique for the detection of myocardial lesions.

In Vivo Imaging of [60]Fullerene-Based Molecular Spherical Nucleic Acids by Positron Emission Tomography.

18F-Labeled [60]fullerene-based molecular spherical nucleic acids (MSNAs), consisting of a human epidermal growth factor receptor 2 (HER2) mRNA antisense oligonucleotide sequence with a native phosphodiester and phosphorothioate backbone, were synthesized, site-specifically labeled with a positron emitting fluorine-18 and intravenously administrated via tail vein to HER2 expressing HCC1954 tumor-bearing mice. The biodistribution of the MSNAs was monitored in vivo by positron emission tomography/computed tomography (PET/CT) imaging. MSNA with a native phosphodiester backbone (MSNA-PO) was prone to rapid nuclease-mediated degradation, whereas the corresponding phosphorothioate analogue (MSNA-PS) with improved enzymatic stability showed an interesting biodistribution profile in vivo. One hour after the injection, majority of the radioactivity was observed in spleen and liver but also in blood with an average tumor-to-muscle ratio of 2. The prolonged radioactivity in blood circulation may open possibilities to the targeted delivery of the MSNAs.

Preclinical Evaluation of 89Zr-Desferrioxamine-Bexmarilimab, a Humanized Antibody Against Common Lymphatic Endothelial and Vascular Endothelial Receptor-1, in a Rabbit Model of Renal Fibrosis.

Bexmarilimab is a new humanized monoclonal antibody against common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) and is in clinical trials for macrophage-guided cancer immunotherapy. In addition being associated with cancer, CLEVER-1 is also associated with fibrosis. To facilitate prospective human PET studies, we preclinically evaluated 89Zr-labeled bexmarilimab in rabbits. Methods: Bexmarilimab was conjugated with desferrioxamine (DFO) and radiolabeled with 89Zr. Retained immunoreactivity was confirmed by flow cytometry. The distribution kinetics of intravenously administered 89Zr-DFO-bexmarilimab (0.1 mg/kg) were determined for up to 7 d in a rabbit model of renal fibrosis mediated by unilateral ureteric obstruction. The in vivo stability of 89Zr-DFO-bexmarilimab was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with autoradiography. Additionally, we estimated the human radiation dose from data obtained in healthy rabbits. Results: 89Zr-DFO-bexmarilimab cleared rapidly from the blood circulation and distributed to the liver and spleen. At 24 h after injection, PET/CT, ex vivo γ-counting, and autoradiography demonstrated that there was significantly higher 89Zr-DFO-bexmarilimab uptake in unilateral ureteric obstruction-operated fibrotic renal cortex, characterized by abundant CLEVER-1-positive cells, than in contralateral or healthy kidneys. The estimated effective dose for a 70-kg human was 0.70 mSv/MBq. Conclusion: The characteristics of 89Zr-DFO-bexmarilimab support future human PET studies to, for example, stratify patients for bexmarilimab treatment, evaluate the efficacy of treatment, or monitor disease progression.

[68Ga]Ga-DOTA-Siglec-9 Detects Pharmacodynamic Changes of FAP-Targeted IL2 Variant Immunotherapy in B16-FAP Melanoma Mice.

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible adhesion molecule, which supports contact between leukocytes and inflamed endothelium. There is evidence that VAP-1 is involved in the recruitment of leukocytes to melanoma tumors. Interleukin-2 (IL-2)-based immunotherapy is an efficient therapy that promotes immune system activity against cancers but is associated with toxicity. In the present study, we evaluated the feasibility of PET/CT imaging using the radiotracer [68Ga]Ga-DOTA-Siglec-9, which is targeted to VAP-1, to monitor pharmacodynamic effects of a novel FAP-IL2v immunocytokine (a genetically engineered variant of IL-2 fused with fibroblast activation protein) in the B16-FAP melanoma model. At 9 days after the inoculation of B16-FAP melanoma cells, mice were studied with [68Ga]Ga-DOTA-Siglec-9 PET/CT as a baseline measurement. Immediately after baseline imaging, mice were treated with FAP-IL2v or vehicle, and treatment was repeated 3 days later. Subsequent PET/CT imaging was performed 3, 5, and 7 days after baseline imaging. In addition to in vivo PET imaging, ex vivo autoradiography, histology, and immunofluorescence staining were performed on excised tumors. B16-FAP tumors were clearly detected with [68Ga]Ga-DOTA-Siglec-9 PET/CT during the follow-up period, without differences in tumor volume between FAP-IL2v-treated and vehicle-treated groups. Tumor-to-muscle uptake of [68Ga]Ga-DOTA-Siglec-9 was significantly higher in the FAP-IL2v-treated group than in the vehicle-treated group 7 days after baseline imaging, and this was confirmed by tumor autoradiography analysis. FAP-IL2v treatment did not affect VAP-1 expression on the tumor vasculature. However, FAP-IL2v treatment increased the number of CD8+ T cells and natural killer cells in tumors. The present study showed that [68Ga]Ga-DOTA-Siglec-9 can detect B16-FAP tumors and allows monitoring of FAP-IL2v treatment.

Association between [68Ga]NODAGA-RGDyK uptake and dynamics of angiogenesis in a human cell-based 3D model.

Radiolabeled RGD peptides targeting expression of αvß3 integrin have been applied to in vivo imaging of angiogenesis. However, there is a need for more information on the quantitative relationships between RGD peptide uptake and the dynamics of angiogenesis. In this study, we sought to measure the binding of [68Ga]NODAGA-RGDyK to αvß3 integrin in a human cell-based three-dimensional (3D) in vitro model of angiogenesis, and to compare the level of binding with the amount of angiogenesis. Experiments were conducted using a human cell-based 3D model of angiogenesis consisting of co-culture of human adipose stem cells (hASCs) and of human umbilical vein endothelial cells (HUVECs). Angiogenesis was induced with four concentrations (25%, 50%, 75%, and 100%) of growth factor cocktail resulting in a gradual increase in the density of the tubule network. Cultures were incubated with [68Ga]NODAGA-RGDyK for 90 min at 37 °C, and binding of radioactivity was measured by gamma counting and digital autoradiography. The results revealed that tracer binding increased gradually with neovasculature density. In comparison with vessels induced with a growth factor concentration of 25%, the uptake of [68Ga]NODAGA-RGDyK was higher at concentrations of 75% and 100%, and correlated with the amount of neovasculature, as determined by visual evaluation of histological staining. Uptake of [68Ga]NODAGA-RGDyK closely reflected the amount of angiogenesis in an in vitro 3D model of angiogenesis. These results support further evaluation of RGD-based approaches for targeted imaging of angiogenesis.

Controlled Monofunctionalization of Molecular Spherical Nucleic Acids on a Buckminster Fullerene Core.

An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleotides. Exposing the intermediate products then to the same reaction (i.e., strain-promoted alkyne-azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs' outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C60-azide scaffold used for the assembly of full-armed SNAs.

Efficacy and tolerability of folate-aminopterin therapy in a rat focal model of multiple sclerosis.

BACKGROUND: Activated macrophages in the experimental model of multiple sclerosis (MS) express folate receptor-ß (FR-ß), representing a promising target for the treatment of MS. Here, we both evaluated the efficacy of a novel folate-aminopterin construct (EC2319) in a rat focal model of multiple sclerosis (MS) and investigated the utility of 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated folate (68Ga-FOL) for assessing inflammatory lesions. In addition, we investigated whether FR-ß is expressed in the brain of patients with MS. METHODS: Focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) was induced in 40 Lewis rats; 20 healthy Lewis rats were used as controls. Rats were divided into six groups according to the duration of disease (control, acute, or chronic) and intervention (vehicle versus EC2319). 68Ga-FOL analyses, histology, and immunofluorescence of the brain were performed to evaluate the efficacy of subcutaneously administered EC2319 on lesion development. Immunofluorescence was used to assess FR-ß expression in postmortem brain samples from 5 patients with MS and 5 healthy controls. RESULTS: Immunofluorescence and histological analyses revealed significant reductions in FR-ß expression (P < 0.05) and lesion size (P < 0.01), as well as improved inducible nitric oxide synthase/mannose receptor C type 1 ratios (P < 0.01) in macrophages and microglia during the chronic but not acute phase of fDTH-EAE in EC2319-treated rats. The uptake of IV-injected 68Ga-FOL in the brain was low and did not differ between the groups, but the in vitro binding of 68Ga-FOL was significantly lower in EC2319-treated rats (P < 0.01). FR-ß positivity was observed in chronically active lesions and in normal-appearing white matter in MS brain samples. CONCLUSIONS: EC2319 was well tolerated and attenuated inflammation and lesion development in a rat model of a chronic progressive form of MS. Human MS patients have FR-ß-positive cells in chronically active plaques, which suggests that these results may have translational relevance.

Evaluation of [68Ga]Ga-NODAGA-RGD for PET Imaging of Rat Autoimmune Myocarditis.

The 68Gallium-labeled 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid conjugated radiolabelled arginine-glycine-aspartic acid peptide ([68Ga]Ga-NODAGA-RGD) is a positron emission tomography (PET) tracer binding to cell surface receptor αvß3 integrin that is upregulated during angiogenesis and inflammation. We studied whether αvß3 targeting PET imaging can detect myocardial inflammation in a rat model of autoimmune myocarditis. To induce myocarditis, rats (n = 8) were immunized with porcine cardiac myosin in complete Freund's adjuvant on days 0 and 7. Control rats (n = 8) received Freund's adjuvant alone. On day 21, in vivo PET/CT imaging with [68Ga]Ga-NODAGA-RGD followed by ex vivo autoradiography and immunohistochemistry were carried out. Inflammatory lesions were detected histologically in the myocardium of 7 out of 8 immunized rats. In vivo PET images showed higher [68Ga]Ga-NODAGA-RGD accumulation in the myocardium of rats with inflammation than the non-inflamed myocardium of control rats (SUVmean 0.4 ± 0.1 vs. 0.1 ± 0.02; P = 0.00006). Ex vivo autoradiography and histology confirmed that [68Ga]Ga-NODAGA-RGD uptake co-localized with inflammatory lesions containing αvß3 integrin-positive capillary-like structures. A non-specific [68Ga]Ga-DOTA-(RGE)2 tracer showed 76% lower uptake than [68Ga]Ga-NODAGA-RGD in the inflamed myocardium. Our results indicate that αvß3 integrin-targeting [68Ga]Ga-NODAGA-RGD is a potential PET tracer for the specific detection of active inflammatory lesions in autoimmune myocarditis.

First-in-Humans Study of 68Ga-DOTA-Siglec-9, a PET Ligand Targeting Vascular Adhesion Protein 1.

Sialic acid-binding immunoglubulinlike lectin 9 (Siglec-9) is a ligand of vascular adhesion protein 1. A 68Ga-labeled peptide of Siglec-9, 68Ga-DOTA-Siglec-9, holds promise as a novel PET tracer for imaging of inflammation. This first-in-humans study investigated the safety, tolerability, biodistribution, and radiation dosimetry of this radiopharmaceutical. Methods: Six healthy men underwent dynamic whole-body PET/CT. Serial venous blood samples were drawn from 1 to 240 min after intravenous injection of 162 ± 4 MBq of 68Ga-DOTA-Siglec-9. In addition to γ-counting, the plasma samples were analyzed by high-performance liquid chromatography to detect intact tracer and radioactive metabolites. Radiation doses were calculated using the OLINDA/EXM software, version 2.2. In addition, a patient with early rheumatoid arthritis was studied with both 68Ga-DOTA-Siglec-9 and 18F-FDG PET/CT to determine the ability of the new tracer to detect arthritis. Results:68Ga-DOTA-Siglec-9 was well tolerated by all subjects. 68Ga-DOTA-Siglec-9 was rapidly cleared from the blood circulation, and several radioactive metabolites were detected. The organs with the highest absorbed doses were the urinary bladder wall (0.38 mSv/MBq) and kidneys (0.054 mSv/MBq). The mean effective dose was 0.022 mSv/MBq (range, 0.020-0.024 mSv/MBq). Most importantly, however, 68Ga-DOTA-Siglec-9 was comparable to 18F-FDG in detecting arthritis. Conclusion: Intravenous injection of 68Ga-DOTA-Siglec-9 was safe and biodistribution was favorable for testing of the tracer in larger group of patients with rheumatoid arthritis, as is planned for the next phase of clinical trials. The effective radiation dose of 68Ga-DOTA-Siglec-9 was within the same range as the effective radiation doses of other 68Ga-labeled tracers. Injection of 150 MBq of 68Ga-DOTA-Siglec-9 would expose a subject to 3.3 mSv. These findings support the possible repeated clinical use of 68Ga-DOTA-Siglec-9, such as in trials to elucidate the treatment efficacy of novel drug candidates.

Evaluation of image quality with four positron emitters and three preclinical PET/CT systems.

BACKGROUND: We investigated the image quality of 11C, 68Ga, 18F and 89Zr, which have different positron fractions, physical half-lifes and positron ranges. Three small animal positron emission tomography/computed tomography (PET/CT) systems were used in the evaluation, including the Siemens Inveon, RAYCAN X5 and Molecubes ß-cube. The evaluation was performed on a single scanner level using the national electrical manufacturers association (NEMA) image quality phantom and analysis protocol. Acquisitions were performed with the standard NEMA protocol for 18F and using a radionuclide-specific acquisition time for 11C, 68Ga and 89Zr. Images were assessed using percent recovery coefficient (%RC), percentage standard deviation (%STD), image uniformity (%SD), spill-over ratio (SOR) and evaluation of image quantification. RESULTS: 68Ga had the lowest %RC (< 62%) across all systems. 18F had the highest maximum %RC (> 85%) and lowest %STD for the 5 mm rod across all systems. For 11C and 89Zr, the maximum %RC was close (> 76%) to the %RC with 18F. A larger SOR were measured in water with 11C and 68Ga compared to 18F on all systems. SOR in air reflected image reconstruction and data correction performance. Large variation in image quantification was observed, with maximal errors of 22.73% (89Zr, Inveon), 17.54% (89Zr, RAYCAN) and - 14.87% (68Ga, Molecubes). CONCLUSIONS: The systems performed most optimal in terms of NEMA image quality parameters when using 18F, where 11C and 89Zr performed slightly worse than 18F. The performance was least optimal when using 68Ga, due to large positron range. The large quantification differences prompt optimization not only by terms of image quality but also quantification. Further investigation should be performed to find an appropriate calibration and harmonization protocol and the evaluation should be conducted on a multi-scanner and multi-center level.

Radiosynthesis and preclinical evaluation of [68Ga]Ga-NOTA-folate for PET imaging of folate receptor ß-positive macrophages.

Folate receptor ß (FR-ß), a marker expressed on macrophages, is a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [68Ga]Ga-NOTA-folate (68Ga-FOL). After determining the affinity of 68Ga-FOL using cells expressing FR-ß, we studied atherosclerotic mice with 68Ga-FOL and 18F-FDG PET/CT. In addition, we studied tracer distribution and co-localization with macrophages in aorta cryosections using autoradiography, histology, and immunostaining. The specificity of 68Ga-FOL was assessed in a blocking study with folate glucosamine. As a final step, human radiation doses were extrapolated from rat PET data. We were able to produce 68Ga-FOL with high radiochemical purity and moderate molar activity. Cell binding studies revealed that 68Ga-FOL had 5.1 nM affinity for FR-ß. Myocardial uptake of 68Ga-FOL was 20-fold lower than that of 18F-FDG. Autoradiography and immunohistochemistry of the aorta revealed that 68Ga-FOL radioactivity co-localized with Mac-3-positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of 68Ga-FOL was significantly higher than that of 18F-FDG. Blocking studies verified that 68Ga-FOL was specific for FR. Based on estimations from rat data, the human effective dose was 0.0105 mSv/MBq. Together, these findings show that 68Ga-FOL represents a promising new FR-ß-targeted tracer for imaging macrophage-associated inflammation.

Exploring Alternative Radiolabeling Strategies for Sialic Acid-Binding Immunoglobulin-Like Lectin 9 Peptide: [68Ga]Ga- and [18F]AlF-NOTA-Siglec-9.

Amino acid residues 283-297 from sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) form a cyclic peptide ligand targeting vascular adhesion protein-1 (VAP-1). VAP-1 is associated with the transfer of leukocytes from blood to tissues upon inflammation. Therefore, analogs of Siglec-9 peptide are good candidates for visualizing inflammation non-invasively using positron emission tomography (PET). Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA)-conjugated Siglec-9 has been evaluated extensively for this purpose. Here, we explored two alternative strategies for radiolabeling Siglec-9 peptide using a 1,4,7-triazacyclononane-triacetic acid (NOTA)-chelator to bind [68Ga]Ga or [18F]AlF. The radioligands were evaluated by in vivo PET imaging and ex vivo γ-counting of turpentine-induced sterile skin/muscle inflammation in Sprague-Dawley rats. Both tracers showed clear accumulation in the inflamed tissues. The whole-body biodistribution patterns of the tracers were similar.

Exploring the radiosynthesis and in vitro characteristics of [68 Ga]Ga-DOTA-Siglec-9.

Vascular adhesion protein 1 is a leukocyte homing-associated glycoprotein, which upon inflammation rapidly translocates from intracellular sources to the endothelial cell surface. It has been discovered that the cyclic peptide residues 283-297 of sialic acid-binding IgG-like lectin 9 (Siglec-9) "CARLSLSWRGLTLCPSK" bind to vascular adhesion protein 1 and hence makes the radioactive analogues of this compound ([68 Ga]Ga-DOTA-Siglec-9) interesting as a noninvasive visualizing marker of inflammation. Three different approaches to the radiosynthesis of [68 Ga]Ga-DOTA-Siglec-9 are presented and compared with previously published methods. A simple, robust radiosynthesis of [68 Ga]Ga-DOTA-Siglec-9 with a yield of 62% (non decay-corrected) was identified, and it had a radiochemical purity >98% and a specific radioactivity of 35 MBq/nmol. Furthermore, the protein binding and stability of [68 Ga]Ga-DOTA-Siglec-9 were analyzed in vitro in mouse, rat, rabbit, pig, and human plasma and compared with in vivo pig results. The plasma in vitro protein binding of [68 Ga]Ga-DOTA-Siglec-9 was the lowest in the pig followed by rabbit, human, rat, and mouse. It was considerably higher in the in vivo pig experiments. The in vivo stability in pigs was lower than the in vitro stability. Despite considerable species differences, the observed characteristics of [68 Ga]Ga-DOTA-Siglec-9 are suitable as a positron emission tomography tracer.