Development of radiopharmaceuticals for PET imaging of Inflammation - VAP-1, FR-beta and CLEVER-1 as target molecules

Positron emission tomography (PET) is a non-invasive technology widely used in oncology, cardiology, neurology and inflammation imaging. PET can detect the expression of receptors, genes and molecular pathways in both patients and experimental animals. Thus far, the radiopharmaceutical 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has been employed in the clinic for several indications involving inflammation. While [18F]FDG uptake in regions of inflammation is increased, it is not specific for inflammation, which can lead to difficulty in analysis as well as false positive findings. Therefore, development of alternative, inflammation-specific PET radiotracers is justified. Our aim is to develop and evaluate new PET radiopharmaceuticals for imaging inflammation-specific targets. We propose these tools may be valuable for both clinical practice and medical research of the inflammatory conditions such as atherosclerosis and rheumatoid arthritis. In this work, three molecules associated with inflammation were selected as targets: vascular adhesion protein-1 (VAP-1), folate receptor β (FR- β) and common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1). These receptors are associated with leukocyte recruitment into inflamed tissues as well as activated macrophages at sites of inflammation. Five different radiopharmaceuticals targeting these receptors were evaluated in experimental disease models or in humans. The results showed that VAP-1-targeting tracers [68Ga]Ga- and [18F]AlF-NOTA-Siglec-9 could visualize sterile skin inflammation in rats. Compared to each other, the tracers showed similar uptake characteristics. Another Siglec-9-derived tracer, [68Ga]Ga-DOTA-Siglec-9 was shown to be safe and well tolerated in healthy volunteers and was able to visualize inflammation in arthritic joints. Further, [68Ga]Ga-NOTA-Folate was successfully used to target FR-β positive macrophages in inflamed atherosclerotic lesion in mice. Finally, [89Zr]Zr-DFO-bexmarilimab showed increased uptake in fibrotic rabbit kidneys expressing CLEVER-1

Arvioita arvioinnin etiikasta

Hyvä, paha arviointi / Päivi Atjonen. Helsinki, 2007

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.</p

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

Radiolabeled RGD peptides targeting expression of alpha(v)beta(3) 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 [Ga-68]NODAGA-RGDyK to alpha(v)beta(3) 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 [Ga-68]NODAGA-RGDyK for 90 min at 37 degrees 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 [Ga-68]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 [Ga-68]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

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 αvβ3 integrin that is upregulated during angiogenesis and inflammation. We studied whether αvβ3 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 αvβ3 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 αvβ3 integrin-targeting [68Ga]Ga-NODAGA-RGD is a potential PET tracer for the specific detection of active inflammatory lesions in autoimmune myocarditis.</p

Preclinical Evaluation of a Humanized Antibody Against Common Lymphatic Endothelial and Vascular Endothelial Receptor-1, 89Zr-Desferrioxamine-Bexmarilimab, 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 to 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. Distribution kinetics of intravenously administered 89Zr-DFO-bexmarilimab (0.1 mg/kg) for up to 7 days in a rabbit model of renal fibrosis mediated by unilateral ureteric obstruction (UUO). 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 hours post-injection, PET/CT, ex-vivo gamma counting and autoradiography demonstrated that there was significantly higher 89Zr-DFO-bexmarilimab uptake in UUO-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.</p

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 oligonucleo-tides. 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

BackgroundActivated macrophages in the experimental model of multiple sclerosis (MS) express folate receptor-beta (FR-beta), 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 Ga-68-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated folate (Ga-68-FOL) for assessing inflammatory lesions. In addition, we investigated whether FR-beta is expressed in the brain of patients with MS.MethodsFocal 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). Ga-68-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-beta expression in postmortem brain samples from 5 patients with MS and 5 healthy controls.ResultsImmunofluorescence and histological analyses revealed significant reductions in FR-beta 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 Ga-68-FOL in the brain was low and did not differ between the groups, but the in vitro binding of Ga-68-FOL was significantly lower in EC2319-treated rats (P < 0.01). FR-beta positivity was observed in chronically active lesions and in normal-appearing white matter in MS brain samples.ConclusionsEC2319 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-beta -positive cells in chronically active plaques, which suggests that these results may have translational relevance

Comprehensive development of nearly zero-energy municipal service buildings (COMBI). Tutkimushankkeen johdanto- ja yhteenvetoraportti.

Tässä COMBI-tutkimushankkeen johdanto- ja yhteenvetoraportissa esitetään vuosina 2015—2018 toteutetun tutkimushankkeen keskeiset suositukset ja johtopäätökset. Hankkeen tavoitteena on ollut parantaa julkisten palvelurakennusten, kuten koulujen, päiväkotien ja vanhainkotien energiatehokkuutta turvallisesti ja kustannustehokkaasti. Hankkeessa on tarkasteltu sekä uudis- että korjausrakentamista.Tutkittuja aihepiirejä on hankkeessa ollut suuri määrä. Arkkitehtuurin osalta on tarkasteltu palvelurakennusten arkkitehtisuunnittelun kehittämistä energiatehokkuuden ja tilasuunnittelun näkökulmista sekä ympäristöystävällisyyden ja kestävyyden huomioon ottamista arkkitehtisuunnittelussa. Rakenteiden osalta on tutkittu niiden lämpö- ja kosteusteknistä toimintaa ja määritetty kosteusteknisiä materiaaliominaisuuksia. Kenttätutkimuksissa on tarkasteltu sisäilman olosuhteita 24 palvelurakennuksessa Tampereen ja ympäristökuntien sekä Helsingin alueella. Myös palvelurakennusten laskennallista ja toteutunutta energiankulutusta on tutkittu Tampereen, Helsingin ja Oulun kohteista. Taloteknisten järjestelmien osalta on tarkasteltu niiden kustannusoptimaalisuutta, uusiutuvan energian etätuotantoa, taloautomaatiojärjestelmien toimintaa, aurinkosuojausta ja valaistusta. Rakennusprosessin osalta näkökulmina ovat olleet päätöksenteon prosessit, talotekniikan käytännön toteutus, rakennuksen toimivuuden varmistus sekä olosuhteiden ja energiankulutuksen seuranta. Lisäksi on kehitetty työkaluja rakennushankkeen taloudellisuustarkasteluihin.Tämän johdanto- ja yhteenvetoraportin liitteenä on hankkeen tulosten pohjalta koottu COMBI 8 suosituslista, jossa esitettyjen toimenpiteiden katsotaan laajasti edesauttavan julkisten palvelurakennusten toimivuutta ja energiatehokkuutta. Raportin liitteenä on myös 45 kpl lyhyitä tuloskortteja sekä niihin liittyvät esitysaineistot, joiden tarkoituksena on helpottaa hankkeessa kerätyn tiedon leviämistä. Hankkeen alkuperäisjulkaisut on listattu tämän raportin liitteenä olevassa julkaisuluettelossa.Keskeisenä johtopäätöksenä todetaan, että hyvä energiatehokkuus on ainoastaan yksi laadukkaan rakentamisen monista ominaisuuksista. Laadukas rakentaminen edellyttää kokonaisvaltaista ja oikeaaikaista asioiden tarkastelua sekä ehjän ketjun rakentamista suunnittelusta toteutukseen ja käyttöön. Tässä onnistumisen edellytyksenä ovat rakennushankkeessa ja sen jälkeen rakennuksen parissa toimivien henkilöiden hyvä ammattitaito ja yhteistyö sekä riittävät resurssit. COMBI-hankkeen tulosten tavoitteena on antaa eri osapuolille tietoa ja työkaluja turvallisen, taloudellisen ja energiatehokkaan lopputuloksen saavuttamiseksi

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

Sialic acid-binding immunoglubulin-like lectin 9 (Siglec-9) is a ligand of vascular adhesion protein 1 (VAP-1). A gallium 68-labeled peptide of Siglec-9, 68Ga-DOTA-Siglec-9, holds promise as a novel PET tracer for imaging of inflammation. This first-in-human study investigated the safety, tolerability, biodistribution, and radiation dosimetry of this radiopharmaceutical. Methods: Six healthy males underwent dynamic whole-body PET/CT. Serial venous blood samples were drawn from 1-240 min after intravenous injection of 162 ± 4 MBq of 68Ga-DOTA-Siglec-9. In addition to gamma 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 2.2 software. 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 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 able to detect arthritis comparable to 18F-FDG. Conclusion: Intravenous injection of 68Ga-DOTA-Siglec-9 was safe and biodistribution is favorable for testing of the tracer in larger group of patients with rheumatoid arthritis planned in the next phase of clinical trials. The effective radiation dose of 68Ga-DOTA-Siglec-9 was within the same range as those 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, e.g., in trials aiming to elucidate the treatment efficacy of novel drug candidates