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Background: Interstitial lung disease (ILD) is a common and severe complication in rheumatic diseases. Folate receptor-β is expressed on activated, but not resting macrophages which play a key role in dysregulated tissue repair including ILD. We therefore aimed to pre-clinically evaluate the potential of 18F-AzaFol-based PET/CT (positron emission computed tomography/computed tomography) for the specific detection of macrophage-driven pathophysiologic processes in experimental ILD. Methods: The pulmonary expression of folate receptor-β was analyzed in patients with different subtypes of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. PET/CT was performed at days 3, 7, and 14 after BLM instillation using the 18F-based folate radiotracer 18F-AzaFol. The specific pulmonary accumulation of the radiotracer was assessed by ex vivo PET/CT scans and quantified by ex vivo biodistribution studies. Results: Folate receptor-β expression was 3- to 4-fold increased in patients with fibrotic ILD, including idiopathic pulmonary fibrosis and connective tissue disease-related ILD, and significantly correlated with the degree of lung remodeling. A similar increase in the expression of folate receptor-β was observed in experimental lung fibrosis, where it also correlated with disease extent. In the mouse model of BLM-induced ILD, pulmonary accumulation of 18F-AzaFol reflected macrophage-related disease development with good correlation of folate receptor-β positivity with radiotracer uptake. In the ex vivo imaging and biodistribution studies, the maximum lung accumulation was observed at day 7 with a mean accumulation of 1.01 ± 0.30% injected activity/lung in BLM-treated vs. control animals (0.31 ± 0.06% % injected activity/lung; p < 0.01). Conclusion: Our preclinical proof-of-concept study demonstrated the potential of 18F-AzaFol as a novel imaging tool for the visualization of macrophage-driven fibrotic lung diseases.

Background: Interstitial lung disease (ILD) is a common and severe complication in rheumatic diseases. Folate receptor-β is expressed on activated, but not resting macrophages which play a key role in dysregulated tissue repair including ILD. We therefore aimed to pre-clinically evaluate the potential of 18F-AzaFol-based PET/CT (positron emission computed tomography/computed tomography) for the specific detection of macrophage-driven pathophysiologic processes in experimental ILD. Methods: The pulmonary expression of folate receptor-β was analyzed in patients with different subtypes of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. PET/CT was performed at days 3, 7, and 14 after BLM instillation using the 18F-based folate radiotracer 18F-AzaFol. The specific pulmonary accumulation of the radiotracer was assessed by ex vivo PET/CT scans and quantified by ex vivo biodistribution studies. Results: Folate receptor-β expression was 3- to 4-fold increased in patients with fibrotic ILD, including idiopathic pulmonary fibrosis and connective tissue disease-related ILD, and significantly correlated with the degree of lung remodeling. A similar increase in the expression of folate receptor-β was observed in experimental lung fibrosis, where it also correlated with disease extent. In the mouse model of BLM-induced ILD, pulmonary accumulation of 18F-AzaFol reflected macrophage-related disease development with good correlation of folate receptor-β positivity with radiotracer uptake. In the ex vivo imaging and biodistribution studies, the maximum lung accumulation was observed at day 7 with a mean accumulation of 1.01 ± 0.30% injected activity/lung in BLM-treated vs. control animals (0.31 ± 0.06% % injected activity/lung; p < 0.01). Conclusion: Our preclinical proof-of-concept study demonstrated the potential of 18F-AzaFol as a novel imaging tool for the visualization of macrophage-driven fibrotic lung diseases., Frontiers in Immunology, 10, ISSN:1664-3224

Formula for the calculation of the 47Sc activity in Bq (s−1), accessible under the applied irradiation conditions. σ = nuclear cross section of the 46Ca(n,γ)47Ca reaction in cm−2, NT = number of 46Ca atoms, Φth = thermal neutron flux in n * cm−2 * s−1, λSc and λCa = decay constants of 47Sc and 47Ca in s−1, tirr = irradiation time and twait = post irradiation waiting time in s. b Formula for the calculation of the optimal post irradiation waiting time (topt) in s, accessible at the applied irradiation time (tirr in s). The decay constants of 47Sc (λSc) and 47Ca (λCa) are given in s−1. c Formula for the calculation of the optimal relative 47Sc activity (a(47Sc)opt) (dimensionless), accessible under the applied irradiation conditions. σ = nuclear cross section of the 46Ca(n,γ)47Ca reaction in cm−2, NT = number of 46Ca atoms, Φth = thermal neutron flux in n * cm−2 * s−1, λSc and λCa = decay constants of 47Sc and 47Ca in s−1. d Formula for the maximal obtainable 47Sc activity (dimensionless). The irradiation time (tirr) is given in s and the decay constants of 47Sc (λSc) and 47Ca (λCa) in s−1. Figure S2. γ-Ray spectra of 47Sc and 47Ca from the neutron-irradiated 46Ca ampoule, obtained 71 h after the end of irradiation (measurement time: 10 s) (a) and of the pure 47Sc eluate after separation (Method B), obtained 1 h after the end of separation (measurement time: 250 s) (b). Figure S3. γ-Ray spectrum of the neutron-irradiated 47Ti ampoule at SINQ, obtained 21 d after the end of irradiation (measurement time: 9600 s). Figure S4. Measured cross section values (squares, retrieved from the EXFOR-database) (Zerkin 2016) as well as the theoretical calculations from the TENDL-2015 library (straight line) (Koning, Rochman et al. 2015) for the 47Ti(n,p)47Sc (blue) and the 47Ti(n,p + n)46Sc (black) nuclear reactions. Table S5. Trace metal analysis of the reduced 46Ti metal by ICP-OES. Only the elements determined at a concentration higher than the detection limit are listed below. (DOCX 415 kb)

Folate receptor (FR)-targeted radionuclide therapy using folate radioconjugates is of interest due to the expression of the FR in a variety of tumor types. The high renal accumulation of radiofolates presents, however, a risk of radionephropathy. A potential option to address this challenge would be to use radioprotectants, such as amifostine. Methods for early detection of kidney damage that—in this case—cannot be predicted based on dose estimations, would facilitate the development of novel therapies. The aim of this study was, therefore, to assess potentially changing levels of plasma and urine biomarkers and to determine DNA damage at an early stage after radiofolate application. The identification of an early indicator for renal damage in mice would be useful since histological changes become apparent only several months after treatment. Mice were injected with different quantities of 177Lu-folate (10 MBq, 20 MBq and 30 MBq), resulting in mean absorbed kidney doses of ~23 Gy, ~46 Gy and ~69 Gy, respectively, followed by euthanasia two weeks (>85% of the mean renal radiation dose absorbed) or three months later. Whereas all investigated biomarkers remained unchanged, the number of γ-H2AX-positive nuclei in the renal cortex showed an evident dose-dependent increase as compared to control values two weeks after treatment. Comparison with the extent of kidney injury determined by histological changes five to eight months after administration of the same 177Lu-folate activities suggested that the quantitative assessment of double-strand breaks can be used as a biological indicator for long-term radiation effects in the kidneys. This method may, thus, enable faster assessment of radiopharmaceuticals and protective measures by preventing logistically challenging long-term investigations to detect kidney damage., Pharmaceuticals, 10 (2), ISSN:1424-8247

Terbium-149 is among the most interesting therapeutic nuclides for medical applications. It decays by emission of short-range α-particles (Eα = 3.967 MeV) with a half-life of 4.12 h. The goal of this study was to investigate the anticancer efficacy of a 149Tb-labeled DOTA-folate conjugate (cm09) using folate receptor (FR)-positive cancer cells in vitro and in tumor-bearing mice. 149Tb was produced at the ISOLDE facility at CERN. Radiolabeling of cm09 with purified 149Tb resulted in a specific activity of ~1.2 MBq/nmol. In vitro assays performed with 149Tb-cm09 revealed a reduced KB cell viability in a FR-specific and activity concentration-dependent manner. Tumor-bearing mice were injected with saline only (group A) or with 149Tb-cm09 (group B: 2.2 MBq; group C: 3.0 MBq). A significant tumor growth delay was found in treated animals resulting in an increased average survival time of mice which received 149Tb-cm09 (B: 30.5 d; C: 43 d) compared to untreated controls (A: 21 d). Analysis of blood parameters revealed no signs of acute toxicity to the kidneys or liver in treated mice over the time of investigation. These results demonstrated the potential of folate-based α-radionuclide therapy in tumor-bearing mice.

Terbium-149 is among the most interesting therapeutic nuclides for medical applications. It decays by emission of short-range α-particles (Eα = 3.967 MeV) with a half-life of 4.12 h. The goal of this study was to investigate the anticancer efficacy of a 149Tb-labeled DOTA-folate conjugate (cm09) using folate receptor (FR)-positive cancer cells in vitro and in tumor-bearing mice. 149Tb was produced at the ISOLDE facility at CERN. Radiolabeling of cm09 with purified 149Tb resulted in a specific activity of ~1.2 MBq/nmol. In vitro assays performed with 149Tb-cm09 revealed a reduced KB cell viability in a FR-specific and activity concentration-dependent manner. Tumor-bearing mice were injected with saline only (group A) or with 149Tb-cm09 (group B: 2.2 MBq; group C: 3.0 MBq). A significant tumor growth delay was found in treated animals resulting in an increased average survival time of mice which received 149Tb-cm09 (B: 30.5 d; C: 43 d) compared to untreated controls (A: 21 d). Analysis of blood parameters revealed no signs of acute toxicity to the kidneys or liver in treated mice over the time of investigation. These results demonstrated the potential of folate-based α-radionuclide therapy in tumor-bearing mice., Pharmaceuticals, 7 (3), ISSN:1424-8247

Background The radiolanthanide 161Tb has, in recent years, attracted increasing interest due to its favorable characteristics for medical application. 161Tb exhibits similar properties to the widely-used therapeutic radionuclide 177Lu. In contrast to 177Lu, 161Tb yields a significant number of short-ranging Auger/conversion electrons (≤50 keV) during its decay process. 161Tb has been shown to be more effective for tumor therapy than 177Lu if applied using the same activity. The purpose of this study was to investigate long-term damage to the kidneys after application of 161Tb-folate and compare it to the renal effects caused by 177Lu-folate. Methods Renal side effects were investigated in nude mice after the application of different activities of 161Tb-folate (10, 20, and 30 MBq per mouse) over a period of 8 months. Renal function was monitored by the determination of 99mTc-DMSA uptake in the kidneys and by measuring blood urea nitrogen and creatinine levels in the plasma. Histopathological analysis was performed by scoring of the tissue damage observed in HE-stained kidney sections from euthanized mice. Results Due to the co-emitted Auger/conversion electrons, the mean absorbed renal dose of 161Tb-folate (3.0 Gy/MBq) was about 24 % higher than that of 177Lu-folate (2.3 Gy/MBq). After application of 161Tb-folate, kidney function was reduced in a dose- and time-dependent manner, as indicated by the decreased renal uptake of 99mTc-DMSA and the increased levels of blood urea nitrogen and creatinine. Similar results were obtained when 177Lu-folate was applied at the same activity. Histopathological investigations confirmed comparable renal cortical damage after application of the same activities of 161Tb-folate and 177Lu-folate. This was characterized by collapsed tubules and enlarged glomeruli with fibrin deposition in moderately injured kidneys and glomerulosclerosis in severely damaged kidneys. Conclusions Tb-folate induced dose-dependent radionephropathy over time, but did not result in more severe damage than 177Lu-folate when applied at the same activity. These data are an indication that Auger/conversion electrons do not exacerbate overall renal damage after application with 161Tb-folate as compared to 177Lu-folate, even though they result in an increased dose deposition in the renal tissue. Global toxicity affecting other tissues than kidneys remains to be investigated after 161Tb-based therapy, however., EJNMMI Research, 6 (1), ISSN:2191-219X

Formula for the calculation of the 47Sc activity in Bq (s−1), accessible under the applied irradiation conditions. σ = nuclear cross section of the 46Ca(n,γ)47Ca reaction in cm−2, NT = number of 46Ca atoms, Φth = thermal neutron flux in n * cm−2 * s−1, λSc and λCa = decay constants of 47Sc and 47Ca in s−1, tirr = irradiation time and twait = post irradiation waiting time in s. b Formula for the calculation of the optimal post irradiation waiting time (topt) in s, accessible at the applied irradiation time (tirr in s). The decay constants of 47Sc (λSc) and 47Ca (λCa) are given in s−1. c Formula for the calculation of the optimal relative 47Sc activity (a(47Sc)opt) (dimensionless), accessible under the applied irradiation conditions. σ = nuclear cross section of the 46Ca(n,γ)47Ca reaction in cm−2, NT = number of 46Ca atoms, Φth = thermal neutron flux in n * cm−2 * s−1, λSc and λCa = decay constants of 47Sc and 47Ca in s−1. d Formula for the maximal obtainable 47Sc activity (dimensionless). The irradiation time (tirr) is given in s and the decay constants of 47Sc (λSc) and 47Ca (λCa) in s−1. Figure S2. γ-Ray spectra of 47Sc and 47Ca from the neutron-irradiated 46Ca ampoule, obtained 71 h after the end of irradiation (measurement time: 10 s) (a) and of the pure 47Sc eluate after separation (Method B), obtained 1 h after the end of separation (measurement time: 250 s) (b). Figure S3. γ-Ray spectrum of the neutron-irradiated 47Ti ampoule at SINQ, obtained 21 d after the end of irradiation (measurement time: 9600 s). Figure S4. Measured cross section values (squares, retrieved from the EXFOR-database) (Zerkin 2016) as well as the theoretical calculations from the TENDL-2015 library (straight line) (Koning, Rochman et al. 2015) for the 47Ti(n,p)47Sc (blue) and the 47Ti(n,p + n)46Sc (black) nuclear reactions. Table S5. Trace metal analysis of the reduced 46Ti metal by ICP-OES. Only the elements determined at a concentration higher than the detection limit are listed below. (DOCX 415 kb)