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Introduction Affibody molecules are small (58 amino acids) high-affinity proteins based on a tri-helix non-immunoglobulin scaffold. A clinical study has demonstrated that PET imaging using Affibody molecules labeled with 68 Ga (T ½ = 68 min) can visualize metastases of breast cancer expressing human epidermal growth factor receptor type 2 (HER2) and provide discrimination between tumors with high and low expression level. This may help to identify breast cancer patients benefiting from HER2-targeting therapies. The best discrimination was at 4 h post injection. Due to longer half-life, a positron-emitting radionuclide 44 Sc (T ½ = 4.04 h) might be a preferable label for Affibody molecules for imaging at several hours after injection. Methods A synthetic second-generation anti-HER2 Affibody molecule Z HER2:2891 was labeled with 44 Sc via a DOTA-chelator conjugated to the N-terminal amino group. Binding specificity, affinity and cellular processing 44 Sc-DOTA-Z HER2:2891 and 68 Ga-DOTA-Z HER2:2891 were compared in vitro using HER2-expressing cells. Biodistribution and imaging properties of 44 Sc-DOTA-Z HER2:2891 and 68 Ga-DOTA-Z HER2:2891 were evaluated in Balb/c nude mice bearing HER2-expression xenografts. Results The labeling yield of 98 ± 2% and specific activity of 7.8 GBq/μmol were obtained. The conjugate demonstrated specific binding to HER2-expressing SKOV3.ip cells in vitro and to SKOV3.ip xenografts in nude mice. The distribution of radioactivity at 3 h post injection was similar for 44 Sc-DOTA-Z HER2:2891 and 68 Ga-DOTA-Z HER2:2891 , but the blood clearance of the 44 Sc-labeled variant was slower and the tumor-to-blood ratio was reduced (15 ± 2 for 44 Sc-DOTA-Z HER2:2891 vs 46 ± 9 for 68 Ga-DOTA-Z HER2:2891 ). At 6 h after injection of 44 Sc-DOTA-Z HER2:2891 the tumor uptake was 8 ± 2% IA/g and the tumor-to-blood ratio was 51 ± 8. Imaging using small-animal PET/CT demonstrated that 44 Sc-DOTA-Z HER2:2891 provides specific and high-contrast imaging of HER2-expressing xenografts. Conclusion The 44 Sc- DOTA-Z HER2:2891 Affibody molecule is a promising probe for imaging of HER2-expression in malignant tumors. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Use of folic acid radioconjugates for folate receptor (FR) targeting is a promising strategy for imaging purposes as well as for potential therapy of cancer and inflammatory diseases due to the frequent FR overexpression found on cancer cells and activated macrophages. Herein, we report on preclinical results using a novel DOTA-Bz-EDA-folate conjugate radiolabeled with [67Ga]-gallium. Methods: DOTA-Bz-EDA-folate was prepared by conjugation of ethylenediamine-(γ)-folate with 2-(p-isothiocyanobenzyl)-DOTA. Radiolabeling was carried out with 67GaCl3 according to standard procedures. Biodistribution studies of the tracer were performed in mice bearing FR-positive KB tumor xenografts. The effects on radiofolate biodistribution with coadministered renal uptake-blocking amino acids, diuretic agents, antifolates as well as different routes of administration were likewise investigated. Supportive imaging studies were performed using a small-animal single photon emission computed tomography (SPECT)/CT scanner. Results: 67Ga-DOTA-Bz-EDA-folate showed a high and specific accumulation in tumors (6.30%±0.75% ID/g, 1 h pi and 6.08%±0.89% ID/g, 4 h pi). Nonspecific radioactivity uptake in nontargeted tissues was negligible, but significant accumulation was found in FR-positive kidneys, which resulted in unfavorably low tumor-to-kidney ratios (<0.1). Coadministered amino acids or diuretics did not effectively reduce renal accumulation; in contrast, predosed pemetrexed did significantly reduce kidney uptake (<29% of control values). The SPECT/CT studies confirmed the excellent tumor-to-background contrast of 67Ga-radiofolate and the favorable reduction in kidney uptake (with improved imaging quality) resulting from pemetrexed administration. Conclusion: Conventional methods to reduce kidney uptake of radiofolates fail. However, the novel 67Ga-radiolabeled DOTA-Bz-EDA-folate can effectively be used to image FR-positive cancer and potentially inflammatory diseases. Due to its rapid blood clearance properties, this tracer is also a promising candidate for positron emission tomography imaging if radiolabeled with the short-lived [68Ga]-gallium radionuclide. [Copyright &y& Elsevier]

Abstract: Introduction: The folate receptor (FR) is frequently overexpressed in tumors and can be targeted with folate-based (radio)pharmaceuticals. However, significant accumulation of radiofolates in FR-positive kidneys represents a drawback. We have shown that preadministration of the antifolate pemetrexed (PMX) significantly improved the tumor-to-kidney ratio of radiofolates in mice. The aim of this study was to investigate the dose dependence of these effects and whether the same results could be achieved with folic acid (FA) or 5-methyl-tetrahydrofolate (5-Me-THF). Methods: Biodistribution was assessed 4 h postinjection of the organometallic 99mTc-picolylamine monoacetic acid folate in nude mice bearing FR-positive KB tumor xenografts. PMX (50–400 μg/mouse) was injected 1 h previous to radioactivity. The effects of FA and 5-Me-THF (0.5–50 μg/mouse) were investigated likewise. Tissues and organs were collected and counted for radioactivity and the values tabulated as percentage of injected dose per gram tissue (% ID/g). Results: PMX administration reduced renal retention (<1.6% ID/g vs. control: >10% ID/g), while the tumor uptake (average 1.35%±0.40% ID/g vs. control: 1.79%±0.49% ID/g) was only slightly affected independent of the PMX dose. Replacement of PMX by FA or 5-Me-THF (50 μg/mouse) resulted in a significant renal blockade (<0.1% ID/g) but at the same time in an undesired reduction of tumor uptake (<0.2% ID/g). Conclusions: Selective reduction of radiofolate uptake in kidneys under retention of high tumor accumulation could be achieved in combination with PMX over a broad dose range but not with FA or 5-Me-THF. [Copyright &y& Elsevier]

Abstract: 99mTc-technetium (99mTc) and 188Re-rhenium (188Re) represent an interesting pair of radionuclides for diagnosis and therapy. The aim of this study was to synthesize and characterize in vitro/in vivo the first 188Re-folate derivative [188Re(CO)3–picolylamine monoacetic acid 188Re-PAMA-folate (2)] for potential targeted radionuclide therapy of FR-positive tumors. The data were compared with those of the isostructural 99mTc-analog [99mTc–PAMA folate (1)] reported previously. Methods: In vitro stability of compound 2 was tested in phosphate-buffered saline and human plasma. Cell binding experiments were performed with FR-positive human KB cells. Biodistribution was assessed in female nude mice, bearing KB tumor xenografts. Results: Cell binding experiments showed high and FR-specific uptake. In vivo, compound 2 accumulated specifically in the FR-positive tumors with maximal values 4 h post injection (p.i.) [ 2 : 1.87±0.04 percent injected dose per gram of weight tissue (% ID/g) vs. 1 : 2.33±0.36% ID/g]. Unfavorably high retention of radioactivity was found in FR-positive kidneys (12.04±0.62% ID/g; 4 h p.i.). Tumor-to-blood ratio of radioactivity ( 2 : 14.5±1.32, 4 h p.i.) was lower than for compound 1 (58.0±12.2, 4 h p.i.), whereas tumor-to-kidney ratios were in the same range ( 2 : 0.15±0.01 vs. 1 : 0.13±0.02, 4 h p.i.). Preadministration of the antifolate pemetrexed significantly improved the tumor-to-kidney ratio ( 2 : 1.59±0.30, 4 h p.i.). Conclusions: The isostructural radiofolates 1 and 2 displayed almost identical pharmacokinetic profiles and accumulated both specifically in FR-positive tumors. However, only the coapplication of the antifolate pemetrexed improved the biodistribution of the radiotracers in such ways that a potential therapeutic application of compound 2 can be envisaged in the future. [Copyright &y& Elsevier]

Abstract: Introduction: We assessed the suitability of the radiolanthanide 155Tb (t1/2 =5.32days, Eγ =87keV (32%), 105keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging. Methods: 155Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~1.4GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. 155Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules – a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) – were radiolabeled with 155Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner. Results: The total yield of the two-step separation process of 155Tb was 86%. 155Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The 155Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (>95%). 155Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of 155Tb-DOTATATE and 155Tb-MD, respectively. The relatively long physical half-life of 155Tb matched in particular the biological half-lives of 155Tb-cm09 and 155Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration. Conclusions: The radiolanthanide 155Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the β--emitting radiolanthanides 177Lu, 161Tb, 166Ho, and the pseudo-radiolanthanide 90Y. [Copyright &y& Elsevier]

Introduction Application of therapeutic folate radioconjugates is a promising option for the treatment of folate receptor (FR)-positive tumors, although high uptake of radiofolates in the kidneys remains a critical issue. Recently, it was shown that enhancing the blood circulation of radiofolates results in increased tumor uptake and reduced retention of radioactivity in the kidneys. In this study, we investigated and compared the anti-tumor effects and potential long-term damage to the kidneys after application of an albumin-binding ( 177 Lu-cm09), and a conventional ( 177 Lu-EC0800) folate radioconjugate. Methods In vivo studies were performed with KB tumor-bearing nude mice. 177 Lu-EC0800 and 177 Lu-cm09 were applied at variable quantities (10–30 MBq/mouse), and the tumor growth was monitored over time. Mice without tumors were injected with the same radiofolates and investigated over eight months by determination of creatinine and blood urea nitrogen plasma levels and by measuring renal uptake of 99m Tc-DMSA using SPECT. At the study end, the morphological changes were examined on renal tissue sections using variable staining methods. Results Compared to untreated controls, dose-dependent tumor growth inhibition and prolonged survival was observed in all treated mice. In line with the resulting absorbed dose, the treatment was more effective with 177 Lu-cm09 than with 177 Lu-EC0800, enabling complete tumor remission after application of ≥ 20 MBq (≥ 28 Gy). Application of radiofolates with an absorbed renal dose ≥ 23 Gy showed increased levels of renal plasma parameters and reduced renal uptake of 99m Tc-DSMA. Morphological changes observed on tissue sections confirmed radionephropathy of variable stages. Conclusions 177 Lu-cm09 showed more favorable anti-tumor effects and significantly less damage to the kidneys compared to 177 Lu-EC0800 as was expected based on improved tumor-to-kidney ratios. It was demonstrated that enhancing the blood circulation time of radiofolates was favorable regarding the risk–benefit profile of a therapeutic application. These results hold promise for future translation of the albumin-binder concept to the clinics, potentially enabling FR-targeted radionuclide therapy in patients. [ABSTRACT FROM AUTHOR]

Introduction 44 Sc, a PET radionuclide, has promising decay characteristics (T 1/2 = 3.97 h, Eβ + av = 632 keV) for nuclear imaging and is an attractive alternative to the short-lived 68 Ga (T 1/2 = 68 min, Eβ + av = 830 keV). The aim of this study was the optimization of the 44 Sc production process at an accelerator, allowing its use for preclinical and clinical PET imaging. Methods 44 CaCO 3 targets were prepared and irradiated with protons (~ 11 MeV) at a beam current of 50 μA for 90 min. 44 Sc was separated from its target material using DGA extraction resin and concentrated using SCX cation exchange resin. Radiolabeling experiments at activities up to 500 MBq and stability tests were performed with DOTANOC by investigating different scavengers, including gentisic acid. Dynamic PET of an AR42J tumor-bearing mouse was performed after injection of 44 Sc-DOTANOC. Results The optimized chemical separation method yielded up to 2 GBq 44 Sc of high radionuclidic purity. In the presence of gentisic acid, radiolabeling of 44 Sc with DOTANOC was achieved with a radiochemical yield of ~ 99% at high specific activity (10 MBq/nmol) and quantities which would allow clinical application. The dynamic PET images visualized increasing uptake of 44 Sc-DOTANOC into AR42J tumors and excretion of radioactivity through the kidneys of the investigated mouse. Conclusions The concept “from-bench-to-bedside” was clearly demonstrated in this extended study using cyclotron-produced 44 Sc. Sufficiently high activities of 44 Sc of excellent radionuclidic purity are obtainable for clinical application, by irradiation of enriched calcium at a cyclotron. This work demonstrates a promising basis for introducing 44 Sc to clinical routine of nuclear imaging using PET. [ABSTRACT FROM AUTHOR]

Introduction The chick embryo is an emerging in vivo model in several areas of pre-clinical research including radiopharmaceutical sciences. Herein, it was evaluated as a potential test system for assessing the biodistribution and in vivo stability of radiopharmaceuticals. For this purpose, a number of radiopharmaceuticals labeled with 18 F, 125 I, 99m Tc, and 177 Lu were investigated in the chick embryo and compared with the data obtained in mice. Methods Chick embryos were cultivated ex ovo for 17–19 days before application of the radiopharmaceutical directly into the peritoneum or intravenously using a vein of the chorioallantoic membrane (CAM). At a defined time point after application of radioactivity, the embryos were euthanized by shock-freezing using liquid nitrogen. Afterwards they were separated from residual egg components for post mortem imaging purposes using positron emission tomography (PET) or single photon emission computed tomography (SPECT). Results SPECT images revealed uptake of [ 99m Tc]pertechnetate and [ 125 I]iodide in the thyroid of chick embryos and mice, whereas [ 177 Lu]lutetium, [ 18 F]fluoride and [ 99m Tc]-methylene diphosphonate ([ 99m Tc]-MDP) were accumulated in the bones. [ 99m Tc]-dimercaptosuccinic acid ( 99m Tc-DMSA) and the somatostatin analog [ 177 Lu]-DOTATOC, as well as the folic acid derivative [ 177 Lu]-DOTA-folate showed accumulation in the renal tissue whereas [ 99m Tc]-mebrofenin accumulated in the gall bladder and intestine of both species. In vivo dehalogenation of [ 18 F]fallypride and of the folic acid derivative [ 125 I]iodo-tyrosine-folate was observed in both species. In contrast, the 3′-aza-2′-[ 18 F]fluorofolic acid ([ 18 F]-AzaFol) was stable in the chick embryo as well as in the mouse. Conclusions Our results revealed the same tissue distribution profile and in vivo stability of radiopharmaceuticals in the chick embryo and the mouse. This observation is promising with regard to a potential use of the chick embryo as an inexpensive and simple test model for preclinical screening of novel radiopharmaceuticals. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Radiolabeled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumors in which BN2/gastrin-releasing peptide (GRP) receptors are overexpressed. However, the low in vivo stability of BN conjugates may limit their clinical application. In an attempt to improve their pharmacokinetics and counteract their rapid enzymatic degradation, we prepared a series of polyethylene glycol (PEG)-ylated BN(7-14) analogues for radiolabeling with 99mTc(CO)3 and evaluated them in vitro and in vivo. Methods: Derivatization of a stabilized (NαHis)Ac-BN(7-14)[Cha13,Nle14] analogue with linear PEG molecules of various sizes [5 kDa (PEG5), 10 kDa (PEG10) and 20 kDa (PEG20)] was performed by PEGylation of the ɛ-amino group of a β3hLys-βAla-βAla spacer between the stabilized BN sequence and the (NαHis)Ac chelator. The analogues were then radiolabeled by employing the 99mTc-tricarbonyl technique. Binding affinity and internalization/externalization studies were performed in vitro in human prostate carcinoma PC-3 cells. Stability was investigated in vitro in human plasma and in vivo in Balb/c mice. Finally, single photon emission computed tomography (SPECT)/X-ray computed tomography studies were performed in nude mice bearing PC-3 tumor xenografts. Results: PEGylation did not affect the binding affinity of BN analogues, as the binding affinity for BN2/GRP receptors remained high (K d<0.9 nM). However, in vitro binding kinetics of the PEGylated analogues were slower. Steady-state condition was reached after 4 h, and the total cell binding was 10 times lower than that for the non-PEGylated counterpart. Besides, PEGylation improved the stability of BN conjugates in vitro and in vivo. The BN derivative conjugated with a PEG5 molecule showed the best pharmacokinetics in vivo, i.e., faster blood clearance and preferential renal excretion. The tumor uptake of the 99mTc-PEG5-Lys-BN conjugate was slightly higher compared to that of the non-PEGylated analogue (3.91%±0.44% vs. 2.80%±0.28% injected dose per gram 1 h postinjection, p.i.). Tumor retention was also increased, resulting in a threefold higher amount of radioactivity in the tumor at 24 h p.i. Furthermore, decreased hepatobiliary excretion and increased tumor-to-nontarget ratios (tumor-to-blood: 17.1 vs. 2.1; tumor-to-kidney: 1.1 vs. 0.4; tumor-to-liver: 5.8 vs. 1.0, 24 h p.i.) were observed and further confirmed via small-animal SPECT images 1 h p.i. Conclusion: PEGylation proved to be an effective strategy to enhance the tumor-targeting potential of 99mTc-labeled BN-based radiopharmaceuticals and probably other radiolabeled peptides. [Copyright &y& Elsevier]

Abstract: Introduction: The most successful clinical studies of immunotherapy in patients with non-Hodgkin''s lymphoma (NHL) use the antibody rituximab (RTX) targeting CD20+ B-cell tumors. Rituximab radiolabeled with β− emitters could potentiate the therapeutic efficacy of the antibody by virtue of the particle radiation. Here, we report on a direct radiolabeling approach of rituximab with the 99mTc- and 188Re-tricarbonyl core (IsoLink technology). Methods: The native format of the antibody (RTXwt) as well as a reduced form (RTXred) was labeled with 99mTc/188Re(CO)3. The partial reduction of the disulfide bonds to produce free sulfhydryl groups (–SH) was achieved with 2-mercaptoethanol. Radiolabeling efficiency, in vitro human plasma stability as well as transchelation toward cysteine and histidine was investigated. The immunoreactivity and binding affinity were determined on Ramos and/or Raji cells expressing CD20. Biodistribution was performed in mice bearing subcutaneous Ramos lymphoma xenografts. Results: The radiolabeling efficiency and kinetics of RTXred were superior to that of RTXwt (99mTc: 98% after 3 h for RTXred vs. 70% after 24 h for RTXwt). 99mTc(CO)3-RTXred was used without purification for in vitro and in vivo studies whereas 188Re(CO)3-RTXred was purified to eliminate free 188Re-precursor. Both radioimmunoconjugates were stable in human plasma for 24 h at 37°C. In contrast, displacement experiments with excess cysteine/histidine showed significant transchelation in the case of 99mTc(CO)3-RTXred but not with pre-purified 188Re(CO)3-RTXred. Both conjugates revealed high binding affinity to the CD20 antigen (K d=5–6 nM). Tumor uptake of 188Re(CO)3-RTXred was 2.5 %ID/g and 0.8 %ID/g for 99mTc(CO)3-RTXred 48 h after injection. The values for other organs and tissues were similar for both compounds, for example the tumor-to-blood and tumor-to-liver ratios were 0.4 and 0.3 for 99mTc(CO)3-RTXred and for 188Re(CO)3-RTXred 0.5 and 0.5 (24 h pi). Conclusion: Rituximab could be directly and stably labeled with the matched pair 99mTc/188Re using the IsoLink technology under retention of the biological activity. Labeling kinetics and yields need further improvement for potential routine application in radioimmunodiagnosis and therapy. [Copyright &y& Elsevier]