Design and Evaluation of Novel Albumin-Binding Folate Radioconjugates: Systematic Approach of Varying the Linker Entities.

Tumor targeting using folate radioconjugates is a promising strategy for theragnostics of folate receptor-positive tumors. The aim of this study was to investigate the impact of structural modifications of folate radioconjugates on their pharmacokinetic properties. Four novel folate radioconjugates ([177Lu]Lu-OxFol-2, [177Lu]Lu-OxFol-3, [177Lu]Lu-OxFol-4, and [177Lu]Lu-OxFol-5), modified with a lipophilic or hydrophilic linker entity in close proximity to the albumin-binding 4-(p-iodophenyl)butanoate entity or the DOTA chelator, respectively, were designed and evaluated for comparison with the previously developed [177Lu]Lu-OxFol-1. A hydrophobic 4-(aminomethyl)benzoic acid linker, incorporated in close proximity to the 4-(p-iodophenyl)butanoate entity, enhanced the albumin-binding properties (relative affinity 7.3) of [177Lu]Lu-OxFol-3 as compared to those of [177Lu]Lu-OxFol-1 (relative affinity set as 1.0). On the other hand, a hydrophilic d-glutamic acid (d-Glu) linker entity used in [177Lu]Lu-OxFol-2 compromised the albumin-binding properties. [177Lu]Lu-OxFol-4 and [177Lu]Lu-OxFol-5, in which the respective linker entities were incorporated adjacent to the DOTA chelator, showed similar albumin-binding properties (0.6 and 1.0, respectively) as [177Lu]Lu-OxFol-1. Biodistribution studies in KB tumor-bearing nude mice revealed twofold higher tumor-to-kidney ratios at 4 h and 24 h after injection of [177Lu]Lu-OxFol-3 (∼1.2) than after injection of [177Lu]Lu-OxFol-1 (∼0.6). The tumor-to-kidney ratios of [177Lu]Lu-OxFol-2 were, however, much lower (∼0.2) due to the high kidney retention of this radioconjugate. The tumor-to-kidney ratios of [177Lu]Lu-OxFol-5 were only slightly increased (∼0.9), and the ratios for [177Lu]Lu-OxFol-4 (∼0.7) were in the same range as for [177Lu]Lu-OxFol-1. SPECT/CT imaging studies demonstrated similar tumor uptake of all radioconjugates but a clearly improved tumor-to-kidney ratio for [177Lu]Lu-OxFol-3 as compared to that for [177Lu]Lu-OxFol-1. Based on these data, it can be concluded that the linker entity in close proximity to the 4-(p-iodophenyl)butanoate entity affects the radioconjugate's pharmacokinetic profile considerably due to the altered affinity to albumin. Changes in the linker entity, which connects the DOTA chelator with the folate molecule, do not have a major impact on the radioconjugate's tissue distribution profile, however. As a result of these findings, [177Lu]Lu-OxFol-3 had a comparable therapeutic effect to that of [177Lu]Lu-OxFol-1 but appeared advantageous in preventing kidney damage. Provided that the kidneys will present the dose-limiting organs in patients, [177Lu]Lu-OxFol-3 would be the preferred candidate for a clinical translation.

Novel Synthetic Strategies Enable the Efficient Development of Folate Conjugates for Cancer Radiotheranostics.

The folate receptor (FR) is an interesting target for radiotheranostics due to its overexpression in several tumor types. The progress in developing novel folate radioconjugates is, however, slow due to the synthetic challenges that folate chemistry presents. The goal of this study was, thus, to establish versatile solid-phase synthetic strategies for a convenient preparation of novel folate conjugates. Two approaches were established based on an orthogonal fluorenylmethyloxycarbonyl (Fmoc)-protection strategy to enable a modular buildup of an albumin-binding DOTA conjugate (known as OxFol-1) using folic acid (oxidized folate version) as a targeting agent. The main difference between the two approaches was the sequence of conjugating the single structural units. The approach that introduced the folate entity as the last unit appeared particularly useful for the preparation of conjugates based on 6R- or 6S-5-methyltetrahydrofolic acid (5-MTHF; a reduced folate version) as targeting entity. Three types of folate conjugates were synthesized either with a p-iodophenyl-based albumin binder (OxFol-1, 6R-RedFol-1, and 6S-RedFol-1) or without an albumin-binding entity (OxFol-14, 6R-RedFol-14, and 6S-RedFol-14). All six conjugates were obtained with high chemical purity (>98%) after 9-13 synthesis steps and a single final HPLC purification. Radiolabeling with lutetium-177 was feasible at high molar activity, and the resulting radioconjugates were stable over at least 24 h. Biodistribution and SPECT/CT imaging studies confirmed the favorable effect of an albumin-binding entity to increase the tumor uptake and reduce kidney retention of folate radioconjugates. The increased tumor-to-kidney ratios obtained with [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 as compared to [177Lu]Lu-OxFol-1 indicated that 5-MTHF is the preferred FR-targeting agent for albumin-binding radioconjugates. This was, however, not the case for folate radioconjugates without an albumin binder. Thanks to the established synthesis strategy, the preparation of further folate radioconjugates will be facilitated, potentially enabling the optimization of the tissue distribution characteristics even more.

Preclinical evaluation of 5-methyltetrahydrofolate-based radioconjugates-new perspectives for folate receptor-targeted radionuclide therapy.

PURPOSE: The folate receptor (FR) is frequently overexpressed in a variety of tumor types and, hence, an interesting target for radionuclide therapy. The aim of this study was to evaluate a new class of albumin-binding radioconjugates comprising 5-methyltetrahydrofolate (5-MTHF) as a targeting agent and to compare their properties with those of the previously established folic acid-based [177Lu]Lu-OxFol-1. METHODS: [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 were investigated in vitro using FR-positive KB tumor cells. Biodistribution studies were performed in KB tumor-bearing mice, and the areas under the curve (AUC0 → 120h) were determined for the uptake in tumors and kidneys. [177Lu]Lu-6R-RedFol-1 was compared with [177Lu]Lu-OxFol-1 in a therapy study over 8 weeks using KB tumor-bearing mice. RESULTS: Both radioconjugates demonstrated similar in vitro properties as [177Lu]Lu-OxFol-1; however, the tumor uptake of [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 was significantly increased in comparison with [177Lu]Lu-OxFol-1. In the case of [177Lu]Lu-6S-RedFol-1, also the kidney uptake was increased; however, renal retention of [177Lu]Lu-6R-RedFol-1 was similar to that of [177Lu]Lu-OxFol-1. This led to an almost 4-fold increased tumor-to-kidney AUC0 → 120h ratio of [177Lu]Lu-6R-RedFol-1 as compared with [177Lu]Lu-6S-RedFol-1 and [177Lu]Lu-OxFol-1. At equal activity, the therapeutic effect of [177Lu]Lu-6R-RedFol-1 was better than that of [177Lu]Lu-OxFol-1, reflected by a slower tumor growth and, consequently, an increased median survival time (49 days vs. 34 days). CONCLUSION: This study demonstrated the promising potential of 5-MTHF-based radioconjugates for FR-targeting. Application of [177Lu]Lu-6R-RedFol-1 resulted in unprecedentedly high tumor-to-kidney ratios and, as a consequence, a superior therapeutic effect as compared with [177Lu]Lu-OxFol-1. These findings, together with the absence of early side effects, make [177Lu]Lu-6R-RedFol-1 attractive in view of a future clinical translation.

Biodistribution and dosimetry of a single dose of albumin-binding ligand [177Lu]Lu-PSMA-ALB-56 in patients with mCRPC.

INTRODUCTION: PSMA-targeted radionuclide therapy with lutetium-177 has emerged as an effective treatment option for metastatic, castration-resistant prostate cancer (mCRPC). Recently, the concept of modifying PSMA radioligands with an albumin-binding entity was demonstrated as a promising measure to increase the tumor uptake in preclinical experiments. The aim of this study was to translate the concept to a clinical setting and evaluate the safety and dosimetry of [177Lu]Lu-PSMA-ALB-56, a novel PSMA radioligand with albumin-binding properties. METHODS: Ten patients (71.8 ± 8.2 years) with mCRPC received an activity of 3360 ± 393 MBq (120-160 µg) [177Lu]Lu-PSMA-ALB-56 followed by whole-body SPECT/CT imaging over 7 days. Volumes of interest were defined on the SPECT/CT images for dosimetric evaluation for healthy tissue and tumor lesions. General safety and therapeutic efficacy were assessed by measuring blood biomarkers. RESULTS: [177Lu]Lu-PSMA-ALB-56 was well tolerated, and no severe adverse events were observed. SPECT images revealed longer circulation of [177Lu]Lu-PSMA-ALB-56 in the blood with the highest uptake in tumor lesions at 48 h post injection. Compared with published data for other therapeutic PSMA radioligands (e.g. PSMA-617 and PSMA I&T), normalized absorbed doses of [177Lu]Lu-PSMA-ALB-56 were up to 2.3-fold higher in tumor lesions (6.64 ± 6.92 Gy/GBq) and similar in salivary glands (0.87 ± 0.43 Gy/GBq). Doses to the kidneys and red marrow (2.54 ± 0.94 Gy/GBq and 0.29 ± 0.07 Gy/GBq, respectively) were increased. CONCLUSION: Our data demonstrated that the concept of albumin-binding PSMA-radioligands is feasible and leads to increased tumor doses. After further optimization of the ligand design, the therapeutic outcomes may be improved for patients with prostate cancer.

Visualisation of interstitial lung disease by molecular imaging of integrin αvß3 and somatostatin receptor 2.

OBJECTIVE: To evaluate integrin αvß3 (alpha-v-beta-3)-targeted and somatostatin receptor 2 (SSTR2)-targeted nuclear imaging for the visualisation of interstitial lung disease (ILD). METHODS: The pulmonary expression of integrin αvß3 and SSTR2 was analysed in patients with different forms of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. Single photon emission CT/CT (SPECT/CT) was performed on days 3, 7 and 14 after BLM instillation using the integrin αvß3-targeting 177Lu-DOTA-RGD and the SSTR2-targeting 177Lu-DOTA-NOC radiotracer. The specific pulmonary accumulation of the radiotracers over time was assessed by in vivo and ex vivo SPECT/CT scans and by biodistribution studies. RESULTS: Expression of integrin αvß3 and SSTR2 was substantially increased in human ILD regardless of the subtype. Similarly, in lungs of BLM-challenged mice, but not of controls, both imaging targets were stage-specifically overexpressed. While integrin αvß3 was most abundantly upregulated on day 7, the inflammatory stage of BLM-induced lung fibrosis, SSTR2 expression peaked on day 14, the established fibrotic stage. In agreement with the findings on tissue level, targeted nuclear imaging using SPECT/CT specifically detected both imaging targets ex vivo and in vivo, and thus visualised different stages of experimental ILD. CONCLUSION: Our preclinical proof-of-concept study suggests that specific visualisation of molecular processes in ILD by targeted nuclear imaging is feasible. If transferred into clinics, where imaging is considered an integral part of patients' management, the additional information derived from specific imaging tools could represent a first step towards precision medicine in ILD.

Implementation of a new separation method to produce qualitatively improved 64 Cu.

BACKGROUND: 64 Cu (T1/2  = 12.7 h) is an important radionuclide for diagnostic purposes and used for positron emission tomography (PET). A previous method utilized at Paul Scherrer Institute (PSI) proved to be unreliable and, while a method using anion exchange chromatography is a popular choice worldwide, it was felt a different approach was required to obtain a robust chemical separation method. METHODS: Enriched 64 Ni targets were created by electroplating on gold foil. The targets were irradiated with protons degraded to approximately 11 MeV at PSI's Injector 2 72 MeV research cyclotron and subsequently dissolved in HCl. The resultant solution was loaded onto AG MP-50 cation exchange resin and the 64 Cu separated from its target material and radiocobalt impurities, produced as part of the irradiation process, using various specific mixtures of HCl/acetone solution. The eluted product was evaporated and picked up in dilute HCl (0.05 M). The chemical purity of 64 Cu was determined by radiolabeling experiments at the highest possible molar activities. RESULTS: Reproducible results were obtained, yielding 3.6 to 8.3 GBq 64 Cu of high radionuclidic and radiochemical purity. The product was labeled to NODAGA-RGD, achieved at up to 500 MBq/nmol, indicating the high chemical purity. In a proof-of-concept in vivo study, 64 Cu-NODAGA-RGD was used for PET imaging of a tumor-bearing mouse. CONCLUSION: The chemical separation devised to produce high-quality 64 Cu proved to be robust and reproducible. The concept can be used at medical cyclotrons utilizing a solid target station, such that 64 Cu can be used at hospitals for PET imaging.

Preclinical Development of Novel PSMA-Targeting Radioligands: Modulation of Albumin-Binding Properties To Improve Prostate Cancer Therapy.

The treatment of metastatic castration-resistant prostate cancer (mCRPC) remains challenging with current treatment options. The development of more effective therapies is, therefore, urgently needed. Targeted radionuclide therapy with prostate-specific membrane antigen (PSMA)-targeting ligands has revealed promising clinical results. In an effort to optimize this concept, it was the aim of this study to design and investigate PSMA ligands comprising different types of albumin binders. PSMA-ALB-53 and PSMA-ALB-56 were designed by combining the glutamate-urea-based PSMA-binding entity, a DOTA chelator and an albumin binder based on the 4-( p-iodophenyl)-moiety or p-(tolyl)-moiety. The compounds were labeled with 177Lu (50 MBq/nmol) resulting in radioligands of high radiochemical purity (≥98%). Both radioligands were stable (≥98%) over 24 h in the presence of l-ascorbic acid. The uptake into PSMA-positive PC-3 PIP tumor cells in vitro was in the same range (54-58%) for both radioligands; however, 177Lu-PSMA-ALB-53 showed a 15-fold enhanced binding to human plasma proteins. Biodistribution studies performed in PC-3 PIP/flu tumor-bearing mice revealed high tumor uptake of 177Lu-PSMA-ALB-53 and 177Lu-PSMA-ALB-56, respectively, demonstrated by equal areas under the curves (AUCs) for both radioligands. The increased retention of 177Lu-PSMA-ALB-53 in the blood resulted in almost 5-fold lower tumor-to-blood AUC ratios when compared to 177Lu-PSMA-ALB-56. Kidney clearance of 177Lu-PSMA-ALB-56 was faster, and hence, the tumor-to-kidney AUC ratio was 3-fold higher than in the case of 177Lu-PSMA-ALB-53. Due to the more favorable tissue distribution profile, 177Lu-PSMA-ALB-56 was selected for a preclinical therapy study in PC-3 PIP tumor-bearing mice. The tumor growth delay after application of 177Lu-PSMA-ALB-56 and 177Lu-PSMA-617 applied at the same activities (2 or 5 MBq per mouse) revealed better antitumor effects in the case of 177Lu-PSMA-ALB-56. As a consequence, the survival of mice treated with 177Lu-PSMA-ALB-56 was prolonged when compared to the mice, which received the same activity of 177Lu-PSMA-617. Our results demonstrated the superiority of 177Lu-PSMA-ALB-56 over 177Lu-PSMA-ALB-53 indicating that the p-(tolyl)-moiety was more suited as an albumin binder to optimize the tissue distribution profile. 177Lu-PSMA-ALB-56 was more effective to treat tumors than 177Lu-PSMA-617 resulting in complete tumor remission in four out of six mice. This promising results warrant further investigations to assess the potential for clinical application of 177Lu-PSMA-ALB-56.

Albumin-Binding PSMA Ligands: Optimization of the Tissue Distribution Profile.

The prostate-specific membrane antigen (PSMA) has emerged as an attractive prostate cancer associated target for radiotheragnostic application using PSMA-specific radioligands. The aim of this study was to design new PSMA ligands modified with an albumin-binding moiety in order to optimize their tissue distribution profile. The compounds were prepared by conjugation of a urea-based PSMA-binding entity, a DOTA chelator, and 4-( p-iodophenyl)butyric acid using multistep solid phase synthesis. The three ligands (PSMA-ALB-02, PSMA-ALB-05, and PSMA-ALB-07) were designed with varying linker entities. Radiolabeling with 177Lu was performed at a specific activity of up to 50 MBq/nmol resulting in radioligands of >98% radiochemical purity and high stability. In vitro investigations revealed high binding of all three PSMA radioligands to mouse (>64%) and human plasma proteins (>94%). Uptake and internalization into PSMA-positive PC-3 PIP tumor cells was equally high for all radioligands. Negligible accumulation was found in PSMA-negative PC-3 flu cells, indicating PSMA-specific binding of all radioligands. Biodistribution and imaging studies performed in PC-3 PIP/flu tumor-bearing mice showed enhanced blood circulation of the new radioligands when compared to the clinically employed 177Lu-PSMA-617. The PC-3 PIP tumor uptake of all three radioligands was very high (76.4 ± 2.5% IA/g, 79.4 ± 11.1% IA/g, and 84.6 ± 14.2% IA/g, respectively) at 24 h post injection (p.i.) resulting in tumor-to-blood ratios of ∼176, ∼48, and ∼107, respectively, whereas uptake into PC-3 flu tumors was negligible. Kidney uptake at 24 h p.i. was lowest for 177Lu-PSMA-ALB-02 (10.7 ± 0.92% IA/g), while 177Lu-PSMA-ALB-05 and 177Lu-PSMA-ALB-07 showed higher renal retention (23.9 ± 4.02% IA/g and 51.9 ± 6.34% IA/g, respectively). Tumor-to-background ratios calculated from values of the area under the curve (AUC) of time-dependent biodistribution data were in favor of 177Lu-PSMA-ALB-02 (tumor-to-blood, 46; tumor-to-kidney, 5.9) when compared to 177Lu-PSMA-ALB-05 (17 and 3.7, respectively) and 177Lu-PSMA-ALB-07 (39 and 2.1, respectively). The high accumulation of the radioligands in PC-3 PIP tumors was visualized on SPECT/CT images demonstrating increasing tumor-to-kidney ratios over time. Taking all of the characteristics into account, 177Lu-PSMA-ALB-02 emerged as the most promising candidate. The applied concept may be attractive for future clinical translation potentially enabling more potent and convenient prostate cancer radionuclide therapy.

Design and Preclinical Evaluation of an Albumin-Binding PSMA Ligand for 64Cu-Based PET Imaging.

Recently, we developed an albumin-binding radioligand (177Lu-PSMA-ALB-56), which showed higher PSMA-specific tumor uptake in mice than the previously developed 177Lu-PSMA-617 under the same experimental conditions. Such a radioligand may be of interest also for PET imaging, possibly enabling better visualization of even small metastases at late time-points after injection. The aim of this study was, therefore, to modify PSMA-ALB-56 by exchanging the DOTA chelator with a NODAGA chelator for stable coordination of 64Cu ( T1/2 = 12.7 h; Eß+av = 278 keV). The resulting NODAGA-functionalized PSMA-ALB-89 ligand, and the previously establish DOTA-functionalized PSMA-ALB-56 ligand were labeled with 64Cu and evaluated in vitro and in vivo. Both radioligands showed plasma protein-binding properties in vitro and PSMA-specific uptake in PC-3 PIP cells. Biodistribution studies, performed in tumor-bearing mice, revealed high accumulation of 64Cu-PSMA-ALB-89 in PSMA-positive PC-3 PIP tumor xenografts (25.9 ± 3.41% IA/g at 1 h p.i.), which was further increased at later time-points (65.1 ± 7.82% IA/g at 4 h p.i. and 97.1 ± 7.01% IA/g at 24 h p.i.). High uptake of 64Cu-PSMA-ALB-89 was also seen in the kidneys, however, 64Cu-PSMA-ALB-89 was efficiently excreted over time. Mice injected with 64Cu-PSMA-ALB-56 showed increased accumulation of radioactivity in the liver (25.3 ± 4.20% IA/g) when compared to the liver uptake of 64Cu-PSMA-ALB-89 (4.88 ± 0.21% IA/g, at 4 h p.i.). This was most probably due to in vivo instability of the 64Cu-DOTA complex, which was also the reason for lower tumor uptake (49.7 ± 16.1% IA/g at 4 h p.i. and 28.3 ± 3.59% IA/g at 24 h p.i.). PET/CT imaging studies confirmed these findings and enabled excellent visualization of the PSMA-positive tumor xenografts in vivo after injection of 64Cu-PSMA-ALB-89. These data indicate that 64Cu-PSMA-ALB-89 is favorable over 64Cu-PSMA-ALB-56 with regard to the in vivo stability and tissue distribution profile. Moreover, 64Cu-PSMA-ALB-89 outperformed previously developed 64Cu-labeled PSMA ligands. Further optimization of long-circulating PSMA-targeting PET radioligands will be necessary before translating this concept to the clinics.

In Vivo Labeling of Plasma Proteins for Imaging of Enhanced Vascular Permeability in the Lungs.

Increased vascular permeability is an important hallmark of many diseases, including cancer, cerebral ischemia, and severe inflammatory disorders. In this regard, the noninvasive assessment of pathologically increased vascular permeability in vivo is of great interest. In this study, the potential of albumin- and transthyretin-binding radioligands was evaluated for imaging of vascular hyperpermeability. For this purpose, the bleomycin-induced lung injury model was used as a model of inflammation-associated vascular leakage. The plasma protein-binding ligands, which bind to albumin (DOTA-PPB-01) and transthyretin (DOTA-PPB-03), were radiolabeled and used for nuclear imaging and biodistribution studies. In this regard, 177Lu was employed as a surrogate nuclide for detailed preclinical investigations, including single-photon emission computed tomography (SPECT) studies, whereas 44Sc was proposed as a radionuclide for positron emission tomography (PET), which may be relevant for future clinical translation. Mice were administered with these radioligands 6-9 days after intratracheal instillation of bleomycin or saline. Bleomycin-treated mice developed pronounced lung inflammation with enhanced vascular permeability that was reflected in significantly increased lung size and weight due to edema and infiltration with inflammatory cells. Biodistribution studies revealed significantly higher accumulation of 177Lu-DOTA-PPB-01 in injured lungs as compared to lungs of control animals at all investigated time points (4-48 h p.i.). The best contrast was achieved at late time points (16.1 ± 2.91% IA/g vs 2.03 ± 1.22% IA/g, 48 h p.i.) when the blood activity levels were ∼7.5% IA/g. Injection of 177Lu-DOTA-PPB-03 also resulted in increased lung accumulation in bleomycin-treated mice at all investigated time points (2-8 h p.i.). The pharmacokinetics was significantly faster, however, resulting in good contrast already at 8 h p.i. (4.32 ± 0.85% IA/g vs 1.06 ± 0.10% IA/g) when blood activity levels were ∼2% IA/g. The absolute lung accumulation of 177Lu-DOTA-PPB-03 was significantly lower than that of 177Lu-DOTA-PPB-01. PET/CT scans performed with 44Sc-DOTA-PPB-01 distinguished injured from healthy lungs only at late time points (20 h p.i.), whereas 44Sc-DOTA-PPB-03 already allowed the differentiation at 4 h p.i. due to its faster clearance. The investigated radioligands, 44Sc/177Lu-DOTA-PPB-01 and 44Sc/177Lu-DOTA-PPB-03, hold promise for the visualization of vascular leakage in a variety of pathological conditions. 44Sc would be the radionuclide of choice for clinical application as it can be stably coordinated with a DOTA chelator and enables PET imaging over extended periods.

DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas.

BACKGROUND: Syncytin-1 and 2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors. RESULTS: We found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas. CONCLUSIONS: We conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.

Synthesis, Radiolabeling, and Characterization of Plasma Protein-Binding Ligands: Potential Tools for Modulation of the Pharmacokinetic Properties of (Radio)Pharmaceuticals.

The development of (radio)pharmaceuticals with favorable pharmacokinetic profiles is crucial for allowing the optimization of the imaging or therapeutic potential and the minimization of undesired side effects. The aim of this study was, therefore, to evaluate and compare three different plasma protein binders (PPB-01, PPB-02, and PPB-03) that are potentially useful in combination with (radio)pharmaceuticals to enhance their half-life in the blood. The entities were functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator via a l-lysine and ß-alanine linker moiety using solid-phase peptide chemistry and labeled with 177Lu (T1/2 = 6.65 days), a clinically established radiometal. The binding capacities of these radioligands and 177Lu-DOTA were evaluated using human plasma and solutions of human serum albumin (HSA), human α1-acid glycoprotein (α1-AGP), and human transthyretin (hTTR) by applying an ultrafiltration assay. 177Lu-DOTA-PPB-01 and 177Lu-DOTA-PPB-02 bound to a high and moderate extent to human plasma proteins (>90% and ∼70%, respectively), whereas the binding to hTTR was considered negligible (<10%). 177Lu-DOTA-PPB-03 showed almost complete binding to human plasma proteins (>90%) with a high fraction bound to hTTR (∼50%). Plasma protein binding of the 177Lu-DOTA complex, which was used as a control, was not observed (<1%). 177Lu-DOTA-PPB-01 and 177Lu-DOTA-PPB-02 were both displaced (>80%) from HSA by ibuprofen, specific for Sudlow's binding site II and coherent with the aromatic structures, and >80% by their respective binding entities. 177Lu-DOTA-PPB-03 was displaced from hTTR by the site-marker l-thyroxine (>60%) and by its binding entity PPB-03* (>80%). All three radioligands were investigated with regard to the in vivo blood clearance in normal mice. 177Lu-DOTA-PPB-01 showed the slowest blood clearance (T1/2,ß: >15 h) followed by 177Lu-DOTA-PPB-03 (T1/2,ß: ∼2.33 h) and 177Lu-DOTA-PPB-02 (T1/2,ß: ∼1.14 h), which was excreted relatively fast. Our results confirmed the high affinity of the 4-(4-iodophenyl)-butyric acid entity (PPB-01) to plasma proteins, while replacement of the halogen by an ethynyl entity (PPB-02) reduced the plasma protein binding significantly. An attractive approach is the application of the transthyretin binder (PPB-03), which shows high affinity to hTTR. Future studies in our laboratory will be focused on the application of these binding entities in combination with clinically relevant targeting agents for diagnostic and therapeutic purposes in nuclear medicine.

Preclinical Comparison of Albumin-Binding Radiofolates: Impact of Linker Entities on the in Vitro and in Vivo Properties.

Tumor targeting with folic acid radioconjugates has been proposed as a promising strategy for radionuclide therapy of folate receptor α (FR)-positive cancer. Recently, it was shown that modification of radiofolates with an albumin-binding entity increased the tumor-to-kidney ratios of accumulated radioactivity in mice. The goal of this study was to evaluate the lead compound cm10 and compare it with new albumin-binding folate conjugates. Compound cm12 was designed with a long spacer consisting of a PEG-11 entity, and compound cm13 contained a short alkane chain between the albumin-binding moiety and folic acid. All of the derivatives were labeled with 177Lu (t1/2 = 6.65 days, Eß-,average = 134 keV; Eγ = 113 keV, 208 keV), a clinically established radionuclide for therapeutic purposes. The evaluation revealed that all of the albumin-binding radiofolates exhibited increased in vitro stability compared with the reference compound (177Lu-cm14) without albumin binder. Serum protein binding, determined with an ultrafiltration assay, was high (>88%) for the derivatives with albumin-binding entities. The FR-binding affinity was in the same range (KD = 4.0-7.5 nM) for all of the radiofolates, independent of the albumin-binding entity and spacer length. FR-specific uptake was proven in vitro using FR-positive KB tumor cells. In vivo studies with KB-tumor-bearing mice were performed in order to assess the tissue distribution profile of the novel radiofolates. 177Lu-cm13 showed high tumor uptake at late time points (13.3 ± 2.94% IA/g, 48 h p.i.) and tumor-to-kidney ratios (0.59 ± 0.03, 48 h p.i.) in the same range as 177Lu-cm10 (0.55 ± 0.07, 48 h p.i.). However, the tumor-to-kidney ratio of 177Lu-cm12 (0.28 ± 0.07, 48 h p.i.) was reduced compared with 177Lu-cm10 and 177Lu-cm13. The results of this study indicate that the spacer entity between folic acid and the albumin binder is of critical importance with regard to the tissue distribution profile of the radiofolate. The PEG spacer compromised the beneficial effects of the lead compound, but the design with a short alkane spacer appeared to be promising. Future studies will focus on the design of radiofolates with lipophilic and more rigid spacer entities, which may allow a further improvement of their tissue distribution profiles.

Toll-like receptors expressed on embryonic macrophages couple inflammatory signals to iron metabolism during early ontogenesis.

Mammalian TLRs in adult animals serve indispensable functions in establishing innate and adaptive immunity and contributing to the homeostasis of surrounding tissues. However, the expression and function of TLRs during mammalian embryonic development has not been studied so far. Here, we show that CD45(+) CD11b(+) F4/80(+) macrophages from 10.5-day embryo (E10.5) co-express TLRs and CD14. These macrophages, which have the capability to engulf both apoptotic cells and bacteria, secrete a broad spectrum of proinflammatory cytokines and chemokines upon TLR stimulation, demonstrating that their TLRs are functional. Comparative microarray analysis revealed an additional set of genes that were significantly upregulated in E10.5 TLR2(+) CD11b(+) macrophages. This analysis, together with our genetic, microscopic, and biochemical evidence, showed that embryonic phagocytes express protein machinery that is essential for the recycling of cellular iron and that this expression can be regulated by TLR engagement in a MyD88-dependent manner, leading to typical inflammatory M1 responses. These results characterize the utility of TLRs as suitable markers for early embryonic phagocytes as well as molecular triggers of cellular responses, the latter being demonstrated by the involvement of TLRs in an inflammation-mediated regulation of embryonic homeostasis via iron metabolism.

Quantitative proteomics analysis of macrophage-derived lipid rafts reveals induction of autophagy pathway at the early time of Francisella tularensis LVS infection.

Francisella tularensis is a highly infectious intracellular pathogen that has evolved an efficient strategy to subvert host defense response to survive inside the host. The molecular mechanisms regulating these host-pathogen interactions and especially those that are initiated at the time of the bacterial entry via its attachment to the host plasma membrane likely predetermine the intracellular fate of pathogen. Here, we provide the evidence that infection of macrophages with F. tularensis leads to changes in protein composition of macrophage-derived lipid rafts, isolated as detergent-resistant membranes (DRMs). Using SILAC-based quantitative proteomic approach, we observed the accumulation of autophagic adaptor protein p62 at the early stages of microbe-host cell interaction. We confirmed the colocalization of the p62 with ubiquitinated and LC3-decorated intracellular F. tularensis microbes with its maximum at 1 h postinfection. Furthermore, the infection of p62-knockdown host cells led to the transient increase in the intracellular number of microbes up to 4 h after in vitro infection. Together, these data suggest that the activation of the autophagy pathway in F. tularensis infected macrophages, which impacts the early phase of microbial proliferation, is subsequently circumvented by ongoing infection.

Eosinophils from patients with type 1 diabetes mellitus express high level of myeloid alpha-defensins and myeloperoxidase.

Type 1 diabetes (T1D) is an autoimmune disease caused by T-cell mediated destruction of pancreatic beta cells. Recently, small cationic α-defensin molecules have been implicated in the pathogenesis of certain inflammatory and autoimmune diseases. The purpose of this study was to assess the α-defensin expression in patients with T1D and elucidate the cellular source of their production. Our results show that 30% of patients exhibit increased levels of α-defensin mRNAs in their capillary blood. Quantitative RT-PCR performed on FACS-sorted granulocytes identified CD15(dull)/CD14(weak) population as the cellular source of α-defensins. Surprisingly, this granulocyte subpopulation displayed augmentation of α-defensin expression in all T1D patients tested. The determination of cell surface markers, expression of cell-specific genes and confocal microscopy identified CD15(dull)/CD14(weak) cells as eosinophils. The presence of transcriptionally active eosinophils in diabetic patients suggests that eosinophils could be a part of an intricate innate immune cellular network involved in the development of diabetes.

Cyclam with a phosphinate-bis(phosphonate) pendant arm is a bone-targeting carrier of copper radionuclides.

Ligands combining a bis(phosphonate) group with a macrocycle function as metal isotope carriers for radionuclide-based imaging and for treating bone metastases associated with several cancers. However, bis(phosphonate) pendant arms often slow down complex formation and decrease radiochemical yields. Nevertheless, their negative effect on complexation rates may be mitigated by using a suitable spacer between bis(phosphonate) and the macrocycle. To demonstrate the potential of bis(phosphonate) bearing macrocyclic ligands as a copper radioisotope carrier, we report the synthesis of a new cyclam derivative bearing a phosphinate-bis(phosphonate) pendant (H5te1PBP). The ligand showed a high selectivity to CuII over ZnII and NiII ions, and the bis(phosphonate) group was not coordinated in the CuII complex, strongly interacting with other metal ions in solution. The CuII complex formed quickly, in 1 s, at pH 5 and at a millimolar scale. The complexation rates significantly differed under a ligand or metal ion excess due to the formation of reaction intermediates differing in their metal-to-ligand ratio and protonation state, respectively. The CuII-te1PBP complex also showed a high resistance to acid-assisted hydrolysis (t1/2 2.7 h; 1 M HClO4, 25 °C) and was effectively adsorbed on the hydroxyapatite surface. H5te1PBP radiolabeling with [64Cu]CuCl2 was fast and efficient, with specific activities of approximately 30 GBq 64Cu per 1 µmol of ligand (pH 5.5, room temperature, 30 min). In a pilot experiment, we further demonstrated the excellent suitability of [64Cu]CuII-te1PBP for imaging active bone compartments by dedicated small animal PET/CT in healthy mice and subsequently in a rat femoral defect model, in direct comparison with [18F]fluoride. Moreover, [64Cu]CuII-te1PBP showed a higher uptake in critical bone defect regions. Therefore, our study highlights the potential of [64Cu]CuII-te1PBP as a PET radiotracer for evaluating bone healing in preclinical and clinical settings with a diagnostic value similar to that of [18F]fluoride, albeit with a longer half-life (12.7 h) than 18F (1.8 h), thereby enabling extended observation times.

Identification of a PET Radiotracer for Imaging of the Folate Receptor-α: A Potential Tool to Select Patients for Targeted Tumor Therapy.

The aim of this study was to identify a folate receptor-α (FRα)-selective PET agent potentially suitable for the selection of patients who might profit from FRα-targeted therapies. The 6R and 6S isomers of 18F-aza-5-methyltetrahydrofolate (MTHF) were assessed regarding their binding to FRα and FRß, expressed on cancer and inflammatory cells, respectively, and compared with 18F-AzaFol, the folic acid-based analog. Methods: FR selectivity was investigated using FRα-transfected (RT16) and FRß-transfected (D4) CHO cells. The cell uptake of 18F-folate tracers was investigated, and receptor-binding affinities were determined with the nonradioactive analogs. In vitro autoradiography of the 18F-folate tracers was performed using RT16 and D4 tissue sections. Biodistribution studies and PET/CT imaging of the radiotracers were performed on mice bearing RT16 and D4 xenografts. Results: The uptake of 18F-6R-aza-5-MTHF was high when using RT16 cells (62% ± 10% of added activity) but much lower when using D4 cells (5% ± 2%). The FRα selectivity of 18F-6R-aza-5-MTHF was further demonstrated by its approximately 43-fold higher binding affinity to FRα (half-maximal inhibitory concentration [IC50], 1.8 ± 0.1 nM) than to FRß (IC50, 77 ± 27 nM). The uptake of 18F-6S-aza-5-MTHF and 18F-AzaFol was equal in both cell lines (52%-70%), with similar affinities to FRα (IC50, 2.1 ± 0.4 nM and 0.6 ± 0.3 nM, respectively) and FRß (0.8 ± 0.2 nM and 0.3 ± 0.1 nM, respectively). The autoradiography signal obtained with 18F-6R-aza-5-MTHF was 11-fold more intense for RT16 than for D4 tissue sections. Biodistribution data showed high uptake of 18F-6R-aza-5-MTHF in RT16 xenografts (81% ± 20% injected activity per gram [IA]/g 1 h after injection) but significantly lower accumulation in D4 xenografts (7.3% ± 2.1% IA/g 1 h after injection), which was also visualized using PET. The uptake of 18F-6S-aza-5-MTHF and 18F-AzaFol was similar in RT16 (53% ± 10% IA/g and 45% ± 2% IA/g, respectively) and D4 xenografts (77% ± 10% IA/g and 52% ± 7% IA/g, respectively). Conclusion: This study demonstrated FRα selectivity for 18F-6R-aza-5-MTHF but not for 18F-6S-aza-5-MTHF or 18F-AzaFol. This characteristic, together with its favorable tissue distribution, makes 18F-6R-aza-5-MTHF attractive for clinical translation to enable detection of FRα-positive cancer while preventing undesired accumulation in FRß-expressing inflammatory cells.