Cyclohexanediamine Triazole (CHDT) Functionalization Enables Labeling of Target Molecules with Al18F/68Ga/111In.

The Al18F-labeling approach offers a one-step access to radiofluorinated biomolecules by mimicking the labeling process for radiometals. Although these labeling conditions are considered to be mild compared to classic radiofluorinations, improvements of the chelating units have led to the discovery of (±)-H3RESCA, which allows Al18F-labeling already at ambient temperature. While the suitability of (±)-H3RESCA for functionalization and radiofluorination of proteins is well established, its use for small molecules or peptides is less explored. Herein, we advanced this acyclic pentadentate ligand by introducing an alkyne moiety for the late-stage functionalization of biomolecules via click chemistry. We show that in addition to Al18F-labeling, the cyclohexanediamine triazole (CHDT) moiety allows stable complexation of 68Ga and 111In. Three novel CHDT-functionalized PSMA inhibitors were synthesized and their Al18F-, 68Ga-, and 111In-labeled analogs were subjected to a detailed in vitro radiopharmacological characterization. Stability studies in vitro in human serum revealed among others a high kinetic inertness of all radiometal complexes. Furthermore, the Al18F-labeled PSMA ligands were characterized for their biodistribution in a LNCaP derived tumor xenograft mouse model by PET imaging. One radioligand, Al[18F]F-CHDT-PSMA-1, bearing a small azidoacetyl linker at the glutamate-urea-lysine motif, provided an in vivo performance comparable to that of [18F]PSMA-1007 but with even higher tumor-to-blood and tumor-to-muscle ratios at 120 min p.i. Overall, our results highlight the suitability of the novel CHDT moiety for functionalization and radiolabeling of small molecules or peptides with Al18F, 68Ga, and 111In and the triazole ring seems to entail favorable pharmacokinetic properties for molecular imaging purposes.

64Cu tumor labeling with hexadentate picolinic acid-based bispidine immunoconjugates.

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities >200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed specific accumulation of the 64Cu-labeled TMs in PSCA- and FAP-overexpressing tumors (standardized uptake value (SUV) for PC3: 2.7±0.6 and HT1080: 7.2±1.25) with almost no uptake in wild type tumors.

Immunotheranostic target modules for imaging and navigation of UniCAR T-cells to strike FAP-expressing cells and the tumor microenvironment.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells are a promising approach in cancer immunotherapy, particularly for treating hematologic malignancies. Yet, their effectiveness is limited when tackling solid tumors, where immune cell infiltration and immunosuppressive tumor microenvironments (TME) are major hurdles. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) and various tumor cells, playing an important role in tumor growth and immunosuppression. Aiming to modulate the TME with increased clinical safety and effectiveness, we developed novel small and size-extended immunotheranostic UniCAR target modules (TMs) targeting FAP. METHODS: The specific binding and functionality of the αFAP-scFv TM and the size-extended αFAP-IgG4 TM were assessed using 2D and 3D in vitro models as well as in vivo. Their specific tumor accumulation and diagnostic potential were evaluated using PET studies after functionalization with a chelator and suitable radionuclide. RESULTS: The αFAP-scFv and -IgG4 TMs effectively and specifically redirected UniCAR T-cells using 2D, 3D, and in vivo models. Moreover, a remarkably high and specific accumulation of radiolabeled FAP-targeting TMs at the tumor site of xenograft mouse models was observed. CONCLUSIONS: These findings demonstrate that the novel αFAP TMs are promising immunotheranostic tools to foster cancer imaging and treatment, paving the way for a more convenient, individualized, and safer treatment of cancer patients.

Lysyl Oxidases as Targets for Cancer Therapy and Diagnostic Imaging.

The understanding of the contribution of the tumour microenvironment to cancer progression and metastasis, in particular the interplay between tumour cells, fibroblasts and the extracellular matrix has grown tremendously over the last years. Lysyl oxidases are increasingly recognised as key players in this context, in addition to their function as drivers of fibrotic diseases. These insights have considerably stimulated drug discovery efforts towards lysyl oxidases as targets over the last decade. This review article summarises the biochemical and structural properties of theses enzymes. Their involvement in tumour progression and metastasis is highlighted from a biochemical point of view, taking into consideration both the extracellular and intracellular action of lysyl oxidases. More recently reported inhibitor compounds are discussed with an emphasis on their discovery, structure-activity relationships and the results of their biological characterisation. Molecular probes developed for imaging of lysyl oxidase activity are reviewed from the perspective of their detection principles, performance and biomedical applications.

Preclinical Characterization of the 177Lu-Labeled Prostate Stem Cell Antigen (PSCA)-Specific Monoclonal Antibody 7F5.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hormone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor progression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The resulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images revealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

A novel ACE2 decoy for both neutralization of SARS-CoV-2 variants and killing of infected cells.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Specific and safe targeting of glioblastoma using switchable and logic-gated RevCAR T cells.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.

Microdialysis Reveals Anti-Inflammatory Effects of Sulfated Glycosaminoglycanes in the Early Phase of Bone Healing.

Although chronic inflammation inhibits bone healing, the healing process is initiated by an inflammatory phase. In a well-tuned sequence of molecular events, pro-inflammatory cytokines are secreted to orchestrate the inflammation response to injury and the recruitment of progenitor cells. These events in turn activate the secretion of anti-inflammatory signaling molecules and attract cells and mediators that antagonize the inflammation and initiate the repair phase. Sulfated glycosaminoglycanes (sGAG) are known to interact with cytokines, chemokines and growth factors and, thus, alter the availability, duration and impact of those mediators on the local molecular level. sGAG-coated polycaprolactone-co-lactide (PCL) scaffolds were inserted into critical-size femur defects in adult male Wistar rats. The femur was stabilized with a plate, and the defect was filled with either sGAG-containing PCL scaffolds or autologous bone (positive control). Wound fluid samples obtained by microdialysis were characterized regarding alterations of cytokine concentrations over the first 24 h after surgery. The analyses revealed the inhibition of the pro-inflammatory cytokines IL-1ß and MIP-2 in the sGAG-treated groups compared to the positive control. A simultaneous increase of IL-6 and TNF-α indicated advanced regenerative capacity of sGAG, suggesting their potential to improve bone healing.

Impact of Sulfated Hyaluronan on Bone Metabolism in Diabetic Charcot Neuroarthropathy and Degenerative Arthritis.

Bone in diabetes mellitus is characterized by an altered microarchitecture caused by abnormal metabolism of bone cells. Together with diabetic neuropathy, this is associated with serious complications including impaired bone healing culminating in complicated fractures and dislocations, especially in the lower extremities, so-called Charcot neuroarthropathy (CN). The underlying mechanisms are not yet fully understood, and treatment of CN is challenging. Several in vitro and in vivo investigations have suggested positive effects on bone regeneration by modifying biomaterials with sulfated glycosaminoglycans (sGAG). Recent findings described a beneficial effect of sGAG for bone healing in diabetic animal models compared to healthy animals. We therefore aimed at studying the effects of low- and high-sulfated hyaluronan derivatives on osteoclast markers as well as gene expression patterns of osteoclasts and osteoblasts from patients with diabetic CN compared to non-diabetic patients with arthritis at the foot and ankle. Exposure to sulfated hyaluronan (sHA) derivatives reduced the exaggerated calcium phosphate resorption as well as the expression of genes associated with bone resorption in both groups, but more pronounced in patients with CN. Moreover, sHA derivatives reduced the release of pro-inflammatory cytokines in osteoclasts of patients with CN. The effects of sHA on osteoblasts differed only marginally between patients with CN and non-diabetic patients with arthritis. These results suggest balancing effects of sHA on osteoclastic bone resorption parameters in 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.

Development and Functional Characterization of a Versatile Radio-/Immunotheranostic Tool for Prostate Cancer Management.

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as a target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic positron emission tomography (PET) imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+, the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.

Men who stare at bone: multimodal monitoring of bone healing.

Knowledge of the physiological and pathological processes, taking place in bone during fracture healing or defect regeneration, is essential in order to develop strategies to enhance bone healing under normal and critical conditions. Preclinical testing allows a wide range of imaging modalities that may be applied both simultaneously and longitudinally, which will in turn lower the number of animals needed to allow a comprehensive assessment of the healing process. This work provides an up-to-date review on morphological, functional, optical, biochemical, and biophysical imaging techniques including their advantages, disadvantages and potential for combining them in a multimodal and multiscale manner. The focus lies on preclinical testing of biomaterials modified with artificial extracellular matrices in various animal models to enhance bone remodeling and regeneration.

Development of an 18F-Labeled Irreversible Inhibitor of Transglutaminase 2 as Radiometric Tool for Quantitative Expression Profiling in Cells and Tissues.

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy cells this enzymatic function is predominantly latent. Methods that enable the highly sensitive detection of TGase 2, such as application of radiolabeled activity-based probes, will support the exploration of the enzyme's function in various diseases. In this context, the radiosynthesis and detailed in vitro radiopharmacological evaluation of an 18F-labeled Nε-acryloyllysine piperazide are reported. Robust and facile detection of the radiotracer-TGase 2 complex by autoradiography of thin layer plates and polyacrylamide gels after chromatographic and electrophoretic separation owing to irreversible covalent bond formation was demonstrated for the isolated protein, cell lysates, and living cells. By use of this radiotracer, quantitative data on the expression profile of activatable TGase 2 in mouse organs and selected tumors were obtained for the first time by autoradiography of tissue sections.

The Pyrazolo[3,4-d]pyrimidine-Based Kinase Inhibitor NVP-BHG712: Effects of Regioisomers on Tumor Growth, Perfusion, and Hypoxia in EphB4-Positive A375 Melanoma Xenografts.

In a previous study, EphB4 was demonstrated to be a positive regulator of A375-melanoma growth but a negative regulator of tumor vascularization and perfusion. To distinguish between EphB4 forward and ephrinB2 reverse signaling, we used the commercially available EphB4 kinase inhibitor NVP-BHG712 (NVP), which was later identified as its regioisomer NVPiso. Since there have been reported significant differences between the inhibition profiles of NVP and NVPiso, we compared the influence of NVP and NVPiso on tumor characteristics under the same experimental conditions. Despite the different inhibitory profiles of NVP and NVPiso, the comparative study conducted here showed the same EphB4-induced effects in vivo as in the previous investigation. This confirmed the conclusion that EphB4-ephrinB2 reverse signaling is responsible for increased tumor growth as well as decreased tumor vascularization and perfusion. These results are further substantiated by microarrays showing differences between mock-transfected and EphB4-transfected (A375-EphB4) cells with respect to at least 9 angiogenesis-related proteins. Decreased expression of vascular endothelial growth factor (VEGF), angiotensin 1 (Ang-1), and protein kinase B (Akt/PKB), together with the increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta-2 (TGF-ß2), is consistent with the impaired vascularization of A375-EphB4 xenografts. Functional overexpression of EphB4 in A375-EphB4 cells was confirmed by activation of a variety of signaling pathways, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT), rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen activated protein kinase kinase (Ras/Raf/MEK), and nuclear factor kappa-B (NFkB) pathways.

The influence of different artificial extracellular matrix implant coatings on the regeneration of a critical size femur defect in rats.

Resorbable biomaterials based on artificial extracellular matrices (aECM) represent promising scaffolds for the treatment of large bone defects. Here, we investigated various glycosaminoglycan (GAG) derivatives of varying sulfation degree with respect to their influence on in vivo bone healing. The materials used in this study consisted of GAG-coated degradable polycaprolactone-co-lactide (PCL). Critical size femur defects in rats were filled with autologous bone serving as positive control or the respective coated or uncoated PCL scaffolds. After 2 and 12 weeks, progress in the healing process was investigated by analyzing the new bone matrix formation, the collagen content and hydroxyapatite formation by using micro-computed tomography (µCT), biomechanical testing, nuclear magnetic resonance spectroscopy (NMR) and histology. The sulfated GAG coating contributed substantially to bone regeneration, increased collagen synthesis and initiated mineralization of the organic matrix. Most substantial collagen production was detected in scaffolds coated with chondroitin sulfate. Scaffolds coated with hypersulfated hyaluronan induced formation of new bone volume comparable to what was observed in the positive control. GAG differing in the sugar backbone and degree of sulfation modulate the healing process at different times, eventually leading to improved bone healing.

Radiopharmacologist's and Radiochemist's View on Targeting the Eph/Ephrin Receptor Tyrosine Kinase System.

In the past decade, there have been extensive efforts to open up the Eph/ephrin subfamily of the receptor tyrosine kinase family for diagnostic and therapeutic applications. Besides classical pharmaceutical developments, which focus either on drugs targeting the extracellular ligand binding domains or on the intracellular tyrosine kinase domains of these receptors, there also have been first radiopharmaceutical approaches. Here the focus is on the development of specific and selective probes for molecular imaging, particularly by means of positron emission tomography, and the functional characterization of the Eph/ephrin subfamily in certain target tissues. The aim of this mini-review is to summarize the different approaches toward Eph-targeting radiotracers by using antibodies, peptides, and small molecules and to discuss their radiopharmacological characterization. With regard to the small molecules, further considerations will focus on the design and synthesis of nonradioactive reference compounds and precursors as well as on radiolabeling strategies.

Adjuvant Drug-Assisted Bone Healing: Advances and Challenges in Drug Delivery Approaches.

Bone defects of critical size after compound fractures, infections, or tumor resections are a challenge in treatment. Particularly, this applies to bone defects in patients with impaired bone healing due to frequently occurring metabolic diseases (above all diabetes mellitus and osteoporosis), chronic inflammation, and cancer. Adjuvant therapeutic agents such as recombinant growth factors, lipid mediators, antibiotics, antiphlogistics, and proangiogenics as well as other promising anti-resorptive and anabolic molecules contribute to improving bone healing in these disorders, especially when they are released in a targeted and controlled manner during crucial bone healing phases. In this regard, the development of smart biocompatible and biostable polymers such as implant coatings, scaffolds, or particle-based materials for drug release is crucial. Innovative chemical, physico- and biochemical approaches for controlled tailor-made degradation or the stimulus-responsive release of substances from these materials, and more, are advantageous. In this review, we discuss current developments, progress, but also pitfalls and setbacks of such approaches in supporting or controlling bone healing. The focus is on the critical evaluation of recent preclinical studies investigating different carrier systems, dual- or co-delivery systems as well as triggered- or targeted delivery systems for release of a panoply of drugs.

Synthesis, radiolabelling and initial biological characterisation of 18F-labelled xanthine derivatives for PET imaging of Eph receptors.

Eph receptor tyrosine kinases, particularly EphA2 and EphB4, represent promising candidates for molecular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives were identified to be potent Eph receptor inhibitors with IC50 values in the low nanomolar range (1-40 nm). These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase domain. Based on lead compound 1, we designed two fluorine-18-labelled receptor tyrosine kinase inhibitors ([18F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-binding site allowed us to find the best position for radiolabelling. The replacement of the methyl group at the uracil residue ([18F]3) rather than the methyl group of the phenoxy moiety ([18F]2) by a fluoropropyl group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis route to [18F]2 and [18F]3 and the respective tosylate precursors as well as a labelling procedure to insert fluorine-18. After radiolabelling, both radiotracers were obtained in approximately 5% radiochemical yield with high radiochemical purity (>98%) and a molar activity of >10 GBq µmol-1. In line with the docking studies, first cell experiments revealed specific, time-dependent binding and uptake of [18F]3 to EphA2 and EphB4-overexpressing A375 human melanoma cells, whereas [18F]2 did not accumulate at these cells. Since both tracers [18F]3 and [18F]2 are stable in rat blood, the novel radiotracers might be suitable for in vivo molecular imaging of Eph receptors with PET.

Active Targeting of Dendritic Polyglycerols for Diagnostic Cancer Imaging.

Active tumor targeting involves the decoration of nanomaterials (NMs) with oncotropic vector biomolecules that selectively recognize certain antigens on malignant cells or in the tumor microenvironment. This strategy can facilitate intracellular uptake of NM through specific interactions such as receptor-mediated endocytosis and can lead to prolonged retention in the malignant tissues by preventing rapid efflux from the tumor. Here, the design of actively targeting, renally excretible bimodal dendritic polyglycerols (dPGs) for diagnostic cancer imaging is described. Single-domain antibodies (sdAbs) specifically binding to the epidermal growth factor receptor (EGFR) are employed herein as targeting warheads owing to their small size and high affinity for their corresponding antigen. The dPGs equipped with EGFR-targeting feature are compared head-to-head with their nontargeting counterparts in terms of interaction with EGFR-overexpressing cells in vitro as well as accumulation at receptor-positive tumors in vivo. Experimental results reveal a higher specificity and preferential tumor accumulation for the α-EGFR dPGs, resulting from the introduction of active targeting capabilities on their backbone. These results highlight the potential for improving the tumor uptake properties of dPGs by strategic use of sdAb functionalization, which can ultimately prove useful to the development of ultrasmall NM with highly specific tumor accumulation.

Deuteration versus ethylation - strategies to improve the metabolic fate of an 18F-labeled celecoxib derivative.

The inducible isoenzyme cyclooxygenase-2 (COX-2) is closely associated with chemo-/radioresistance and poor prognosis of solid tumors. Therefore, COX-2 represents an attractive target for functional characterization of tumors by positron emission tomography (PET). In this study, the celecoxib derivative 3-([18F]fluoromethyl)-1-[4-(methylsulfonyl)phenyl]-5-(p-tolyl)-1H-pyrazole ([18F]5a) was chosen as a lead compound having a reported high COX-2 inhibitory potency and a potentially low carbonic anhydrase binding tendency. The respective deuterated analog [D2,18F]5a and the fluoroethyl-substituted derivative [18F]5b were selected to study the influence of these modifications with respect to COX inhibition potency in vitro and metabolic stability of the radiolabeled tracers in vivo. COX-2 inhibitory potency was found to be influenced by elongation of the side chain but, as expected, not by deuteration. An automated radiosynthesis comprising 18F-fluorination and purification under comparable conditions provided the radiotracers [18F]5a,b and [D2,18F]5a in good radiochemical yields (RCY) and high radiochemical purity (RCP). Biodistribution and PET studies comparing all three compounds revealed bone accumulation of 18F-activity to be lowest for the ethyl derivative [18F]5b. However, the deuterated analog [D2,18F]5a turned out to be the most stable compound of the three derivatives studied here. Time-dependent degradation of [18F]5a,b and [D2,18F]5a after incubation in murine liver microsomes was in accordance with the data on metabolism in vivo. Furthermore, metabolites were identified based on UPLC-MS/MS.