Chelating Moiety Research Articles

Abstract 5200: Preclinical pharmacology of the PSMA-targeted thorium-227 conjugate PSMA-TTC: a novel targeted alpha therapeutic for the treatment of prostate cancer

Abstract Prostate-specific membrane antigen (PSMA, FOLH1) is a type II transmembrane glycoprotein of the M28 peptidase family that acts as a glutamate carboxypeptidase on various substrates. PSMA is well established as a target antigen in prostate cancer due to its high and specific overexpression on the surface of prostate cancer cells at all tumor stages, including metastatic and hormone-refractory disease. Several PSMA targeting antibodies and ligands are currently in clinical development or compassionate use therapeutically or as imaging agents. Targeted alpha therapy (TAT) has an established clinical profile with the successful transition of Ra223, an alpha-particle emitter, from bench to bedside in prostate cancer. Thorium-227 is the immediate precursor for Ra223 via alpha-particle emission. We herein describe the generation of a novel TAT, a high energy, alpha-particle emitting PSMA-targeted thorium-227 conjugate (PSMA-TTC). PSMA-TTC consists of a fully human PSMA targeting IgG1 antibody covalently linked via an amide bond to a chelator moiety (3,2 HOPO), enabling radiolabeling with thorium-227 (227Th). PSMA-TTC was prepared in high radiochemical yield and purity and tested for binding affinity to PSMA target (ELISA) as well as PSMA expressing cell lines (FACS). In vitro cytotoxicity experiments were carried out on prostate CA cell lines with different PSMA levels (from 3.000 to 150.000 mAbs bound/ cell). In vivo biodistribution and anti-tumor efficacy were analyzed after i.v. injection of 100-500 kBq/kg at protein doses of 0.14 mg/kg to mice bearing s.c. prostate cancer xenograft models. Additionally, anti-tumor efficacy was evaluated in a PSMA expressing orthotopic bone xenograft model (LNCaP-Luc) monitored by bioluminescence imaging, micro CT and x-ray. PSMA-TTC retains binding affinities to PSMA target and PSMA positive cancer cells similar to the PSMA antibody. Strong in vitro potency and selectivity of PSMA-TTC was shown on different PSMA positive cells. Biodistribution studies in C4-2 xenografts demonstrated specific tumor uptake of PSMA-TTC with a maximum of 50 % of ID/g at t = 72h post dose administration. Selective significant antitumor efficacy was shown for PSMA-TTC in s.c. prostate CA xenograft models with high (C4-2) and medium/low (22Rv1) PSMA protein levels at doses of 250 and 500 kBq/kg. Furthermore, statistically significant prevention of tumor growth was observed after treatment with PSMA-TTC at a dose of 100 kBq/kg in an orthotopic bone xenograft model (LNCaP-Luc). The promising preclinical antitumor activity of PSMA-TTC supports its development for the treatment of patients with metastatic prostate cancer. Citation Format: Stefanie Hammer, Aasmund Larssen, Christine Ellingsen, Solene Geraudie, Derek Grant, Baard Indrevoll, Oliver von Ahsen, Alexander Kristian, Urs B Hagemann, Jenny Karlsson, Roger M Bjerke, Olav B Ryan, Dominik Mumberg, Bertolt Kreft, Alan Cuthbertson. Preclinical pharmacology of the PSMA-targeted thorium-227 conjugate PSMA-TTC: a novel targeted alpha therapeutic for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5200. doi:10.1158/1538-7445.AM2017-5200

Abstract 5199: Preclinical activity of the FGFR2-targeted thorium-227 conjugate in preclinical models of colorectal, gastric and triple-negative breast cancer

Abstract FGFR2 is a transmembrane tyrosine kinase receptor, consisting of three extracellular N-terminal immunoglobulin-like domains which are involved in ligand-binding as well as in receptor dimerization. Ligand-independent activation of FGFR2 signaling either via genomic amplification, gene fusion events, mRNA overexpression, or mutations has been observed e.g. in gastric cancer, colorectal cancer (CRC), and triple-negative breast cancer (TNBC). As such, FGFR2 has been described to be involved in cancer progression, promotion of oncogenesis, neoangiogenesis, as well as resistance to targeted therapies. Overexpression of FGFR2 and relatively low levels of cell surface expression of FGFR2 in normal human tissues renders FGFR2 an attractive candidate to explore targeted alpha therapy (TAT). We describe the generation of a high energy, alpha-particle emitting FGFR2 targeted thorium-227 conjugate (FGFR2-TTC). The FGFR2-TTC consists of a fully human FGFR2 binding IgG1 antibody (BAY 1179470) cross-reactive with mouse FGFR2, covalently linked via an amide bond to a chelator moiety (3,2 HOPO), enabling radiolabeling with the alpha particle emitting thorium-227 (227Th). In vitro cytotoxicity experiments with FGFR2-TTC demonstrated potency in the sub-picomolar range compared to a non-targeting control-TTC and a correlation between decrease in cell viability and increasing number of anti-FGFR2 antibodies bound per cell (ABC counts) in a panel of FGFR2-positive cancer cell lines. Upon treatment of cells with FGFR2-TTC, the DNA damage response marker protein γH2AX was up-regulated indicating that the mode-of-action involves induction of DNA double strand breaks. Furthermore, induction of the immunogenic cell death marker calreticulin was observed Biodistribution studies of the FGFR2-TTC in mouse models, evaluated by whole body autoradiography and acquisition of gamma-spectra specific for thorium-227, demonstrated specific accumulation of thorium-227 in FGFR2-positive tumors and very limited signal in murine organs and tissues. FGFR2-TTC exhibited in vivo tumor growth inhibition after a single dose in mouse xenograft models of CRC (NCI-H716) and gastric cancer (SNU-16). In addition, FGFR2-TTC showed anti-tumor activity in the aggressive murine syngeneic orthotopic 4T1 TNBC model. In summary, FGFR2-TTC has been established as a promising targeted alpha therapy (TAT) for efficacious and selective delivery of alpha emitter-based radiotherapy in several FGFR2-positive cancer indications. Further exploration for cancer therapy may thus be of interest. Citation Format: Urs B. Hagemann, Anette Sommer, Alexander Kristian, Ellen Wang, Åsmund Larsen, Uta Wirnitzer, Heidrun Ellinger-Ziegelbauer, Steffen Sandmann, Thorsten Poethko, Jenny Karlsson, Roger M. Bjerke, Lars Linden, Bertolt Kreft, Hanno Wild, Alan S. Cuthbertson. Preclinical activity of the FGFR2-targeted thorium-227 conjugate in preclinical models of colorectal, gastric and triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2017-5199

A porphyrin-based fluorescent probe for optical detection of toxic Cd2+ ion in aqueous solution and living cells

A porphyrin-based water-soluble fluorescent probe was reported for ratiometric detection of cadmium ion (Cd2+) in aqueous solution. This probe bearing two 2,2′-dipyridylamine (DPA) groups as metal chelating moieties which show chelation-produced ratiometric fluorescence effect with Cd2+via the interruption of intramolecular charge transfer (ICT) process. Exhibiting a large ratiometric fluorescence signal, the probe shows high sensitivity, high selectivity as well as quick response when detecting Cd2+ in the physiological pH window with a detection limit of part per billion (ppb) level. Moreover, it displays very low cytotoxicity and can be employed to visualize intracellular Cd2+ in living cells by utilizing laser scanning confocal microscopy.

Color-Tunable Heterodinuclear Pt(II)/B(III) and Pt(II)/Ir(III) Arrays with N^O-julolidine Ligands.

The synthesis and photophysical properties of two multichromophoric systems, Pt(II)/B(III) and Pt(II)/Ir(III), based on novel N^O-julolidine ligands are reported. The functionalization of the julolidine core enables the introduction of two different anchoring sites, a terminal acetylene and an N^O chelating moiety, which allow the assembling of two different chromophoric centers. The complex photophysical behavior of these multicomponent arrays is rationalized by investigating a series of model compounds, which are prepared through specific synthetic pathways. The photophysical properties of the final multicomponent arrays are investigated in parallel with the models. The multichromophoric system, composed by a platinum(II) and an iridium(III) chromophore connected through a modified julolidine ditopic ligand, displays a peculiar excitation wavelength dependent luminescence behavior. It exhibits tuning of the emission color from blue to orange, passing through pure and warm white.

Targeted alpha therapy using a novel CD70 targeted thorium-227 conjugate in in vitro and in vivo models of renal cell carcinoma.

The cell surface receptor CD70 has been previously reported as a promising target for B-cell lymphomas and several solid cancers including renal cell carcinoma. We describe herein the characterization and efficacy of a novel CD70 targeted thorium-227 conjugate (CD70-TTC) comprising the combination of the three components, a CD70 targeting antibody, a chelator moiety and the short-range, high-energy alpha-emitting radionuclide thorium-227 (227Th). In vitro analysis demonstrated that the CD70-TTC retained binding affinity to its target and displayed potent and specific cytotoxicity compared to an isotype control-TTC. A biodistribution study in subcutaneous tumor-bearing nude mice using the human renal cell carcinoma cell line 786-O demonstrated significant uptake and retention with 122 ± 42% of the injected dose of 227Th per gram (% ID/g) remaining in the tumor seven days post dose administration compared to only 3% ID/g for the isotype control-TTC. Tumor accumulation correlated with a dose dependent and statistically significant inhibition in tumor growth compared to vehicle and isotype control-TTC groups at radioactivity doses as low as 50 kBq/kg. The CD70-TTC was well tolerated as evidenced by only modest changes in hematology and normal gain in body weight of the mice. To our knowledge, this is the first report describing molecular targeting of CD70 expressing tumors using a targeted alpha-therapy (TAT).

Rhenium(I) complexes with bidentate carbohydrazide Schiff bases: Synthesis, characterization, computational and DNA interaction studies

In this research study, the formation of rhenium(I) complexes with bidentate carbohydrazide Schiff bases are reported. More specifically, the respective coordination reactions of [Re(CO)5Cl] with N-[1,3-benzothiazol-2-ylmethylidene]pyridine-2-carbohydrazide (bzpc), N-[1,3-benzothiazol-2-ylmethylidene]thiophene-2-carbohydrazide (bztc) and N-[(4-oxo-4H-chromen-3-yl)methylidene]thiophene-2-carbohydrazide (chrtc) afforded the rhenium(I) complexes, fac-[Re(CO)3Cl(L)] {L = bzpc for 1, bztc for 2 and chrtc for 3). Single crystal X-ray studies supported by spectroscopic characterization confirmed that the ligands coordinated mutually as bidentate neutral chelating moieties to their fac-[Re(CO)3Cl] cores. The DNA interactions of the metal complexes were evaluated using UV–Vis DNA binding titrations and gel electrophoresis. The redox properties of the metal complexes were probed using voltammetry and DFT studies were used to rationalize the variable coordination modes of 1 and 2 compared to 3.

Structural Investigations of Quiet Outer Membrane Protein G Nanopores

Interest in nanopore technology has been growing due to their unique capabilities in small molecule sensing, measurement of protein folding, and low cost DNA and RNA sequencing. The E. coli outer membrane protein OmpG is an excellent alternative to the heptameric α-hemolysin (αHL) because its pore is formed by a single polypeptide chain. OmpG is a 14-stranded β-barrel protein with seven flexible extracellular loops. It has been shown that loop 6 controls pore access in a pH-dependent manner and its dynamics result in spontaneous gating of the open pore current. This phenomenon ultimately limits the applications of OmpG in traditional biosensing. In this work, we modified the OmpG nanopore by selectively deleting several residues in and near loop 6. Electrophysiological measurements show that these modifications eliminate the flickering between open and closed states across wide pH range. We used nuclear magnetic resonance (NMR) spectroscopy to assess the structural role of these modifications on nanopore function by solving the structure of our quiet OmpG. Our data show that chemical shift perturbations are more prominent near the modification site indicating possible structural rearrangements. We further modified the inside of the quiet OmpG derivative with a copper chelating moiety to facilitate the detection of small molecules such as catecholamines, amino acids, and nucleotides. For instance, we can concurrently detect glutamic acid and ATP using this approach.

Synthesis and biological evaluation of deferiprone-resveratrol hybrids as antioxidants, Aβ1–42 aggregation inhibitors and metal-chelating agents for Alzheimer's disease

A series of deferiprone-resveratrol hybrids have been designed and synthesized as multitarget-directed ligands (MTDLs) through merging the chelating moiety 3-hydroxypyridin-4-one into the structure of resveratrol, a natural antioxidant agent and β-amyloid peptide (Aβ) aggregation inhibitor. The in vitro biological evaluation revealed that most of these newly synthesized compounds exhibited good inhibitory activity against self-induced Aβ1–42 aggregation, excellent antioxidant activity and potent metal chelating capability. Compounds 3i and 4f were identified as the most promising MTDLs with triple functions, possessing micromolar IC50 values for Aβ1–42 aggregation inhibition, greater 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) scavenging activity than Trolox and similar pFe(III) values to that of deferiprone.

Novel enterobactin analogues as potential therapeutic chelating agents: Synthesis, thermodynamic and antioxidant studies.

A series of novel hexadentate enterobactin analogues, which contain three catechol chelating moieties attached to different molecular scaffolds with flexible alkyl chain lengths, were prepared. The solution thermodynamic stabilities of the complexes with uranyl, ferric(III), and zinc(II) ions were then investigated. The hexadentate ligands demonstrate effective binding ability to uranyl ion, and the average uranyl affinities are two orders of magnitude higher than 2,3-dihydroxy-N1,N4-bis[(1,2-hydroxypyridinone-6-carboxamide)ethyl]terephthalamide [TMA(2Li-1,2-HOPO)2] ligand with similar denticity. The high affinity of hexadentate ligands could be due to the presence of the flexible scaffold, which favors the geometric agreement between the ligand and the uranyl coordination preference. The hexadentate ligands also exhibit higher antiradical efficiency than butylated hydroxyanisole (BHA). These results provide a basis for further studies on the potential applications of hexadentate ligands as therapeutic chelating agents.

Dual action Smac mimetics–zinc chelators as pro-apoptotic antitumoral agents

Dual action compounds (DACs) based on 4-substituted aza-bicyclo[5.3.0]decane Smac mimetic scaffolds (ABDs) linked to a Zn2+-chelating moiety (DPA, o-hydroxy, m-allyl, N-acyl (E)-phenylhydrazone) through their 10 position are reported and characterized. Their synthesis, their target affinity (XIAP BIR3, Zn2+) in cell-free assays, their pro-apoptotic effects and cytotoxicity in tumor cells with varying sensitivity to Smac mimetics are described. The results are interpreted to evaluate the influence of Zn2+ chelators on cell-free potency and on cellular permeability of DACs, and to propose novel avenues towards more potent antitumoral DACs based on Smac mimetics and Zn2+ chelation.

Abstract 591: A novel high energy alpha-pharmaceutical: In vitro and in vivo potency of a mesothelin-targeted thorium-227 conjugate (TTC) in a model of bone disease

Abstract Mesothelin (MSLN) is a 40 kDa membrane-anchored glycoprotein, involved in mediating cell-cell adhesion, metastatic spread, promotion of cell proliferation and resistance to apoptosis. Overexpression of MSLN is most prominent in mesothelioma, ovarian, lung, triple-negative breast (TNBC) and pancreatic cancers, while in healthy tissue, MSLN is confined mainly to the mesothelial cells of the peritoneum and pericardium. Several MSLN-targeting approaches are currently being investigated, including antibody drug conjugates. We describe herein a high energy, alpha-particle emitting MSLN Targeted Thorium Conjugate (MSLN-TTC). Thorium-227 (227Th) has a half-life of 18.7 days and decays via emission of an alpha particle to radium-223 (half-life 11.4 days), a calcium-mimetic used in the treatment of CRPC [1]. The MSLN-TTC comprises an anti-mesothelin monoclonal antibody covalently linked via an amide bond to a chelator moiety possessing high affinity for thorium-227. We present data from in vitro cytotoxicity assays demonstrating selective cell killing on MSLN positive cell-lines as well as in vivo efficacy in a mouse orthotopic bone xenograft model using NCI-H226 luciferase labeled cells. Experimental procedures: MSLN-TTC was prepared in high radiochemical yields and purity. In vitro cytotoxicity experiments were performed on the mesothelin-positive cell lines Ovcar-3 (ovarian), NCI-H226 (lung mesothelioma) and mesothelin-transfected HT29 (HT29MSLN/colorectal) cells. An in vivo model was established by orthotopic intratibial inoculation of luciferase-transfected NCI-H226 cells in athymic mice. Development of bone disease was monitored by luciferase activity and the extent of bone lesions determined by x-ray imaging and microCT. Results: MSLN-TTC induced specific in vitro cytotoxicity via induction of DNA double strand breaks as determined by phosphorylated histone protein H2AX. MSLN-TTC demonstrated statistical significant in vivo potency administered as a single dose of either 250 or 500 kBq/ kg in the orthotopic bone xenograft model. Animals treated with MSLN-TTC showed a) significantly reduced disease in the bone metastatic lesions b) decreased metastatic disease in the lungs and c) significant reduction in osteolytic/ osteoblastic bone lesions as evidenced by X-Ray and microCT compared to the vehicle control group. Furthermore, no significant loss in body weight was observed during the course of the study demonstrating that the MSLN-TTC was well tolerated. The data presented support the further investigation of the MSLN-TTC in bone metastatic disease.

A reversible “turn-on” fluorescent sensor for selective detection of Zn2+

A novel Schiff-base oxime sensor H2L with the ONON chelating moieties was designed and synthesized based on condensation reaction. Different methods such as elemental analyses, FT-IR, 1H and 13C NMR were fully utilized in characterizing the structure of sensor H2L. The UV–vis spectra of the Zn2+ complex exhibit four clear isosbestic points which may be assigned to the ONON moieties binding to Zn2+. Sensor H2L operates on the CHEF and PET mechanisms, and the tendency of Zn2+ to strongly enhance fluorescence of sensor H2L was explored. Fluorescence measurements show that sensor H2L has excellent fluorescent selectivity for Zn2+ over many other metal ions based on intramolecular charge-transfer, and the results demonstrate that the binding between sensor H2L and Zn2+ is chemically reversible. The crystal structure of the Zn2+ complex has been characterized by X-ray crystallography.

Design of a Small-Molecule Drug Conjugate for Prostate Cancer Targeted Theranostics.

Targeted therapy has become an effective strategy of precision medicine for cancer treatment. Based on the success of antibody-drug conjugates (ADCs), here we report a theranostic design of small-molecule drug conjugates (T-SMDCs) for targeted imaging and chemotherapy of prostate cancer. The structure of T-SMDCs built upon a polyethylene glycol (PEG) scaffold consists of (i) a chelating moiety for positron emission tomography (PET) imaging when labeled with (68)Ga, a positron-emitting radioisotope; (ii) a prostate specific membrane antigen (PSMA) specific ligand for prostate cancer targeting; and (iii) a cytotoxic drug (DM1) for chemotherapy. For proof-of-concept, such a T-SMDC, NO3A-DM1-Lys-Urea-Glu, was synthesized and evaluated. The chemical modification of Lys-Urea-Glu for the construction of the conjugate did not compromise its specific binding affinity to PSMA. The PSMA-mediated internalization of (68)Ga-labeled NO3A-DM1-Lys-Urea-Glu displayed a time-dependent manner, allowing the desired drug delivery and release within tumor cells. The antiproliferative activity of the T-SMDC showed a positive correlation with the PSMA expression level. Small animal PET imaging with (68)Ga-labeled NO3A-DM1-Lys-Urea-Glu exhibited significantly higher uptake (p < 0.01) in the PSMA positive PC3-PIP tumors (4.30 ± 0.20%ID/g) at 1 h postinjection than in the PSMA negative PC3-Flu tumors (1.12 ± 0.42%ID/g). Taken together, we have successfully designed and synthesized a T-SMDC system for prostate cancer targeted imaging and therapy.

Thickness of functionalized graphene oxide sheets plays critical role in tissue accumulation and urinary excretion: A pilot PET/CT study

We have recently reported that administration of thin graphene oxide (GO) sheets in the systemic circulation of rodents leads to rapid urinary excretion for the majority of injected dose and accumulation by the reticuloendothelial system organs for the remaining dose. In this study, graphene oxide was functionalized with a chelating moiety (DOTA, (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)) and labeled with [64Cu] for positron emission computed tomography (PET/CT) imaging. The thin functionalized graphene oxide material (f-GO-thin) consisted of a few layers (∼5nm) in thickness. Aging of the f-GO-thin material led to re-stacking of the flakes that resulted in materials of increased thickness (f-GO-thick) without altering their lateral dimension. These two types of f-GOs were comparatively studied pharmacologically to reveal the previously unexplored in vivo role of graphene oxide sheet thickness. Our results showed that a significantly larger fraction of the thicker GO sheets (47.5% of injected dose) remained within the body of living animals 24h after intravenous administration, residing mainly in the spleen and liver. The thinner GO sheets were predominantly (76.9% of injected dose) excreted through the glomerular filter into the urine. This pilot study provides an initial correlation between graphene-based material structure and pharmacological profile that is imperative towards understanding of how 2D structures behave in vivo to give information on potential biomedical applications.

Oxidation-Induced Degradable Nanogels for Iron Chelation.

Iron overload can increase cellular oxidative stress levels due to formation of reactive oxygen species (ROS); untreated, it can be extremely destructive to organs and fatal to patients. Since elevated oxidative stress levels are inherent to the condition in such patients, oxidation-induced degradable nanogels for iron chelation were rationally designed by simultaneously polymerizing oxidation-sensitive host-guest crosslinkers between β-cyclodextrin (β-CD) and ferrocene (Fc) and iron chelating moieties composed of deferoxamine (DFO) into the final gel scaffold in reverse emulsion reaction chambers. UV-Vis absorption and atomic absorption spectroscopy (AAS) was used to verify iron chelating capability of nanogels. These materials can degrade into smaller chelating fragments at rates proportional to the level of oxidative stress present. Conjugating DFO reduces the cytotoxicity of the chelator in the macrophage cells. Importantly, the nanogel can effectively reduce cellular ferritin expression in iron overloaded cells and regulate intracellular iron levels at the same time, which is important for maintaining a homeostatic level of this critical metal in cells.

Atomistic Simulation of Protein Encapsulation in Metal-Organic Frameworks.

Fabrication of metal-organic frameworks (MOFs) with large apertures triggers a brand-new research area for selective encapsulation of biomolecules within MOF nanopores. The underlying inclusion mechanism is yet to be clarified however. Here we report a molecular dynamics study on the mechanism of protein encapsulation in MOFs. Evaluation for the binding of amino acid side chain analogues reveals that van der Waals interaction is the main driving force for the binding and that guest size acts as a key factor predicting protein binding with MOFs. Analysis on the conformation and thermodynamic stability of the miniprotein Trp-cage encapsulated in a series of MOFs with varying pore apertures and surface chemistries indicates that protein encapsulation can be achieved via maintaining a polar/nonpolar balance in the MOF surface through tunable modification of organic linkers and Mg-O chelating moieties. Such modifications endow MOFs with a more biocompatible confinement. This work provides guidelines for selective inclusion of biomolecules within MOFs and facilitates MOF functions as a new class of host materials and molecular chaperones.

Design of Internalizing PSMA-specific Glu-ureido-based Radiotherapeuticals.

Despite the progress in diagnosis and treatment, prostate cancer (PCa) is one of the main causes for cancer-associated deaths among men. Recently, prostate-specific membrane antigen (PSMA) binding tracers have revolutionized the molecular imaging of this disease. The translation of these tracers into therapeutic applications is challenging because of high PSMA-associated kidney uptake. While both the tumor uptake and the uptake in the kidneys are PSMA-specific, the kidneys show a more rapid clearance than tumor lesions. Consequently, the potential of endoradiotherapeutic drugs targeting PSMA is highly dependent on a sustained retention in the tumor - ideally achieved by predominant internalization of the respective tracer. Previously, we were able to show that the pharmacokinetics of the tracers containing the Glu-urea-based binding motif can be further enhanced with a specifically designed linker. Here, we evaluate an eventual influence of the chelator moiety on the pharmacokinetics, including the tumor internalization. A series of tracers modified by different chelators were synthesized using solid phase chemistry. The conjugates were radiolabeled to evaluate the influence on the receptor binding affinity, the ligand-induced internalization and the biodistribution behavior. Competitive binding and internalization assays were performed on PSMA positive LNCaP cells and the biodistribution of the most promising compound was evaluated by positron emission tomography (PET) in LNCaP-tumor-bearing mice. Interestingly, conjugation of the different chelators did not cause significant differences: all compounds showed nanomolar binding affinities with only minor differences. PET imaging of the 68Ga-labeled CHX-A''-DTPA conjugate revealed that the chelator moiety does not impair the specificity of tumor uptake when compared to the gold standard PSMA-617. However, strong differences of the internalization ratios caused by the chelator moiety were observed: differences in internalization between 15% and 65% were observed, with the CHX-A''-DTPA conjugate displaying the highest internalization ratio. A first-in-man PET/CT study proved the high tumor uptake of this 68Ga-labeled PSMA-targeting compound. These data indicate that hydrophobic entities at the chelator mediate the internalization efficacy. Based on its specific tumor uptake in combination with its very high internalization ratio, the clinical performance of the chelator-conjugated Glu-urea-based PSMA inhibitors will be further elucidated.

A Comparative Study on Surface Treatments in the Immobilization Improvement of Hexahistidine-tagged Protein on the Indium Tin Oxide Surface

Biosensors are becoming increasingly important in various sectors. However, choosing transducer type and surface treatment still faces severe limitations. Indium-tin oxide is widely used in electrochemical sensing, though surface cleaning and functionalization are not always straightforward and often poorly controlled. Here, we have covalently immobilized a hexahistidine-tagged model protein on three different treated ITO surfaces using a Ni2+ chelator moiety. The study shows that compared to two other treatments (H2O/H2O2/NH4OH and KOH treatments), the ITO Piranha treatment gives satisfactory results in term of yields of EC12 protein immobilized on the surface. The study in detail of treatments effect was realized by applying a combination of local and global techniques such X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), contact angle measurements, and electrochemical impedance spectroscopy (EIS). The biofunctionalization allowed proteins to move freely around their anchoring bond. As the polyhistidine tags are widely used in protein engineering, such substrates offer a large panel of applications. This covalent and oriented immobilization process of recombinant proteins applied to ITO could provide transparent electroactive surfaces of high quality for electrochemical detection of soluble compounds, as well as cells.

Synthesis and characterisation of bismacrocyclic DO3A-amide derivatives - an approach towards metal-responsive PARACEST agents.

Three new bismacrocyclic Ln(3+) chelates consisting of triamide derivatives of cyclen with glycine, methyl and tert-butyl substituents (, respectively) linked to an acyclic EGTA-derived calcium chelator were synthesised as potential MRI contrast agents (EGTA - ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Eu(3+) and Yb(3+) complexes of were investigated as chemical exchange saturation transfer (CEST) agents. Moderate to minor CEST effects were observed for , and complexes in the absence of Ca(2+), with negligible changes upon addition of this metal ion. Luminescence steady-state emission and lifetime experiments did not reveal any changes in the coordination environment of the complexes, while the number of inner-sphere water molecules remained constant in the absence and presence of Ca(2+). The protonation constants of and and stability constants of their complexes with Ca(2+), Mg(2+) and Zn(2+) were determined by means of potentiometric titrations. The results show that the charge of the complex dramatically affects the protonation constants of the EGTA-binding unit. The stability constants of the complexes formed with Ca(2+), Mg(2+) and Zn(2+) are several orders of magnitude lower than those of EGTA. These findings indicate that the nature of Ln(3+) chelates and their charge are the main reasons for the observed results and weaker response of these EGTA-derived triamide derivatives compared to their tricarboxylate analogues.

Rhenium(V)-nitrido complexes with different nitrogen donor bidentate ligands

Rhenium(V)-nitridocyano complexes with 2,2′bipyridine (bipy), 1,10-phenanthroline (phen) and 2,9-dimethyl-1,10-phenanthroline (Me2phen) are isolated for the first time and characterized by IR and X-ray crystallography as (AsPh4)[ReN(CN)3(2,2′bipy)]·2H2O·0.5bipy, 2{(AsPh4)[ReN(CN)3(phen)]·0.5H2O·0.54EtOH·0.46MeOH and (PPh4)[ReN(CN)3(Me2phen]·H2O·Me2phen. The rhenium anions in all three structures have a distorted octahedral geometry with the Re(V) atom coordinated by a terminal nitrido ligand, three carbon atoms of the cyanide ligands and bidentate ligands that are bonded bidentally via two pyridine nitrogen atoms. The bidentate ligands coordinate to the Re(V) metal centre via two nitrogen atoms and form five-membered chelate rings with bite angles of 71.37(9)°, 72.7(2)° and 70.7(3)° for the bipy, phen and Me2phen complexes, respectively. Principal dimensions are: bipy: Re≡N = 1.651(3), Re–N(11) = 2.144(1), Re–N(12) = 2.374(2), Re–C(1) = 2.025(4), Re–C(2) = 2.142(4) and Re–C(3) 2.080(3) A; phen: Re≡N = 1.674(5), Re–N(11) = 2.164(5) A, Re–N(12) = 2.374(5) A, Re–C(1) = 2.112(6), Re–C(2) = 2.082(6) and Re–C(3) 2.102(6) A; Me2phen: Re≡N = 1.634(9), Re–N(11) = 2.190(7) A, Re–N(22) = 2.454(8), Re–C(1) = 2.074(9) A, Re–C(2) = 2.095(10) A and Re–C(3) 2.094(10) A. One of the major differences between the complexes of phen and Me2phen is that the latter exhibits a fluxional behaviour, while the phenanthroline plane in the phen complex is almost coplanar with the coordination plane. This pronounced distortion in the Me2phen complex is illustrated by the dihedral angle of 117°(2) between the plane of the chelating moiety and that of the square-planar coordination plane.