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Malignant melanoma has a steadily increasing incidence, but treatment options are still limited, and the prognosis for patients, especially for men, is poor. To investigate whether targeting estrogen receptor (ER) signaling is a valid therapeutic approach, we retrospectively analyzed ER gene expression profiles in 448 patients with melanoma. High ERα gene (ESR1) expression was associated with improved overall survival (hazard ratio = 0.881; 95% confidence interval = 0.793-0.979; P = 0.018) and increased with tumor stage, whereas ERβ gene (ESR2) expression did not change with tumor progression. This seemingly protective function of ERα led us to speculate that specific targeting of ERβ has a therapeutic benefit in malignant melanoma. An ERβ-selective ER modulator with nitric oxide‒releasing moiety (nitric oxide‒releasing selective ER modulator 4d [NO-SERM 4d]) significantly reduced the prometastatic behavior of two melanoma cell lines (A2058 and MEL-JUSO). Epithelial‒mesenchymal transition in melanoma is consistent with a switch from E- to N-cadherin expression, mediating the invasive phenotype. NO-SERM 4d reduced N-cadherin expression and impaired spheroid formation in A2058 cells. In addition, the growth of A2058 spheroids was significantly reduced, confirming the antitumorigenic potential of NO-SERM 4d. Targeting ERβ signaling combined with targeted nitric oxide release represents a promising therapeutic approach in malignant melanoma that has the potential to prevent metastatic spread and reduce tumor growth.

Objectives: Finding optimal 18F-fluorination conditions represents a critical and often time-consuming step in radiotracer development. Microfluidic or lab-on-a-chip devices are modern tools for that purpose but not accessible everywhere. Inspired by 18F-chemistry in low volumes based on a MAX/MCX-trapping technique for one complete 18F-batch,[1] we herein present an 18F-labeling approach using low volume aliquots (50 reactions/day) using minimal amounts of precursor. Utilizing mixtures of 2-3% water in acetonitrile for 18F-elution allowed for radiolabeling even without azeotropic drying in certain cases, which can further accelerate the entire labeling process. For more demanding radiolabeling reactions, drying of only 3 min with helium furnished highly reactive [18F]fluoride with comparable reactivity even several hours after the elution step. This optimization workflow was used successfully to identify high yielding reaction conditions for a variety of precursor molecules like aliphatic tosylates or mesylates as well as aromatic sydnones, boronic acids or trimethylammonium salts. As examples, optimization results for 18F-labeling of the FDG mannose triflate precursor and the tosylate precursor of celecoxib-derivative [18F]A[3] are shown in Figure 1. Transfer into isolated RCY at activity levels suitable for preclinical evaluations was achieved (e.g. for [18F]A: 25-30% after 18F-labeling in acetonitrile at 90°C and purification) and turned out to be comparable to results from conventional automated radiosyntheses. Conclusion: This microliter scale optimization and labeling procedure can easily be adapted and transferred to the synthesis of known and new radiotracers. The straightforward approach represents a valuable radiochemistry tool to enter rapidly into subsequent preclinical studies. References: [1] Iwata (2018), J.Labelled.Comp.Radiopharm., 61, 540; [2] Kniess (2017), Appl.Radiat.Isot 127, 260; [3] Laube (2020), RSC Advances, 10, 38601. Figure 1. Setup of HPLC vial experiments and representative results of optimization.

High extracellular S100A4 level proves a specific characteristic of some cancer cases, including malignant melanoma. Concerning the latter, extracellular S100A4 in an autocrine manner was shown to promote prometastatic activation of A375 cells by interaction with the receptor for advanced glycation endproducts (RAGE). We hypothesized that interaction of extracellular S100A4 with RAGE in a paracrine manner will affect endothelial cell (EC) integrity thus further promoting melanoma metastasis. We investigated the influence of recombinant and cell (A375)-derived S100A4 on junction protein expression and EC (hCMEC/D3) integrity by measuring transendothelial electrical resistance (TEER). Decrease of TEER and diminished expression of both occludin and VE-cadherin revealed the loss of EC integrity. Transmigration of transgenic A375 cells (A375-hS100A4/A375-hRAGE) through the EC monolayer was significantly higher compared to wild-type A375 cells, and was substantially decreased by sRAGE. A pilot study in mice, intracardially injected with A375-hS100A4 or A375-hRAGE cells, showed lower survival rates and a higher incidence of metastases compared to wild-type A375 cells. Tumor development was mostly located in the brain, bones, and ovaries. These findings provide further evidence on extracellular S100A4 as paracrine mediator of prometastatic endothelial dysfunction involving its interaction with RAGE.

Biomaterials with attenuated adverse host tissue reactions, and meanwhile, combining biocompatibility with mimicry of mechanical and biochemical cues of native extracellular matrices (ECM) to promote integration and regeneration of tissues are important for many biomedical applications. Further, the materials should also be tailorable to feature desired application-related functions, like tunable degradability, injectability, or controlled release of bioactive molecules. Herein, a non-covalently assembled, injectable hydrogel system based on oligopeptides interacting with sulphated polysaccharides is reported, showing high tolerability and biocompatibility in immunocompetent hairless mice. Altering the peptide or polysaccharide component considerably varies the in vivo degradation rate of the hydrogels, ranging from a half-life of three weeks to no detectable degradation after three months. The hydrogel with sulphated low molecular weight hyaluronic acid exhibits sustained degradation-mediated release of heparin-binding molecules in vivo, as shown by small animal magnetic resonance imaging and fluorescence imaging, and enhances the expression of vascular endothelial growth factor in hydrogel surrounding. In vitro investigations indicate that M2-macrophages could be responsible for the moderate difference in pro-angiogenic effects. The ECM-mimetic and injectable hydrogels represent tunable bioactive scaffolds for tissue engineering, also enabling controlled release of heparin-binding signalling molecules including many growth factors.

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.

Hydrogels prepared from gelatin and lysine diisocyanate ethyl ester provide tailorable elastic properties and degradation behavior. Their interaction with human aortic endothelial cells (HAEC) as well as human macrophages (Mɸ) and granulocytes (Gɸ) were explored. The experiments revealed a good biocompatibility, appropriate cell adhesion, and cell infiltration. Direct contact to hydrogels, but not contact to hydrolytic or enzymatic hydrogel degradation products, resulted in enhanced cyclooxygenase-2 (COX-2) expression in all cell types, indicating a weak inflammatory activation in vitro. Only Mɸ altered their cytokine secretion profile after direct hydrogel contact, indicating a comparably pronounced inflammatory activation. On the other hand, in HAEC the expression of tight junction proteins, as well as cytokine and matrix metalloproteinase secretion were not influenced by the hydrogels, suggesting a maintained endothelial cell function. This was in line with the finding that in HAEC increased thrombomodulin synthesis but no thrombomodulin membrane shedding occurred. First in vivo data obtained after subcutaneous implantation of the materials in immunocompetent mice revealed good integration of implants in the surrounding tissue, no progredient fibrous capsule formation, and no inflammatory tissue reaction in vivo. Overall, the study demonstrates the potential of gelatin-based hydrogels for temporal replacement and functional regeneration of damaged soft tissue.

The bifunctional chelating agent 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid, DMPTACN-COOH, has been found to bind strongly to copper(II), resulting in a radiocopper(II)-ligand complex that exhibits high in vivo stability. The pendant carboxylic acid group enables this derivative to be conjugated to the N-terminal amino acid residues of peptides. Exploiting this, two stabilized bombesin (BBN) derivatives, βAla-βAla-[Cha13,Nle14]BBN(7–14) and βhomo-Glu-βAla-βAla-[Cha13,Nle14]BBN(7–14) have been coupled to DMPTACN-COOH and radiolabeled with the positron emitter copper-64 (64Cu-1 and 64Cu-3). The in vitro binding characteristics of the [64Cu]Cu-labeled bombesin conjugates in gastrin-releasing peptide receptor (GRPR) over-expressing prostate cancer (PC-3) cells have been evaluated. Biodistribution studies performed in Wistar rats indicate a specific uptake in the GRPR-rich pancreas and rapid renal elimination for both 64Cu-1 and 64Cu-3. Small animal PET imaging studies performed in NMRI nu/nu mice bearing the human prostate tumor PC-3 demonstrated a very high degree of tumor accumulation for 64Cu-1 and 64Cu-3. Incorporation of a single additional glutamic acid residue within the spacer between bombesin and the radiolabeled complex (64Cu-3) leads to a higher tumor-to-muscle uptake ratio (amounting to >30 at 100 min post injection) compared to 64Cu-1.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

Introduction Phosphopeptides represent interesting compounds to study and elucidate cellular protein phosphorylation/dephosphorylation processes underlying various signal transduction pathways. However, studies of phosphopeptide action in cells are severely constrained by the negatively charged phosphate moiety of the phosphopeptide resulting in poor transport through the cell membrane. The following study describes the synthesis and radiopharmacological evaluation of two 18 F-labeled phosphopeptide-cell-penetrating peptide dimers. The polo-like kinase-1-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH was coupled to cell-penetrating peptides (CPPs), either sC18, a cathelicidin-derived peptide, or the human calcitonin derivative hCT(18-32)-k7. Methods Radiolabeling was accomplished with the prosthetic group N -succinimidyl 4-[ 18 F]fluorobenzoate ([ 18 F]SFB) using both, conventional and microfluidic-based bioconjugation of [ 18 F]SFB to N -terminal end of phosphopeptide part of the peptide dimers. Cellular uptake studies in human cancer cell lines HT-29 and FaDu cells at 4 °C and 37 °C and small animal PET in BALB/c mice were utilized for radiopharmacological characterization. Results Isolated radiochemical yields ranged from 2% to 4% for conventional bioconjugation with [ 18 F]SFB. Significantly improved isolated radiochemical yields of up to 26% were achieved using microfluidic technology. Cellular uptake studies of radiolabeled phosphopeptide and phosphopeptide-CPP dimers indicate enhanced internalization of 50% ID/mg protein after 2 h for both phosphopeptide dimers compared to the phosphopeptide alone ( In vivo biodistribution of 18 F-labeled peptide dimers was determined with small animal PET revealing a superior biodistribution pattern of sC18-containing peptide dimer MQSpTPL-sC18 [ 18 F]4 . Conclusion [18 F]SFB labeling of the phosphopeptide-CPP dimers using a microfluidic system leads to an improved chemoselectivity towards the N -terminal NH 2 group compared to the conventional labeling approach. Cell-penetrating peptide sC18 can be considered as an ideal molecular shuttle for intracellular delivery of the Plk1-PBD-binding hexaphosphopeptide as demonstrated by its favourable radiopharmacological profile.

The radiosynthesis of 3-(4-[ 18 F]fluorophenyl)-2-(4-methylsulfonylphenyl)-1 H -indole [ 18 F]- 3 as potential PET radiotracer for functional characterization of cyclooxygenase-2 (COX-2) in vitro and in vivo is described. [ 18 F]- 3 was prepared by McMurry cyclization of a 18 F-labeled intermediate with low valent titanium and zinc via a two-step procedure in a remote controlled synthesizer unit including HPLC purification and solid phase extraction. In this way [ 18 F]- 3 was synthesized in 80 min synthesis time in 10% total decay corrected yield from [ 18 F]fluoride in radiochemical purity >98% and a specific activity of 74–91 GBq/μmol (EOS). [ 18 F]- 3 was evaluated in vitro using pro-inflammatory stimulated THP-1 and COX-2 expressing tumor cell lines (FaDu, A2058, HT-29), where the radiotracer uptake was shown to be consistent with up regulated COX-2 expression. The stability of [ 18 F]- 3 was determined by incubation in rat whole blood and plasma in vitro and by metabolite analysis of arterial blood samples in vivo, showing with 75% of original compound after 60 min an acceptable high metabolic stability. However, no substantial tumor accumulation of [ 18 F]- 3 could be observed by dynamic small animal PET studies on HT-29 tumor-bearing mice in vivo. This may be due to the only moderate COX-1/COX-2 selectivity of 3 as demonstrated by both cellular and enzymatic cyclooxygenase inhibition assay in vitro. Nevertheless, the new approach first using McMurry cyclization in 18 F-chemistry gives access to 18 F-labeled diarylsubstituted heterocycles that hold promise as radiolabeled COX-2 inhibitors.

Tumor cells are characterized by their loss of growth control resulting from alterations in regulating pathways of the cell cycle, such as a deregulated cyclin-dependent kinase (Cdk) activity and/or Cdk expression. Appropriately radiolabeled Cdk4 inhibitors are discussed as promising molecular probes for imaging cell proliferation processes and tumor visualization by PET. This work describes the design, synthesis and radiopharmacological evaluation of two 124I-labeled Cdk4 inhibitors as potential radiotracers for imaging of Cdk4 in vivo. Treatment of a solution containing labeling precursors with [124I]NaI gave radiolabeled Cdk4 inhibitors [124I]CKIA and [124I]CKIB in radiochemical yields of up to 35%. 124I-labeled radiotracers [124I]CKIA and [124I]CKIB were used in cell uptake studies as well as biodistribution studies in Wistar rats and small-animal PET in tumor-bearing mice. In vitro radiotracer uptake studies in adherent tumor cells using [124I]CKIA showed substantial uptake in HT-29 and FaDu cells (750–850 %ID/mg protein [124I]CKIA and 900–1000 %ID/mg protein [124I]CKIB) after 1 h at 37 °C. Biodistribution of [124I]CKIA and [124I]CKIB showed rapid blood clearance of radioactivity and an accumulation as well as metabolization in the liver. Both radiotracers were administered intravenously to mouse FaDu xenograft tumor model and imaging studies were performed on a small-animal PET scanner. Both imaging techniques showed only little uptake of both radiotracers in the FaDu tumor xenografts.

Introduction Our group has synthesized technetium-labeled fatty acids (FA) that are extracted into the myocardium and sequestered due to heart-type fatty acid binding protein (H-FABP) binding. In this article, we further address the detailed subcellular distribution and potential myocardial metabolism of [ 99m Tc]"4+1" FA. Methods Experiments were conducted using isolated hearts of Wistar rats, as well as of wild-type and H-FABP −/− mice. Myocardium samples underwent subcellular fractionation [subsarcolemmal mitochondria (SM), intermyofibrillar mitochondria (IM), cytosol with microsomes, and nuclei and crude membranes] and analysis by thin-layer chromatography and high-performance liquid chromatography. Results The largest fraction of tissue radioactivity was associated with cytosol [79.69±8.88% of infused dose]. About 9.07±0.95% and 3.43±1.38% of the infused dose were associated with SM and IM fractions, respectively. In the rat heart, etomoxir, an inhibitor of carnitin-palmitoyl transferase I, did not significantly decrease radioactivity associated with mitochondrial fractions, whereas myocardial extraction of [ 123 I]-labeled 15-( p -iodophenyl)-pentadecanoic acid (13.26% vs. 49.49% in controls) and the radioactivity associated with the SM and IM fractions were blunted. The percentage of the infused dose in the mitochondrial and crude fractions increased with the number of NH-amide groups of the FA derivative. Absence of H-FABP significantly decreased radioactivity count in the cytosolic fraction ( P 99m Tc]"4+1" FA could be detected in any isolated heart. Conclusions Myocardial [ 99m Tc]"4+1" FA extraction reflects binding to H-FABP and membrane structures (including the mitochondrial membrane). However, the compounds do not undergo mitochondrial metabolism because they do not reach the mitochondrial matrix.

The radiosynthesis of [N-methyl-(11)C]Org 34850 as a potential brain glucocorticoid receptor (GR)-binding radiotracer is described. The radiosynthesis was accomplished via N-methylation of the corresponding desmethyl precursor with [(11)C]methyl triflate in a remotely controlled synthesis module to give the desired compound in a radiochemical yield of 23+/-5% (decay-corrected, based upon [(11)C]CO(2)) at a specific activity of 47+/-12 GBq/micromol (n=15) at the end-of-synthesis (EOS). The radiochemical purity after semi-preparative HPLC purification exceeded 95%. The total synthesis time was 35-40 min after end-of-bombardment (EOB). The radiotracer is rapidly metabolized in rat plasma leading to the formation of two more hydrophilic metabolites as the major metabolites. Radiopharmacological evaluation involving biodistribution and small animal PET imaging in normal Wistar rats showed that the compound [N-methyl-(11)C]Org 34850 is not able to sufficiently penetrate the blood-brain barrier. Therefore, compound [N-methyl-(11)C]Org 34850 seems not to be a suitable PET radiotracer for imaging rat brain GRs. However, involvement of Pgp or species differences requires further clarification to establish whether the radiotracer [N-methyl-(11)C]Org 34850 may still represent a suitable candidate for imaging GRs in humans.

A series of 1,4- and 1,5-diaryl substituted 1,2,3-triazoles was synthesized by either Cu(I)-catalyzed or Ru(II)-catalyzed 1,3-dipolar cycloaddition reactions between 1-azido-4-methane-sulfonylbenzene 9 and a panel of various para-substituted phenyl acetylenes (4-H, 4-Me, 4-OMe, 4-NMe2, 4-Cl, 4-F). All compounds were used in in vitro cyclooxygenase (COX) assays to determine the combined electronic and steric effects upon COX-1 and COX-2 inhibitory potency and selectivity. Structure-activity relationship studies showed that compounds having a vicinal diaryl substitution pattern showed more potent COX-2 inhibition (IC50 = 0.03–0.36 µM) compared to their corresponding 1,3-diaryl-substituted counterparts (IC50 = 0.15 to >10.0 µM). In both series, compounds possessing an electron-withdrawing group (Cl and F) at the para-position of one of the aryl rings displayed higher COX-2 inhibition potency and selectivity as determined for compounds containing electron-donating groups (Me, OMe, NMe2). The obtained data show, that the central carbocyclic or heterocyclic ring system as found in many COX-2 inhibitors can be replaced by a central 1,2,3-triazole unit without losing COX-2 inhibition potency and selectivity. The high COX-2 inhibition potency of some 1,2,3-triazoles having a vicinal diaryl substitution pattern along with their ease in synthesis through versatile Ru(II)-catalyzed click chemistry make this class of compounds interesting candidates for further design and synthesis of highly selective and potent COX-2 inhibitors.

Background We analyzed the efficacy and possible synergistic actions of pioglitazone and simvastatin monotherapy versus their combination on LDL subfractions from a subpopulation from the PIOSTAT three-arm randomized controlled trial. PPARγ agonists, such as pioglitazone, improve insulin sensitivity and glycemic control and appear to lower the concentration of atherogenic small dense LDL particles. Insulin resistance frequently occurs in non-diabetic patients with cardiovascular disease. Statins, such as simvastatin, reduce cardiovascular events by lowering LDL-C. So far, only scarce information exists for comparative efficacy and possible synergistic effects of combination therapy on LDL subfractions, cholesterol particle load, and particle number of atherogenic small dense LDL. Methods 125 non-diabetic patients with high cardiovascular risk were randomized to therapy with pioglitazone 45 mg/day, simvastatin 40 mg/day, or the combination of both, for 12 weeks. In the present sub-study, LDL subfractions from 88 patients were separated by very-fast ultracentrifugation. Results Simvastatin monotherapy significantly reduced cholesterol and triglyceride concentrations in IDL, LDL1, and LDL2. The lipid concentrations and lipid loads in LDL3 remained unchanged. By contrast, treatment with pioglitazone reduced the cholesterol concentration in LDL3 (density 1.040–1.066 kg/l) from 0.38 to 0.31 mmol/l ( p = 0.0004) and of the cholesterol load per particle from 1058 to 934 mol/mol ( p = 0.0149). Even greater reductions of cholesterol in LDL3 were observed with the combination of pioglitazone and simvastatin: from 0.38 to 0.29 mmol/l ( p = 0.0006) and from 1021 to 903 mol/mol ( p = 0.0011), respectively. In addition, combination therapy reduced the particle number of LDL3 from 356 to 316 nmol/l ( p = 0.0074). Conclusions Simvastatin preferentially lowered LDL1 and LDL2 subfractions, whereas pioglitazone reduced LDL3 cholesterol and cholesterol load. In addition, the combination reduced the LDL3 particle number. Thus, our data suggest a synergistic action of pioglitazone and simvastatin on atherogenicity of small dense LDL particles.

The radiosynthesis and radiopharmacological evaluation of 1-[(11)C]methoxy-4-(2-(4-(methanesulfonyl)phenyl)cyclopent-1-enyl)-benzene [(11)C]5 as novel PET radiotracer for imaging of COX-2 expression is described. The radiotracer was prepared via O-methylation reaction with [(11)C]methyl iodide in 19% decay-corrected radiochemical yield at a specific activity of 20-25GBq/mumol at the end-of-synthesis within 35 min. The radiotracer [(11)C]5 was evaluated in vitro using various pro-inflammatory and tumor cell lines showing high functional expression of COX-2 at baseline or after induction. In vivo biodistribution of compound [(11)C]5 was characterized in male Wistar rats. Compound [(11)C]5 was rapidly metabolized in rat plasma, and more pronounced, in mouse plasma. In vivo kinetics and tumor uptake were demonstrated by dynamic small animal PET studies in a mouse tumor xenograft model. Tumor uptake of radioactivity was clearly visible overtime. However, radioactivity uptake in the tumor could not be blocked by the pre-injection of nonradioactive compound 5. Therefore, it can be concluded that radioactivity uptake in the tumor was not COX-2 mediated.

Data concerning the pathophysiological role of the interaction of circulating S100 proteins, a multigenic family of Ca(2+)-modulated proteins, with the receptor for advanced glycation endproducts (RAGE) in cardiovascular diseases, inflammatory processes, and tumorigenesis in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A1, S100B, and S100A12 as potential probes for molecular imaging of this interaction. Therefore, human S100 proteins were cloned as GST fusion proteins in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100 proteins were radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radiolabeled recombinant S100 proteins ((18)F-S100) were used in biodistribution experiments and small animal positron emission tomography (PET) studies in rats. The tissue-specific distribution of (18)F-S100 proteins in vivo correlated well with the anatomical localization of RAGE, e.g., in lungs and in the vascular system. These findings indicate circulating S100A1, S100B, and S100A12 proteins to be ligands for RAGE in rats in vivo. The approach allows the use of small animal PET and provides novel probes to delineate functional expression of RAGE under normal and pathophysiological conditions in rodent models of disease.

The radiosynthesis and the radiopharmacological characterization of [(11)C]AL-438 as a nonsteroidal ligand for the glucocorticoid receptor (GR) is described. Radiolabeling of the corresponding desmethyl precursor 10 with [(11)C]MeI gave [(11)C]AL-438 in decay-corrected radiochemical yields of 30+/-4% (based upon [(11)C]CO(2)) within 35 min at a specific radioactivity of 10-15 GBq/micromol at the end-of-synthesis. The radiopharmacological evaluation of [(11)C]AL-438 involved biodistribution and small animal PET imaging in rats, and autoradiography studies using rat brain sections. Biodistribution studies were performed in male Wistar rats and demonstrated high radioactivity uptake in pituitary and brain. However, the inability of high dose corticosterone to block binding would suggest that the radioactivity accumulation in the brain was not receptor-mediated.

The novel thiol-group-selective bifunctional 18F-labeling agent N-[6-(4-[18F]fluoro-benzylidene)aminooxyhexyl]maleimide ([18F]FBAM) has been developed. The bifunctional labeling precursor N-(6-aminoxyhexyl)maleimide containing a thiol-reactive maleimide group and a carbonyl-group-reactive aminooxy group was prepared in only three steps in a total chemical yield of 59%. Subsequent radiolabeling with 4-[18F]fluorobenzaldehyde gave the bifunctional 18F-labeling agent [18F]FBAM in a radiochemical yield of 29%. In a typical experiment, 3.88 GBq of [18F]fluoride could be converted into 723 MBq of [18F]FBAM within 69 min. Conjugation of [18F]FBAM with thiol groups was exemplified with the cysteine-containing tripeptide glutathione and with various apolipoproteins of human low-density lipoprotein (LDL) subfractions. The latter was evaluated with respect to the uptake of [18F]FBAM-LDL subfractions in human hepatoma cells (HepG2) in vitro. In vivo biodistribution studies in rats revealed high stability for [18F]FBAM-LDL subfractions. Moreover, the metabolic fate of [18F]FBAM-LDL subfractions in vivo was delineated by dynamic positron emission tomography studies using a dedicated small animal tomograph. Data were compared to former studies that used the NH2-reactive 18F-labeling agent N-succinimidyl-4-[18F]fluorobenzoate. The compound [18F]FBAM can be considered as an excellent prosthetic group for the selective and mild 18F labeling of thiol-group-containing biomolecules suitable for subsequent investigations in vitro and in vivo.

Oxidative modification of low-density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Assessing the metabolic fate of oxidized LDL (oxLDL) in vivo with radiotracer techniques is hindered by the lack of suitable sensitive and specific radiolabeling methods. We evaluated an improved methodology based on the radiolabeling of native LDL (nLDL) and oxLDL with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). We investigated whether radiolabeling of LDL induces adverse structural modifications. Results suggest that radiolabeling of both nLDL and oxLDL using [(18)F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively. Thus, radiolabeling of LDL using [(18)F]SFB could prove to be a promising approach for studying the kinetics of oxLDL in vivo.

Objective: In an in vitro biosensor model (PCT/ET 97/05212), the interplay between different lipoproteins in arteriosclerotic nanoplaque formation, as well as aqueous garlic extract (0.2-5.0 g/l from LI 111 powder) as a possible candidate drug against arterio/atherosclerosis were tested within the frame of a high throughput screening. Methods: The processes described below were studied by ellipsometric techniques quantifying the adsorbed amount (nanoplaque formation) and layer thickness (nanoplaque size). A thorough description of the experimental setup has been given previously. Results: Proteoheparan sulfate (HS-PG) absorption to hydrophobic silica was monoexponential and after approximately 30 min constant. The addition of 2.52 mmol/l Ca2+ led to a further increase in HS-PG adsorption because Ca2+ was bound to the polyanionic glycosaminoglycan (GAG) chains thus screening their negative fixed charges and turning the whole molecule more hydrophobic. Incubation with 0.2 g/l aqueous garlic extract (GE) for 30 min did not change the adsorption of HS-PG. However, the following addition of Ca2+ ions reduced the increase in adsorption by 50.8 % within 40 min. The adsorption of a second Ca2+ step to 10.08 mmol/l was reduced by even 82.1 % within the next 40 min. Having detected this inhibition of receptor calcification, it could be expected that the build-up of the ternary nanoplaque complex is also affected by garlic. The LDL plasma fraction (100 mg/dl) from a healthy probationer showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca2+ concentration, with a strong increase at higher Ca2+ concentration. GE, preferably in a concentration of 1 g/l, applied acutely in the experiment, markedly slowed down this process of ternary aggregational nanoplaque complexation at all Ca2+ concentrations used. In a normal blood Ca2+ concentration of 2.52 mmol/l, the garlic induced reduction of nanoplaque formation and molecular size amounted to 14.8 % and 3.9 %, respectively, as compared to the controls. Furthermore, after ternary complex build-up. GE similar to HLD, was able to reduce nanoplaque formation and size. The incubation time for HDL and garlic was only 30 min each in these experiments. Nevertheless, after this short time the deposition of the ternary complex decreased by 6.2 % resp. 16.5 %, i.e. the complex aggregates were basically resolvable. Conclusions: These experiments clearly proved that garlic extract strongly inhibits Ca2+ binding to HS-PG. In consequence, the formation of the ternary HS-PG/LDL/Ca2+ complex, initially responsible for the nanoplaque´composition and ultimately for the arteriosclerotic plaque generation, is decisively blunted.

An alteration of apolipoprotein (apo) B-100 structure by direct oxidative modification is supposed to be an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification owing to a lack of specific assays. We evaluated a methodology based on the oxidation of protein arginine and proline to γ-glutamyl semialdehyde which by reduction forms 5-hydroxy-2-aminovaleric acid (HAVA). We determined HAVA by using derivatization to N(O)-ethoxycarbonyl ethyl esters and gas chromatography–mass spectrometry in different low-density lipoprotein preparations subjected to oxidative damage in the presence of iron. Results suggest that apoB-100 proline and arginine residues are highly reactive toward oxygen radicals ex vivo. Femtomole levels of HAVA can be reproducible measured. HAVA determination compares well with the measurement of carbonyl group formation used as a generally accepted but nonspecific index of protein oxidation. Thus, HAVA could prove to be a sensitive assay for studying specific modification of apoB-100.

A short run-time in separation of lipoproteins by preparative ultracentrifugation is desirable for several reasons. Recently, a method was described that needs only 100 min for one run in a centrifugal field of 625,000 x g. It is assumed that lipoprotein separation depends on the rotor speed but this has not been systematically studied in centrifugal fields of this order. We performed such a study. Rotor speeds of 120, 90, 60 and 30 x 10(3) rev./min and run-times of 100 min, 3 h, 6.7 h and 27 h were selected in such a way that the product of centrifugal field and run-time remained constant. The first conditions correspond to the 'very fast ultracentrifugation' (VFU) procedure with a centrifugal field of 625,000 x g. The Optima tabletop ultracentrifuge, the rotor TLA-120.2 and thick wall open tubes for 1 ml were used. Thirteen different plasma samples covering a wide range of cholesterol and triglyceride concentrations were separated into VLDL, LDL and HDL in the course of two centrifugal runs at densities of 1.006 and 1.063. The constituents of the lipoproteins were calculated considering the mass of the tube contents after slicing. Recoveries of cholesterol, triglycerides and protein were 97%, 98% and 90%, respectively. The influence of the rotor speed on the apparent composition of the lipoproteins was small. With increasing rotor speed, VLDL-cholesterol became higher (by 14%, P < 0.001), VLDL-triglyceride became lower (by 6%, P < 0.012), LDL-cholesterol became lower (by 9%, P < 0.000). The effects on LDL-triglyceride and on HDL-cholesterol and HDL-triglyceride, did not reach statistical significance. Protein in VLDL and in LDL decreased and increased in 'HDL' (the subnatant of the LDL run). As checked by SDS-PAGE the protein effects were due to complete disappearance of albumin from VLDL and LDL while the apolipoproteins B-100, E and C-I to C-III remained unaffected. It is concluded that the main advantages of VFU are the short run-time and the disappearance of albumin from VLDL and LDL. The other compositional changes need to be further investigated.

The melanocortin-1 receptor is known to be overexpressed in melanoma. Thus, it is a potential target for novel α-MSH peptide derivatives aiming at diagnosis and therapy of melanoma. In this study, NOTA-NCS was conjugated with two peptides: NAP-NS1, a linear peptide with 9 amino acids (Ahx-βAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2) and NAP-NS2, a lactam bridge-cyclized peptide with 12 amino acids (ε-Ahx-β-Ala-cyclo(Lys-Glu-His-D-Phe-Arg-Trp-Glu)-Arg-Pro-Val-NH2) each with the sequence His-Phe-Arg-Trp for biological activity. Four α-MSH derivatives were investigated in competition assays in murine B16-F10 and human MeWo melanoma cells. (S)-p-NH2-Bn-NOTA (NOTA-NAP-NS1) labeled with 64Cu and 68Ga, showing no transchelation in the cysteine and histidine challenge, was applied in saturation assays. Determination of octanol/water partition coefficients suggested that [64Cu]Cu-NOTA-NAP-NS1 had high hydrophilicity, and in buffer and serum it was stable after 1 h and 24 h. NAP-NS1 and NOTA-NAP-NS1 showed higher affinity than the cyclic derivatives. Linking the chelate unit at the peptide was accompanied by some loss of affinity. Saturation studies with the labeled peptide resulted in Kd values in the lower nanomolar range for [64Cu]Cu-NOTA-NAP-NS1 and [68Ga]Ga-NOTA-NAP-NS1, respectively. Thus, both radiolabeled peptides appear to be promising for further investigations in animal melanoma models. This research was supported by MIUR (PRIN 2008F5A3AF_002).

Ligands based on 3,7-diazabicyclo[3.3.1]nonane (bispidine) form very stable coordination compounds, in particular with first row transition metal ions. Considering multiple functionalization, bispidines are promising candidates for pharmaceutical targeting and multimodal imaging. Due to the formation of thermodynamically stable and kinetically inert CuII complexes, penta- and hexadentate bispidine ligands are well suited for 64Cu positron emission tomography imaging and radiotherapy (64Cu/67Cu). The bispidine scaffold paves the way for introducing further functionalities, such as targeting units and fluorescence labels, which broadens the scope regarding pharmaceutical targeting and dual labelling (PET and optical imaging). Several bispidine ligands have been developed in order to improve the radiopharmaceutical behavior as well as possibilities for further beneficial functionalization. Variable denticity (tetra-, penta- and hexadentate) with different donor groups, such as amino, amido, pyridine and/or methoxypyridine functionalities allows for tuning properties such as complexation and lipophilicity. These ligands and the important properties of their CuII complexes, e.g., stabilities, ligand exchange kinetics, serum stability, partition coefficients ([64Cu]Cu-bispidine: n-octanol/water) and biodistribution studies will be reported.