Increased energy expenditure and leptin sensitivity account for low fat mass in myostatin-deficient mice.

Myostatin deficiency causes dramatically increased skeletal muscle mass and reduced fat mass. Previously, myostatin-deficient mice were reported to have unexpectedly low total energy expenditure (EE) after normalizing to body mass, and thus, a metabolic cause for low fat mass was discounted. To clarify how myostatin deficiency affects the control of body fat mass and energy balance, we compared rates of oxygen consumption, body composition, and food intake in young myostatin-deficient mice relative to wild-type (WT) and heterozygous (HET) controls. We report that after adjusting for total body mass using regression analysis, young myostatin-deficient mice display significantly increased EE relative to both WT (+0.81 ± 0.28 kcal/day, P = 0.004) and HET controls (+0.92 ± 0.31 kcal/day, P = 0.005). Since food intake was not different between groups, increased EE likely accounts for the reduced body fat mass (KO: 8.8 ± 1.1% vs. WT: 14.5 ± 1.3%, P = 0.003) and circulating leptin levels (KO: 0.7 ± 0.2 ng/ml vs. WT: 1.9 ± 0.3 ng/ml, P = 0.008). Interestingly, the observed increase in adjusted EE in myostatin-deficient mice occurred despite dramatically reduced ambulatory activity levels (-50% vs. WT, P < 0.05). The absence of hyperphagia together with increased EE in myostatin-deficient mice suggests that increased leptin sensitivity may contribute to their lean phenotype. Indeed, leptin-induced anorexia (KO: -17 ± 1.2% vs. WT: -5 ± 0.3%) and weight loss (KO: -2.2 ± 0.2 g vs. WT: -1.6 ± 0.1, P < 0.05) were increased in myostatin-deficient mice compared with WT controls. We conclude that increased EE, together with increased leptin sensitivity, contributes to low fat mass in mice lacking myostatin.

Hypothalamic inflammation and energy homeostasis: resolving the paradox.

Determining the effect of hypothalamic inflammatory signals on energy balance presents a paradox. On the one hand, a large body of work has identified inflammatory signaling in the hypothalamus as an essential mediator of the sickness response--the anorexia, cachexia, fever, inactivity, lethargy, anhedonia and adipsia that are triggered by systemic inflammatory stimuli and promote negative energy balance. On the other hand, numerous recent studies implicate inflammatory activation within the hypothalamus as a key factor whereby high-fat diets--and saturated fats in particular--cause central leptin and insulin resistance and thereby promote the defense of elevated body weight. This paradox will likely remain unresolved until several issues have been addressed. Firstly, the hypothalamus--unlike many peripheral inflamed tissues--is an extremely heterogeneous tissue comprised of astrocytes, oligodendrocytes, microglia, endothelial cells, ependymal cells as well as numerous neuronal subgroups. Determining exactly which cells activate defined inflammatory signals in response to a particular stimulus--i.e. sepsis vs. nutrient excess--may yield critical clues. Secondly, for the sake of simplicity many studies evaluate inflammation as an on/off phenomenon. More realistically, inflammatory signaling occurs as a cascade or cycle that changes and progresses over time. Accordingly, even within the same cell type, the low-grade, chronic signal induced by nutrient excess may invoke a different cascade of signals than a strong, acute signal such as sepsis. In addition, because tolerance can develop to certain inflammatory mediators, physiological outcomes may not correlate with early biochemical markers. Lastly, the neuroanatomical location, magnitude, and duration of the inflammatory stimulus can undoubtedly influence the net CNS response. Rigorously evaluating the progression of the inflammatory signaling cascade within specific hypothalamic cell types is a key next step towards resolving the paradox surrounding the effect of inflammatory signaling on energy homeostasis.

Cultured hypothalamic neurons are resistant to inflammation and insulin resistance induced by saturated fatty acids.

Hypothalamic inflammation induced by high-fat feeding causes insulin and leptin resistance and contributes to the pathogenesis of obesity. Since in vitro exposure to saturated fatty acids causes inflammation and insulin resistance in many cultured cell types, we determined how cultured hypothalamic neurons respond to this stimulus. Two murine hypothalamic neuronal cell cultures, N43/5 and GT1-7, were exposed to escalating concentrations of saturated fatty acids for up to 24 h. Harvested cells were evaluated for activation of inflammation by gene expression and protein content. Insulin-treated cells were evaluated for induction of markers of insulin receptor signaling (p-IRS, p-Akt). In both hypothalamic cell lines, inflammation was induced by prototypical inflammatory mediators LPS and TNFalpha, as judged by induction of IkappaBalpha (3- to 5-fold) and IL-6 (3- to 7-fold) mRNA and p-IkappaBalpha protein, and TNFalpha pretreatment reduced insulin-mediated p-Akt activation by 30% (P < 0.05). By comparison, neither mixed saturated fatty acid (100, 250, or 500 microM for

Double-stranded RNA mediates interferon regulatory factor 3 activation and interleukin-6 production by engaging Toll-like receptor 3 in human brain astrocytes.

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. In this study, human brain astrocytes were found to constitutively express TLR3, and this expression was increased by interferon-gamma (IFN-gamma) or double-stranded RNA (dsRNA). Treatment employing dsRNA in astrocytes induced IFN regulatory factor 3 (IRF3) phosphorylation, dimer formation and nuclear translocation followed by STAT1 activation. This treatment also activated nuclear factor-kappaB, p38 and c-Jun N-terminal kinase significantly, while activating extracellular signal-regulated kinase to a lesser extent. Treatment with anti-TLR3 antibody inhibited dsRNA-mediated interleukin-6 (IL-6) production. In the presence of mitogen-activated protein kinase inhibitors, astrocytes failed to secrete IL-6 in response to dsRNA treatment. Therefore, dsRNA-induced IL-6 production is dependent on mitogen-activated protein kinases and type I IFN production is dependent on IRF3 in brain astrocytes. These results suggest that brain inflammation, which produces inflammatory cytokines and type I IFNs, may enhance TLR3 expression in astrocytes. Additionally, upregulated TLR3 might modulate inflammatory processes by producing proinflammatory cytokines.

Central venous catheterization-induced right brachiocephalic vein pseudoaneurysm: Successfully treated with stent-assisted coiling.

INTRODUCTION: Central venous catheterization-induced central vein pseudoaneurysm is rare. Several treatment options have been recommended. We describe a case of central venous catheterization-induced right brachiocephalic vein pseudoaneurysm successfully treated with an uncovered self-expandable stent-assisted coil embolization and discuss the imaging findings, treatment strategy, and review of literature associated with thoracic venous pseudoaneurysm. CASE REPORT: A 77-year-old woman was referred to our trauma center to undergo treatment for central venous catheterization-induced central vein pseudoaneurysm. The initial contrast-enhanced chest computed tomography revealed a 3.4-cm pseudoaneurysm arising from the right brachiocephalic vein and a surrounding mediastinal hematoma. The pseudoaneurysm was successfully embolized with stent-assisted coiling. Computed tomography angiography was performed 10 days after the procedure and demonstrated a completely embolized pseudoaneurysm and resolved mediastinal hematoma. Blood flow from the right subclavian and left innominate veins was not disturbed by the stent-assisted coils. CONCLUSION: To our knowledge, this is the first report of treatment of a right brachiocephalic vein pseudoaneurysm with stent-assisted coil embolization. We think that uncovered stent-assisted coil embolization is the safest and most fundamental treatment for wide-neck venous pseudoaneurysm especially in a hemodynamically unstable setting.

Femoral versus jugular access for Denali Vena Cava Filter placement: Analysis of fluoroscopic time, filter tilt and retrieval outcomes.

PURPOSE: To analyze relevant metrics involved in Denali Vena Cava Filter placement via different venous access sites. MATERIALS AND METHODS: Patients with Denali filters inserted between March 2017 and February 2018 were retrospectively analyzed. Pre-procedural and pre-retrieval computed tomography (CT) were reviewed. We compared inferior vena cava (IVC) diameter, filter tilt angle, filter tip IVC wall abutment, fluoroscopy time, and retrieval outcomes by venous access site. Filter tip abutment/limb penetration and procedure-related complications were investigated. RESULTS: Seventy-eight patients had successfully-placed Denali filters. Seventy-one of 78 (91%) patients had both pre-procedural and pre-retrieval CT. The majority (35 [49%]) were placed via the right femoral vein (left femoral vein: 22 [31%]; right internal jugular vein: 14 [20%]). The jugular approach involved a longer fluoroscopy time (mean 117 ±â€¯37 s [s]) than the right and left femoral approaches (mean 64 ±â€¯21 s, mean 67 ±â€¯15 s, respectively [p < 0.05]). Filter tilt and filter tip abutment were not significantly different between the 3 access routes. Filter tip abutment and limb penetration were observed in 8/71 (11%) and 2/71 (3%) patients, respectively. Filter retrieval was attempted in 68 of 78 (87%) cases, and all filters were successfully retrieved. One filter arm fractured during advanced retrieval; no other procedure related complications were recorded. CONCLUSIONS: Both femoral venous approaches can be safely used for placement of the Denali filter. Femoral venous access involved a shorter fluoroscopy time without any differences in filter tilt and filter tip abutment compared to transjugular access.

Proteus vulgaris and Proteus mirabilis Decrease Candida albicans Biofilm Formation by Suppressing Morphological Transition to Its Hyphal Form.

PURPOSE: Candida albicans (C. albicans) and Proteus species are causative agents in a variety of opportunistic nosocomial infections, and their ability to form biofilms is known to be a virulence factor. In this study, the influence of co-cultivation with Proteus vulgaris (P. vulgaris) and Proteus mirabilis (P. mirabilis) on C. albicans biofilm formation and its underlying mechanisms were examined. MATERIALS AND METHODS: XTT reduction assays were adopted to measure biofilm formation, and viable colony counts were performed to quantify yeast growth. Real-time reverse transcriptase polymerase chain reaction was used to evaluate the expression of yeast-specific genes (rhd1 and rbe1), filament formation inhibiting genes (tup1 and nrg1), and hyphae-related genes (als3, ece1, hwp1, and sap5). RESULTS: Candida biofilm formation was markedly inhibited by treatment with either living or heat-killed P. vulgaris and P. mirabilis. Proteus-cultured supernatant also inhibited Candida biofilm formation. Likewise, treatment with live P. vulgaris or P. mirabilis or with Proteus-cultured supernatant decreased expression of hyphae-related C. albicans genes, while the expression of yeast-specific genes and the filament formation inhibiting genes of C. albicans were increased. Heat-killed P. vulgaris and P. mirabilis treatment, however, did not affect the expression of C. albicans morphology-related genes. CONCLUSION: These results suggest that secretory products from P. vulgaris and P. mirabilis regulate the expression of genes related to morphologic changes in C. albicans such that transition from the yeast form to the hyphal form can be inhibited.

Differential expression, shedding, cytokine regulation and function of TNFR1 and TNFR2 in human fetal astrocytes.

Tumor necrosis factor (TNF)-alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF-alpha receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF- receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.

The availability of contrast media in the application of Holmium-166-DTPA for vascular brachytherapy.

Since coronary angioplasty using a liquid radiation source is performed with computed tomography(CT) angiography, use of a CT contrast agent is a good alternative to see if the balloon has close contact with the blood vessel wall for the delivery of a sufficient radiation dose to the stenotic artery. In order to examine the usefulness of the CT contrast agent as a diluent of a liquid radiation source, various physicochemical studies and in vivo stability studies using animals were implemented using (166)Ho-DTPA for vascular brachytherapy and a PTCA balloon catheter. For this study, three CT contrast agents, Hexabrix (320)(Rx), Iomeron (350)(Rx) and Visipaque (320)(Rx) were used. Results showed that (166)Ho radiolabeled component of Hexabrix (320)(Rx) and the (166)Ho-complex was proposed to be (166)Ho-EDTA. However, in the case of Iomeron (350)(Rx) and Visipaque (320)(Rx), no other (166)Ho-complex was formed except the desired (166)Ho-DTPA. In the case where (166)Ho-EDTA (>98% radiolabeling yield) was administrated to rabbits, only 10% of the administered dose was excreted through the urinary track 30 min after injection. However, in the animal experiment where Hexabrix (320)(Rx) was added to the (166)Ho-DTPA vial with the volume ratio of 1:1, over 80% of the administrated dose accumulated into the bladder within 30 min after injection. Therefore, Hexabrix (320)(Rx) is applicable when it is used as a diluent of a (166)Ho-based liquid radiation source and its volume is applied in a minimal manner to visualize the balloon catheter. In conclusion, the use of a CT contrast agent in the clinical application of a liquid radiation source has beneficiary effects such as visualization of both the position and shape of the balloon are possible and most importantly, whether or not there is a formation of a void volume of liquid inside the balloon as well as the detection of radiation leakage on a real-time basis, on site during the angioplasty.

Holmium-166-DTPA as a liquid source for endovascular brachytherapy.

Liquid radiation sources with beta emitters have advantages of accurate positioning and uniform dose distribution to the vessel walls to prevent the restenosis of coronary artery. As a liquid radiation source, 166Ho-DTPA was prepared and evaluated its in-vivo pharmacokinetic behavior through animal studies.166Ho-DTPA was prepared by simple mixing the Holmium with DTPA at room temperature. The radiolabelling yield was 100% when the DTPA/Holmium molar ratio was >2. Radiolabelling of 166Ho-DTPA was not dependent on the pH range of 1.7-7.5. High radiochemical stability (>98%) was maintained over a period of 6 hours even with a radioactivity ( approximately 11.1 GBq/12 mg of DTPA) stored at room temperature. Biodistribution of 166Ho-DTPA in rats and gamma camera images in rabbits showed that 166Ho-DTPA was quickly excreted via the urinary system. The average of T(max) and T(1/2) of 166Ho-DTPA in the kidneys of rabbits were 3.71 +/- 1.18 min and 9.15 +/- 3.15 min. 166Ho-DTPA is a potential liquid radiation source for radiation brachytherapy to prevent the restenosis of the coronary artery using a liquid-filled balloon.

Characterization of the two genes differentially expressed during development in human fetal astrocytes.

Astrocytes are ubiquitous in the brain and have multiple functions. It is becoming clear that they play an important role in monitoring the neuromicroenvironment, information processing, and signaling in the central nervous system (CNS) in normal conditions and that they respond to CNS injuries. During the development of the CNS, astrocytes play a key role as a substrate for neuronal migration and axonal growth. To identify genes that could participate in astrocyte maturation, we used the differential display reverse transcription-PCR (DDRT-PCR) method. Human fetal astrocytes were cultured and total RNAs were isolated at intervals of 5 days for 50 days. Using 24 primer combinations, we identified a set of 18 candidate cDNAs deriving from the excised DDRT-PCR bands. DNA sequencing revealed 16 genes that have been described already. We found that RTP, TG, hTM-alpha, SPARC, TRIP7, and RPL7 genes were expressed increasingly, while HMGCR, RPL27a, NACA, NPM, and TARBP2 genes were expressed decreasingly, according to their culture stages. We also found two unidentified genes, A3 and C8, which were expressed differently in culture stages; the former was expressed decreasingly and the latter increasingly. These two genes were found in the same amount in genomic DNA from various human cells such as astrocytes, astrocytoma, trophoblasts and lymphocytes. The A3 gene was found only in human genomic DNA, but not in rat (ATr5), mouse (RAW264.7), or monkey (Vero) cells, whereas the C8 gene was found in human genomic DNA and monkey cells, but not in rat or mouse cells. We analysed these two genes for identification. There was >92% nucleotide sequence identity between the A3 gene (3,626 bp) and the Homo sapiens general transcription factor 3 (GTF3), and >96% nucleotide sequence identity between the C8 gene (2,401 bp) and the transmembrane receptor Unc5h2. These findings suggest that these two genes may participate in some functional roles within the cells.

Preparation and in-vivo evaluation of 99mTc-IOTIDA for cholescintigraphy.

The synthesis, radiolabeling and in vivo evaluation of 99mTc-IOIDA(3-iodo 2,4,6-trimethylpheyl carbamoylmethyl iminodiacetic acid) for the assessment of hepatocytic function and the functional status of the cystic duct and the gallbladder are described. For a scintigraphic imaging comparison, three different 99mTc-IDA derivatives, 99mTc-DISIDA, 99mTc-mebrofenin and 99mTc-IOTIDA, were prepared and evaluated for their in vivo pharmacokinetic behavior through animal studies. Serial static image scans of rabbits injected with 99mTc-IOTIDA revealed that none of the tissues except the hepatobiliary system showed radioactivity concentrations. A scintigraphic study in a healthy volunteer showed that most of the administrated radioactivity accumulated in the liver and was rapidly excreted through the hepatobiliary system, visualizing the gallbladder within 15 min. In conclusion, 99mTc-IOTIDA is a potential hepatobiliary imaging agent for the evaluation of the functional status of hepatocytes and the patency of the biliary duct.

166Ho-coated balloon for vascular brachytherapy.

A simple procedure was developed for coating the surface of a conventional percutaneous transluminal coronary angioplasty balloon angioplasty catheter with 166Ho. The absorbed dose delivered by the surface-coated catheter is twice that of a similar catheter filled with a solution containing the same activity of 166Ho. Leakage tests show that <0.6% of the coated activity is removable from the surface of the catheter. The coated catheter removes the risk of release of the 166Ho into the body as a result of rupture of the balloon, and also reduces the radiation exposure to staff.

Influence of bacterial presence on biofilm formation of Candida albicans.

PURPOSE: Candida albicans is an opportunistic pathogen that is commonly found in human microflora. Biofilm formation (BF) is known as a major virulence factor of C. albicans. The aim of this study was to examine the influence of bacterial presence on biofilm formation of C. albicans. MATERIALS AND METHODS: The BF of Candida was investigated when it was co-cultured with C. albicans (C. albicans 53, a yeast with a low BF ability, and C. albicans 163, a yeast with high BF ability) and bacteria. BF was assessed with XTT reduction assay. A scanning electron microscope was used to determine the structure of the biofilm, and real-time reverse transcriptase polymerase chain reaction was used to amplify and quantify hyphae-associated genes. RESULTS: Co-culturing with two different types of bacteria increased the BF value. Co-culturing with C. albicans 53 and 163 also increased the BF value compared to the value that was obtained when the C. albicans was cultured individually. However, co-culturing with bacteria decreased the BF value of C. albicans, and the BF of C. albicans 163 was markedly inhibited. The expression of adherence and morphology transition related genes were significantly inhibited by co-culturing with live bacteria. CONCLUSION: Bacteria have a negative effect on the formation of biofilm by C. albicans. This mechanism is the result of the suppression of genes associated with the hyphae transition of C. albicans, and bacteria particles physically affected the biofilm architecture and biofilm formation.

Synthesis and application of a novel cysteine-based DTPA-NCS for targeted radioimmunotherapy.

INTRODUCTION: For the development of safe and effective protein-based radiolabeled complexes such as radioimmunotherapy (RIT), the selection of the radionuclides and the chelating agents used for the radiolabeling of tumor-targeting molecules is a critical factor. We aim to synthesize a novel bifunctional chelating agent containing the isothiocyanate group for easy conjugation with antibodies having the characteristics of high stable chelation with therapeutic radionuclides. METHODS: We have synthesized the DTPA analogue retaining L-cysteine as a core ligand of the thiol group. The chelating power of cysteine-based DTPA-NCS (cys-DTPA-NCS) was compared with that of commercial ρ-SCN-Bn-DTPA. In an application, the cetuximab was radioimmunoconjugated with (177)Lu using cys-DTPA-NCS. The affinity was tested in a cell line overexpressing EGFR. A therapy study was conducted in nude mice with subcutaneous HT-29 xenografts. RESULTS: The cys-DTPA-NCS presents an excellent ability to chelate as compared to the ρ-SCN-Bn-DTPA. For mean ratio chemical labeling yields of 95%, the result was 0.97. (177)Lu-cys-DTPA-NCS-cetuximab was prepared under ambient condition with a high radiolabeling yield and the radiochemical purity was sustained for at least 6days. The IC50 value of the (177)Lu-labeled cetuximab was 10nM (95% confidence). The stability and therapeutic efficacy of the candidate radiopharmaceutical were verified. CONCLUSION: The new DTPA derivative, cys-DTPA-NCS, is a good bifunctional chelating agent that can be used for protein-based radiopharmaceutical using lanthanides such as (177)Lu and (90)Y. The prepared (177)Lu-cys-DTPA-NCS-cetuximab can be used for the diagnosis and treatment of human colorectal tumor.

Immunological features of macrophages induced by various morphological structures of Candida albicans.

Candida albicans is a dimorphic fungus that commensally colonizes human mucosal surfaces. The aim of this study was to assess the role of different C. albicans morphologies in inducing pattern recognition receptors (PRRs) and cytokines in macrophages. Macrophages may respond to pathogen-associated molecular patterns via TLR2 and TLR4 by expressing cytokines. The hyphal transition of C. albicans was induced by 20% serum (S), RPMI-1640 (R), or 39℃ culture (H). Macrophages were then challenged with either yeast (Y) or different hyphae cultures of C. albicans, followed by RT-PCR and FACS analysis of PRRs expression. In addition, macrophages were stimulated with either yeast or different hyphae cultures of C. albicans used by RT-PCR and Bio-Plex analysis of cytokines production. Macrophages expressed high levels of TLR4 and dectin-1 after stimulation with Y cells. In contrast, stimulation with H or R cells strongly increased the expression of TLR2 and dectin-2. Stimulation with Y cells significantly enhanced the expression of IL-1ß and weakly increased the expression of IL-6 and IL-12. Stimulation with hyphal cells (S, R, and H) strongly increased IL-10 expression, but weakly reduced IL-1ß expression. The phagocytosis activity and NO production of macrophages were decreased upon treatment with hyphal cells compared with yeast, and depended on the length of hyphae. In summary, the yeast and hyphae forms of C. albicans resulted in an induction of different PRRs, with accompanying differences in immune cell cytokine profiles.

Anterior mediastinal fat in Behçet's disease: qualitative and quantitative CT analysis.

The fat-rich anterior mediastinum could be a sensitive window for monitoring minute changes in vascularity induced by systemic vasculitis. To evaluate this hypothesis, an analysis of anterior mediastinal fat in patients with Behçet's disease and a control group was conducted. This study included 43 patients diagnosed with Behçet's disease within the last 11 years who underwent CT scan; 55 patients were selected as a control population. Mediastinal fat was classified according to CT morphology. Comparison of serum inflammatory markers was performed for evaluation of disease activity according to morphologic types, and average Hounsfield unit of the anterior mediastinum was measured. Significantly higher mean CT attenuation was observed in the Behçet's disease group, compared with the control group (-48.5 ± 33.5 vs. -67.7 ± 18.7, respectively, P < 0.05). Mediastinal fat types were classified as follows: pure fatty tissue (2 vs. 31 % [Behçet's disease vs. control group]), diffuse soft tissue infiltration (16 vs. 29 %), tubular structures (21 vs. 4 %), mixed infiltration with tubular structures (42 vs. 15 %), and evident thymic tissue (19 vs. 22 %). The value for mean mediastinal attenuation was significantly higher in the group with a high level of C-reactive protein than in the normal level group. The mean CT attenuation of anterior mediastinal fat is significantly higher in the Behçet's disease group, compared with the normal group. Although pathologic confirmation is needed, the cause is postulated to be either inflammatory neovascularization or minimal thymic hyperplasia induced by Behçet's disease.

Synthesis and biological evaluation of a novel (177)Lu-DOTA-[Gly(3)-cyclized(Dap(4), (d)-Phe(7), Asp(10))-Arg(11)]α-MSH(3-13) analogue for melanocortin-1 receptor-positive tumor targeting.

In this study, a novel α-melanocyte stimulating hormone (α-MSH) analogue 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) coupled [Gly(3)-cyclized(Dap(4), (d)-Phe(7), Asp(10))-Arg(11)]α-MSH(3-13) (DOTA-GMSH) for melanocortin-1 receptor (MC-1R) targeting was newly synthesized, radiolabeled with (177)Lu, and in vitro and in vivo characterized. (177)Lu-labeled peptides were prepared with a high radiolabeling yield (>98%), and its Log p value was -2.89. No degradation was observed not only by serum incubation at 37°C for 7 days but also by an HPLC analysis of radioactive metabolites in urine. A cell binding assay revealed that an inhibitory concentration of 50% (IC(50)) of the peptide was 3.80 nM. The tumor-to-blood ratio, which was 14.27 at 2 hours p.i., was increased to 56.37 at 24 hours p.i., which means that the radiolabeled peptide was highly accumulated in a tumor and was rapidly cleared from the blood pool. We, therefore, conclude that (177)Lu-DOTA-GMSH has promising characteristics for application in nuclear medicine, namely for the diagnosis of MC-1R over-expressing tumors.

Preparation and evaluation of 99mTc-labeled cyclic arginine-glycine-aspartate (RGD) peptide for integrin targeting.

Technetium coordination chemistry has been a subject of interest in the development of radiopharmaceuticals, especially imaging radiotracers. Due to the extensive work done on developing chelates for (99m)Tc, various chelators have been investigated and applied to radiopharmceuticals. Previous studies on the coordination chemistry of the [(99m)Tc=O] core have established peptide-derived sequences as effective chelating ligands. These observations led to the design of tetradentate ligands derived from amino acid sequences. Such amino acid sequences provide a tetradentate coordination site for chelation to the radionuclide and an effective functional group for conjugation to biomolecules using conventional solid-phase synthetic routes. A derivative of a novel tripeptide chelating sequence, Pro-Gly-Cys (PGC) has been developed where it is possible to form stable technetium complexes with the [(99m)Tc=O] via N(3)S(1) tetradentate coordination core that serves this function and can be readily incorporated into biomolecules using solid-phase synthesis techniques. As a model system, the RGD peptide was selected which has been well known to target the integrin receptor for angiogenesis and tumor imaging agents. The results of in vivo studies with these novel radiolabeled compounds in tumor xenografts demonstrated a distribution in tumor targeting and other organs, such as kidney, liver and intestines.