Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [(18)F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.

Non-invasive imaging procedures to identify atherosclerotic lesions in peripheral or coronary vessels are currently limited. A discovery program identified a low molecular weight gadolinium chelate...

Background BMS-747158-02 is a novel fluorine 18-labeled pyridazinone derivative designed for cardiac imaging. The uptake and retention mechanisms of F-18 BMS-747158-02 in cardiac myocytes were studied in vitro, and the biodistribution of F-18 BMS-747158-02 was studied in vivo in mice. Methods and Results Fluorine 19 BMS-747158-01 inhibited mitochondrial complex I (MC-I) in bovine heart submitochondrial particles with an IC 50 of 16.6±3 nmol/L that was comparable to the reference inhibitors of MC-1, rotenone, pyridaben, and deguelin (IC 50 of 18.2±6.7 nmol/L, 19.8±2.6 nmol/L, and 23.1±1.5 nmol/L, respectively). F-18 BMS-747158-02 had high uptake in monolayers of neonatal rat cardiomyocytes (10.3%±0.7% of incubated drug at 60 minutes) that was inhibited by 200 nmol/L of rotenone (91%±2%) and deguelin (89%±3%). In contrast, an inactive pyridaben analog, P-0 (IC 50 value>4 μmol/L in MC-1 assay), did not inhibit the binding of F-18 BMS-747158-02 in cardiomyocytes. Uptake and washout kinetics for F-18 BMS-747158-02 in rat cardiomyocytes indicated that the time to half-maximal (t 1/2 ) uptake was very rapid (approximately 35 seconds), and washout t 1/2 for efflux of F-18 BMS-747158-02 was greater than 120 minutes. In vivo biodistribution studies in mice showed that F-18 BMS-747158-02 had substatial myocardial uptake (9.5%±0.5% of injected dose per gram) at 60 minutes and heart-to-lung and heart-to-liver ratios of 14.1±2.5 and 8.3±0.5, respectively. Positron emission tomography imaging in the mouse allowed clear cardiac visualization and demonstrated sustained myocardial uptake through 55 minutes. Conclusions F-18 BMS-747158-02 is a novel positron emission tomography cardiac tracer targeting MC-I in cardiomyocytes with rapid uptake and slow washout. These characteristics allow fast and sustained accumulation in the heart.

BACKGROUND:Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders.MATERIALS AND METHODS:The antiangiogenic efficacy of the novel alphavbeta3 antagonist TA138 was examined using in vivo and in vitro model systems.RESULTS:The in vitro studies demonstrated the ability of TA138 and RP747 (conjugated TA138) to inhibit endothelial cell migration toward vitronectin, with an IC50=0.04 and 0.045 microM, respectively. Furthermore, utilizing the chick chorioallantoic membrane models, TA138 inhibited basic fibroblast growth factor-induced neovascularization.CONCLUSION:TA138 might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or other pathological neovascularization processes. RP747 demonstrated antitumor efficacy in 1 spontaneous tumor model (c-neu oncomouse model, alphavbeta3 positive cells) and in 1 xenograft model (HCT116 human tumor colon carcinoma, alphavbeta3 negative cells) injected subcutaneously into nude mice.

Computer-assisted procedures are used to measure infarct areas in animal stroke models, but this approach usually follows the less objective manual tracing of the boundaries of the infarct. Building on previously reported methodology using scanned images of triphenyltetrazolium chloride (TTC)-stained rat brains in the intraluminal thread model, we developed an objective method to assess ischemic damage in both the mouse and rat brains. The unique addition to our approach is the use of sham-treated animals, which thereby permits the removal of normal brain white matter from the ipsilateral injured brain. All brain sections per animal were scanned simultaneously using a Microtek Scanmaker 4 flatbed scanner. Color segmentation on full color images of 2 mm coronal brain sections was performed. Using Image Pro Plus (4.0) and color segmentation, ischemic and normal white matter areas were measured in the green channel and the entire brain area in the red channel. The percent of unstained tissue was calculated for sham-treated animals and for those with cerebral ischemia. By subtracting the average unstained area of the sham-treated group from the average unstained area from the ischemic group, the ischemic area was calculated. This methodology was validated using mouse and rat permanent and transient, focal ischemia models and MK-801 in the permanent ischemia models. MK-801, dosed at 3 mg/kg i.p. prior to the injury, reduced the injury by 75% in the mouse and 44% in the rat permanent occlusion models. The benefits of this methodology include: objectivity of the analysis of the ischemic injury, use of readily available software so that costs can be contained and removal of normal subcortical white matter from the calculation. This method should allow more consistent evaluation of changes in the infarct size, therefore, resulting in reduced variability and higher productivity.

Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.

Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.

Recent evidence supports the involvement of integrins in angiogenesis: blockade of alpha v beta 3 and alpha v beta 5 integrins disrupts angiogenesis leading to decreased blood vessel formation and hence decreased tumor growth. We hypothesized that av antagonists could inhibit tumor growth in tumor cells devoid of alpha v beta 3 integrins. We evaluated SM256 and SD983, novel small molecules that are specific av antagonists in mouse models of angiogenesis and tumorigenesis, and compared them with standards: TNP470, a fumagillin analog in the clinic, and flavopiridol, a cell cycle kinase inhibitor. In vitro SM256 was a selective alpha v beta 3 inhibitor with an IC50 = 4nM, and the affinity of SD983 against the mouse endothelial alpha v beta 3 integrin yielded an IC50 = 2nM and an IC50 = 54nM against alpha v beta 5. In the mouse Matrigel model of angiogenesis SM256 decreased blood vessel formation (hemoglobin content) with an ED50 = 0.055 ug/kg/day, tenfold more potent than TNP470. SG545, an ester of SD983, decreased blood vessel formation with an ED50 = 6 ug/kg/day, while flavopiridol ED50 = 18 ug/kg/day. In the mouse xenograft model, using human colon carcinoma RKO cells that do not express alpha v beta 3 but express alpha v beta 5, tumor growth was inhibited by SG545 (10 mg/kg/day) and flavopiridol (5 mg/kg/every other day) 40% and 70%, respectively (p < 0.05). Although the proliferative index (measured by BrdU incorporation) was not significantly changed with SM256, SG545 or flavopiridol (29-32%), the apoptotic index increased significantly (p < 0.05) in the SM256 and SG545-treated groups (2.3-2.7%) compared with controls (1.1%), suggesting increased cell death contributed to decreased tumor volumes. Neovascularization decreased with SM256 and SG545 treatment. The data demonstrate that potent selective av antagonist can target endothelial cells, tumor cells, inhibit angiogenesis and inhibit tumor growth.

Activated endothelium and some dendritic cells express the adhesion molecule VCAM1, a member of the immunoglobulin gene superfamily. Mononuclear leukocytes display the integrin VLA4 that functions as a counter-receptor for VCAM1. The interaction of VCAM1 with VLA4 mediates cell to cell adhesion events believed to be important regulators of inflammation, cancer cell metastasis, and atherosclerosis. This report describes the development of a fluorescent adhesion assay that specifically measures T cell adhesion to recombinant human VCAM1 (rVCAM1) expressed in a baculovirus expression vector system (BEVS). We describe a simple and rapid protocol to partially purify non-denatured rVCAM1 from insect cell membrane preparations (VCAM1 infected Sf9 cells). Jurkat cells, a T cell line expressing VLA4, specifically adhered to the rVCAM1 membrane preparations coated onto 96-well plates. Jurkat cells did not adhere to control membrane preparations that lacked rVCAM1 protein. Both unstimulated and IL-2 stimulated Jurkat cells displayed functional VLA4 capable of binding to immobilized rVCAM1. Monoclonal antibodies recognizing either VCAM1 (E1/6, BBA6) or VLA4 (HP2/1) blocked specific VCAM1/VLA4 adhesion, whereas a monoclonal antibody to the alpha chain of LFA1 did not block adhesion. The methods described here could be applied to develop similar functional assays for other cell surface receptors/counterreceptors expressed in a BEVS.