Enhanced potency of recombinant factor VIIa with increased affinity to activated platelets.

BACKGROUND: Recombinant factor VIIa (rFVIIa) enhances thrombin generation in a platelet-dependent manner; however, rFVIIa binds activated platelets with relatively low affinity. Triggering receptor expressed on myeloid cells (TREM)-like transcript (TLT)-1 is expressed exclusively on activated platelets. OBJECTIVE: To enhance the potency of rFVIIa via binding TLT-1. METHODS: Recombinant FVIIa was conjugated to a TLT-1 binding Fab. In vitro potency of this platelet-targeted rFVIIa (PT-rFVIIa) was evaluated using factor X activation assays and by measuring viscoelastic changes in whole blood. In vivo potency was evaluated using a tail vein transection model in F8-/- mice expressing human TLT-1. RESULTS: PT-rFVIIa and rFVIIa had similar dissociation constant values for tissue factor binding and similar tissue factor-dependent factor X activation. However, PT-rFVIIa had increased catalytic efficiency on TLT-1-loaded vesicles and activated platelets. The in vitro potency in normal human blood with antibody-induced hemophilia A was dependent on assay conditions used; with maximally activated platelets, the half maximal effective concentration for clot time for PT-rFVIIa was 49-fold lower compared with rFVIIa. In the murine bleeding model, a 53-fold lower half maximal effective concentration was observed for blood loss for PT-rFVIIa, supporting the relevance of the assay conditions with maximally activated platelets. In vitro analysis of blood from subjects with hemophilia A confirmed the data obtained with normal blood. CONCLUSIONS: Increasing the affinity of rFVIIa to activated platelets resulted in approximately 50-fold increased potency both in vitro and in the mouse model. The correlation of in vivo with in vitro data using maximally activated platelets supports that these assay conditions are relevant when evaluating platelet-targeted hemostatic concepts.

Long-acting CCK analogue NN9056 lowers food intake and body weight in obese Göttingen Minipigs.

BACKGROUND/OBJECTIVES: Cholecystokinin (CCK) is a regulator of appetite and energy intake in man. The aim of this study was to determine the effect of NN9056, a long-acting CCK-1 receptor-selective CCK analogue, on food intake and body weight (BW) in obese Göttingen Minipigs. SUBJECTS/METHODS: Tolerability of NN9056 and acute effects on food intake, pancreas histology, amylase and lipase levels were assessed in lean domestic pigs in doses up to 100 nmol/kg (n = 3-4). Subsequently, obese Göttingen Minipigs were treated subcutaneously (s.c.) once daily for 13 weeks with vehicle, NN9056 low dose (regulated from 5 to 2 nmol/kg) or NN9056 high dose (10 nmol/kg) (n = 7-8). Food intake was measured daily and BW twice weekly. At the end of the treatment period, an intravenous glucose tolerance test (IVGTT) and a 24-h exposure profile was obtained. Data are mean ± SD. RESULTS: The acute studies in domestic pigs showed significant and dose-dependent effect of NN9056 on food intake, acceptable tolerability and no histopathological signs of pancreatitis. Sub-chronic treatment in obese Göttingen Minipigs was also well tolerated and accumulated food intake was significantly lower in both treated groups compared to vehicle, with no significant difference between the dose levels of NN9056 (41.8 ± 12.6, 51.5 ± 13.8 and 86.5 ± 19.5 kg in high-dose, low-dose and vehicle groups, respectively, p = 0.012 and p < 0.0001 for low and high dose vs. vehicle, respectively). Accordingly, there was a weight loss in both treated groups vs. a weight gain in the vehicle group (-7.2 ± 4.6%, -2.3 ± 3.2% and 12.3 ± 3.9% in the high-dose, low-dose and vehicle groups, respectively, p < 0.0001 for both vs. vehicle). IVGTT data were not significantly different between groups. CONCLUSION: NN9056, a long-acting CCK-1 receptor-selective CCK analogue, significantly reduced food intake and BW in obese Göttingen Minipigs after once daily s.c. dosing for 13 weeks.

Proteomics-Based Comparative Mapping of the Secretomes of Human Brown and White Adipocytes Reveals EPDR1 as a Novel Batokine.

Adipokines secreted from white adipose tissue play a role in metabolic crosstalk and homeostasis, whereas the brown adipose secretome is less explored. We performed high-sensitivity mass-spectrometry-based proteomics on the cell media of human adipocytes derived from the supraclavicular brown adipose and from the subcutaneous white adipose depots of adult humans. We identified 471 potentially secreted proteins covering interesting categories such as hormones, growth factors, extracellular matrix proteins, and proteins of the complement system, which were differentially regulated between brown and white adipocytes. A total of 101 proteins were exclusively quantified in brown adipocytes, and among these was ependymin-related protein 1 (EPDR1). EPDR1 was detected in human plasma, and functional studies suggested a role for EPDR1 in thermogenic determination during adipogenesis. In conclusion, we report substantial differences between the secretomes of brown and white human adipocytes and identify novel candidate batokines that can be important regulators of human metabolism.

TAB1 modulates IL-1alpha mediated cytokine secretion but is dispensable for TAK1 activation.

Biochemical evidence indicates that TGF-beta-activated kinase 1 (TAK1), a key modulator of the inflammatory response, exists in a complex with various adaptor proteins including the TAK1 binding protein 1 (TAB1). However, the physiological importance of TAB1 in TAK1 activation, and in the subsequent induction of proinflammatory mediators, remains unclear. In this study, a critical role for TAK1 in IL-1alpha or TNFalpha stimulated MAPK and NFkappaB activation was confirmed by inhibition of the nuclear accumulation of NFkappaB p65 and phosphorylated forms of c-Jun and p38 following siRNA mediated TAK1 silencing. These effects were associated with significant reductions in IL-1alpha stimulated levels of secreted IL-6, IL-8, MCP-1 and GM-CSF. In contrast, IL-1alpha or TNFalpha dependent cellular redistribution of NFkappaB p65 and phosphorylated c-Jun and p38 was not affected by 80% siRNA mediated knockdown of TAB1 protein levels. Interestingly, IL-6, IL-8 and GM-CSF release from TAB1 siRNA transfected cells was significantly reduced following IL-1alpha treatment, but was unchanged after TNFalpha stimulation, suggesting differential roles for TAB1 in IL-1alpha and TNFalpha signalling pathways. These findings may imply an as yet unidentified role for TAB1 in the inflammatory response, which is independent of the activation of classical TAK1 associated signalling cascades.

Inflammatory pathway analysis using a high content screening platform.

High content cellular screening assays are useful tools to investigate the interplay between signaling pathways and offer valuable platforms to determine the mode of action, potency, and selectivity of potential drug candidates in a biological setting. We describe a cell-based multiplex fluorescent imaging assay that permits concurrent detection and quantification of the distribution of nuclear factor kappaB (NFkappaB) p65/RelA and phosphorylated forms of p38 and c-Jun between the cytosol and nucleus. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). A significant window between untreated and interleukin-1alpha (IL-1alpha) stimulated HeLa cells for all three targets was achieved with low variability. Staining specificity was confirmed with blocking peptides and pharmacological inhibition of p38, c-Jun-N-terminal kinase (JNK), and inhibitory kappaB kinase 2, and channel bleed-through was eliminated or counterbalanced by the use of fixed exposure times together with careful reporter channel selection. The JNK inhibitor SP600125 was used as a demonstration compound because in addition to inhibiting nuclear accumulation of phosphorylated c-Jun it reduced nuclear translocation of phosphorylated p38 and NFkappaB p65/RelA in a dose-dependent manner, indicating a lack of SP600125 selectivity. This was supported by RNA interference where co-transfection of small interfering RNA targeting both JNK1 and JNK2, to limit signaling redundancy, significantly inhibited IL-1alpha-stimulated translocation of phosphorylated c-Jun without altering phosphorylated p38 and NFkappaB p65/RelA redistribution. This image analysis application is a valuable and information-rich screening tool to investigate compound selectivity and/or cross-talk between key signaling pathways involved in the inflammatory response.