Composition comprising PDE-4 inhibitor and H1-receptor antagonist and use thereof for manufacture of medicament for treatment of respiratory diseases
A technology of respiratory system diseases and receptor antagonists, applied in the field of compositions for treating or alleviating the severity of lung diseases, treating lung diseases, compositions for treating lung diseases, capable of solving problems with substantial effects, etc.
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Embodiment 1
[0049] Embodiment 1--Phosphodiesterase and rolipram binding detection experiment
Embodiment 1A
[0051] Ex vivo human monocyte PDE4 and hrPDE (human recombinant PDE4) have been determined to exist mainly in low affinity forms. Therefore, by using 1uM[ 3 H] cAMP as a substrate PDE4 catalytic activity standard assay method (Torphy et al., J.of Biol.Chem., Vol.267, No.3pp1798-1804, 1992) to detect test compounds that antagonize the low-affinity form of PDE4 activity.
[0052] Rat brain high-speed supernatant was used as protein source. preparation[ 3 The enantiomer of H]-rolipram to obtain the specific activity is 25.6Ci / mmol. The published standard experimental conditions were improved to be the same as the PDE detection conditions, except the final cAMP: 50mM Tris HCl (pH 7.5), 5mM MgCl 2 , and 1 nmol of [ 3 H]-rolipram (Torphy et al., Jof Biol. Chem., Vol. 267, No. 3pp 1798-1804, 1992). Experiments were performed at 30°C for 1 hour. The reaction was terminated and free ligand was separated from bound ligand using a Brandel cell harvester. In addition to not contai...
Embodiment 1B
[0054] Detection of phosphodiesterase activity
[0055] use[ 3 H] cAMP scintillation approach assay (SPA) or [ 3 H] The cGMP SPA enzyme assay was used to detect PDE activity as described by the supplier (Amersham Life Sciences). Reactions were performed in 96-well plates at room temperature in 0.1 ml of reaction buffer containing (final concentration): 50 mM Tris-HCl, pH 7.5, 8.3 mM MgCl 2 , 1.7mM EGTA, [ 3 H] cAMP or [ 3 H] cGMP (approximately 2000 dpm / pmol), enzyme and various concentrations of inhibitors. Experiments were run for 1 hour and stopped by adding 50 μl of SPA yttrium silicate beads in the presence of zinc sulfate. The plate was shaken and left at room temperature for 20 minutes. The formation of the radiolabeled product is detected by scintillation spectroscopy. Using 0.05μM [ 3 H]cAMP detects the activity of PDE3 and PDE7, while PDE4 is detected by using 1μM[ 3 H]cAMP was detected as a substrate. The activities of PDE1B, PDE1C, PDE2 and PDE5 were dete...
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