Novel treatment method
A disease and effective dose technology, applied in the field of compositions for treating lung diseases, can solve problems such as identification of difficult disease therapeutic agents
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Embodiment 1
[0045] Example 1 - Phosphodiesterase and Cyclopentyloxymethoxyphenylpyrrolidone Binding Assay
Embodiment 1A
[0047] Determination of PDE4 and hrPDE (human recombinant PDE4) in isolated human monocytes, mainly in the form of low affinity. Therefore, the activity of test compounds against low affinity forms of PDE4 can be assessed using standard PDE4 catalytic activity assays using 1 μM [ 3 H] cAMP as a substrate (Torphy et al., J. of Biol. Chem., Vol. 267, No. 3 pp1798-1804, 1992).
[0048] Use rat brain high-speed supernatant as a protein source. preparation[ 3 The enantiomer of H]-cyclopentyloxymethoxyphenylpyrrolidone with a specific activity of 25.6Ci / mmol. Modify the standard assay conditions according to the published method, which is equivalent to the PDE assay conditions, except that the final cAMP: 50mM Tris-HCl (pH7.5), 5mM MgCl 2 , lnM[ 3 H]-cyclopentyloxymethoxyphenylpyrrolidone (Torphy et al., J. of Biol. Chem., Vol. 267, No. 3 pp1798-1804, 1992). The measurement was performed at 30°C for 1 hour. The reaction was terminated and bound and free ligand was separated us...
Embodiment 1B
[0050] Measurement of phosphodiesterase activity
[0051] Using as described by the supplier (Amersham Life Sciences) [ 3 H] cAMP scintillation approximation assay (SPA) or [ 3 H] cGMP SPA enzyme assay to measure PDE activity. Reactions were carried out in 96-well plates at room temperature in 0.1 ml 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. The assay was allowed to proceed for 1 hour and was terminated by the addition of 50 [mu]l SPA yttrium silicate beads in the presence of zinc sulfate. Shake the plate and let stand at room temperature for 20 minutes. The generation of radiolabeled products was assessed by means of scintillation spectrometry. Use 0.05 μM [ 3 H] cAMP to assess the activity of PDE3 and PDE7, using 1 μM [ 3 H]cAMP was used as a substrate to assess the activity of PDE4. Use 1 μM [ 3 H] ...
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