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Application of maprotiline as soluble guanylate cyclase agonist

A guanylate cyclase, soluble technology for use in medicine and pharmacy

Active Publication Date: 2018-11-02
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]Currently, sGC is the therapeutic target of pulmonary arterial hypertension, and its agonist is only riociguat approved for marketing, but pulmonary arterial hypertension is a progressive disease, and its therapeutic drugs need to be continuously developed

Method used

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  • Application of maprotiline as soluble guanylate cyclase agonist
  • Application of maprotiline as soluble guanylate cyclase agonist
  • Application of maprotiline as soluble guanylate cyclase agonist

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Expression and purification of soluble guanylate cyclase

[0048] Express the full-length sGC recombinant protein using the eukaryotic expression system: first culture Sf9 cells to the logarithmic phase (cell density reaches 2×10 6 cells / mL), Sf9 cells were added to the Erlenmeyer flask containing Sf-900TM II SFM medium to make the density 5×10 5 cells / mL, the total volume is 650mL, and cultured at 27°C, 140rpm for 3 days; at the same time, the obtained P2 virus strain containing the sGC target gene expression vector continued to infect Sf9 cells to obtain a higher titer of P3 virus strain; then add the obtained P3 virus strain with higher titer to logarithmic growth Sf9 cells, continue to culture at 27°C, 140rpm for 3 days; finally, centrifuge the cultured cells at 1790rpm for 5min, discard Collect the cells expressing the sGC recombinant protein.

[0049] Use the AKTA purification system to purify the full-length sGC recombinant protein: first, use affinity chromato...

Embodiment 2

[0051] Drug screening for agonists of soluble guanylate cyclase

[0052] Based on the interaction between proteins and small molecules, the compounds that can bind to sGC were screened by surface plasmon resonance experiments. Operated on a Biacore T200 instrument, the selected chip was a CM7 sensor chip (GE Healthcare); the purified sGC protein (containing 1mM DTT, 2mM ATP and 3mM MgCl) was purified using sodium acetate solution (pH 4.5) 2 ) was diluted to 50 μg / mL, immobilized on the CM7 sensor chip by amino coupling, and the program was run after the coupling was successful; the buffer for program operation was 1.05×PBS (pH 7.4), 3mM EDTA, 0.025% v / v Surfactant P20, 1mM DTT and 10mM MgCl 2 ; Set the sample injection flow rate to 30 μL / min, the binding and dissociation time of the sample and the target to 120 s and 200 s, respectively, and use 10% DMSO to clean the injection needle every time the sample is changed. The initial screening concentration of the compound was 1...

Embodiment 3

[0054] Activity test of maprotiline stimulating sGC (EC 50 determination)

[0055] Drug EC 50Determination of (median effective concentration): The cGMP EnzymeImmunoassay Kit was used to measure the activity of maprotiline in stimulating sGC by means of competitive immunoassay. In the presence of DEA / NO (300nM), maprotiline catalyzed sGC (20μg / mL) protein to convert GTP (2.6mg / mL) into cGMP, and after incubation at room temperature for 10min, the reaction solution was diluted 400 times with 0.1M HCl Then transfer to a 96-well plate coated with anti-rabbit IgG antibody, and add reagents such as Neutralizing reagent, Conjugate and cGMP EIAAntibody, place on a shaker with a rotation speed of 500rpm, and incubate at room temperature for 2h; then suck out the reaction solution, and add 1 Wash the plate three times with ×Wash Buffer, add cGMP-Alkaline Phosphatase Conjugate to TA (Total Activity) wells, then add 200 μL p-Nitrophenyl Phosphate Substrate to each well, and incubate at...

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Abstract

The invention relates to maprotiline of a novel soluble guanylate cyclase agonist and application of the maprotiline. Particularly, the invention relates to application of the maprotiline in preparation of medicines for treating soluble guanylate-cyclase-mediated diseases or agitating the soluble guanylate cyclase. The soluble guanylate-cyclase-mediated diseases include but are not limited to cardiovascular diseases, kidney diseases, diabetes, glaucoma, obesity, osteoporosis, fibrosis diseases, urinary system diseases, inflammatory diseases and neurosis diseases, in particular pulmonary hypertension. The formula (1) is shown in the description.

Description

technical field [0001] The present invention relates to the fields of medicine and pharmacy; in particular, the present invention relates to maprotiline as an agonist of soluble guanylate cyclase (sGC), especially its application in the treatment of pulmonary hypertension. Background technique [0002] Pulmonary hypertension (PAH) is an increase in pulmonary artery pressure and pulmonary vascular resistance caused by a variety of etiologies, which in turn promotes vasoconstriction, vascular wall hyperplasia, vascular remodeling, and in situ thrombus formation, which eventually lead to right heart failure and death. a progressive disease. The diagnostic criteria of hemodynamics is that the mean pulmonary artery pressure detected by the right catheter is ≥25mmHg at resting state at sea level. Although PAH is a rare disease, it is more harmful and has become a public health care problem that seriously threatens human physical and mental health. PAH can be divided into five ca...

Claims

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Application Information

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IPC IPC(8): A61K31/137A61P9/00A61P13/12A61P3/10A61P27/06A61P3/04A61P19/10A61P13/00A61P29/00A61P25/00A61P9/12
CPCA61K31/137
Inventor 李洪林朱丽丽童依焦茜吕剑昆王霞王蕊赵振江
Owner EAST CHINA UNIV OF SCI & TECH
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