Application of recombinant human Rho kinase to preparation of medicaments

A technology of kinase inhibitors and drugs, which is applied in the application field of recombinant human Rho kinase in the preparation of drugs, and can solve the problem that recombinant human ROCK1 functional fragment protein has not been found.

Active Publication Date: 2011-09-21
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, we have not found any reports on the application of recombinant human ROCK1 functional fragment protein in cardiovascular an

Method used

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  • Application of recombinant human Rho kinase to preparation of medicaments
  • Application of recombinant human Rho kinase to preparation of medicaments
  • Application of recombinant human Rho kinase to preparation of medicaments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Evaluation of Kinase Biological Activity of RP1.

[0025] In this example, the activity of RP1 was identified by the Kinase-Glo Luminescent Kinase Assay method. The reaction was carried out in a white opaque 96-well plate, and 5 μL of kinase buffer (25 mmol L -1 Tris-HCl, pH7.5, 10mmol L -1 MgCl 2 , 0.1mg·mL -1 BSA), 10 μL kinase, 10 μL S6 (AKRRLSSLRA) polypeptide substrate and 5 μL ATP. After mixing, incubate at 37°C for a certain period of time, add 50 μL of Kinase-Glo reagent, room temperature for 10 min, and read the relative luminescence value of each well on SpectraMax M5. Add 10 μmol·L to the reaction -1 Y-27632 (a specific inhibitor of Rho kinase), observe whether it can selectively inhibit the activity of RP1, and compare it with the blank control without kinase and the positive control that adds kinase at the same time, to determine the activity of RP1 in Example 1 Rho kinase biological activity.

[0026] There is a significant difference be...

Embodiment 2

[0027] Example 2: Effect of RP1 on vascular reactivity in anesthetized rats.

[0028] The experimental animals in this example were male SD rats (body weight 250-300 g). After the rats were killed by decapitation, the thoracic aorta was quickly taken out, cut into 2-3mm long vascular rings, and placed in a 10mL K-H solution (37°C constant temperature, and continuously fed with 95% oxygen and 5% carbon dioxide). In the bath of mixed gas), the tension change is transmitted and recorded in the BL-420S biological function experiment system. The vascular ring was stabilized at 1.2g tension for 60 minutes, and the K-H solution was changed every 20 minutes during this period. Stimulate the vascular ring with 60mM KCl to shrink the vascular ring. After reaching the maximum amplitude, flush the vascular ring twice to restore the vascular ring to the state before the stimulation, a total of 2 times. Add 1 μM norepinephrine, give 10 μM acetylcholine after reaching the maximum contracti...

Embodiment 3

[0030] Example 3: Effects of various endothelial-derived active substances on the vasoconstrictor effect of RP1.

[0031] The preparation method of the blood vessel ring for tension measurement is the same as that in Example 3.

[0032]In this example, nitric oxide synthase inhibitor L-NAME (100 μM), cyclooxygenase inhibitor indomethacin (5 μM), guanylate cyclase inhibitor methylene blue (10 μM) were added to the reaction system ), adding cumulative concentration of RP1 after pre-incubation for 20 min to observe the effect of endothelial-derived active substances on the vasoconstrictor effect of RP1. L-NAME can significantly enhance the vasoconstrictor effect of RP1 (attached image 3) . It was determined that the regulation of RP1 on vascular function was endothelium-dependent.

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Abstract

The invention discloses application of recombinant human Rho kinase to preparation of medicaments. Recombinant human Rho-associated coiled-coil forming protein kinase (ROCK)1 fragment protein (RP1 for short) has the activity of the Rho kinase, so that corresponding substrates can be phosphorylated; the RP1 has the effect of contracting blood vessels, and the functional regulation effect of the blood vessels is related to the endothelium of the blood vessels, is blocked by a calcium channel blocking agent and is inhibited by Fasudil serving as an Rho kinase inhibitor; after entering bodies of animals in an injection mode, the RP1 can rise the blood pressure of the animals and has the characteristic of quick response; and the RP1 has the effect of accelerating the heart rate of the animals under the condition of providing high dose in the injection mode, and has the effect of improving the blood vessel function reduced due to various factors including disease factors. The RP1 can be applied to the preparation of medicaments for relieving, preventing and/or treating cardiovascular and cerebrovascular diseases or symptoms which are caused by various factors including disease factors and are related to the reduction of the blood vessel function.

Description

technical field [0001] The invention relates to the application of recombinant human Rho kinase in the preparation of medicines. technical background [0002] As we all know, cardiovascular and cerebrovascular system diseases have become the most common type of diseases in humans, accounting for the first in the global mortality rate, and becoming the most important diseases that threaten human health and life. With the development of my country's economy, the acceleration of population aging, and the acceleration of the pace of life, cardiovascular diseases such as hypertension, hyperlipidemia, heart failure, and stroke have increased significantly among the population, directly increasing the burden on families and society. [0003] Small molecule GTP-binding proteins are monomeric G protein molecules that play key roles in the regulation of cellular functions. Rho is a small molecule G protein, which is in an inactive state when combined with GDP, and becomes active when...

Claims

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Application Information

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IPC IPC(8): A61K38/45A61P9/00A61P9/12A61K31/175A61K31/435A61K31/551
Inventor 方莲花杜冠华宫丽丽陈柏年
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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