阅读说明：本技术 氟芬那酸作为有效成分在制备气道平滑肌舒张剂中的应用 (Application of flufenamic acid as active ingredient in preparation of airway smooth muscle relaxant ) 是由 罗明志 倪凯 田姈玉 郭佳 刘磊 李晶晶 潘艳 邓林红 于 2021-09-29 设计创作，主要内容包括：本发明公开了氟芬那酸作为有效成分在制备气道平滑肌舒张剂中的应用。本发明经试验首次揭示了,化合物(CID:3371)能够活化苦味受体,并强效舒张气道平滑肌细胞,其舒张程度接近异丙肾上腺素和沙丁胺醇；提示化合物(CID:3371)可用于制备舒张气道平滑肌药物,也可用于制备气道平滑肌舒张剂,从而用于哮喘、慢性阻塞性肺部疾病等,具有广阔的应用前景。(The invention discloses application of flufenamic acid as an active ingredient in preparation of an airway smooth muscle relaxant. The test of the invention firstly reveals that the compound (CID:3371) can activate bitter receptors and effectively relax airway smooth muscle cells, and the relaxation degree of the compound is close to that of isoproterenol and salbutamol; the prompting compound (CID:3371) can be used for preparing a medicament for relaxing airway smooth muscle, and can also be used for preparing an airway smooth muscle relaxing agent, so that the compound is used for treating asthma, chronic obstructive pulmonary diseases and the like, and has wide application prospect.)

1. The application of flufenamic acid as an active ingredient in preparing an airway smooth muscle relaxant is disclosed, wherein the structure of the flufenamic acid is shown as the formula (I):

2. the use according to claim 1, wherein the compound relaxes airway smooth muscle cells by targeting bitter taste receptors.

3. The application of the flufenamic acid as an active ingredient in preparing the medicine for relaxing airway smooth muscle is disclosed, wherein the structure of the flufenamic acid is shown as a formula (I).

4. The use according to claim 3, wherein the compound relaxes airway smooth muscle cells by targeting bitter taste receptors.

5. The use of claim 3, wherein the diastolic airway smooth muscle drug is an asthma treatment drug.

6. The pharmaceutical composition for relaxing airway smooth muscle is characterized by at least comprising a compound with a structural formula shown as a formula (I).

7. The pharmaceutical preparation for relaxing airway smooth muscle is characterized by comprising a safe and effective amount of a compound shown as a structural formula (I), and the balance of a pharmaceutically acceptable carrier.

8. The pharmaceutical preparation according to claim 7, wherein the pharmaceutical preparation is an injection or a spray.

9. The pharmaceutical formulation of claim 8, wherein the pharmaceutically acceptable carrier is selected from phosphate buffered saline or physiological saline.

10. The pharmaceutical preparation according to any one of claims 7 to 9, wherein the pharmaceutical preparation for relaxing airway smooth muscle is a pharmaceutical preparation for treating asthma.

Technical Field

The invention belongs to the technical field, and particularly relates to application of flufenamic acid as an active ingredient in preparation of an airway smooth muscle relaxant.

Background

Asthma is a common chronic respiratory disease, and as many as 3 hundred million people suffer from asthma worldwide, and even though the pharmaceutical industry has made great progress, the pharmaceutical industry still brings great economic burden to the society. Asthma patients are shared nationally in the vicinity of 5000 million and their prevalence is increasing year by year. Although hospital stays and mortality declines in some areas due to the continuing development of scientific technology, the burden of asthma has increased by 20% over the last 20 years.

Asthma is characterized by recurrent episodes of wheezing, chest distress, pulmonary obstruction, and respiratory insufficiency, which are primarily caused by airway obstruction. Airway smooth muscle cells are present in the middle and periphery of the airway, spirally surrounding the airway. Therefore, airway constriction mediated by airway smooth muscle cell contraction is a significant cause of obstruction in asthma. Therefore, current research efforts are focused on how to relax airway smooth muscle cells in asthma.

Currently, the drugs used to treat obstructive airways disease according to the global asthma control protocol (2020 edition) are largely divided into two categories: firstly, soothing agents (bronchodilators) relieve airway obstruction primarily by relaxing airway smooth muscle cells, and secondly, controlling agents (preventives) primarily inhibit underlying disease and provide long-term control of symptoms. Suction type beta₂Receptor agonists, the most effective bronchodilators currently on the market, are used for short-term relief of dyspnea and other symptoms, but have poor control over symptoms in severe asthma patients. While severe asthma patients account for only 5-10% of all asthmatics, the treatment costs for severe asthma patients account for more than half of the asthma healthcare expenditure because they require more expensive medications, are more likely to be hospitalized, or require additional medical care. Therefore, a novel bronchodilator and airway smooth muscle relaxing with different action mechanisms are foundThe new medicine is urgently needed.

In recent years, Bitter taste receptors (TAS 2Rs) have been found to play a critical role in regulating airway smooth muscle cell contraction, relaxation, and proliferation. Therefore, agonists discovered based on bitter receptors are likely to be potential drugs for treating respiratory diseases such as asthma.

Due to the wide variety of bitter substances, bitter compounds that have been identified in 2019 are updated to over 1000 in the bitterant database (BitterDB). However, to date, there is still a lack of potent TAS2R agonists that can be used as new pharmaceutical agents for patients with severe asthma.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

In view of the above and/or the defects existing in the prior art, the invention firstly reveals that the compound (CID:3371) can be used for preparing the medicine for relaxing the airway smooth muscle and can also be used for preparing the vasodilating agent of the airway smooth muscle, so that the compound can be used for treating asthma, chronic obstructive pulmonary diseases and the like and has wide application prospect.

In order to solve the technical problems, the invention provides the following technical scheme: the application of flufenamic acid as an active ingredient in preparing an airway smooth muscle relaxant is disclosed, wherein the structure of the flufenamic acid is shown as the formula (I):

specifically, the compound (CID:3371) with the structure shown as the formula (I) has the molecular formula C₁₄H₁₀F₃NO₂Molecular weight 281.23g/mol, slightly soluble in water. The International Union of Pure and Applied Chemistry (IUPAC) specifies the names: 2- [3- (trifluoromethylphenyl) anilino]benzoic acid. The compound is named as: flufenamic acid.

As a preferred embodiment of the use of the present invention, wherein the compound relaxes airway smooth muscle cells by targeting bitter taste receptors.

The Bitter taste receptor (TAS 2Rs) is a 7-transmembrane G-protein coupled receptor (GPCR), the transduction of Bitter taste in taste receptor cells is mainly completed by the G protein and the GPCR, and Bitter compounds identified by 2019 are updated to over 1000 in Bitter taste database (BitterDB).

The invention also aims to provide application of flufenamic acid as an effective ingredient in preparing a medicine for relaxing airway smooth muscle, wherein the structure of the flufenamic acid is shown as the formula (I).

As a preferred embodiment of the use of the present invention, wherein the compound relaxes airway smooth muscle cells by targeting bitter taste receptors.

In a preferred embodiment of the use of the present invention, the airway smooth muscle relaxing drug is an asthma treatment drug.

By "treating" is meant reducing, inhibiting and/or reversing the development of asthma in a subject in need thereof. The term "treatment" includes any sign of successful treatment or improvement of asthma, including any objective or subjective parameter, such as remission; moderating; reduced symptoms or making the subject more tolerant to injury, pathology or condition; delay or slow the rate of development, etc. The measurement of treatment or improvement may be based on the results of physical examination, pathological examination, and/or diagnostic examination, for example, as known in the art.

Treatment may also refer to a reduction in the onset or onset of asthma, or a reduction in asthma relapse (e.g., a prolonged time to relapse) as compared to what would occur in the absence of such measures.

The invention also aims to provide a pharmaceutical composition for relaxing airway smooth muscle, which at least comprises a compound shown in a structural formula (I).

The invention also aims to provide a pharmaceutical preparation for relaxing airway smooth muscle, which contains a safe and effective amount of a compound shown as a structural formula (I), and the balance of a pharmaceutically acceptable carrier.

As a preferable scheme of the pharmaceutical preparation for relaxing airway smooth muscle, the pharmaceutical preparation is an injection or a spray.

The term "pharmaceutically acceptable carrier" includes any or all of solvents, dispersion media, coatings, isotonic agents, absorption enhancers, absorption blockers, and the like that are physiologically compatible. Examples of the pharmaceutically acceptable carrier include saccharides such as water, saline solutions, Phosphate Buffered Saline (PBS), monosaccharides, disaccharides, oligosaccharides, polysaccharides (dextrin, dextran, isomaltose dextrin, cellulose, pullulan, chitin, chitosan, guar gum, carrageenan, and the like), derivatives thereof, alcohols such as glycerol, ethanol, and the like, and they may be used alone or in combination as appropriate. When used as an injection or the like, 1 or more kinds of pH adjusting agents, isotonic agents, sugar alcohols such as the above sugars, mannitol, sorbitol, maltitol, or sodium chloride may be used in combination as appropriate.

As a preferable scheme of the pharmaceutical preparation for relaxing airway smooth muscle, the pharmaceutical acceptable carrier in the injection solution is phosphate buffer solution or normal saline.

As a preferable mode of the pharmaceutical preparation for relaxing airway smooth muscle of the present invention, the pharmaceutical preparation for relaxing airway smooth muscle is a pharmaceutical preparation for treating asthma.

Compared with the prior art, the invention has the following beneficial effects:

the invention firstly reveals that the compound (CID:3371) can activate bitter receptors and can effectively relax airway smooth muscle cells, and the relaxation degree of the compound is close to that of isoproterenol and salbutamol. The prompting compound (CID:3371) can be used for preparing a medicament for relaxing airway smooth muscle, and can also be used for preparing an airway smooth muscle relaxing agent, so that the compound is used for treating asthma, chronic obstructive pulmonary diseases and the like, and has wide application prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:

FIG. 1 is a graph comparing the relaxation capacity of 16 different bitter tasting substances of the present invention for ASMCs;

FIG. 2 is a fitting curve of the dose-effect relationship of flufenamic acid according to the present invention;

FIG. 3 is a graph comparing the diastolic effect of flufenamic acid and isoproterenol of the present invention;

FIG. 4 is a graph showing the results of the optical magnetic particle torsion cell analysis according to the present invention;

fig. 5 is a graph showing the results of an evaluation test of the airway diastolic function of flufenamic acid in accordance with the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein are all conventional in the art. These techniques are well described in the literature and are described in detail in:

Ni,K.,Guo,J.,Bu,B.,Pan,Y.,Li,J.,Liu,L.,Luo,M.,and Deng,L.(2021). Naringin as a plant-derived bitter tastant promotes proliferat ion of cultured human airway epithelial cells via activation of TAS2R signaling.Phytomedicine 84,153491.

Luo,M.,Yu,P.,Ni,K.,Jin,Y.,Liu,L.,Li,J.,Pan,Y.,and Deng,L.(2020). Sanguinarine Rapidly Relaxes Rat Airway Smooth Muscle Cells Dependent on TAS2R Signaling.Biological&pharmaceutical bulletin 43,1027-1034.

Luo,M.,Ni,K.,Yu,P.,Jin,Y.,Liu,L.,Li,J.,Pan,Y.,and Deng,L.(2019). Sanguinarine Decreases Cell Stiffness and Traction Force and Inhibits the Reactivity of Airway Smooth Muscle Cells in Culture.molecular cellular biomechanics 16,141-151.

WangY.,Lu Y.,Luo M.,Shi X.,DengL.(2016).Evaluation of pharmacological relaxation effect of the natural product naringin on in vitro cultured airway smooth muscle cells and in vivo ovalbumin-induced asthma Balb/c mice.Biomedical Reports 5(6):715-722.

example 1

(1) Test object

Primary culture of human ASMCs

(2) Laboratory apparatus

Optical magnetic particle torsion cell analysis system (OMTC), living cell workstation, detachable 96-well plate, ultra-clean bench, CO₂Cell culture box, low-speed centrifuge, cell counting plate, constant temperature water bath, autoclave, and confocal culture dish.

(3) Experimental procedure

16 bitter substances (flufenamic acid, kaempferol, 1, 10-phenanthroline, carisoprodol, andrographolide, benethanaminium, hesperetin, quinine, chloroquine, naringenin, benzoin, hesperetin, apigenin, quercetin, cucurbitacin E, artesunate) capable of activating bitter receptors at a concentration of less than 10 μ M are screened from a BitterDB database. After being treated by the 16 different bitter substances, the cells are detected to change the rigidity of the cells by an optical magnetic particle torsion cytometric method, thereby evaluating the relaxation capacity of the bitter substances on the ASMCs.

The dose-dependent effect of flufenamic acid (final concentration 0.01. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M, 100. mu.M, 1000. mu.M) was then analyzed and compared to the effect of flufenamic acid on the contraction induced by 50mM potassium chloride in the relaxation of the classical airway smooth muscle relaxant Isoproterenol (ISO).

The specific method comprises inoculating cells into detachable 96-well plate at an inoculation density of 1 × 10⁴And (3) changing an IT culture medium for culturing for 12 hours after the cells grow adherent to the cells for 24 hours, adding magnetic microbeads coated with the integrin antibody, and then dynamically detecting the change of the cells after the treatment of compounds with different cell rigidity (100 mu M) by using an optical magnetic particle torsion cell measurement technique. The cell stiffness in the basal state was first measured for 1min, then 10. mu.L of different bitter substances were added and the cell stiffness was measured to reflect the degree of cell relaxation. The detection conditions are as follows: the frequency is 0.3Hz, and the detection time is 5 min.

(4) Results of the experiment

Experimental results found that treatment of ASMCs with 16 bitter tasting substances at a concentration of 100 μ M resulted in only 11 of these significantly reduced ASMCs cell stiffness, with flufenamic acid reducing ASMCs cell stiffness the most, to about 60% (see fig. 1).

As a result of detecting the dose-dependent effect of the flufenamic acid, 5 concentrations of the flufenamic acid in different tested concentrations show obvious relaxation effect (0.1 mu M, 1 mu M, 10 mu M, 100 mu M and 1000 mu M), and the EC is calculated by fitting dose-effect relationship curve₅₀The curve was fitted to 10.02 μ M as shown in fig. 2.

Further analysis comparing the relaxation effects of flufenamic acid and isoproterenol revealed that the relaxation effect of flufenamic acid was greater than that of isoproterenol after first causing contraction of airway smooth muscle cells with 50mM potassium chloride (fig. 3).

The above experimental results suggest that flufenamic acid is a potent ASMCs relaxant.

Example 2

(1) Test object

Primary culture of human ASMCs

(2) Laboratory apparatus

Optical magnetic particle torsion cell analysis system (OMTC), living cell workstation, detachable 96-well plate, ultra-clean bench, CO₂Cell culture box, low-speed centrifuge, cell counting plate, constant temperature water bath, autoclave, and confocal culture dish.

(3) Experimental procedure

The method comprises the steps of inhibiting ASMCs bitter receptors and downstream signal channels, treating with flufenamic acid, and detecting the change of cell rigidity by optical magnetic particle torsion cell measurement. The specific method comprises inoculating cells into a detachable 96-well plate with an inoculation density of 1 × 10⁴And/well, culturing for 12h after adherent growth for 24h by changing an IT culture medium, treating ASMCs 48h with siRNA targeting TAS2R14 to reduce bitter receptor 14 expression, inhibiting phospholipase C activity by U73122(20 mu mol/L) and inhibiting IP3R activity by PD98059(20 mu mol/L) to inhibit cell bitter receptor 14 signal pathway, adding magnetic microbeads coated with integrin antibodies, and dynamically detecting the change of cell rigidity along with the treatment of different concentrations of flufenamic acid by optical magnetic particle torsion cytometry.

The siRNA transfection procedure was performed by first adding 125. mu.L of Opti-MEM and 8. mu.L of si-RNA (5nM) to an EP tube; to another EP tube was added 125. mu.L of Opti-MEM and 6. mu.L of Lipofectamine 3000 reagent. Standing for 5min, adding the latter tube into the former tube, and standing at room temperature for 20 min. The incubated reagents are respectively added into the cells to be transfected, after about 12 hours, the cells are washed 1-2 times by 1 XPBS, replaced by a normal culture medium without antibiotics, and cultured for 24 hours.

The cell stiffness was measured by measuring the cell stiffness in the basal state for 1min, followed by the addition of 10. mu.L of flufenamic acid at various concentrations (final concentrations 0.01. mu.M, 0.1. mu.M, 1. mu.M, 10. mu.M, 100. mu.M, 1000. mu.M) to measure the cell stiffness and thus reflect the degree of cell relaxation. The detection conditions are as follows: the frequency is 0.3Hz, and the detection time is 5 min.

(4) Results of the experiment

The RNA interference technology is adopted to reduce the expression of bitter taste receptor 14, U73122 inhibits the activity of phospholipase C, and PD98059 inhibits the activity of IP3R to inhibit the signaling pathway of the bitter taste receptor 14 of cells, and as a result, the relaxation effect of the flufenamic acid is found to be remarkably reduced, which indicates that the flufenamic acid targets the bitter taste receptor 14 to exert the relaxation function (figure 4).

Optical magnetic particle torsion cell assay (OMTC) experiments demonstrated that compound (CID:3371) potently relaxes airway smooth muscle cells by activating bitter receptor 14.

Example 3

(1) Test object

BALB/c mice

(2) Laboratory apparatus

FlexiVent small animal lung function measuring instrument, superclean bench, constant temperature water-bath.

(3) Experimental procedure

The lung function measuring instrument of the FlexiVent small animal is adopted to detect the airway resistance of the normal and asthma model mice and evaluate the airway relaxation function of the flufenamic acid. The experiment was first randomized into 6 groups: a normal control group, an asthma model group (ovalbumin treated) control group, a high, medium and low dose group of flufenamic acid (8mg/kg, 4mg/kg, 2mg/kg) and salbutamol (8mg/kg), with 5 per group.

The establishment process of the asthma model is that 200 mu L of ovalbumin sensitizer is respectively injected into the abdominal cavity of each mouse on the 1 st day and the 7 th day, and 100 mu L of ovalbumin sensitizer is injected into the abdominal cavity on the 14 th day; the normal control group is injected with normal saline instead of the sensitizer, and the site and dosage are the same as those of the experimental group. Atomizing and inhaling with 6mL of 1% ovalbumin exciting solution from day 15 to day 21, 1 time per day, 30min each time, and continuously for 7 days; the normal control group was challenged with nebulization with saline instead of 1% ovalbumin.

After the ovalbumin stress mice induce the asthma model, on the 22 th day, the therapeutic groups respectively inhale 8, 4 and 2mg/kg doses of flufenamic acid and 8mg/kg of salbutamol by atomization, and then the lung function instrument of the FlexiVent small animal is adopted to detect the airway resistance of each experimental group, and the parameters are set as follows: tidal volume 10.72mL/kg (about 250 μ L/time), 150 breaths per minute, and end-of-breath pressure of 3cm H₂O；

The detection process of the FlexiVent small animal lung function instrument is that firstly, after a mouse is connected with an animal respirator to breathe stably, 50 mu L of normal saline and Mch with the concentration of 32.5mg/kg are added into an atomization adapter, so that the resistance of an airway exceeds 4-5 times of a basic value. The effect of flufenamic acid on mouse lung resistance after nebulization inhalation was then evaluated by nebulization of the vasodilator salbutamol or flufenamic acid.

(4) Results of the experiment

After acetylcholine stimulation, salbutamol and flufenamic acid are separately inhaled by atomization, and the result shows that 32.5mg/kg of acetylcholine alone can increase the airway resistance by 502% (normal group) and 560% (asthma group), while 10mg/kg of salbutamol can significantly reduce the airway resistance, while the same concentration of flufenamic acid has the similar relaxation ability of salbutamol in the asthma group, which indicates that the flufenamic acid can also relax airway smooth muscle strongly at animal level (fig. 5).

The lung function experiment of the small animal proves that the compound (CID:3371) can relax the airway smooth muscle of the mouse strongly.

Aiming at bitter receptors mediating relaxation of ASMCs, mainly TAS2R-5, -10 and-14, 16 bitter substances capable of activating the bitter receptors at the concentration of below 10 mu M are screened from a BitterDB database, and the relaxation effect of the drugs on the ASMCs at the concentration of 100 mu M is detected by an optical magnetic particle twisting cell assay (OMTC), so that the flufenamic acid in the 16 bitter substances has the maximum relaxation effect on the ASMCs and reaches about 60 percent. It was subsequently discovered that flufenamic acid has a relaxing effect on ASMCs, EC₅₀The value was 10.02. mu.M and 0.1. mu.M flufenamic acid was able to significantly relax ASMCs. Further comparison of the relaxing effects of flufenamic acid on ASMC with the classical ASMCs relaxants Isoproterenol (ISO) and albuterol revealed that flufenamic acid was more pronounced than ISO at the same concentration, similar to the effect of albuterol, indicating that flufenamic acid is a potent ASMCs relaxant.

The invention firstly reveals that the compound (CID:3371) can activate bitter receptors and can effectively relax airway smooth muscle cells, and the relaxation degree of the compound is close to that of isoproterenol and salbutamol. The prompting compound (CID:3371) can be used for preparing a medicament for relaxing airway smooth muscle, and can also be used for preparing an airway smooth muscle relaxing agent, so that the compound is used for treating asthma, chronic obstructive pulmonary diseases and the like, and has wide application prospect.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.