CN112915082B

CN112915082B - Application of compound in preparation of antiallergic drugs

Info

Publication number: CN112915082B
Application number: CN202110099786.7A
Authority: CN
Inventors: 陈家杰; 王慧娜; 吉坤美
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2022-07-05
Anticipated expiration: 2041-01-25
Also published as: CN112915082A

Abstract

The invention discloses application of a compound in preparing an antiallergic medicine, which is characterized in that molecules of the compoundFormula is C₂₉H₂₇NO₈The chemical structural formula is as follows:

the research of the invention finds that the compound T-5224 can obviously inhibit the release of beta-aminoglycoside in mast cells, has an obvious effect of inhibiting degranulation of the mast cells, and can obviously inhibit anaphylactic reaction in mice. The T-5224 compound can be used for preparing antiallergic drugs and has wide application prospect.

Description

Application of compound in preparation of antiallergic drugs

Technical Field

The invention relates to the technical field of anti-allergic medicines, in particular to application of a compound in preparing an anti-allergic medicine.

Background

Allergy is a hypersensitive reaction state of the immune system to substances in the normal environment, can occur in a plurality of organs and tissues, clinically common diseases comprise pollinosis, food allergy, atopic dermatitis, allergic asthma and the like, and symptoms of the hypersensitive reaction state mainly comprise red and swollen skin, itching, watery nasal discharge, shortness of breath and the like, so that persistent health threats are brought to the declaration of people.

Mast cells play a key role in the induction and maintenance of inflammation and are distributed primarily in areas of the body that are exposed to the external environment, such as the skin, the gut, the intestinal and reproductive tract mucosa, among the host cells that are first exposed to the allergen. The surface of the antigen has high affinity IgE receptor Fc-RI, when a specific allergen and Fc-RI-IgE are crosslinked, the antigen can quickly induce the degranulation of mast cells, and release pre-existing media, lipid media, cytokines, chemotactic factors and the like. The pre-stored medium histamine is the main reason for vasodilatation, increase of vascular permeability, low temperature and leukocyte aggregation; lipid mediators including leukotrienes and prostaglandins, which are released relatively rapidly, can enhance vascular permeability and induce the recruitment of immune effector cells to the site of the inflammatory lesion; cytokines and chemokines are produced several hours after mast cell activation and can further contribute to the development of inflammatory responses. Therefore, inhibiting the activation of mast cells can reduce the occurrence of anaphylactic reaction, and is also an important reference for searching new antiallergic drugs.

However, currently, symptomatic treatment methods are mainly adopted for treating allergic diseases clinically, such as antihistamine drugs, hormones and the like and Allergen-specific immunotherapy (Allergen-specific immunotherapy), the former can only temporarily relieve symptoms, and symptoms such as drowsiness, somnolence, mental confusion and the like appear after use, while the latter has long treatment course, high cost and sometimes unobvious effect, so that the mast cell inhibitor becomes a recent research hotspot.

Accordingly, the prior art is yet to be improved and developed.

Disclosure of Invention

The invention aims to solve the technical problems that the application of a compound in preparing an antiallergic medicament is provided aiming at overcoming the defects of the prior art, and the problems of less species, poorer curative effect and larger side effect of the conventional antiallergic medicament are solved.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

application of compound in preparation of antiallergic drugs, wherein the molecular formula of the compound is C₂₉H₂₇NO₈The chemical structural formula is as follows:

the use of the compound for the manufacture of an anti-allergic medicament, wherein the compound is for inhibiting mast cell activation.

Has the advantages that: the invention provides application of a compound T-5224 in preparing an antiallergic medicament, and researches show that the compound T-5224 can obviously inhibit the release of beta-aminoglycoside in mast cells, has an obvious effect of inhibiting degranulation of the mast cells, and can obviously inhibit allergic reactions in mice. The T-5224 compound can be used for preparing antiallergic drugs and has wide application prospect.

Drawings

FIG. 1 is a graph showing the results of relative expression amounts of cFos in RBL-2H3 cells under negative and positive conditions for DNP-HSA in example 1 of the present invention.

FIG. 2 is a graph showing the results of p-c Fos protein expression levels of RBL-2H3 cells under different conditions in example 1 of the present invention.

FIG. 3 is a graph showing the results of cell viability tests of RBL-2H3 cells cultured in example 1 with different concentrations of T-5224.

FIG. 4 is a graph showing the results of the release rate of aminoglycoside from RBL-2H3 cells under various conditions in example 1 of the present invention.

FIG. 5 is a graph showing the results of cell viability tests of BMMC cells cultured in example 2 of the present invention with different concentrations of T-5224.

FIG. 6 is a graph showing the results of the release rate of aminoglycoside from BMMC cells under different conditions in example 2 of the present invention.

FIG. 7 is a graph showing the results of the PCA model for inhibiting T-5224 in example 3 of the present invention.

FIG. 8 is a graph of an ASA model inhibited by T-5224 in example 4 of the present invention.

FIG. 9 is a graph showing the inhibition of ASA by T-5224 in example 4 of the present invention.

Detailed Description

The invention provides an application of a compound in preparing an antiallergic medicament, and in order to make the purpose, technical scheme and effect of the invention clearer and clearer, the invention is further described in detail by referring to the attached drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

As used herein, the singular forms "a", "an", "the" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It will be understood that when an element is referred to as being "connected" or "coupled" to another element, it can be directly connected or coupled to the other element or intervening elements may also be present. Further, "connected" or "coupled" as used herein may include wirelessly connected or wirelessly coupled. As used herein, the term "and/or" includes all or any element and all combinations of one or more of the associated listed items.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

At present, the clinical treatment of allergic diseases mainly adopts symptomatic treatment methods, such as antihistamine drugs, hormones and the like and Allergen-specific immunotherapy (allergy-specific immunotherapy), wherein the former can only temporarily relieve symptoms, and symptoms such as drowsiness, somnolence, mental confusion and the like can appear after the use, while the latter has long treatment course, high cost and sometimes unobvious effect.

Based on the above, the invention provides application of a compound in preparing an antiallergic medicament, wherein the molecular formula of the compound is C₂₉H₂₇NO₈The chemical structural formula is as follows:

the molecular formula of the compound provided by the invention is C₂₉H₂₇NO₈And may be referred to as T-5224 for short, and has a molecular weight of 517.53. Specifically, the development of new drugs is a time-consuming, labor-consuming, expensive and high-risk process, while old drugs have the characteristics of high efficiency, low cost and the like. The invention utilizes bioinformatics to find a targeted drug T-5224 for inhibiting the activation of mast cells. The research proves that T-5224 can inhibit the activation of mast cells and further inhibit the occurrence of anaphylactic reaction. The invention aims to disclose the new application of the compound T-5224, namely the application of the T-5224 in treating type I hypersensitivity caused by activating mast cellsThe application is as follows. That is, the compound T-5224 in this example is useful for inhibiting mast cell activation.

The use of a compound of the present invention for the preparation of an antiallergic agent is further illustrated by the following specific examples:

example 1

Inhibition of T-5224 in degranulation of rat basophils (RBL-2H 3):

basophils from rats have a good allergic immune response in the diagnosis of research allergy and immunotherapy. RBL-2H3 has a high affinity IgE receptor. They can be activated to secrete histamine and other transmitters by either aggregating these receptors or acting synergistically with calcium ionophores. This cell line is widely used to study the biochemical pathways of mast cell FcERI and secretion. The specific implementation mode is as follows:

1. RT-PCR: mixing RBL-2H3 (5X 10)⁵Per well) were inoculated in 6-well plates and sensitized overnight by incubation with anti-DNP IgE, incubated for one hour with T-5224, and stimulated with DNP-HSA (dinitrophenylated human serum albumin) for 4 h. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Dusseldoff, Germany) according to the manufacturer's instructions. Using the Honor^TMII Strand cDNA Synthesis Kit Synthesis of c DNA. Use of TB

Premix Ex Taq^TMThe m RNA level was quantified using (Tokyo, Japan) reagent.

2. Immunoblotting for p-c Fos protein expression level: mixing RBL-2H3 (5X 10)⁵Per well) were inoculated in 6-well plates and sensitized overnight by incubation with anti-DNP IgE, incubated for one hour with T-5224, and stimulated with DNP-HSA (sigma) for 30 min. Cells were lysed with 200 μ l RIPA lysate containing protease inhibitors (MedChem, new jersey, usa) and total protein extracted from the cells. After protein quantification by the BCA method, proteins were transferred to nitrocellulose membranes by electrophoresis on a 10% SDS-PAGE after loading with 20 μ g, primary antibody incubation, overnight at 4 ℃, 1: HRP-labeled anti-rabbit IgG diluted 2000 as a secondary antibody, incubated for one hour, washed three times with TBST for 10min each, developed by chemiluminescence toGAPDH was used as an internal control and the results are shown in fig. 1 and 2.

3. And (3) cell culture: RBL-2H3 (purchased from Guangzhou Seiki Biotechnology Ltd.) has a high affinity IgE receptor and is activated with DNP-HSA after overnight incubation with 50ng/ml anti-Dinitrophenyl (DNP) IgE (sig). RBL-2H3 was cultured in DMEM (Hyclone), 10% Fetal Bovine Serum (FBS), 1% diabody, 1% non-essential amino acids. BMMCs were isolated from Balb/c mice (purchased from Guangdong provincial animal center for medicine), cultured in RPMI (Hyclone) 10% FBS (Gibico), 1% diabody, 10ng/ml IL-3 and SCF for 4-6 weeks.

4. CCK8 tests the toxic effect of cells on T-5224: RBL-2H3 (2X 103/well) was inoculated into 96-well plates, and T-5224 was added after 24 hours, respectively, and incubation was continued for 24 hours. Then, 10ul of CCK8 reagent was added thereto, and the absorbance (OD value) was measured in a microplate reader having a wavelength of 450nm after 1 hour of incubation, and the results are shown in FIG. 3.

5. Release of β -aminoglycosidase: RBL-2H3 cells were seeded in 24-well plates and incubated overnight with anti-DNP IgE for sensitization, T-5224 for one hour, and after 30min of stimulation with DNP-HSA (sigma), 50ul of the supernatant was reacted with 50ul of substrate at 37 ℃ for 90min and absorbance was measured at 405 nm. Discarding supernatant, adding 500ul 0.1% T-X100, cracking for 20min, centrifuging, and reacting 50ul supernatant with 50ul substrate for 90min to determine OD value. The release rate was 100% of the first OD value/(first OD value + second OD value), and the results are shown in fig. 4.

The results of the above experiments are shown in FIGS. 1 to 4, and it can be seen from FIGS. 1 to 2 that the expression level of Fos gene is increased after the activation of RBL-2H3 cells via the IgE-DNP mediated pathway. T-5224 is an inhibitor T-5224 of c Fos/AP 1, and experiments prove that the expression level of c Fos can be obviously inhibited. As can be seen in FIG. 3, T-5224 has no toxic effect on RBL-2H3 cells. As can be seen from FIG. 4, this example further measured the amount of beta-aminoglycoside, a marker of mast cell activation, and found that T-5224 significantly inhibited degranulation of RBL-2H3 in a dose-dependent manner.

Example 2

T-5224 inhibits bone marrow-derived mast cell (BMMCs) degranulation

1. Cell culture: BMMCs were isolated from Balb/c mice (purchased from Guangdong provincial animal center for medical laboratory) and cultured in RPMI (Hyclone) 10% FBS (Gibico), 1% diabody, 10ng/ml IL-3 and SCF for 4-6 weeks.

2. CCK8 tests the toxic effect of cells on T-5224: BMMC (1X 104 per well) were plated in 96-well plates and T-5224 was added after 24 hours each and incubation continued for 24 hours. Then, 10ul of CCK8 reagent was added thereto, and the resultant was incubated for 1 hour and then the absorbance (OD value) was measured in a microplate reader having a wavelength of 450 nm.

3. Release of β -aminoglycosidase: BMMC were inoculated in 24-well plates, sensitized overnight by anti-DNP IgE, incubated for one hour with T-5224, stimulated with DNP-HSA (sigma) for 30min, 50ul of the supernatant was reacted with 50ul of substrate at 37 ℃ for 90min and absorbance was measured at 405 nm. Discarding the supernatant, adding 500ul 0.1% T-X100, lysing for 20min, centrifuging, taking 50ul supernatant, reacting with 50ul substrate for 90min, and determining OD value. The release rate was 100% of the first OD value/(first OD value + second OD value).

To further validate the pharmacological effect of T-5224 on mast cell activation, we used bone marrow derived mast cells (BMMCs). The effect of T-5224 on the cell viability of BMMCs was first measured and T-5224 ≦ 50 μ M was found to have no toxic effect on cells, as shown in FIG. 5. Next, we measured the release amount of beta-aminoglycoside, a marker of mast cell activation, which significantly inhibited the release amount of beta-aminoglycoside from BMMCs, as compared to the control group, and the detailed results are shown in FIG. 6. These data indicate that T-5224 can degranulate BMMCs.

Example 3

Effect of T-5224 on Passive cutaneous anaphylaxis in mice:

1. feeding of BALB/c mice: female mice (4-5 weeks old BALB/C) were purchased from Guangdong provincial medical laboratory animal center (Guangdong Foshan) and placed in a relatively stable SPF environment at temperature (24 + -1 deg.C) and humidity (55 + -10%) for 1 week. Mice were used to isolate bone marrow-derived mast cells (BMMC) and a passive skin allergy (PCA) model and an Active System Allergy (ASA) model were performed.

2. Treatment of mice: each mouse ear was injected with 0.5. mu.g of Anti-IgE and infiltrated for 24 h. The drugs were injected intraperitoneally, and DNP-HSA and evans blue were injected in the tail vein after one hour of action. After 1h, the mice were sacrificed by cervical dislocation, the color change of the ears was observed, the ears were extracted with formamide solution at 62 ℃ for 24h, and the OD value was measured at 620 nm. The thickness of the mouse ear was measured with a micrometer, the section was stained with toluidine blue, and the change in the activated number of mast cells was observed, and the result is shown in FIG. 7.

The PCA model is an animal model that can be used to evaluate the effectiveness of drugs well. In the IgE-DNP induced passive skin allergy in mice, the development of local skin allergy resulted in enhanced skin penetration, and we could monitor the development of allergy by injecting evans blue dye. As shown in FIG. 7, the degree of pigment accumulation was significantly increased in the experimental control group compared to the blank control group by visually observing the pigment accumulation in the skin of the mouse ear. Whereas the group treated with T-5224 showed a significant reduction in pigment accumulation (ketotifen as a positive control).

Example 4

Effect of T-5224 on OVA-induced systemic anaphylaxis:

1. treatment of mice: mice sensitized by intraperitoneal injection of OVA mixture [100 μ g OVA and 2mg alum-adjuvanted 200 μ L Phosphate Buffered Saline (PBS) ] 7 on days 0 and 7 (n 6/group, total n 24) followed by intraperitoneal administration of T-5224(50mg/kg) on

days

9, 11 and 13. On day 14, 200ug OVA was injected intraperitoneally and rectal temperature was measured every 10 minutes for 90 minutes. After 90 minutes, blood samples were taken from the tail of each mouse.

The OVA-induced systemic anaphylaxis model is a mature model for researching antiallergic drugs and is closely related to the activation of mast cells. After successful induction of the allergic reaction, significant body temperature fluctuations are caused. FIG. 8 is the construction of the experimental protocol. The results of the above experiment are shown in FIG. 9, and it can be seen from FIG. 9 that OVA can induce a significant decrease in body temperature compared to the blank control group. Compared with an experimental control group, T-5224 can obviously inhibit the fluctuation of body temperature (ketotifen is used as a positive control medicament). These data all indicate that T-5224 can inhibit OVA-induced systemic anaphylaxis.

In conclusion, the invention aims to disclose the new application of T-5224, namely the effect in the type I hypersensitivity medicament caused by the activation of mast cells, and provides a novel method which is non-toxic, efficient and cheap for preparing the antiallergic medicament. In vitro experiments, RBL-2H3 and BMMCs were degranulated by comparison. In vivo experiments, the temperature was fluctuated by comparing the degree of swelling of the ear. T-5224 was determined to reduce degranulation of mast cells and to be effective in reducing local and systemic anaphylactic reaction symptoms in mice.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. The application of a compound in preparing an antiallergic medicine is characterized in that the molecular formula of the compound is C₂₉H₂₇NO₈The chemical structural formula is as follows:

the compounds are useful for inhibiting mast cell activation.