CN118161503A - Application of small molecular compound BTZ043 in preparation of wound healing promoting medicine - Google Patents

Abstract

The invention relates to application of a small molecular compound BTZ043 in preparing a medicine for promoting wound healing, which is developed for the new application of the small molecular compound BTZ043, particularly, the migration and invasion penetration capacity of human foreskin fibroblast HFF-1 can be obviously improved at the concentration lower than 2 mu M, and meanwhile, the small molecular compound BTZ043 has small toxicity to normal human foreskin fibroblast HFF-1 and mouse fibroblast L-929, can realize the effect of promoting wound healing, accelerates wound healing, can reduce the time of chronic wound healing particularly for diabetics with poor wound healing capacity, and can be widely applied to the wound healing for promoting trauma, burns, ulcers, dermatitis, chronic wounds and delivery.

Description

Application of small molecular compound BTZ043 in preparation of wound healing promoting medicine

Technical Field

The invention belongs to the technical field of medicines and preparations, and particularly relates to application of a small molecular compound BTZ043 in preparation of a medicine for promoting wound healing.

Background

Wounds are mainly caused by wounds, burns, surgery, vascular insufficiency, neuropathy, diabetes, venous disease, ischemia, etc. In the wound healing process, the normal repair of wounds can be influenced by whole body and local factors such as immune function deficiency of individuals, growth factor deficiency in tissues, wound infection, insufficient nutrition supply of skin and the like, so that the wounds can not be recovered, and finally chronic wounds are formed. Compared with acute wounds, chronic wounds exhibit the characteristic of delayed healing processes, even failure to heal, and arrest in the inflammatory phase. Chronic wounds usually have a disease course of more than 1 month, are clinically common, and have slow wound healing processes without intervention, are most likely to produce serious conditions such as inflammation, ischemia or necrosis, and can cause abnormal functions such as scars, pigmentation, ulcers and the like. The non-healing wounds not only increase pain and discomfort to the patient, but also increase amputation risk and medical costs, causing serious physiological, psychological and economic burden to the patient.

For healing of chronic wounds, traditional skin healing such as laser, treatment auxiliary materials, negative pressure treatment, skin transplantation and other treatment means have unsatisfactory effects and limited clinical application. Therefore, development of new drugs for accelerating wound healing, especially chronic wound healing for diabetics and the like, is a highly-needed problem. At present, the development difficulty of new drugs for promoting wound healing is high, the wound healing activity is still low, and cytotoxicity is difficult to control, so that the development of new applications by adopting known drugs is a rapid and effective drug development way.

Disclosure of Invention

Aiming at the prior art problems, the invention provides application of a small molecular compound BTZ043 in preparing a medicine for promoting wound healing, and the small molecular compound BTZ043 can remarkably improve migration and invasion capacities of cells and can be used for promoting wound healing.

In a first aspect, the invention provides application of a small molecular compound BTZ043 in preparing a medicine for promoting wound healing, wherein the molecular structural formula of the small molecular compound BTZ043 is shown as a formula (I) and a formula (II);

Formula (I): 2- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one;

Formula (II): 2- [ (2S) -2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl ] -8-nitro-6-trifluoromethyl-4H-1, 3-benzothiazin-4-one;

in a second aspect, the invention provides a medicament for promoting wound healing, wherein the medicament component comprises a small molecular compound BTZ043 with a molecular structural formula (I) and a geometric isomer thereof or a pharmaceutically acceptable salt thereof and/or a solvate thereof and/or a hydrate thereof.

Further, the medicine for promoting wound healing also comprises pharmaceutically acceptable auxiliary materials.

Further, the dosage forms of the medicament include, but are not limited to, suspensions, granules, capsules, powders, tablets, emulsions, solutions, drop pills, injections, aerosols, gels, patches, drops or liniments.

Further, the wound healing promoting pharmaceutical indications include, but are not limited to, trauma, burns, ulcers, dermatitis, chronic wounds, childbirth, or diabetic wounds.

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

The invention develops a new application of a small molecular compound BTZ043, and discovers that the small molecular compound BTZ043 can promote migration and invasion of cells, wherein the small molecular compound BTZ043 has remarkable activity on migration of human foreskin fibroblast HFF-1 at a concentration lower than 2 mu M, and has remarkable invasion activity on human foreskin fibroblast HFF-1 and mouse embryo fibroblast 3T3 at a concentration lower than 2 mu M, therefore, the small molecular compound BTZ043 can remarkably improve the movement capability and invasion penetration capability of the human foreskin fibroblast HFF-1 and mouse embryo fibroblast 3T3 at a very low concentration, and meanwhile, the small molecular compound BTZ043 has small toxicity on the cells, has small toxicity on the human foreskin fibroblast HFF-1, cannot be detected, has no remarkable drug toxicity, has a IC ₅₀ of L-929 for the mouse fibroblast HFF-1 as high as 216.4 mu M/L, can obviously improve the healing capability of the small molecular compound BTZ 3, can promote healing of wounds, especially burns and wounds, has poor healing capability, and can promote the healing of wounds, especially burns and wounds, and the chronic wounds can be healed.

Drawings

FIG. 1 is an in vitro toxicity test of the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one of the present invention on human foreskin fibroblast HFF-1.

FIG. 2 is an in vitro toxicity test of the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one of the present invention on mouse embryo fibroblast L-929.

FIG. 3 is a migration chart of the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one of the present invention against human foreskin fibroblast HFF-1.

FIG. 4 is a statistical analysis of the migration of the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one of the present invention to human foreskin fibroblast HFF-1.

FIG. 5 is a graph showing the invasion of the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one 48H of the present invention on human foreskin fibroblast HFF-1.

FIG. 6 is a statistical result of the invasion of human foreskin fibroblast HFF-1 by the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one 48H of the present invention.

FIG. 7 is a graph showing the invasion of the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one of the present invention for 3T3 in mouse embryonic fibroblasts for 48H.

FIG. 8 is a statistical result of data of invasion of mouse embryo fibroblasts 3T3 by the small molecule compound BTZ 043- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decan-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one of the present invention for 48H.

Detailed Description

The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless defined otherwise, all 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.

The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

The specific embodiment of the invention adopts a small molecular compound BTZ043 2- (2-methyl-1, 4-dioxa-8-azaspiro [4.5] decane-8-yl) -8-nitro-6- (trifluoromethyl) -4H-benzo [ E ] [1,3] thiazin-4-one for experiments, and the molecular structural formula is as follows:

Example 1 in vitro cytotoxicity assay

(1) The experimental object: human foreskin fibroblast HFF-1, mouse embryo fibroblast L-929;

(2) Experimental drugs: small molecule compound BTZ043, solvent DMSO (dimethyl sulfoxide);

(3) The experimental method comprises the following steps:

the first pm plating: collecting logarithmic phase cells, regulating cell suspension concentration, adding 90uL,5000 human foreskin fibroblasts HFF-1 and 8000 mouse embryo fibroblasts L-929 into each hole;

Dosing the next morning: adding 10 mu L of medicine with concentration gradient into each hole, arranging 3 compound holes for each medicine concentration, placing into 5% CO ₂, and incubating in a 37 ℃ incubator;

plate collection after 48h of dosing: firstly, carrying out visual observation under an inverted microscope, then adding 10uLCCK solution into each hole, and after incubation for 1h at 37 ℃, stopping the reaction;

OD value detection: detecting the light absorption value of each hole at the wavelength of 450nm of the enzyme label instrument, and calculating the relative survival rate or the drug inhibition rate of the cells according to the formula (1);

drug inhibition%1-relative survival%

In the experiment, a cell-free culture medium is used as a blank control group, a DMSO solution with the same dilution ratio as that of the small molecular compound BTZ043 is added into each hole to be used as a negative control group, and the small molecular compound BTZ043 is used as a compound experimental group.

The effect of the small molecular compound BTZ043 on the growth activity of human foreskin fibroblast HFF-1 and mouse embryo fibroblast L-929 cells is tested according to the above experimental method (CCK 8 toxicity test, 48 h), the calculated result is shown in fig. 1 and 2, the small molecular compound BTZ043 has no obvious drug toxicity trend on human foreskin fibroblast HFF-1, and IC ₅₀ can not be detected; the small molecule compound BTZ043 has IC ₁₀＝2.860μm/L、IC₂₀＝14.572μm/L、IC₃₀＝43.003μm/L、IC₅₀ =216.4 μm/L to mouse embryo fibroblast L-929, thus the result shows that the small molecule compound BTZ043 has little toxicity to normal cells.

Example 2 cell migration assay (cell scratch assay)

(1) The experimental object: human foreskin fibroblast HFF-1;

(2) Experimental drugs: small molecule compound BTZ043, solvent DMSO (dimethyl sulfoxide);

(3) The experimental method comprises the following steps:

Before plating, a marker pen thin head is used for uniformly drawing a transverse line behind a 6-hole plate, a straight ruler is used for comparison, a hole is traversed approximately every 0.5-1 cm, three lines are generally drawn, the line is sequentially named as a line, b and c, the line is traversed to the center, two other lines are drawn at equal intervals on two sides of the line b, six-hole plates are plated, 2mL of complete culture medium containing 10% FBS (fetal bovine serum) is added into each hole, 2X 10 ⁶ cells are respectively arranged in 2 multiple holes, the culture is carried out for about 24 hours, the cell number is preferably equal to or greater than 90% after overnight, the cover of the hole plate is properly adjusted, the old culture medium is sucked off the next day, a straight ruler vertical b line frame is arranged on the hole plate, a 200uL gun head is closely attached to the straight ruler, cell scratch lines are manufactured, similarly, two parallel lines are respectively drawn on two sides of the line at equal intervals, line 2 and line 3 are respectively named as line 1, cell culture medium is washed 3 times by sterile 1X (phosphate buffer solution diluted to 1 time), the cell culture medium containing 10% FBS (fetal serum) is removed, the cell culture medium is completely dissolved at a concentration of 5 mu.0 ℃ after the cell culture medium is completely dissolved, and the cell culture medium is completely dissolved at a concentration of 5 mu.0M after the cell culture medium is completely dissolved, and the cell culture medium is completely dissolved at a concentration of 20 mu.5M and 20M is added.

The results obtained by the cell scratch test according to the above test method are shown in fig. 3 and fig. 4, and the test results show that compared with the Control group (DMSO solution), the migration speed of human foreskin fibroblast HFF-1 treated by small molecular compound BTZ043 with low concentration (1 μm, 2 μm) is obviously accelerated, which indicates that the small molecular compound BTZ043 can obviously enhance cell movement and proliferation and promote scratch wound healing, and the result indicates P <0.001, which indicates that the compared with the non-medicated Control group and the medicated group has obvious difference.

Example 3 cell invasion assay (Transwell)

(1) The experimental object: human foreskin fibroblast HFF-1, mouse embryo fibroblast 3T3;

(2) Experimental drugs: small molecule compound BTZ043, solvent DMSO (dimethyl sulfoxide);

(3) The experimental method comprises the following steps:

preparing a cell suspension: digesting cells, centrifuging after stopping digestion, discarding culture solution, washing with PBS (phosphate buffer solution) for 1 time, re-suspending with serum-free culture medium, and adjusting cell density to proper concentration (the plate density of human foreskin fibroblast HFF-1 is 1×10 ⁵/200 μL, the plate density of mouse embryo fibroblast 3T3 is 3.5X10 ⁴/200 μL);

Inoculating cells: each cell is provided with a negative control group (DMSO with the same dilution ratio as that of the small molecular compound BTZ043 is added), a dosing group (the working concentration of the small molecular compound BTZ043 is 1 mu M and 2 mu M), 3 compound holes are respectively formed in each group, a proper amount of cell suspension is taken according to the cell density, a proper amount of 10% BSA (bovine serum albumin) is added to make the final percentage of the cell suspension be 0.1%, the small molecular compound BTZ043 is added, and finally the mixture is supplemented with DMEM, so that the total volume of each hole is 200 mu L, after uniform mixing, the mixture is gently and uniformly added into a Transwell upper chamber, 800 mu L of culture medium containing 20% FBS is generally added into a 24-pore plate lower chamber immediately, and a plate is harvested after 48 hours of dosing;

Cell staining: taking out Transwell chamber, discarding culture solution in the well, gently wiping off non-migrated cells in the upper chamber with cotton bud, placing into clean 24-well plate, washing 1 time with 1 XPBS, fixing with methanol for 30min, sucking methanol, air drying the chamber in fume hood, dyeing for 20min with 0.1% crystal violet, sucking off recovered crystal violet, washing 1 time with PBS, sucking PBS, and air drying in fume hood.

And (3) result statistics: the cells are observed under a 5X microscope, five fields of view are randomly observed under a 10X microscope, the cells are photographed, counted and statistically plotted, the results are shown in fig. 5-8, and the results show that the small molecular compound BTZ043 with the concentration as low as 1 mu m/L-2 mu m/L can remarkably promote migration of fibroblast HFF-1 and mouse embryo fibroblast 3T3, promote wound healing, improve wound healing capacity and accelerate wound healing speed.

It should be noted that, in the present specification, specific features, structures, materials, or characteristics may be arbitrarily combined, and in order to simplify the description, all possible combinations of the features in the foregoing embodiments are not described, and those skilled in the art may combine and combine the features of the different embodiments and the different embodiments described in the present specification without contradiction.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (5)

1. The application of the small molecular compound BTZ043 in preparing the medicine for promoting wound healing is characterized in that the molecular structural formulas of the small molecular compound BTZ043 are shown as (I) and (II);

2. A medicament for promoting wound healing, characterized in that it comprises the small molecule compound BTZ043 according to claim 1 and its geometric isomer or a pharmaceutically acceptable salt and/or solvate and/or hydrate thereof.

3. The wound healing promoting medicament according to claim 2, wherein the medicament further comprises pharmaceutically acceptable excipients.

4. A medicament for promoting wound healing according to claim 3, wherein the dosage form of the medicament comprises a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, an aerosol, a gel, a patch, a drop or a liniment.

5. The wound healing promoting medicament according to claim 4, wherein the pharmaceutical indications comprise trauma, burns, ulcers, dermatitis, chronic wounds, childbirth or diabetic wounds.