CN118178418A - Application of GPR119 agonist in preparation of wound healing promoting drugs - Google Patents

Abstract

The invention relates to an application of GPR119 agonists in preparing medicaments for promoting wound healing, which is developed for the new application of the GPR119 agonists, particularly, the invention finds that the GPR119 agonists can obviously promote human foreskin fibroblast HFF-1 at a concentration lower than 2 mu M, improve the migration and invasion penetration capacity of cells, and simultaneously, the IC ₅₀ of the GPR119 agonists AS126957 on human foreskin fibroblast HFF-1 and mouse fibroblast L-929 respectively reaches 52.62 mu M/L and 36.26 mu M/L, which shows that the GPR119 agonists have small toxicity on cells, have better healing capacity on the wounds of mice when the administration concentration is 1mg/kg, can realize the effect of promoting wound healing, accelerate the wound healing, particularly for diabetics with poor wound healing capacity, can reduce chronic wound healing time, and can be widely applied to promote the wound healing of wounds, burns, ulcers, dermatitis, chronic wounds and delivery wounds.

Description

Application of GPR119 agonist in preparation of wound healing promoting drugs

Technical Field

The invention belongs to the technical field of medicines and preparations, and particularly relates to application of GPR119 agonists in preparation of medicines for promoting wound healing.

Background

The skin is the first important defense line of human body lower than external invasion, contains a large amount of nutrients such as protein, fat, moisture and the like, participates in metabolism of human body, has the functions of immunity, resisting invasion of external pathogens, regulating body temperature and the like, has a protective effect, needs to take timely treatment measures when the skin is wounded, promotes wound healing, has slow wound healing process if no intervention is performed, is very likely to produce serious conditions such as inflammation, ischemia or necrosis and the like, and can cause abnormal functions such as scars, pigmentation, ulcers and the like. However, wound repair is complex and typically requires 4 phases: fibrotic clot formation, inflammatory response, revascularization and connective tissue remodeling, of which revascularization is critical, is an abnormally difficult stage of revascularization for healing of chronic wounds, such as very slow revascularization in wounds of diabetics, where the vascular surface area, branch connection number, total vascular length and total branch number are all significantly reduced, and angiogenesis can effectively support wound closure, while vascular lesions are one of the causes of difficult healing of wounds of diabetics. For healing common skin wounds, especially chronic wounds, traditional skin healing methods such as laser, treatment auxiliary materials, negative pressure treatment, skin transplantation and the like are not ideal in effect, and have limitation in 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 GPR119 agonists in preparing medicaments for promoting wound healing, and the GPR119 agonists 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 GPR119 agonist in preparing a medicine for promoting wound healing, wherein the general molecular structural formula of the GPR119 agonist is shown as (I);

Wherein A is selected from one of halogen, hydrogen and C _1～6 alkane; b is selected from one of C _1～6 straight-chain paraffin and C _1～6 branched-chain paraffin; m is selected from 1 to 6.

Further, the GPR119 agonist has a molecular structural formula shown in (II) to (VI):

Preferably, the GPR119 agonist is AS126957, which has a molecular structural formula shown in (III):

In a second aspect, the present invention provides a medicament for promoting wound healing, the medicament comprising a GPR119 agonist of the general formula (I) and its geometric isomers or a pharmaceutically acceptable salt and/or solvate and/or hydrate thereof.

Preferably, the medicine for promoting wound healing comprises a GPR119 agonist with the molecular structural formulas (II) - (VI) 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 the GPR119 agonist, and discovers that the GPR119 agonist can promote migration and invasion of cells, in particular to the GPR119 agonist AS126957 has obvious migration and invasion effects on human foreskin fibroblast HFF-1, wherein the GPR119 agonist AS126957 has obvious activity on the migration of the cells of the human foreskin fibroblast HFF-1 at the concentration lower than 2 mu M and has obvious invasion activity on the cells of the human foreskin fibroblast HFF-1 at the concentration lower than 0.2 mu M, therefore, the invention discovers that the GPR119 agonist can obviously improve the migration and invasion penetration capability on the human foreskin fibroblast HFF-1 at the extremely low concentration, and meanwhile, the GPR119 agonist AS126957 has little toxicity on the cells, the IC ₅₀ on the human foreskin fibroblast HFF-1 is up to 52.62 mu M/L, the IC ₅₀ on the mouse foreskin fibroblast HFF-1 is up to 36.26 mu M/L, and the invention discovers that the GPR119 agonist AS126957 has little cytotoxicity; meanwhile, the compound has better healing capacity on the wounds of mice when the administration concentration is 1mg/kg, can improve the movement capacity of cells, realize the effect of promoting the wound healing, quicken the wound healing, especially for diabetics with poor wound healing capacity, can reduce the chronic wound healing time, and can be widely applied to promote the wound healing of wounds, burns, scalds, ulcers, dermatitis, chronic wounds and childbirth.

Drawings

FIG. 1 is an in vitro toxicity test of GPR119 agonist AS126957 of the present invention on human foreskin fibroblast HFF-1.

FIG. 2 is an in vitro toxicity assay of GPR119 agonist AS126957 of the invention on mouse fibroblast L-929.

FIG. 3 is a graph showing migration of the GPR119 agonist AS126957 of the present invention to human foreskin fibroblast HFF-1.

FIG. 4 is a data statistic of migration of human foreskin fibroblast HFF-1 by GPR119 agonist AS126957 of the present invention.

FIG. 5 is an invasiveness graph of the GPR119 agonist AS126957 48h of the present invention on human foreskin fibroblast HFF-1.

FIG. 6 is a data statistical result of invasion of human foreskin fibroblast HFF-1 by GPR119 agonist AS126957 48h of the present invention.

FIG. 7 is a graph showing wound healing of mice with the pan-DUB-enzyme inhibitor AS126957 of the present invention.

FIG. 8 is a data statistic of wound healing in mice with the pan-DUB-enzyme inhibitor AS126957 of the present invention.

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 examples of the invention were tested using GPR119 agonist AS126957, which has the following molecular structural formula:

example 1 in vitro cytotoxicity assay

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

(2) Experimental drugs: compound AS126957, 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 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%

The experiment uses a cell-free culture medium AS a blank control group, a DMSO solution with the same dilution ratio AS the compound is added into each hole AS a negative control group, and the compound AS126957 is used AS a compound experiment group.

The results obtained by the above experimental and computational methods are shown in fig. 1 and 2, wherein fig. 1 shows that compound AS126957 has IC ₁₀＝2.367μm/L、IC₂₀＝7.456μm/L、IC₃₀＝15.987μm/L、IC₅₀ =52.62 μm/L for human foreskin fibroblasts HFF-1 and fig. 2 shows that compound AS126957 has IC ₁₀＝1.944μm/L、IC₂₀＝5.67μm/L、IC₃₀＝11.551μm/L、IC₅₀ =36.26 μm/L for mouse fibroblasts L-929.

From this result, it was shown that GPR119 agonist AS126957 was very lethal to both human foreskin fibroblasts HFF-1 and mouse fibroblasts L-929, indicating very 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: compound AS126957, 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, the other two 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 more than 90% after overnight adherence, 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 used for tightly clinging 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 drawn from left to right, cell culture medium containing 10% FBS (serum buffer solution) is diluted to 1 time, 2X 1X (buffer solution of phosphate buffer solution for 1 time), the cell is removed), the cell culture medium containing 5% of fetal serum is completely dissolved at the concentration of 5 mu.3, the buffer solution is added into the buffer solution for 5 mu, and the buffer solution is completely cultured for 5 mu.0 ℃ after the buffer solution is added, and the buffer solution is added for complete culture solution, and after the buffer solution is added.

The results obtained by performing the cell scoring experiments according to the above experimental methods are shown in fig. 3 and fig. 4, and the experimental results show that compared with the control group Negative (DMSO solution), the migration rate of human foreskin fibroblast HFF-1 treated with GPR119 agonist AS126957 at low concentration (1 μm, 2 μm) is significantly accelerated, which indicates that GPR119 agonist AS126957 can effectively promote cell migration and movement, promote scoring wound healing, and the result indicates P <0.001, which indicates that there is a significant difference between the non-medicated control group and the medicated group.

Example 3 cell invasion assay (Transwell)

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

(2) Experimental drugs: compound AS126957, 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 in serum-free culture medium, and adjusting cell density to proper concentration (the plate density of human foreskin fibroblast HFF-1 is 4×10 ⁵/200 μL);

Inoculating cells: each cell is provided with a negative control group (DMSO with the same dilution ratio AS that of the compound AS126957 is added), a dosing group (working concentration of the compound AS126957 of human foreskin fibroblast HFF-1 is 0.1 mu M and 0.2 mu M), 3 compound holes are respectively arranged in each group, a proper amount of cell suspension is taken according to cell density, a proper amount of BSA (bovine serum albumin) with the volume of 10% is added to make the final percentage of the cell suspension be 0.1%, then the compound AS126957 is added, finally, DMEM is used for supplementing, 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-hole plate lower chamber immediately, and plates are harvested after 48 hours;

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 were observed under 5X microscope and five random fields under 10X microscope, the cells were photographed, counted and statistically plotted, and the results are shown in FIG. 5 and FIG. 6, which show that GPR119 agonist AS126957 can significantly promote migration of fibroblast HFF-1, promote wound healing, enhance wound healing capacity and accelerate wound healing rate.

Example 4 test on mice with wound healing promotion

The experiment of the invention for promoting the healing of the wound of the diabetes mice comprises modeling, wound construction and the healing condition of the wound of the mice by drug administration,

The specific experimental design is shown in table 1;

Table 1: design of experiment

(1) Mouse ordering: ordering 13-week-old, male and BALB/c mice from a laboratory animal center, and feeding for 5 days in an SPF feeding room, and performing isolation observation;

(2) Grouping: the mice were randomly divided into Control negative Control groups and compound AS126957 dosing groups;

(3) Anesthesia: injecting 5% chloral hydrate into abdominal cavity for anesthesia (100 uL/20 g), placing the fully anesthetized mice on a pad towel in prone position, removing back hair with depilatory cream, sterilizing back skin with 75% alcohol, respectively making 2 circular skin full-layer wounds with diameters of about 15mm on two sides of the highest position of the back midline with a trephine with diameters of 15mm, removing subcutaneous tissues with surgical scissors and forceps, exposing muscle surface fascia, stopping bleeding and sterilizing the wounds, opening the wounds, taking a ruler as a control, photographing and recording the shape and the size of the wounds;

(4) Administration: each mouse of the control group was injected with the same volume (0.1 mL) of saline containing 10% DMSO-40% PEG 400-5% tween 80-45%; compound AS126957 (1 mg/kg/d) was administered by intraperitoneal injection; mice were injected with D1, D3, D5, D7, D9, D11 on day of mice trauma;

(5) The anesthetized mice are resuscitated under the temperature control plate and transported back to the EVC cage of the clean rearing room.

The results of the wound healing promotion experiments performed on normal mice according to the method are shown in fig. 7 and 8, and the experimental results show that: the mice dosed with compound AS12695 have faster wound healing than the control group without dosing, and the mice can remarkably promote wound healing when the compound AS12695 is dosed at a concentration of 1mg/kg, and the experimental results have remarkable differences.

It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in any suitable manner,

In order to simplify the description, all possible combinations of the technical features of the above embodiments are not described, and those skilled in the art may combine and combine the features of 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 (7)

1. The application of the GPR119 agonist in preparing a medicine for promoting wound healing is characterized in that the general molecular structural formula of the GPR119 agonist is shown as (I);

Wherein A is selected from one of halogen, hydrogen and C _1～6 alkane; b is selected from one of C _1～6 straight-chain paraffin and C _1～6 branched-chain paraffin; m is selected from 1 to 6.

2. The use of a GPR119 agonist according to claim 1 for the manufacture of a medicament for promoting wound healing, wherein the GPR119 agonist has the molecular structural formulae (II) to (VI);

3. The use of a GPR119 agonist according to claim 2 wherein the GPR119 agonist has the molecular structural formula (III);

4. A medicament for promoting wound healing, characterized in that it comprises a GPR119 agonist according to any one of claims 1 to 3 and its geometric isomers or its pharmaceutically acceptable salts and/or its solvates and/or its hydrates.

5. The wound-healing-promoting medicament of claim 4, further comprising a pharmaceutically acceptable adjuvant.

6. The wound-healing-promoting agent according to claim 5, wherein the dosage form of the agent 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.

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