Medicament for treating sports injuries, screening method and use

Abstract

The present application relates to a kind of drugs for treating sports injury and its application, a kind of construction method of tendon injury repair animal model, the treatment sports injury is at least one of acute injury and chronic injury, preferably, the treatment sports injury is the treatment tendon injury, more preferably, the treatment tendon injury is to promote tendon healing and / or inhibit tendon adhesion formation.The advantages of the present application (1) good drug screening effect.(2) the drug screened can prevent and treat tendon adhesion and has less effect on tendon healing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a drug for treating sports injury and its application. BACKGROUND

[0002] Sports injury is very common in daily life and sports field, especially in the field of sports medicine. Among them, the most common is tendon injury. Tendon adhesion is a common complication after tendon injury and repair, with an incidence of up to 60%, which seriously affects the limb movement of patients and even leads to lifelong disability. After tendon injury, how to reduce postoperative adhesion has always been an important problem in clinical practice.

[0003] At present, the treatment of tendon adhesion often needs to be treated by surgery to release and remove the adhesion tissue, but there is still a possibility of re-adhesion after surgery, thereby forming a vicious cycle of "adhesion, release, and re-adhesion". Generally, tendon healing includes two pathways, endogenous healing and exogenous healing. In treatment, it is often desired to promote endogenous healing and inhibit exogenous healing. In the process of release treatment, the use of anti-adhesion membranes made of biological materials to wrap the surgically repaired tendon can play a physical barrier role to prevent tendon adhesion; however, the current biological material anti-adhesion membranes have single function, poor efficacy and can easily cause inflammation, which limits their clinical application. Moreover, there is a lack of related anti-adhesion treatment drugs in clinical practice. During the healing process after tendon injury, exogenous healing is mainly the growth of peritendinous tissue into the tendon or on the surface of the tendon, forming granulation tissue to help tendon repair; endogenous healing is the proliferation of tendon cells / tendon stem cells themselves under sufficient synovial nutrition, and self-repair through the synthesis and secretion of collagen. Exogenous healing is the main cause of tendon adhesion. The existing anti-adhesion membranes and drugs all have their limitations, so there is an urgent need for new drugs with strong specificity and effective means to find new drugs.

[0004] In the application of drugs, there are mainly three kinds of drugs currently used for tendon anti-adhesion. One is anti-inflammatory drugs, such as ibuprofen, indomethacin, and hyaluronic acid. These drugs act on the inflammatory period of tendon healing. Two is antitumor drugs, such as mannose-6-phosphate and 5-fluorouracil. Three is antifibrotic drugs, such as trypsin inhibitors. The latter two drugs have the effect of inhibiting the growth and differentiation of fibroblasts, thereby reducing tendon adhesion.

[0005] At present, the demand for drugs that can inhibit exogenous healing without inhibiting endogenous healing is still huge, and there has always been a lack of an effective way to find such ideal drugs. SUMMARY

[0006] The present application aims to provide a drug for treating sports injuries, wherein the treatment of sports injuries is at least one of treating acute injuries and chronic injuries, preferably, the treatment of sports injuries is treating tendon injuries, more preferably, the treatment of tendon injuries is promoting tendon healing and / or inhibiting tendon adhesion formation.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0008] The use of the compound of formula 1 or a pharmaceutically acceptable salt thereof is characterized in that it is used for preparing a drug composition or preparation for treating and promoting tendon healing and inhibiting tendon adhesion formation.

[0009]

[0010] R1-R4 are independently selected from substituted or unsubstituted H, halogen, CN, OH, NH2, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,

[0011] R5, R7 are independently selected from substituted or unsubstituted H, halogen, CN, OH, NH2, C1-C6 alkyl, and R5, R7 are optionally substituted with one or more optional substituents of alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,

[0012] R6 is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, -(CH)n-COOH, -(CH)n-COO-R10, -(CH)n-CO-R10, -(CH)n-CO-NH-R10, -(CH)n-O-CO-NH-R10, -(CH)n-CO-NR10R11, -(CH)n-O-CO-NR10R11, -(CH)n-O-CO-R10, -(CH)n-O-CO-NR10R11, -(CH)n-O-COO-R10, -C(=O)SR10, -C(=S)-R10,

[0013] R8-R9 are independently selected from C, N, O, S,

[0014] n is selected from 1-12,

[0015] R10, R11 are independently selected from substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, heterocycloalkyl, aryl, heteroaryl,

[0016] The above-mentioned substituents can be substituted with one or more optional substituents of alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl.

[0017] The compound of formula I is

[0018] The English name of the Orantinib is Orantinib, the alias is SU 6668, SU6668, TSU-68, the CAS number is 210644-62-5.

[0019] Another technical solution, the use of tioxolone or its pharmaceutically acceptable salt is characterized in that, for the preparation of the treatment of sports injury in the preparation of the treatment of sports injury drug composition or preparation.

[0020] The English name of the tioxolone is tioxolone, and the CAS number is 4991-65-5.

[0021] Further, the treatment of sports injury is at least one of the treatment of acute injury and chronic injury, preferably, the treatment of sports injury is the treatment of tendon injury, more preferably, the treatment of tendon injury is to promote tendon healing and / or inhibit tendon adhesion formation.

[0022] The pharmaceutical composition or preparation contains 0.001-99wt% of the compound of formula I or its pharmaceutically acceptable salt, based on the total weight of the composition.

[0023] The dosage form of the pharmaceutical composition or preparation is oral dosage form or injection.

[0024] The oral dosage form includes tablets, capsules, films and granules.

[0025] The pharmaceutical composition or preparation also contains a pharmaceutically acceptable carrier.

[0026] The pharmaceutical composition also contains other active ingredients for promoting tendon healing and / or inhibiting tendon adhesion formation.

[0027] The present application is based on the tendon injury repair animal model, using primary cell culture and flow cytometry technology to extract tendon stem cells and myofibroblasts, and for drug screening, the screened tioxolone and orantinib can prevent and treat tendon adhesion and have less effect on tendon healing. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the schematic diagram of the construction of rat tendon adhesion provided by the present application.

[0029] Figure 2 It is the flow cytometry sorting myofibroblast results provided by the present application.

[0030] Figure 3 It is the identification results of tendon stem cells provided by the present application.

[0031] Figure 4 is the result of drug screening.

[0032] Figure 5 is the HE staining result of untreated (wt) and treated groups (medicine) 14 days after modeling of mice.

[0033] Figure 6 is the type I collagen immunofluorescence staining result of untreated (wt) and treated groups (medicine) 14 days after modeling of mice.

[0034] Figure 7 is the scx immunofluorescence staining result of untreated (wt) and treated groups (medicine) 14 days after modeling of mice.

[0035] Figure 8 is the type III collagen immunofluorescence staining result of untreated (wt) and treated groups (medicine) 14 days after modeling of mice.

[0036] Figure 9 is the sma immunofluorescence staining result of untreated (wt) and treated groups (medicine) 14 days after modeling of mice. DETAILED DESCRIPTION

[0037] In order for those skilled in the art to better understand the technical solutions of the present application, the following examples further describe the present application in detail, and the following examples are only used to illustrate the present application, but not to limit the scope of the present application.

[0038] Example 1 Construction of rat tendon injury repair animal model

[0039] All animal research protocols were approved by the Institutional Review Board of Shanghai Jiao Tong University (SYXK (Hu) 2016-0020). Rats weighing 250-300 g were anesthetized by intraperitoneal injection of pentobarbital, then fixed and prepared for skin. A longitudinal incision was made at the Achilles tendon to expose the Achilles tendon. The peritenon tissue was separated, and the Achilles tendon was cut transversely. The Achilles tendon was sutured using a 6-0 suture using a modified Kessler suture method. The skin was sutured with a 4-0 suture and sterilized.

[0040] As shown in Figure 1 A, after anesthesia and skin preparation, exposure and separation of the Achilles tendon, cutting of the Achilles tendon, suture of the Achilles tendon, and suture of the skin and sterilization of the five steps, the model of rat Achilles tendon adhesion was constructed. Ten days after the operation, the skin at the Achilles tendon was incised, and the granulation tissue and adhesion tissue attached around the Achilles tendon were visible, and could be separated from the peritenon only with a sharp instrument Figure 1 B).

[0041] Example 2 Tendon stem cells and myofibroblasts for drug screening

[0042] Ten days after operation, rats were sacrificed and their hind limbs were harvested. After 1 hour of 75% alcohol disinfection, the skin at the Achilles tendon was cut open and the tendon was exposed. The newly formed adhesion tissue around the tendon was carefully cut off and placed in 3 mg / ml collagenase I for 3 hours of digestion at 37°C. After the digestion, the supernatant was discarded by centrifugation, and the cells in the lower layer were resuspended and inoculated in a 10 cm culture dish, which was then placed in a 37°C incubator with a 5% carbon dioxide concentration.

[0043] After the cells were in the logarithmic growth phase, the α-SMA positive cells were screened by flow sorting and continued to be cultured. Briefly, the original culture medium was discarded, and the cells were washed once with 1 ml of PBS. Trypsin digestion was performed, and the supernatant was discarded by centrifugation. The cells were resuspended with 2 ml of PBS, and the supernatant was discarded by centrifugation. The cells were resuspended with 500 μl of staining buffer, and the cell concentration was adjusted to 10 7 / ml. The α-SMA flow antibody was added, the cell suspension was mixed by blowing, and the cells were incubated in the dark for 15 minutes. The cells were centrifuged for 5 minutes with 2 ml of PBS, and the washing was repeated twice. The machine was started, and the α-SMA positive cells were sorted by flow cytometry. The myofibroblasts were inoculated in a 10 cm culture dish, which was then placed in a 37°C incubator with a 5% carbon dioxide concentration.

[0044] The hind limbs of normal rats were harvested, and 75% alcohol was used for disinfection for 1 hour. The skin at the Achilles tendon was cut open and the tendon was exposed in a clean bench. The tendon was carefully removed and cut into 1 mm*1 mm*1 mm pieces, taking care not to mix muscle and tendon tissue. The cut tendon tissue was placed in 4 mg / ml collagenase I for 4 hours of digestion at 37°C. After the digestion, the supernatant was discarded by centrifugation, and the cells in the lower layer were resuspended and inoculated in a 10 cm culture dish, which was then placed in a 37°C incubator with a 5% carbon dioxide concentration.

[0045] The tendon stem cells were identified by immunofluorescence. The cells were washed with PBS for 3 times, 3 minutes each time. Fixed with 4% paraformaldehyde for 15 minutes, washed with PBS for 3 times, 3 minutes each time. Permeated with 0.5% Triton X-100 for 20 minutes at room temperature, washed with PBS for 3 times, 3 minutes each time. Add blocking solution on the slide, block for 30 minutes at room temperature. Remove the blocking solution, add enough diluted Col I and Scx primary antibodies to each slide and put it in a wet box, incubate overnight at 4°C. The next day, wash with PBST for 3 times, 3 minutes each time, add diluted fluorescent secondary antibody, incubate in a wet box at 37°C for 1 hour, wash with PBST for 3 times, 3 minutes each time. Add DAPI and incubate in the dark for 5 minutes, wash with PBST for 3 times. Mount the slide with an anti-fluorescence quencher-containing mounting medium, and then observe and collect images under a fluorescence microscope.

[0046] Primary adhesion tissue cells pre-stained with antibodies were sorted using flow cytometry. α-SMA-positive cells, i.e., myofibroblasts, accounted for 1.30% of the total adhesion tissue cells. Figure 2 The target cells are collected, cultured, and expanded for subsequent drug screening.

[0047] Primary tendon stem cells were identified using Scx and ColI, specific markers for tendon stem cells. DAPI showed blue fluorescence, Scx showed green fluorescence, and ColI showed red fluorescence, indicating that both Scx and ColI were expressed intracellularly. Figure 3 This indicates that the primary cells extracted from the tendon are indeed tendon stem cells, which can be used for subsequent drug screening experiments.

[0048] Example 3 Drug Screening and Drug Screening Analysis

[0049] Using a Multidrop automated dispensing system, well-grown tendon stem cells and myofibroblasts were evenly seeded into clear 384-well U-plates, with 400 cells per well and 100 μL of complete DMEM medium per well. Next, small molecules from a compound library were added to the seeded 384-well plates using a JANUS automated pipetting workstation. The final concentration of the selected compounds was controlled at 10 μM. After drug addition, the cell culture plates were placed in a 37°C CO2 cell culture incubator for 3 days. After drug treatment, CellProof solution was added, and the results were analyzed using an EnSpire microplate reader.

[0050] Some results of drug screening are presented in the form of a heatmap. Figure 4 Cells treated with DMSO were used as a control. Cells with a relative inhibition rate higher than 50% were plotted in red, and those with a relative inhibition rate lower than 50% were plotted in blue. It can be seen that L-ascorbic acid had a relative inhibition rate of less than 50% for both cell types, while the other four drugs—WAY-600, GSK1070916, Ispinoxetine, and SRT1720—had relative inhibition rates higher than 50% for both cell types. Thiacoxone showed a relative inhibition rate higher than 50% for myofibroblasts but a relative inhibition rate lower than 50% for tendon stem cells, making it a potentially ideal drug. Olantinib showed a relative inhibition rate of 57% for myofibroblasts but a relative proliferation rate of 15% for tendon stem cells. Therefore, olantinib could be considered a potentially ideal drug, inhibiting tendon adhesion formation without affecting tendon healing.

[0051] Example 4: Construction of a classic mouse adhesion model

[0052] Mice were anesthetized with 2.5% avertin 0.2ml, the proximal tendon of the deep flexor muscle was cut, the plantar surface was exposed dorsally under the stereomicroscope, the skin was incised, bluntly dissected and the tendon was fully exposed, the tendon was sutured with 8-0 suture, kessler suture after cutting the tendon, the skin was sutured with 6-0 suture, and the mice were freely fed without fixation.

[0053] Example 5: Experiment with classic mouse adhesion model

[0054] We carried out the classic mouse adhesion model experiment on the better Orlamatin selected by cell screening.

[0055] As shown in Figure 5 , 14 days after modeling of mice, HE staining of untreated (wt) and treated groups (medicine).▲ represents adhesion tissue, the part circled by dotted line is the modeling area, and T represents tendon. According to the adhesion semi-quantitative score, the adhesion around the tendon is reduced after treatment, t=5.814 df=6,**P<0.01. Scale: 1cm. The continuity of the whole tendon is also better than that of the untreated group.

[0056] As shown in Figure 6 , 14 days after modeling of mice, type I collagen immunofluorescence staining of untreated (wt) and treated groups (medicine). T represents tendon. It can be seen that the expression of type I collagen in the tendon of the treated group is more, but there is no statistical significance. Scale: 500um

[0057] As shown in Figure 7 , 14 days after modeling of mice, scx immunofluorescence staining of untreated (wt) and treated groups (medicine). T represents tendon. It can be seen that the expression of SCX in the tendon of the treated group is more, but there is no statistical significance. Scale: 500um

[0058] As shown in Figure 8 , 14 days after modeling of mice, type III collagen immunofluorescence staining of untreated (wt) and treated groups (medicine). T represents tendon, and M represents muscle. The expression of type III collagen in the treated group is reduced, but there is no statistical significance. Scale: 500um

[0059] As shown in Figure 9 , 14 days after modeling of mice, sma immunofluorescence staining of untreated (wt) and treated groups (medicine). T represents tendon, and M represents muscle. The expression of sma in the treated group is reduced, but there is no statistical significance. Scale: 500um

[0060] The results show that Orlamatin is an ideal drug for treating tendon injury, which can promote tendon healing while inhibiting tendon adhesion formation.

[0061] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0062] In addition, it should be noted that each specific technical feature and step described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

[0063] In addition, various different embodiments of the present application can also be combined in any manner, as long as they do not deviate from the idea of the present application, and they should also be considered as disclosed by the present application.

Claims

1. Use of olantinib in the preparation of a medicament for treating tendon injury, said treating tendon injury is promoting tendon healing and inhibiting tendon adhesion formation; said olantinib has the following structure: 。 2. Use according to claim 1, characterized in that, The medicament contains 0.001-99wt% of olantinib or its pharmaceutically acceptable salt, based on the total weight of the composition.

3. Use according to claim 1, characterized in that, The dosage form of the medicament is oral dosage form or injection.

4. Use according to claim 3, characterized in that, The oral dosage form is tablet, capsule, film or granule.

5. The use according to claim 1, characterized in that, The medicament contains a pharmaceutically acceptable carrier.

6. The use according to claim 1, characterized in that, The medicament contains other active ingredients that can promote tendon healing and / or inhibit tendon adhesion formation.

2. The use of claim 1, wherein the oral dosage form is tablet, capsule, film or granule.

3. The use of claim 1, wherein the injection is injection solution or injection suspension.

4. The use of claim 1, wherein the medicament contains 0.001-99wt% of olantinib or its pharmaceutically acceptable salt, based on the total weight of the composition.

5. The use of claim 1, wherein the medicament contains a pharmaceutically acceptable carrier.

6. The use of claim 1, wherein the medicament contains other active ingredients that can promote tendon healing and / or inhibit tendon adhesion formation.

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