Application of polylactic acid and copolymer thereof in preparation of tendon / ligament growth promoting medicine

Abstract

The invention provides application of polylactic acid and a copolymer thereof in preparation of a medicine for promoting tendon / ligament growth, and the molecular weight of the polylactic acid is 400-300000Da, preferably 5000-20000Da. The polylactic acid copolymer comprises one or more of a polylactic acid-glycolic acid copolymer, a polylactic acid-polyethylene glycol copolymer, a polyethylene glycol polylactic acid-glycolic acid copolymer, a polylactic acid-chitosan copolymer and a lactide-caprolactone copolymer, and preferably, the polylactic acid-glycolic acid copolymer. The effect of promoting ligament / tendon growth of polylactic acid and the copolymer thereof is found for the first time, and a new way is provided for treatment of ligament / tendon injuries.

Description

Technical Field

[0001] The invention relates to the technical field of polymer drugs, in particular to the application of polylactic acid and its copolymers in the preparation of drugs for promoting tendon / ligament growth. Background Art

[0002] Joint ligament injury is one of the most common sports injuries, with a large number of new cases every year in the world, and has become a global health problem. Tendons / ligaments are fibrous connective tissues that connect muscles to bones or bones to bones, and play a role in traction and stability. Once injured, it will greatly reduce the patient's quality of life, and severe cases will lead to functional disability.

[0003] In order to restore the function of the knee joint after joint ligament injury, ACL reconstruction surgery is often required. The current clinical reconstruction grafts include autologous tendons, allogeneic tendons and artificial ligaments. Among them, autologous tendons are taken from the patient's own hamstrings, gracilis muscles, etc. Autologous tendon transplantation will cause secondary damage to the patient, which is not conducive to later rehabilitation. At the same time, the length and diameter of the autologous tendon are limited by the sampling site, and it is difficult to meet the use standard; allogeneic tendons have the risk of immune rejection and infection in clinical use, and the donor source is limited; artificial ligaments are usually high-molecular organic materials, and their degradation products will change the local microenvironment of the joint and easily cause aseptic inflammation. In addition, tendons, cartilage and other tissues lack blood supply, so tissue regeneration is difficult, and grafts are mainly functional replacements and have no ability to promote tissue regeneration. Therefore, the biological integration of tissues and grafts is difficult, and long-term implantation still has the risk of re-rupture.

[0004] The molecular formula of polylactic acid (PLA) is (C3H4O2) n , mainly prepared by lactic acid polymerization or lactide ring-opening polymerization. PLA and its copolymers not only have excellent mechanical strength and chemical stability, but also have good biocompatibility and biodegradability. In recent years, a lot of research has been done on its application in biomedicine at home and abroad. It has been widely used in surgical sutures, bone repair materials, drug controlled sustained release systems, and tissue functional scaffolds (such as artificial bones and artificial skin).

[0005] Biodegradable and biocompatible L-polylactic acid microparticles (trade name SculptraTM) were approved by the U.S. FDA in 2004 for filling sagging skin areas caused by lipoatrophy in the face of AIDS patients. Clinicians also use them for aging-related lipoatrophy and localized lipoatrophy in healthy people. In 2009, the U.S. FDA officially approved L-polylactic acid fillers for improving nasolabial folds. However, there are no reports in the prior art on the effects of polylactic acid and its copolymers on ligament or tendon repair. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides the use of polylactic acid and its copolymers in the preparation of drugs for promoting tendon / ligament growth, which provides a new approach for promoting tendon / ligament growth.

[0007] The purpose of the present invention is to provide the use of polylactic acid and its copolymers in the preparation of drugs for promoting tendon / ligament growth, wherein the molecular weight of the polylactic acid is 400-300000Da, preferably, the molecular weight is 5000-20000Da.

[0008] Furthermore, the polylactic acid is poly-L-lactic acid.

[0009] Furthermore, the polylactic acid copolymer includes one or more of polylactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polyethylene glycol-polylactic acid-glycolic acid copolymer, polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer.

[0010] Preferably, the polylactic acid copolymer is a polylactic acid-glycolic acid copolymer.

[0011] Furthermore, the polylactic acid and its copolymers are in the form of preparations.

[0012] Furthermore, the preparation includes microspheres, micelles, and gels.

[0013] The preparation further comprises excipients, which include at least one of a stabilizer, a filler, a binder, a surfactant and a lubricant.

[0014] Furthermore, the surfactant includes one or more of polyethylene glycol, sodium dodecyl sulfate, Tween, and Span; the stabilizer includes one or two of carboxymethyl cellulose and mannitol; the filler includes one or more of lactose, mannitol, cyclodextrin, and sorbitol; the binder includes one or more of hydroxypropyl cellulose, methyl cellulose, and polyvinyl pyrrolidone; and the lubricant includes one or more of magnesium stearate, calcium stearate, and stearic acid.

[0015] Furthermore, the preparation further comprises a first active ingredient, and the first active ingredient has the effect of reducing swelling and relieving pain.

[0016] Furthermore, the first active ingredient includes celecoxib, loxoprofen sodium, phenylbutazone, ibuprofen, and etoricoxib.

[0017] Furthermore, the dosage of L-lactic acid in the poly-L-lactic acid and its copolymer during at least one administration of the preparation is 8 to 3000 mmol / L, preferably 250 to 750 mmol / L.

[0018] In a specific embodiment of the present invention, the preparation is injected into the diseased tendon, and the dosage of the preparation used is calculated according to the mass of the tendon at the diseased site.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the present invention is the first to discover that polylactic acid and its copolymers have the effect of promoting tendon / ligament growth, providing a new approach for the treatment of tendon / ligament injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SEM image of the PLLA microspheres in Example 1 of the present invention.

[0021] Figure 2 This is the SEM image of PLGA microspheres in Example 2 of the present invention.

[0022] Figure 3 This is a graph showing the proliferation results of tendon cells under the action of different concentrations of PLLA microspheres in Example 3 of the present invention.

[0023] Figure 4 The stained images are of the tendons of the experimental side (0.5 mg PLLA microspheres) and the control side (normal saline) in Example 4 of the present invention; red represents collagen fibers.

[0024] Figure 5 The stained images are of the tendons of the experimental side (0.5 mg PLGA microspheres) and the control side (normal saline) in Example 4 of the present invention; red represents collagen fibers. DETAILED DESCRIPTION

[0025] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0026] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0027] Example 1 Preparation of L-polylactic acid microspheres

[0028] 10 g of left-handed polylactic acid (PLLA) (molecular weight 20,000 Da) was dissolved in 150 mL of dichloromethane, and then the above polymer solution was added into 2000 mL of 1.0% polyvinyl alcohol aqueous solution, emulsified at 5000 rpm / min for 5 minutes, and then stirred at 1000 rpm for 6 hours to remove dichloromethane, and finally left-handed polylactic acid microspheres were obtained.

[0029] The prepared L-polylactic acid microspheres were observed by scanning electron microscopy and the particle size was calculated. The photo of the microspheres is shown in Figure 1 As shown. Figure 1 It can be seen that the particle size range of the microspheres is 24.31 to 54.29 μm.

[0030] Example 2 Preparation of polylactic acid-co-glycolic acid microspheres

[0031] 10 g of polylactic acid-co-glycolic acid (PLGA) (molecular weight 20,000 Da) was dissolved in 150 mL of dichloromethane, and then the polymer solution was added into 2000 mL of a 0.5% polyvinyl alcohol aqueous solution, emulsified at 5000 rpm / min for 5 minutes, and then stirred at 1000 rpm for 6 hours to remove dichloromethane, and finally PLGA microspheres were obtained.

[0032] The prepared polylactic acid microspheres were observed by scanning electron microscopy and the particle size was calculated. The photo of PLGA microspheres is shown in Figure 2 As shown. Figure 2 It can be seen that the particle size range of the microspheres is 17.89 to 55.87 μm.

[0033] Example 3 Cell experiment to promote tendon growth

[0034] Tendon cells were extracted from the subscapularis tendon of 2-week-old rabbits. The proliferation of rabbit tenocytes in PLLA microspheres was tested by CCK-8 method. First, 5 mg, 10 mg and 20 mg of the polymer microspheres prepared in Example 1 were added to the bottom of a 96-well plate, and 5 parallel wells were set for each sample. Then, 200 μL of 4×10 4 / mL of cell suspension was placed in a cell culture incubator. After 1, 3, and 5 days of incubation in the dark, the cell culture medium in the 96-well plate was taken out and placed in an empty 96-well plate. 20μL of CCK-8 solution was added to each well. After incubation in the dark for 4 hours, the absorbance at 450nm was tested by an ELISA instrument, and the proliferation results of rabbit tendon cells on PLLA microsphere samples with different concentrations were finally calculated. The results are shown in Figure 3 shown.

[0035] Depend on Figure 3 It can be seen that as the concentration of PLLA microspheres increases, the number of tendon cells also gradually increases. Therefore, PLLA microspheres have a significant effect on promoting the proliferation of tendon cells, and as the concentration of the drug increases, its effect is significantly enhanced. The inventors tested the effect of polymer microspheres on ligament cells in the same way and obtained the same effect, indicating that PLLA microspheres have a significant effect on promoting the proliferation of ligament cells.

[0036] Example 4 Effects of PLLA microspheres and PLGA microspheres on tendon growth in experimental animals

[0037] Experimental animals: C57BL mice, weighing 25±3 g (male and female, 1:1), were divided into 2 groups, 10 mice in each group.

[0038] Experimental site: right Achilles tendon of mice.

[0039] Experimental groups:

[0040] (1) 0.5 mg PLLA microsphere group (Example 1);

[0041] (2) 0.5 mg PLGA microsphere group (Example 2).

[0042] Experimental operation: Determine the anatomical position of the right Achilles tendon of the mouse, disinfect the dorsal skin of the distal tibia with an alcohol cotton ball, and use a 26G needle and a 1mL sterile syringe to inject into the Achilles tendon (the angle between the needle and the Achilles tendon should be between 15-30° to avoid piercing the tendon). The right side is set as the experimental side (in situ injection of PLLA microspheres or PLGA microspheres in saline solution, only once), and the left side is the control side (injection of saline).

[0043] Three mice in each group were killed on days 7, 14, and 28 after surgery, and the experimental and control sides of each mouse were selected for fixation and pathological histological examination.

[0044] 2. Detection method:

[0045] 1) The tissue to be observed was cut and fixed, and then stained with resorcinol fuchsin to observe the effects of injected microspheres on tissues in various parts.

[0046] 2) Randomly select the experimental side and the control side for sectioning, measure the tissue diameter under a 100x optical microscope, and calculate the average value of the experimental side and the control side using the following formula:

[0047] Average value = (3 measurements of animal No. ① + 3 measurements of animal No. ② + 3 measurements of animal No. ③) / 9

[0048] 3. Experimental results:

[0049] The experimental results are as follows Figure 4 and Figure 5 As shown in the figure. After the injection of 0.5mg PLLA microspheres or 0.5mg PLGA microspheres, the experimental side tendon tissue of the two groups of experimental animals showed different degrees of significant thickening. It can be seen from the figure that the experimental side collagen fibers (red) were large on the 7th, 14th and 28th days, indicating that the tendon was significantly thickened and the Achilles tendon diameter increased. Table 1 lists the average diameters of the Achilles tendons on the experimental and control sides of the two groups.

[0050] The values ​​of tendon tissue measured under a 100x optical microscope are shown in Table 1: Compared with the control side, the difference in the diameter of the Achilles tendon on the experimental side treated with 0.5 mg PLLA microspheres on the 14th day and the 28th day was statistically significant (P<0.05), and the diameter of the Achilles tendon increased, showing a significant promotion effect on tendon growth; Compared with the control side, the difference in the diameter of the Achilles tendon on the experimental side treated with 0.5 mg PLGA microspheres on the 14th day and the 28th day was statistically significant (P<0.05), and the diameter of the Achilles tendon increased, showing a significant promotion effect on tendon growth.

[0051] It can be seen from these experimental results that PLLA microspheres or PLGA microspheres have a significant promoting effect on the regeneration of animal tendon tissue. The inventors further used the same method to detect the effect of PLLA microspheres or PLGA microspheres on animal ligament tissue and obtained the same effect, indicating that PLLA microspheres or PLGA microspheres also have a significant promoting effect on the regeneration of animal ligament tissue.

[0052] Table 1 Comparison of measured values ​​between the injection side of PLLA microspheres or PLGA microspheres and the control side

[0053]

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Application of polylactic acid and its copolymers in the preparation of drugs for promoting tendon / ligament growth, characterized in that: The molecular weight of the polylactic acid is 400-300000Da, preferably, the molecular weight is 5000-20000Da.

2. The use according to claim 1, characterized in that: The polylactic acid is poly-L-lactic acid.

3. The use according to claim 1, characterized in that: The polylactic acid copolymer includes one or more of polylactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polyethylene glycol-polylactic acid-glycolic acid copolymer, polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer. Preferably, the polylactic acid copolymer is a polylactic acid-glycolic acid copolymer.

4. The use according to claim 1, characterized in that: The polylactic acid and its copolymer are in the form of preparation.

5. The use according to claim 4, characterized in that: The preparations include microspheres, micelles, and gels.

6. The use according to claim 4, characterized in that: The preparation further comprises excipients, which include at least one of a stabilizer, a filler, a binder, a surfactant and a lubricant.

7. The use according to claim 6, characterized in that: The surfactant includes one or more of polyethylene glycol, sodium lauryl sulfate, Tween, and Span; the stabilizer includes one or two of carboxymethyl cellulose and mannitol; the filler includes one or more of lactose, mannitol, cyclodextrin, and sorbitol; the binder includes one or more of hydroxypropyl cellulose, methyl cellulose, and polyvinyl pyrrolidone; and the lubricant includes one or more of magnesium stearate, calcium stearate, and stearic acid.

8. The use according to claim 4, characterized in that: The preparation further comprises a first active ingredient, which has the effect of reducing swelling and relieving pain.

9. The use according to claim 8, characterized in that: The first active ingredient includes celecoxib, loxoprofen sodium, phenylbutazone, ibuprofen, and etoricoxib.

10. The use according to claim 7, characterized in that: The dosage of L-lactic acid in the poly-L-lactic acid and its copolymer during at least one administration of the preparation is 8 to 3000 mmol / L, preferably 250 to 750 mmol / L.

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