Application of polylactic acid and its copolymers in the preparation of drugs promoting tendon / ligament growth

Abstract

The present invention provides the use of polylactic acid and its copolymers in the preparation of tendon / ligament growth-promoting drugs, wherein the molecular weight of the polylactic acid is 400-300000Da, and the preferred molecular weight is 5000-20000Da. The polylactic acid copolymer includes one or more of polylactic acid-co-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polyethylene glycol-polylactic acid-co-glycolic acid copolymer, polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer, preferably polylactic acid-co-glycolic acid copolymer. The present invention first discovered that polylactic acid and its copolymers have the effect of promoting ligament / tendon growth, providing a new approach for the treatment of ligament / tendon injuries.

Description

Technical Field

[0001] The present 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 and ligament injuries are common sports injuries, with a significant number of new cases reported annually worldwide, making them a global health concern. Tendons and ligaments are fibrous connective tissues that connect muscle to bone, or bone to bone, providing traction and stability. Injury can significantly reduce a patient's quality of life and, in severe cases, 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 recovery. At the same time, the length and diameter of the autologous tendon are limited by the sampling site, making it difficult to meet the usage standards; 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 PLA is primarily prepared by polymerizing lactic acid or lactide through ring-opening polymerization. PLA and its copolymers not only possess excellent mechanical strength and chemical stability, but also possess good biocompatibility and biodegradability. In recent years, extensive research has been conducted both domestically and internationally on its biomedical applications. It has found widespread application in surgical sutures, bone repair materials, controlled drug release systems, and tissue scaffolds (such as artificial bone and skin).

[0005] Biodegradable and biocompatible poly(L-lactic acid) microparticles (trade name Sculptra™) were approved by the US FDA in 2004 for filling sagging skin caused by facial fat atrophy in AIDS patients. Clinicians also use them for aging-related fat atrophy and localized fat atrophy in healthy individuals. In 2009, the US FDA officially approved poly(L-lactic acid) fillers for improving nasolabial folds. However, there are no reports in the prior art regarding the effects of poly(lactic acid) and its copolymers on ligament or tendon repair. Summary of the Invention

[0006] In view of the shortcomings of 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 tendon / ligament growth promoting drugs, 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 formulation further includes 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 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.

[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 copolymers 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 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 the 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 PLLA microspheres at different concentrations 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 solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative 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 encompassed within the scope of protection 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 L-polylactic acid (PLLA) (molecular weight 20,000 Da) was dissolved in 150 mL of dichloromethane, and then the above polymer solution was added to 2000 mL of a 1.0% polyvinyl alcohol aqueous solution. After emulsification at 5000 rpm / min for 5 minutes, the mixture was stirred at 1000 rpm for 6 hours to remove the dichloromethane, and finally L-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 the figure. 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 glycolic acid microspheres

[0031] 10 g of polylactic-co-glycolic acid (PLGA) (molecular weight 20,000 Da) was dissolved in 150 mL of dichloromethane, and then the above polymer solution was added to 2000 mL of 0.5% polyvinyl alcohol aqueous solution. After emulsification at 5000 rpm / min for 5 minutes, the mixture was 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 the figure. Figure 2 As shown. Figure 2 It can be seen that the particle size of the microspheres ranges from 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 using the CCK-8 assay. 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, with five parallel wells set up for each sample. Then, 200 μL of a 4×10 4 After 1, 3, and 5 days of incubation in the dark, the cell culture medium in the 96-well plate was removed and placed in an empty 96-well plate. 20 μL of CCK-8 solution was added to each well. After incubation in the dark for 4 hours, the absorbance at 450 nm was measured by a microplate reader. The proliferation results of rabbit tenocytes on samples with different concentrations of PLLA microspheres were finally calculated. Figure 3 shown.

[0035] Depend on Figure 3 As can be seen, as the concentration of PLLA microspheres increases, the number of tenocytes also gradually increases. Therefore, PLLA microspheres have a significant effect on promoting the proliferation of tenocytes, and this effect is significantly enhanced with increasing drug concentration. The inventors tested the effects of polymer microspheres on ligament cells using the same method 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 ratio) were divided into two groups, with 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 Procedure: The anatomical location of the right Achilles tendon of the mouse was determined. The dorsal skin of the distal tibia was disinfected with an alcohol swab. A 26G needle and a 1mL sterile syringe were used to inject into the Achilles tendon (the needle should be positioned at an angle of 15-30° to the tendon to avoid puncturing the tendon). The right side was designated as the experimental side (in situ injection of PLLA microspheres or PLGA microspheres in saline solution, once), and the left side was designated as the control side (injection of saline).

[0043] Three mice in each group were killed 7, 14, and 28 days after surgery, and the experimental and control sides of each mouse were fixed and subjected to histopathological examination.

[0044] 2. Detection method:

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

[0046] 2) Randomly select the experimental and control sides for sectioning, measure the tissue diameter under a 100x optical microscope, and calculate the average value of the experimental and control sides 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 Figure 1, after injection of 0.5 mg of PLLA microspheres or 0.5 mg of PLGA microspheres, the experimental side tendon tissue showed varying degrees of significant thickening. The figure shows that collagen fibers (red) were extensive on the experimental side at 7, 14, and 28 days, indicating significant tendon thickening and increased Achilles tendon diameter. Table 1 lists the average Achilles tendon diameters of the experimental and control sides in the two groups.

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

[0051] These experimental results demonstrate that PLLA or PLGA microspheres significantly promote tendon regeneration in animals. The inventors further tested the effects of PLLA or PLGA microspheres on animal ligament tissue using the same method and obtained the same results, demonstrating that PLLA or PLGA microspheres also significantly promote ligament regeneration in animals.

[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 solutions of the present invention and are not limiting. 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 solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of poly-L-lactic acid or poly(lactic-co-glycolic acid) as the sole active ingredient in the preparation of a drug for treating tendon injury, characterized in that: The molecular weight of the poly-L-lactic acid or poly(lactic-co-glycolic acid) copolymer is 20,000 Da; the drug can promote tendon growth, and the drug is a microsphere preparation.

2. The use according to claim 1, characterized in that: The formulation further comprises excipients, which include at least one of a stabilizer, a filler, a binder, a surfactant, and a lubricant.

3. The use according to claim 2, characterized in that: The surfactant includes one or more of polyethylene glycol, sodium lauryl sulfate, Tween, and Span; the stabilizer includes one or both 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.

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