Application of polylactic acid and copolymer thereof in preparation of medicine for promoting nerve growth and repairing

Abstract

The invention provides an application of polylactic acid and a copolymer thereof in preparation of a medicine for promoting nerve growth and repairing, the molecular weight of the polylactic acid and the copolymer thereof is 400-300k Da, and the medicine has the following effects: 1) promoting growth of nerve cells and nerve tissues; 2) promoting the repair of nerve cells and nerve tissues; 3) treating or preventing peripheral nerve injury, spinal cord injury, peripheral neuropathy and neuritis; preferably, the molecular weight is in the range of 5000 to 100 k Da. The invention discovers that polylactic acid and the copolymer thereof have a protection effect on nerves for the first time, and can promote growth of the nerves and repair of nerve injury.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer medicine, and in particular to the application of a polymer containing lactic acid repeating units in the preparation of a drug for promoting nerve growth and repairing. Background Art

[0002] The molecular formula of polylactic acid (PLA) is (C3H4O2) n , mainly prepared by lactic acid polymerization or lactide ring-opening polymerization, with the structural formula

[0003]

[0004] 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] Among them, biodegradable L-polylactic acid microparticles (trade name SculptraTM) with good biocompatibility were approved by the US FDA in 2004 for filling the sagging skin areas caused by facial fat atrophy in AIDS patients. At the same time, clinicians also use it for aging fat atrophy and local fat atrophy in healthy people. In 2009, the US FDA officially approved L-polylactic acid fillers for improving nasolabial folds. The mechanism of action of L-polylactic acid fillers is achieved by stimulating collagen proliferation. The filling effect of the initial injection can last for about 1 week, after which the L-polylactic acid filler is phagocytosed and dissolved by macrophages in the body, destroying its polymerization state, and finally degrading into lactic acid, water and carbon dioxide through non-enzymatic hydrolysis. The lactic acid remaining at the injection site stimulates the surrounding tissues to produce collagen. After a few months, the dermis at the injection site gradually thickens, and finally achieves a cosmetic effect.

[0006] The non-patent document "Materials for peripheral nerve repair constructs: Natural proteins or synthetic polymers?" discloses a method for repairing nerve damage, including using an electrospinning process to make materials such as polylactic acid into stents or catheters and nerve regeneration agents (such as neurotrophic factors) to promote the repair of nerve function. The polylactic acid stents or catheters simply support the nerves, guide directional growth, and bridge them.

[0007] However, there is no report in the prior art as to whether polylactic acid and its copolymers can be directly used as polymer drugs for nerve protection, repair and regeneration. Summary of the invention

[0008] 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 nerve growth and repair, which provides a new approach for nerve protection, repair and regeneration.

[0009] The object of the present invention is to provide the use of polylactic acid and its copolymers in the preparation of drugs for promoting nerve growth and repair, wherein the molecular weight of the polylactic acid and its copolymers is 400-300k Da, and the drugs include the following effects 1)-3):

[0010] 1) Promote the growth of nerve cells and nerve tissue;

[0011] 2) Promote the repair of nerve cells and nerve tissue;

[0012] 3) Treat or prevent peripheral nerve damage, spinal cord injury, peripheral neuropathy and neuritis;

[0013] Preferably, the molecular weight is 5000-100 kDa.

[0014] Furthermore, the polylactic acid and its copolymer are selected from the following combinations: poly-L-lactic acid, poly-D,L-lactic acid, and a copolymer containing L-lactic acid but not D-lactic acid.

[0015] Furthermore, the poly (L-lactic acid) has a crystallinity of 0 to 85%, preferably 10 to 40%, and a molecular weight distribution of 1 to 3, preferably 1.1 to 1.8.

[0016] Furthermore, the concentration of L-lactic acid in the poly D,L-lactic acid is at least 50%; more preferably, the ratio of L-lactic acid to D-lactic acid in the poly D,L-lactic acid is about 1:1.

[0017] Further, the concentration of L-lactic acid in the copolymer containing L-lactic acid but not D-lactic acid is at least 20%;

[0018] Preferably, the copolymer containing L-lactic acid but not D-lactic acid 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.

[0019] Furthermore, the polylactic acid and its copolymers are in the form of preparations.

[0020] Furthermore, the preparation includes microspheres, micelles, and gels.

[0021] Preferably, the microspheres are spherical with a smooth surface; the particle size of the microspheres is distributed in the range of 1-100 μ, preferably 30-60 μ.

[0022] Furthermore, the preparation further comprises excipients, and the excipients comprise surfactants and stabilizers.

[0023] 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.

[0024] The preparation further includes a first active ingredient, which has a neuroprotective effect. Preferably, the first active ingredient includes one or more of piracetam, aniracetam, oxiracetam, methylcobalamin, adenosylcobalamin, ganglioside, mannitol, edaravone, methylcobalamin, rapamycin, nerve growth factor, and vitamin B.

[0025] Furthermore, the concentration of L-lactic acid in the polylactic acid and its copolymer during at least one administration of the preparation is 5 to 2500 mmol / L, preferably 100 to 1200 mmol / L.

[0026] The term "smooth surface" in the present invention means that on the surfaces of 10 balls, there are no more than 50 protrusions with a height of 1 to 3 μm, and there are no protrusions larger than 5 μm.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention first discovered that a polymer containing lactic acid repeating units has a protective and repairing effect on nerves, and can promote nerve growth and repair of nerve damage.

[0029] (2) The polylactic acid and copolymer microspheres prepared by the present invention can reduce the level of malondialdehyde in the stump spinal cord after complete spinal cord transection injury, reduce the damage caused by lipid peroxidation, and thus protect the spinal cord tissue. The drug of the present invention has an early protective effect on the complete cross-section of the spinal cord, thereby extending the time window for early treatment.

[0030] (3) The polylactic acid and copolymer microspheres prepared by the present invention can achieve the effect of sustained release of lactic acid, improve the therapeutic effect and prolong the action time.

[0031] (4) The polylactic acid and copolymer microspheres prepared by the present invention reduce cytotoxicity and improve cell survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the SEM image of the PLLA microspheres in Example 1 of the present invention.

[0033] Figure 2This is the SEM image of PLGA microspheres in Example 2 of the present invention.

[0034] Figure 3 This is a graph showing the lactic acid release results of PLLA microspheres and PLGA microspheres in Example 3 of the present invention.

[0035] Figure 4 This is a graph showing the cell viability results of PLLA microspheres and lactic acid in Example 4 of the present invention.

[0036] Figure 5 This is a diagram showing the repair effect of nerve damage in Example 6 of the present invention. DETAILED DESCRIPTION

[0037] 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.

[0038] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Polylactic acid glycolic acid polymer (LA to GA ratio of 75:25, molecular weight 15000). Levorotatory polylactic acid (PLLA), crystalline polymer.

[0039] Example 1 Preparation of polylactic acid microspheres

[0040] 9 g of left-handed polylactic acid (PLLA) (molecular weight of 15000 g / mol, molecular weight distribution of 1.1, crystallinity of 30%) was dissolved in 135 mL of dichloromethane, and then the above polymer solution was added into 1800 mL of polyvinyl alcohol aqueous solution with a mass concentration of 0.5%, and after emulsification at 3000 rpm / min for 10 minutes, the mixture was stirred at 500 rpm for 3 hours to remove dichloromethane, and finally left-handed polylactic acid microspheres were obtained.

[0041] The prepared PLLA 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, the particle size of the microspheres ranges from 3.72 to 33.12 μm.

[0042] Example 2 Preparation of polylactic acid-co-glycolic acid polymer microspheres

[0043] 9 g of lactic acid glycolic acid polymer (PLGA) (molecular weight 15000 g / mol, molecular weight distribution 1.1, crystallinity 30%) was dissolved in 135 mL of dichloromethane, and then the above polymer solution was added into 1800 mL of polyvinyl alcohol aqueous solution with a mass concentration of 0.5%, and after emulsification at 3000 rpm / min for 10 minutes, the dichloromethane was removed by stirring at 500 rpm for 3 hours to finally obtain lactic acid glycolic acid polymer microspheres.

[0044] 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, the particle size of the microspheres ranges from 2.94 to 36.92 μm.

[0045] Example 3: Release of L-lactic acid from polylactic acid microspheres and polylactic acid copolymer microspheres

[0046] 12 portions (70 mg each) of the PLLA and PLGA microspheres prepared in Examples 1 and 2 were weighed and suspended in 10 ml of pH 7.4 phosphate buffer, and then placed in a 37% saline constant temperature water bath oscillator at a speed of 60 rpm / min. Three samples were taken every 7 days and the liquid portion was collected.

[0047] The change of lactic acid concentration in the medium over time was determined by high performance liquid chromatography. Chromatographic conditions: mobile phase was 0.1% H3PO4 aqueous solution, detection wavelength was 210nm, flow rate was 1.0mL / min, injection volume was 10μL. The experimental results are shown in Figure 3 As shown in the figure, it can be seen that in the first 21 days of degradation, the concentration of lactic acid produced by the degradation of microspheres showed a steady increase trend, and then lactic acid was released rapidly, indicating that the degradation of microspheres accelerated after 21 days of degradation. The release rate of lactic acid from PLLA microspheres was significantly higher than that from PLGA microspheres.

[0048] Example 4 Cytotoxicity Experiment

[0049] The rat Schwann cells (RSC96) in the logarithmic growth phase were used to prepare a concentration of 7×10 4 Cell suspension. Inoculate the cell suspension in a 96-well plate, 200 μl per well. Place the culture plate in an incubator and culture. When the cells cover more than 95% of the bottom of the well, add 100 μL of 5 mg / mL PLLA microspheres prepared in Example 1 and 5 mg / mL untreated lactic acid API to each well of the culture plate. Set up at least 5 parallel wells for each sample, and continue to place the culture plate in an incubator and incubate in the dark for 24, 48 and 72 hours. Then add 10 μL of CCK-8 solution to each well, place the culture plate in an incubator and incubate for 1 hour, use an enzyme reader to measure the absorbance (OD) at 450 nm, and calculate the cell survival rate. The results are as follows Figure 4 shown.

[0050] As the results show, the survival rate of nerve cells added with microspheres prepared by the method of the present invention is significantly higher than that added with lactic acid API; it can be seen that the microspheres prepared by the method of the present invention have higher pharmacodynamic activity than lactic acid API. The sustained-release microspheres have a better promoting effect on nerve cells than lactic acid API, which may be because the activity of nerve cells is more sensitive to lactic acid concentration.

[0051] Example 5 Promoting nerve injury recovery in vivo

[0052] 1. Establishment of animal model of spinal cord injury

[0053] Healthy SD rats, weighing 220-250 g, were randomly divided into 4 groups, namely, normal saline control group, low-concentration PLLA microsphere group, medium-concentration PLLA microsphere group, and high-concentration PLLA microsphere group, with 6 rats in each group.

[0054] Rats were anesthetized by intraperitoneal injection of 3.0% (w / v) sodium pentobarbital (0.2 ml / 100 g). After complete anesthesia, the rats were fixed in a prone position, and routine disinfection was performed after skin preparation. A longitudinal incision (2-3 cm) was made on the dorsal midline based on the twelfth thoracic vertebra (T12) and cut from the outside to the inside until the subcutaneous fascia. The dorsal lamina of T9-11 of the rats was accurately removed to expose the dura mater of the spinal cord. The middle part of the exposed spinal cord was clamped with an aneurysm clip for 40 seconds to prepare a spinal cord injury (SCI) model.

[0055] 2. Experimental Grouping

[0056] Group A: Normal saline without PLLA microspheres, serving as the control group

[0057] Group B: low concentration PLLA microsphere group, normal saline plus PLLA microspheres of Example 1, microsphere concentration (10 mg / mL)

[0058] Group C: medium concentration PLLA microsphere group, normal saline plus PLLA microspheres of Example 1, microsphere concentration (50 mg / mL)

[0059] Group D: high concentration PLLA microsphere group, normal saline plus PLLA microspheres of Example 1, microsphere concentration (150 mg / mL)

[0060] 3. PLLA microspheres promote nerve injury recovery

[0061] Each group was administrated according to body weight ratio immediately, 1 hour, 3 hours, 8 hours, 24 hours, 3 days, 7 days, 14 days, 21 days and 28 days after injury (20 μL of samples of control group and experimental group were injected epidurally at one time), and the motor recovery of both lower limbs of animals was observed. Samples were taken for macroscopic and pathological examination at 8 hours, 24 hours, 7 days, 28 days and 3 months after injury. The results showed that (C group had significantly better motor recovery of both lower limbs of animals than the other groups, while there was no obvious recovery in the control group A. Macroscopic and pathological observations showed that the local inflammatory response of spinal cord injury was the least in (B, C groups).

[0062] 4. Effect of polylactic acid microspheres on lipid peroxides after acute spinal cord transection injury in rats

[0063] 15 minutes after the establishment of the spinal cord complete transection injury model, the control group was given 83 μL / kg of normal saline at the spinal cord defect. The experimental group was divided into three groups and each group was given 100 μL of the above three different concentrations of PLLA microsphere suspension. The content of malondialdehyde, a lipid peroxidation product, in the spinal cord tissues proximal and distal to the stump was determined by the thiobarbituric acid method 1, 4, and 24 hours after injury. The results are shown in Table 1.

[0064] Table 1 Malondialdehyde content in rat spinal cord tissue (unit: nmol / mg)

[0065] Group 1h 4h 24h Normal saline group C 1.36±0.06 7.28±0.15 10.23±0.11 Low dose group B1% 0.91±0.04 6.52±0.13 8.52±0.21 Medium dose group A4% 0.53±0.03 5.46±0.15 7.16±0.17 High dose group 12% 0.59±0.03 5.97±0.12 7.41±0.09

[0066] The above experimental results show that the PLLA microspheres of the present invention can reduce the level of malondialdehyde in the stump spinal cord after complete spinal cord transection injury, reduce the damage caused by lipid peroxidation, and thus protect the spinal cord tissue. This experiment verified the early protective effect of the drug of the present invention on the complete cross-section of the spinal cord by observing the changes in the content of malondialdehyde in the stump spinal cord after complete spinal cord transection injury in rats, thereby extending the time window for early treatment.

[0067] Example 6 Promoting peripheral nerve regeneration in vivo

[0068] 1. Establishment of sciatic nerve injury model

[0069] SD rats were divided into three groups, 10 in each group, namely, sham operation group, control group, and microsphere group. After the rats were anesthetized with isoflurane inhalation, the right sciatic nerve was exposed under sterile conditions, the sciatic nerve was transected with a blade 5 mm proximal to the bifurcation of the sciatic nerve, and sutured with 8-0 absorbable sutures. After that, the anastomosis was processed according to the grouping, the anastomosis was marked with 10-0 non-absorbable sutures, and the rats were fed normally after suturing layer by layer.

[0070] 2. Experimental Grouping

[0071] Sham operation group: no nerve cutting

[0072] Control group: After nerve cutting, the anastomosis was sutured with only one stitch of 8-0 absorbable suture

[0073] Microsphere group: After nerve cutting, the anastomosis was sutured with 8-0 absorbable suture, and then 10 mg of PLGA microspheres were added, wherein the PLGA microspheres were prepared according to the process of Example 2.

[0074] 3. Evaluation of functional recovery after nerve injury

[0075] At 4, 8, and 12 weeks after surgery, the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were measured with an electromyographic instrument to evaluate the functional recovery after nerve injury. The specific operation was as follows: the rats were anesthetized by inhalation of isoflurane, and the sciatic nerve was exposed by incision according to the original incision. The receiving electrodes were connected and inserted into the Achilles tendon and the middle end of the gastrocnemius muscle, and the ground electrode was inserted subcutaneously. The stimulating electrode was adjusted to 10 mA, and the distal and proximal ends of the sciatic nerve anastomosis were stimulated respectively, and the potential was recorded, and the CMAP and NCV values ​​were calculated.

[0076] CMAP reflects the number of new axons, and its test results are as follows Figure 5 (A) (*p<0.05). It can be seen that there was no statistical significance between the control group and the microsphere group at 4 weeks after surgery. At 8 weeks after surgery, the CMAP in the microsphere group was significantly higher than that in the control group. At 12 weeks after surgery, the CMAP in the microsphere group was significantly higher than that in the control group, and its CMAP value reached 28±3mV. NCV reflects the maturity of the myelin sheath. The NCV test results of the three groups of samples are shown as follows Figure 5 (B) (#p<0.01). It can be seen that at 4, 8, and 12 weeks after surgery, the microsphere group was higher than the control group, with significant differences. At 12 weeks, the NCV value of the microsphere group reached 39±2m / s. The above test results prove that PLGA microspheres have a significant effect in promoting the regeneration and repair of damaged nerves.

[0077] 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 nerve growth and repair, wherein: The molecular weight of the polylactic acid and its copolymer is 400-300k Da, and the drug has the effect of any one of the following 1)-3): 1) Promote the growth of nerve cells and nerve tissue; 2) Promote the repair of nerve cells and nerve tissue; 3) Treat or prevent peripheral nerve damage, spinal cord injury, peripheral neuropathy and neuritis; Preferably, the molecular weight is 5000-100 kDa.

2. The use according to claim 1, characterized in that The polylactic acid and its copolymer are selected from the following group: poly L-lactic acid, poly D, L-lactic acid, and a copolymer containing L-lactic acid but not D-lactic acid.

3. The use according to claim 2, characterized in that The poly-L-lactic acid has a crystallinity of 0 to 85%, preferably 10 to 40%, and a molecular weight distribution of 1 to 3, preferably 1.1 to 1.

8.

4. The use according to claim 2, characterized in that The concentration of L-lactic acid in the poly D,L-lactic acid is at least 50%; more preferably, the ratio of L-lactic acid to D-lactic acid in the poly D,L-lactic acid is about 1:

1.

5. The use according to claim 2, characterized in that The concentration of L-lactic acid in the copolymer containing L-lactic acid but not D-lactic acid is at least 20%; Preferably, the copolymer containing L-lactic acid but not D-lactic acid 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 polylactide-caprolactone copolymer.

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

7. The use according to claim 6, characterized in that The preparations include microspheres, micelles, and gels. Preferably, the microspheres are spherical with a smooth surface; the particle size of the microspheres is distributed in the range of 1-100 μm, preferably 30-60 μm.

8. The use according to claim 6, characterized in that The preparation further includes excipients, which include surfactants and stabilizers; 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.

9. The use according to claim 6, characterized in that The preparation further includes a first active ingredient, which has a neuroprotective effect. Preferably, the first active ingredient includes one or more of piracetam, aniracetam, oxiracetam, methylcobalamin, adenosylcobalamin, ganglioside, mannitol, edaravone, methylcobalamin, rapamycin, nerve growth factor, and vitamin B.

10. The use according to claim 6, characterized in that The concentration of L-lactic acid in the polylactic acid and its copolymer during at least one administration of the preparation is 5 to 2500 mmol / L, preferably 100 to 1200 mmol / L.

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