Polylactic acid and use of copolymer thereof in preparation of drug for promoting nerve growth and repair

Abstract

Provided are a polylactic acid and a use of a copolymer thereof in the preparation of a drug for promoting nerve growth and repair. The molecular weight of the polylactic acid and the copolymer thereof is 400-300 k Da. The drug has the following effects: 1) promoting the growth of nerve cells and nervous tissue; 2) promoting repair of nerve cells and nervous tissue; and 3) treating or preventing a peripheral nerve injury, a spinal cord injury, peripheral neuropathy, and neuritis. The molecular weight is preferably 5000-100 k Da. It is found for the first time that the polylactic acid and the copolymer thereof have a protective effect on nerves, and can promote nerve growth and a nerve injury repair.

Description

Application of polylactic acid and its copolymers in the preparation of drugs for promoting nerve growth and repair

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310854812.1 filed on July 12, 2023, entitled “Application of polylactic acid and its copolymers in the preparation of drugs for promoting nerve growth and repair”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of polymer medicine, and in particular to the use of a polymer containing lactic acid repeating units in the preparation of a drug for promoting nerve growth and repair. Background Art

[0004] 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

[0005] 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 their biomedical applications. They have found widespread application in surgical sutures, bone repair materials, controlled drug release systems, and tissue scaffolds (such as artificial bone and skin).

[0006] Among them, 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 HIV 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. The mechanism of action of poly(L-lactic acid) fillers is by stimulating collagen production. The initial filling effect after injection lasts for about a week. Afterward, the poly(L-lactic acid) filler is phagocytosed and dissolved by macrophages in the body, disrupting its aggregated state. It is ultimately degraded by non-enzymatic hydrolysis into lactic acid, water, and carbon dioxide. The residual lactic acid at the injection site stimulates collagen production in the surrounding tissue. Over several months, the dermis at the injection site gradually thickens, ultimately achieving a cosmetic effect.

[0007] The non-patent document "Materials for peripheral nerve repair constructs: Natural proteins or synthetic polymers?" discloses a method for repairing damaged nerves. It involves using electrospinning to create materials like polylactic acid (PLA) into stents or conduits combined with nerve regeneration agents (such as neurotrophic factors) to promote nerve function repair. The PLA stents or conduits simply provide support, guide directional growth, and bridge the nerves.

[0008] 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.

[0009] Summary of the Invention

[0010] 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.

[0011] The present invention aims 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 400-300 kDa, and the drugs have the following effects 1)-3):

[0012] 1) Promote the growth of nerve cells and nerve tissue;

[0013] 2) Promote the repair of nerve cells and nerve tissue;

[0014] 3) Treat or prevent peripheral nerve injury, spinal cord injury, peripheral neuropathy and neuritis;

[0015] Preferably, the molecular weight is 5000-100 kDa.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] Further, the concentration of L-lactic acid in the copolymer comprising L-lactic acid but not D-lactic acid is at least 20%;

[0020] 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.

[0021] Furthermore, the polylactic acid and its copolymers exist in the form of preparations.

[0022] Furthermore, the preparation includes microspheres, micelles, and gels.

[0023] 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.

[0024] Furthermore, the preparation further includes excipients, which include surfactants and stabilizers.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention first discovered that polymers containing lactic acid repeating units have protective and repairing effects on nerves, and can promote nerve growth and repair of nerve damage.

[0031] (2) The polylactic acid and copolymer microspheres prepared by the present invention can reduce the level of malondialdehyde in the spinal cord stump 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 transection of the spinal cord, thereby extending the time window for early treatment.

[0032] (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 duration of action.

[0033] (4) The polylactic acid and its copolymer microspheres prepared by the present invention reduce cytotoxicity and improve cell survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG1 is a SEM image of PLLA microspheres in Example 1 of the present invention.

[0035] FIG2 is a SEM image of PLGA microspheres in Example 2 of the present invention.

[0036] FIG3 is a graph showing the lactic acid release results of PLLA microspheres and PLGA microspheres in Example 3 of the present invention.

[0037] FIG4 is a graph showing the cell viability results of PLLA microspheres and lactic acid in Example 4 of the present invention.

[0038] FIG5 is a diagram showing the repair effect of nerve damage in Example 6 of the present invention. DETAILED DESCRIPTION

[0039] 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.

[0040] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Poly(lactic-co-glycolic acid) polymer (LA:GA ratio of 75:25, molecular weight 15,000). Poly(L-lactic acid) (PLLA), a crystalline polymer.

[0041] Example 1 Preparation of polylactic acid microspheres

[0042] 9 g of L-polylactic acid (PLLA) (molecular weight of 15,000 g / mol, molecular weight distribution of 1.1, and crystallinity of 30%) was dissolved in 135 mL of dichloromethane. The polymer solution was then added to 1800 mL of a 0.5% polyvinyl alcohol aqueous solution. The mixture was emulsified at 3,000 rpm / min for 10 minutes and then stirred at 500 rpm for 3 hours to remove dichloromethane, thereby obtaining L-polylactic acid microspheres.

[0043] The prepared PLLA microspheres were observed by scanning electron microscopy and the particle size was calculated. A photo of the microspheres is shown in FIG1 . The particle size of the microspheres ranged from 3.72 to 33.12 μm.

[0044] Example 2 Preparation of polylactic acid glycolic acid polymer microspheres

[0045] 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 to 1800 mL of 0.5% polyvinyl alcohol aqueous solution. After emulsification at 3000 rpm / min for 10 minutes, the mixture was stirred at 500 rpm for 3 hours to remove dichloromethane, and finally lactic acid glycolic acid polymer microspheres were obtained.

[0046] The prepared polylactic acid microspheres were observed by scanning electron microscopy and the particle size was calculated. The photo of the PLGA microspheres is shown in FIG2 . The particle size of the microspheres ranged from 2.94 to 36.92 μm.

[0047] Example 3: Release of L-lactic acid from polylactic acid microspheres and polylactic acid copolymer microspheres

[0048] Twelve 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. The suspensions were then placed in a 37°C constant-temperature water bath and shaken at 60 rpm / min. Three samples were removed every seven days, and the liquid fraction was collected.

[0049] High-performance liquid chromatography (HPLC) was used to determine the change in lactic acid concentration in the medium over time. Chromatographic conditions: mobile phase: 0.1% H₃PO₄ aqueous solution, detection wavelength: 210 nm, flow rate: 1.0 mL / min, injection volume: 10 μL. The experimental results are shown in Figure 3. As can be seen, during the first 21 days of degradation, the lactic acid concentration generated by microsphere degradation showed a steady increase, followed by rapid lactic acid release, indicating accelerated microsphere degradation after 21 days. The lactic acid release rate from PLLA microspheres was significantly higher than that from PLGA microspheres.

[0050] Example 4 Cytotoxicity Experiment

[0051] The rat Schwann cells (RSC96) in the logarithmic growth phase were used to prepare a concentration of 7×10 4The cell suspension was inoculated in a 96-well plate, 200 μl per well. The culture plate was placed in an incubator for culture. When the cells covered more than 95% of the bottom of the well, 100 μL of PLLA microspheres prepared in Example 1 at a concentration of 5 mg / mL and 5 mg / mL of untreated lactic acid bulk drug were added to each well of the culture plate. At least 5 parallel wells were set for each sample, and the culture plate was placed in an incubator for incubation in the dark for 24, 48 and 72 hours. 10 μL of CCK-8 solution was then added to each well, the culture plate was placed in an incubator and incubated for 1 hour, the absorbance (OD) at 450 nm was measured with an enzyme reader, and the cell survival rate was calculated. The results are shown in Figure 4.

[0052] As the results show, the survival rate of neurons in the microspheres prepared by the present invention was significantly higher than that in the microspheres prepared with the lactic acid API; thus, the microspheres prepared by the present invention have higher pharmacodynamic activity than the lactic acid API. The sustained-release microspheres have a greater neuronal activation effect than the lactic acid API, likely due to the sensitivity of neuronal activity to lactic acid concentration.

[0053] Example 5 Promoting nerve injury recovery in vivo

[0054] 1. Establishment of an animal model of spinal cord injury

[0055] Healthy SD rats, weighing 220-250 g, were randomly divided into four groups: normal saline control group, low-concentration PLLA microsphere group, medium-concentration PLLA microsphere group, and high-concentration PLLA microsphere group, with six rats in each group.

[0056] Rats were anesthetized with 3.0% (w / v) sodium pentobarbital (0.2 ml / 100 g) intraperitoneally. After complete anesthesia, the rats were placed in a prone position. After skin preparation and routine disinfection, a longitudinal incision (2–3 cm) was made along the dorsal midline, starting at the twelfth thoracic vertebra (T12). This incision was then made from the outside inward until the subcutaneous fascia was reached. The dorsal lamina of T9–11 was precisely excised to expose the dura mater of the spinal cord. An aneurysm clip was applied to the exposed midsection of the spinal cord for 40 s to establish a spinal cord injury (SCI) model.

[0057] 2. Experimental Grouping

[0058] Group A: normal saline without PLLA microspheres, serving as the control group

[0059] Group B: low concentration PLLA microsphere group, normal saline plus PLLA microspheres of Example 1, microsphere concentration (10 mg / mL)

[0060] Group C: medium concentration PLLA microsphere group, normal saline plus PLLA microspheres of Example 1, microsphere concentration (50 mg / mL)

[0061] Group D: High concentration PLLA microsphere group, normal saline plus PLLA microspheres of Example 1, microsphere concentration (150 mg / mL)

[0062] 3. PLLA microspheres promote nerve injury recovery

[0063] Each group was administered drugs according to body weight ratio immediately after injury, 1 hour, 3 hours, 8 hours, 24 hours, 3 days, 7 days, 14 days, 21 days, and 28 days after injury (20 μL of control and experimental group samples were injected epidurally once). The recovery of lower limb movement of the animals was observed, and samples were collected for gross and pathological examinations at 8 hours, 24 hours, 7 days, 28 days, and 3 months after injury. The results showed that (Group C) had significantly better recovery of lower limb movement than the other groups, while there was no significant recovery in the control group A. Gross and pathological observations showed that the local inflammatory response of the spinal cord injury (Groups B and C) was the least.

[0064] 4. Effects of polylactic acid microspheres on lipid peroxides after acute spinal cord transection injury in rats

[0065] Fifteen minutes after the establishment of the complete spinal cord transection injury model, the control group was given 83 μL / kg of normal saline at the spinal cord defect site. The experimental group was divided into three groups and each was given 100 μL of the three different concentrations of PLLA microsphere suspension. The content of the lipid peroxidation product malondialdehyde 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.

[0066] Table 1 Malondialdehyde content in rat spinal cord tissue (unit: nmol / mg)

[0067] The above experimental results demonstrate that the PLLA microspheres of the present invention can reduce the level of malondialdehyde (MDA) in the spinal cord stump after complete spinal cord transection, alleviating damage caused by lipid peroxidation and thus protecting spinal cord tissue. This experiment, by observing changes in MDA levels in the spinal cord stump after complete spinal cord transection in rats, validated the early protective effect of the present drug on complete spinal cord transection, thereby extending the window for early treatment.

[0068] Example 6 Promoting peripheral nerve regeneration in vivo

[0069] 1. Establishment of sciatic nerve injury model

[0070] Sprague-Dawley rats were divided into three groups, 10 in each: a sham-operated group, a control group, and a microsphere-treated group. After isoflurane inhalation anesthesia, the right sciatic nerve was exposed under sterile conditions. The sciatic nerve was transected with a blade 5 mm proximal to the bifurcation and sutured with a single stitch of 8-0 absorbable suture. The anastomosis was then treated according to grouping, marked with 10-0 non-absorbable suture, and sutured layer by layer before routine maintenance.

[0071] 2. Experimental Grouping

[0072] Sham operation group: no nerve cutting

[0073] Control group: After nerve cutting, the anastomosis was sutured with only one stitch of 8-0 absorbable suture

[0074] Microsphere group: After nerve transection, the anastomosis was sutured with 8-0 absorbable suture, and then 10 mg of PLGA microspheres were added. The PLGA microspheres were prepared according to the process of Example 2.

[0075] 3. Assessment of functional recovery after nerve injury

[0076] At 4, 8, and 12 weeks postoperatively, electromyography (EMG) was used to measure the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) to assess functional recovery after nerve injury. The procedure was as follows: rats were anesthetized with isoflurane inhalation, and the sciatic nerve was exposed by incision at the original incision. Receiving electrodes were connected and inserted into the Achilles tendon and mid-gastrocnemius muscle, respectively, with the ground electrode inserted subcutaneously. The stimulating electrode was adjusted to 10 mA and stimulated both distal and proximal to the sciatic nerve anastomosis. The potentials were recorded, and the CMAP and NCV values ​​were calculated.

[0077] CMAP reflects the number of newly formed axons, and the test results are shown in 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 in 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, and there were 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.

[0078] 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. 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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