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

Abstract

This invention provides the use of polylactic acid and its copolymers in the preparation of a drug for promoting nerve growth and repair, wherein the polylactic acid and its copolymers have a molecular weight of 400-300 kDa, and the drug has the following effects: 1) promoting the growth of nerve cells and nerve tissue; 2) promoting the repair of nerve cells and nerve tissue; 3) treating or preventing peripheral nerve injury, spinal cord injury, peripheral neuropathy, and neuritis; preferably, the molecular weight is 5000-1000 kDa. This invention is the first to discover that polylactic acid and its copolymers have a protective effect on nerves and can promote nerve growth and repair of nerve damage.

Description

Technical Field

[0001] This invention relates to the field of polymer pharmaceutical technology, and more particularly to the use of polymers containing lactate repeating units in the preparation of drugs for promoting nerve growth and repair. Background Technology

[0002] The molecular formula of polylactic acid (PLA) is (C3H4O2). n It is mainly prepared by lactic acid polymerization or lactide ring-opening polymerization, and its structural formula is:

[0003]

[0004] PLA and its copolymers not only possess excellent mechanical strength and chemical stability, but also exhibit good biocompatibility and biodegradability. In recent years, extensive research has been conducted both domestically and internationally on their applications in biomedicine. They have already found wide application in surgical sutures, bone repair materials, controlled-release drug systems, and tissue functional scaffolds (such as artificial bone and artificial skin).

[0005] Among them, biodegradable and biocompatible polylactic acid (PLA) microparticles (trade name Sculptra™) were approved by the US FDA in 2004 for filling sagging skin areas caused by facial fat atrophy in AIDS patients. Clinicians also use them for age-related fat atrophy and localized fat atrophy in healthy individuals. In 2009, the US FDA officially approved PLA fillers for improving nasolabial folds. The mechanism of action of PLA fillers is through stimulating collagen proliferation. The initial filling effect lasts for about one week, after which the PLA filler is phagocytosed and dissolved by macrophages in the body, disrupting its polymeric state, and ultimately degraded into lactic acid, water, and carbon dioxide through non-enzymatic hydrolysis. Residual lactic acid at the injection site stimulates collagen production in surrounding tissues. Several months later, the dermis at the injection site gradually thickens, ultimately achieving the cosmetic effect.

[0006] The non-patent literature "Materials for peripheral nerve repair constructs: Natural proteins or synthetic polymers?" discloses a method for repairing nerve damage, including using electrospinning to make scaffolds or conduits from materials such as polylactic acid and nerve regeneration agents (such as neurotrophic factors) to promote the repair of nerve function. In this case, the polylactic acid scaffolds or conduits simply play a supporting, guiding, and bridging role for the nerve.

[0007] However, there are no reports in the existing technology regarding whether polylactic acid and its copolymers can be directly used as polymeric drugs for neuroprotection, repair and regeneration. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides the application of polylactic acid and its copolymers in the preparation of drugs for promoting nerve growth and repair, offering a novel approach for neuroprotection, repair, and regeneration.

[0009] The purpose of this 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 400-300 kDa, and the drugs include the effects described in 1)-3) below:

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

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

[0012] 3) Treatment or prevention of peripheral nerve injury, spinal cord injury, peripheral neuropathy, and neuritis;

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

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

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

[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 approximately 1:1.

[0017] Furthermore, 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-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polyethylene glycol-polylactic acid-glycolic acid copolymer, polylactic acid-chitosan copolymer, and lactide-caprolactone copolymer.

[0019] Furthermore, the polylactic acid and its copolymers exist in the form of a formulation.

[0020] Furthermore, the formulation 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 μm, preferably 30-60 μm.

[0022] Furthermore, the formulation further includes excipients, including surfactants and stabilizers.

[0023] The surfactant includes one or more of polyethylene glycol, sodium dodecyl sulfonate, Tween, and Span; the stabilizer includes one or two of carboxymethyl cellulose and mannitol.

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

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

[0026] In this invention, the term "smooth surface" means that the surface of 10 spheres has no more than 50 protrusions with a height of 1 to 3 μm and no protrusions larger than 5 μm.

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

[0028] (1) This invention is the first to discover that polymers containing lactic acid repeating units have protective and repairing effects on nerves, and can promote nerve growth and repair of nerve damage.

[0029] (2) The polylactic acid and its copolymer microspheres prepared in this invention can reduce the level of malondialdehyde in the spinal cord at the fracture site after a complete spinal cord transection injury, alleviate the damage caused by lipid peroxidation, and thus protect the spinal cord tissue. The drug of this invention has an early protective effect on the complete spinal cord transection, thereby prolonging the time window for early treatment.

[0030] (3) The polylactic acid and its 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 its copolymer microspheres prepared in this invention reduce cytotoxicity and improve cell survival rate. Attached Figure Description

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

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

[0034] Figure 3 This is a diagram 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 survival rate 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 after nerve injury in Embodiment 6 of the present invention. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0038] Unless otherwise stated, 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:GA ratio 75:25, molecular weight 15000). L-polylactic acid (PLLA), a crystalline polymer.

[0039] Example 1: Preparation of polylactic acid microspheres

[0040] 9 g of polylactic acid (PLLA) (molecular weight 15000 g / mol, molecular weight distribution 1.1, crystallinity 30%) was dissolved in 135 mL of dichloromethane. The polymer solution was then 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 the dichloromethane, finally obtaining polylactic acid microspheres.

[0041] The prepared PLLA microspheres were observed and their particle size was calculated using a scanning electron microscope. The microsphere images are shown below. Figure 1 As shown, the microsphere particle size ranges from 3.72 to 33.12 μm.

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

[0043] 9 g of lactoglycolic acid polymer (PLGA) (molecular weight 15000 g / mol, molecular weight distribution 1.1, crystallinity 30%) was dissolved in 135 mL of dichloromethane. Then, the 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 the dichloromethane, and finally, lactoglycolic acid polymer microspheres were obtained.

[0044] The prepared polylactic acid (PLGA) microspheres were observed and their particle size was calculated using a scanning electron microscope. Images of the PLGA microspheres are shown below. Figure 2 As shown, the microsphere particle size ranges from 2.94 to 36.92 μm.

[0045] Example 3: L-lactic acid microspheres and polylactic acid copolymer microspheres release L-lactic acid.

[0046] Weigh out 12 portions (70 mg each) of PLLA and PLGA microspheres prepared in Examples 1 and 2 respectively, and suspend each portion in 10 ml of pH 7.4 phosphate buffer. Then place the suspensions on a 37°C constant temperature water bath shaker and shake at 60 rpm / min. Take out 3 samples every 7 days and collect the liquid fraction.

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

[0048] Example 4 Cytotoxicity Experiment

[0049] Prepare a solution with a concentration of 7×10⁻⁶ using nerve cells (rat Schwann cells (RSC96)) in the logarithmic growth phase. 4 Cell suspension was seeded into 96-well plates at a concentration of 200 μl per well. The plates were then incubated in an incubator until cells covered more than 95% of the bottom area of ​​each well. Then, 100 μL of PLLA microspheres (5 mg / mL, prepared in Example 1) and 5 mg / mL of untreated lactic acid were added to each well, with at least five parallel wells per sample. The plates were then incubated in the dark for 24, 48, and 72 hours. Next, 10 μL of LCK-8 solution was added to each well, and the plates were incubated for 1 hour. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated. The results are shown below. Figure 4 As shown.

[0050] The results showed that the nerve cell survival rate of the microspheres prepared by the method of this invention was significantly higher than that of the microspheres prepared by the method of this invention. This indicates that the microspheres prepared by the method of this invention have higher pharmacological activity compared to the microspheres prepared by the method of lactic acid. The sustained-release microspheres showed a better promoting effect on nerve cells than the microspheres prepared by the method of lactic acid, which may be because the activity of nerve cells is more sensitive to the concentration of lactic acid.

[0051] Example 5: Promoting Nerve Injury Recovery in Vivo

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

[0053] Healthy SD rats, weighing 220-250g, were randomly divided into four groups: a saline control group, a low-concentration PLLA microsphere group, a medium-concentration PLLA microsphere group, and a high-concentration PLLA microsphere group, with six 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. After skin preparation and routine disinfection, a longitudinal incision (2-3 cm) was made along the midline of the back, using the twelfth thoracic vertebra (T12) as the base point. The incision was made from the outside inward until the subcutaneous fascia was reached. The dorsal lamina of the T9-11 vertebrae 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 establish a spinal cord injury (SCI) model.

[0055] 2. Experimental Grouping

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

[0057] Group B: Low-concentration PLLA microspheres group, physiological saline with PLLA microspheres from Example 1, microsphere concentration (10 mg / mL).

[0058] Group C: Medium-concentration PLLA microspheres group, physiological saline plus PLLA microspheres from Example 1, microsphere concentration was 50 mg / mL.

[0059] Group D: High-concentration PLLA microspheres group, physiological saline with PLLA microspheres from Example 1, microsphere concentration was 150 mg / mL.

[0060] 3. PLLA microspheres promote nerve damage recovery

[0061] Each group was administered medication according to body weight at the following times: immediately after injury, 1 hour after injury, 3 hours after injury, 8 hours after injury, 24 hours after injury, 3 days after injury, 7 days after injury, 14 days after injury, 21 days after injury, and 28 days after injury (20 μL of the control and experimental group samples were injected epidurally once). The recovery of motor function in both lower limbs was observed. Samples were taken at 8 hours, 24 hours, 7 days, 28 days, and 3 months after injury for gross and pathological examination. Results showed that group C showed significantly better recovery of motor function in both lower limbs than the other groups, while group A (the control group) showed no significant recovery. Gross and pathological observations revealed the least inflammatory response at the site of spinal cord injury in groups B and C.

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

[0063] Fifteen minutes after establishing the complete spinal cord transection injury model, the control group was given 83 μL / kg of physiological saline at the spinal cord defect site, while the experimental group was divided into three groups and given 100 μL of the above three different concentrations of PLLA microsphere suspension. At 1, 4 and 24 hours after injury, the content of malondialdehyde, a lipid peroxidation product, in the proximal and distal segments of the spinal cord was determined by the thiobarbituric acid method. 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 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 indicate that the PLLA microspheres of this invention can reduce the level of malondialdehyde (MDA) in the spinal cord at the fracture site after a complete spinal cord transection injury, alleviate the damage caused by lipid peroxidation, and thus protect spinal cord tissue. This experiment, by observing the changes in MDA content in the spinal cord at the fracture site in rats after a complete spinal cord transection injury, verified the early protective effect of the drug of this invention on the complete spinal cord transection, thereby prolonging the time window for early treatment.

[0067] Example 6: Promoting Peripheral Nerve Regeneration in Vivo

[0068] 1. Establishment of a sciatic nerve injury model

[0069] SD rats were divided into three groups of 10 rats each: a sham-operated group, a control group, and a microsphere group. After anesthetizing the rats with isoflurane inhalation, the right sciatic nerve was exposed under aseptic conditions. The sciatic nerve was transected 5 mm proximal to the bifurcation point with a scalpel and sutured with one stitch using 8-0 absorbable suture. The anastomosis was then treated according to the grouping, and marked with 10-0 non-absorbable suture. After suturing layer by layer, the rats were fed routinely.

[0070] 2. Experimental Grouping

[0071] Sham surgery group: No nerve transection performed

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

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

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

[0075] At 4, 8, and 12 weeks post-surgery, compound action potentials (CMAP) and nerve conduction velocity (NCV) were measured using electromyography to assess functional recovery following nerve injury. The procedure was as follows: Rats were anesthetized with isoflurane, and the sciatic nerve was exposed by reopening the original incision. Receiving electrodes were connected and inserted into the Achilles tendon and the mid-gastrocnemius muscle, respectively, with the ground electrode inserted subcutaneously. The stimulation electrode was adjusted to 10 mA, and stimulation was applied to the distal and proximal ends of the sciatic nerve anastomosis. Potentials were recorded, and CMAP and NCV values ​​were calculated.

[0076] CMAP reflects the number of newly generated axons, and the test results are as follows: Figure 5 As shown in (A) (*p<0.05). It can be seen that at 4 weeks post-operation, there was no statistically significant difference between the control group and the microsphere group. At 8 weeks post-operation, the CMAP in the microsphere group was significantly higher than that in the control group. At 12 weeks post-operation, the CMAP in the microsphere group was significantly higher than that in the control group, reaching a CMAP value of 28±3 mV. NCV reflects the maturity of myelin sheath; the NCV test results of the three groups are shown below. Figure 5 As shown in (B) (#p<0.01). It is evident that at weeks 4, 8, and 12 post-surgery, the microsphere group showed significantly higher values ​​than the control group. At week 12, the NCV value of the microsphere group reached 39±2 m / s. These test results demonstrate that PLGA microspheres have a significant effect on 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 solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of polylactic acid or polylactic-co-glycolic acid as an active pharmaceutical ingredient in the preparation of a medicament for the treatment of spinal cord injury or sciatic nerve injury, wherein, The molecular weight of the polylactic acid or poly(lactic-co-glycolic acid) is 5000 Da to 100 k Da, and the drug includes the effects of any one of the following 1) to 3): 1) reducing lipid peroxidation; 2) extending the treatment time window; 3) promoting axonal regeneration or myelin maturation; The crystallinity of the polylactic acid or poly(lactic-co-glycolic acid) is 10% to 40%, and the molecular weight distribution is 1.1 to 1.8; the polylactic acid or poly(lactic-co-glycolic acid) exists in the form of microspheres; the particle size distribution of the microspheres is 1 to 100 μm.

2. Use according to claim 1, wherein The polylactic acid is from the following combination: poly-L-lactic acid or poly-D, L-lactic acid.

3. Use according to claim 2, wherein the compound is ###0002### The proportion of L-lactic acid in the poly-D, L-lactic acid is at least 50%.

4. The use according to claim 2, wherein The proportion of L-lactic acid and D-lactic acid in the poly-D, L-lactic acid is 1:

1.

5. The use according to claim 1, wherein The particle size distribution of the microspheres is 30 to 60 μm.

6. The use according to claim 1, wherein The preparation further includes an excipient, which includes a surfactant, a stabilizer; The surfactant includes one or more of sodium dodecyl sulfonate, Tween, Span; the stabilizer includes carboxymethyl cellulose.

7. The use according to claim 1, wherein The preparation further includes a first active ingredient, which has a neuroprotective effect.

8. Use according to claim 7, wherein the compound is ###0002### The first active ingredient includes one or more of pramipexole, alniditan, oxiracetam, mecobalamin, adenosine cobalamin, ganglioside, mannitol, edaravone, rapamycin, and nerve growth factor.

9. The use according to claim 1, wherein The concentration of L-lactic acid in the polylactic acid or poly(lactic-co-glycolic acid) during at least one administration of the preparation is 5 to 2500 mmol / L.

10. Use according to claim 9, wherein The concentration of L-lactic acid is 100 to 1200 mmol / L.

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