Nanosphere containing betahistine and preparation method thereof
By encapsulating brain-derived neurotrophic factor and betahistine in nanospheres, combined with inner ear blood-labyrinth barrier-penetrating peptides, the problem of existing drugs being unable to simultaneously load small molecules and large molecules of protein was solved, achieving the dual effects of inner ear circulation and hair cell repair, and significantly reducing the recurrence rate of Meniere's disease.
Patent Information
- Application Number
- CN202511039907.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-11-07
AI Technical Summary
Existing drugs for treating Meniere's disease cannot efficiently load both small and large molecular proteins simultaneously, lacking the ability to actively target the inner ear, resulting in a high relapse rate. Conventional nanocarriers are insufficient to achieve the dual effects of drugs.
Using polyacetic acid-glycolic acid copolymer as a carrier, a hydrophilic core of brain-derived neurotrophic factor and hyaluronic acid and a hydrophobic shell of betahistine are encapsulated, combined with an inner ear blood-labyrinth barrier-penetrating peptide, to form a spatiotemporally synergistic core-shell structure, achieving targeted drug delivery and dual action.
It achieves the dual effects of the drug, improving inner ear circulation and repairing damaged hair cells, significantly reducing the disease recurrence rate, and providing a new targeted treatment strategy through intelligent response targeted penetration.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biological medicine, more particularly to a betahistine-containing nanomicrosphere and a preparation method thereof. BACKGROUND
[0002] The mainstream drug for treating Meniere's disease, betahistine, mainly expands the inner ear blood vessels through histamine H3 receptor antagonism, only improves the inner ear circulation and relieves the symptoms of dizziness, and cannot repair or regenerate damaged hair cells and nerve endings, resulting in a high disease recurrence rate of 60-80%. Conventional nanocarriers are difficult to simultaneously and efficiently load small molecules and large molecule proteins, and lack the ability to actively target the inner ear, and thus further improvement and development are needed. SUMMARY
[0003] In view of the various deficiencies of the prior art, in order to solve the above problems, a betahistine-containing nanomicrosphere and a preparation method thereof are provided, and the following technical solutions are provided. A betahistine-containing nanomicrosphere comprises: a carrier composed of polyglycolic acid-hydroxyacetic acid copolymer; a hydrophilic core loaded in the interior of the carrier, the hydrophilic core comprising brain-derived neurotrophic factor and hyaluronic acid; and a hydrophobic shell layer loaded on the surface of the carrier, the hydrophobic shell layer comprising betahistine or a pharmaceutically acceptable salt / derivative thereof.
[0004] Further, the amount of brain-derived neurotrophic factor is 1-10 mg per 100 mg of polyglycolic acid-hydroxyacetic acid copolymer carrier; the amount of hyaluronic acid is 1-5 mg; and the amount of betahistine or its salt / derivative is 5-30 mg.
[0005] Further, the mass ratio of brain-derived neurotrophic factor to hyaluronic acid is 1-5:1.
[0006] Further, the hydrophilic core further comprises an antioxidant curcumin, and the amount of curcumin is 0.5-5 mg per 100 mg of polyglycolic acid-hydroxyacetic acid copolymer carrier.
[0007] Further, the molar ratio of lactic acid to hydroxyacetic acid in the polyglycolic acid-hydroxyacetic acid copolymer is 60-80:20-40.
[0008] Further, the surface of the multifunctional nanomicrosphere is modified with an inner ear blood-labyrinth barrier penetrating peptide.
[0009] Further, the modification amount of the inner ear blood-labyrinth barrier penetrating peptide is 0.1-2 mg of peptide per 100 mg of polyglycolic acid-hydroxyacetic acid copolymer carrier.
[0010] In addition, the application further provides a preparation method of the above-mentioned betahistine-containing nanomicrospheres, wherein hyaluronic acid aqueous solution is mixed with brain-derived neurotrophic factor to obtain a compound W1, the W1 is added into an ethyl acetate solvent containing polyglycolic acid-hydroxyacetic acid copolymer to form a W1 / O emulsion through primary emulsification; the W1 / O emulsion is dispersed in an external water phase containing betahistine or a pharmaceutically acceptable salt / derivative thereof and polyglycolic acid-hydroxyacetic acid copolymer to form a (W1 / O) / W2 emulsion through secondary emulsification, and the multifunctional nanomicrospheres are obtained through stirring and solvent volatilization and solidification.
[0011] Further, the specific operation of stirring and solvent volatilization and solidification is that the temperature is increased to 20-25 °C after stirring at 3-5 °C for 1.5-2.5 hours, and the stirring is performed at 20-25 °C for 0.5-1.5 hours.
[0012] Further, in the ethyl acetate solvent containing polyglycolic acid-hydroxyacetic acid copolymer, the concentration of the polyglycolic acid-hydroxyacetic acid copolymer is 5-15 mg / mL.
[0013] Due to the adoption of the above technical solutions, the application has the following beneficial technical effects: 1. The application co-encapsulates betahistine and a neurotrophic factor having a vestibular hair cell protection / regeneration effect in a carrier, so as to realize the dual effects of "improving circulation + repairing function"; 2. The application innovatively integrates a spatiotemporal synergistic core-shell structure and intelligent response target penetration, and successfully overcomes the key barriers of co-delivery of double-effect drugs; 3. The system provides a new strategy for targeted treatment of vestibular disorders and the like through synergistic effects (brain-derived neurotrophic factor repairing neurons + betahistine improving inner ear microcirculation + curcumin antioxidant). DETAILED DESCRIPTION
[0014] In order to enable personnel in the art to better understand the technical solutions of the application, the technical solutions of the application are described clearly and completely below in combination with the embodiments of the application, and other similar embodiments obtained by personnel in the art without making creative efforts on the basis of the embodiments in the application shall all belong to the protection scope of the application.
[0015] A betahistine-containing nanomicrosphere comprises: a carrier composed of polyacetic acid-hydroxyacetic acid copolymer; a hydrophilic core loaded in the interior of the carrier, the hydrophilic core comprising brain-derived neurotrophic factor and hyaluronic acid; and a hydrophobic shell layer loaded on the surface of the carrier, the hydrophobic shell layer comprising betahistine or a pharmaceutically acceptable salt / derivative thereof. The present application co-encapsulates betahistine and neurotrophic factor having a vestibular hair cell protection / regeneration effect in the carrier, realizes the dual effects of 'improved circulation + repair function', and innovatively integrates the spatiotemporal synergistic core-shell structure and intelligent response target penetration, successfully overcoming the key barriers of co-delivery of double-effect drugs.
[0016] The polyacetic acid-hydroxyacetic acid copolymer is abbreviated as PLGA; the brain-derived neurotrophic factor is abbreviated as BDNF; and the hyaluronic acid is abbreviated as HA.
[0017] Embodiment 1 Formulation (per 100 mg of PLGA): BDNF 5 mg + HA 2.5 mg (core) Betahistine dihydrochloride 15 mg (shell) The molar ratio of lactic acid to hydroxyacetic acid in the PLGA is 70:30, and the PLGA is dissolved in ethyl acetate at a concentration of 10 mg / mL.
[0018] The concentration of the PLGA hydrophobic segment in the external water phase is 0.5-3% w / v.
[0019] Steps: Mix the HA aqueous solution with the BDNF to obtain a W1 phase; Add the W1 phase to a PLGA / ethyl acetate solution, and ultrasonically emulsify (100 W, 30 s) to form a W1 / O emulsion; Inject the W1 / O emulsion into an external water phase containing the PLGA hydrophobic segment and betahistine, and perform secondary emulsification (200 W, 60 s) to obtain a (W1 / O) / W2 emulsion; Stir at 4°C for 2 h, then warm to 22°C and stir for 1 h, and finally centrifuge and wash.
[0020] Embodiment 2 Formulation (per 100 mg of PLGA): BDNF 5 mg + HA 2.5 mg + curcumin 2 mg (core) Betahistine dihydrochloride 15 mg (shell) The molar ratio of lactic acid to hydroxyacetic acid in the PLGA is 70:30, and the PLGA is dissolved in ethyl acetate at a concentration of 10 mg / mL.
[0021] The concentration of the PLGA hydrophobic segment in the external water phase is 0.5-3% w / v.
[0022] Step: Mixing HA aqueous solution with BDNF, curcumin to get W1 phase; W1 phase is added into PLGA / ethyl acetate solution, ultrasonic emulsification (100W, 30s) to form W1 / O emulsion; W1 / O emulsion is injected into external water phase containing PLGA hydrophobic segment and betahistine to form (W1 / O) / W2 emulsion by secondary emulsification (200W, 60s); Stirring at 4℃ for 2h, then warming to 22℃ for 1h, finally centrifugation and washing.
[0023] Example 3 Formulation (per 100mg PLGA): BDNF 5mg + HA 2.5mg (core) Betahistine dihydrochloride 15mg (shell) The molar ratio of lactic acid: glycolic acid in PLGA is 70:30, and PLGA is dissolved in ethyl acetate with a concentration of 10mg / mL. The concentration of PLGA hydrophobic segment in external water phase is 0.5-3% w / v. RGD-TAT peptide 1mg.
[0024] Step: Mixing HA aqueous solution with BDNF to get W1 phase; W1 phase is added into PLGA / ethyl acetate solution, ultrasonic emulsification (100W, 30s) to form W1 / O emulsion; W1 / O emulsion is injected into external water phase containing PLGA hydrophobic segment and betahistine to form (W1 / O) / W2 emulsion by secondary emulsification (200W, 60s); Stirring at 4℃ for 2h, then warming to 22℃ for 1h, finally centrifugation and washing.
[0025] Example 4 Formulation (per 100mg PLGA): BDNF 5mg + HA 2.5mg (core) Betahistine dihydrochloride 15mg (shell) The molar ratio of lactic acid: glycolic acid in PLGA is 80:20, and PLGA is dissolved in ethyl acetate with a concentration of 10mg / mL.
[0026] The concentration of PLGA hydrophobic segment in external water phase is 0.5-3% w / v.
[0027] Step: Mixing HA aqueous solution with BDNF to get W1 phase; W1 phase is added into PLGA / ethyl acetate solution, ultrasonic emulsification (100W, 30s) to form W1 / O emulsion; W1 / O emulsion is injected into external water phase containing PLGA hydrophobic segment and betahistine to form (W1 / O) / W2 emulsion by secondary emulsification (200W, 60s); Stirring at 4℃ for 2h, then warming to 22℃ for 1h, finally centrifugation and washing.
[0028] Example 5 Formulation (per 100mg PLGA): BDNF 5mg + HA 2.5mg + curcumin 2mg (core) Betahistine dihydrochloride 15mg (shell layer) The molar ratio of lactic acid:glycolic acid in PLGA is 70:30, and PLGA is dissolved in ethyl acetate with a concentration of 10mg / mL.
[0029] The concentration of PLGA hydrophobic segment in external water phase is 0.5-3% w / v. RGD-TAT peptide 1mg.
[0030] Steps: Mixing HA aqueous solution with BDNF and curcumin to get W1 phase; W1 phase is added into PLGA / ethyl acetate solution, ultrasonic emulsification (100W, 30s) to form W1 / O emulsion; W1 / O emulsion is injected into external water phase containing PLGA hydrophobic segment and betahistine to form (W1 / O) / W2 emulsion by secondary emulsification (200W, 60s); Stirring at 4℃ for 2h, then warming to 22℃ for 1h, finally centrifugation and washing.
[0031] Example 6 Formulation (per 100mg PLGA): BDNF 10mg + HA 2mg + curcumin 0.5mg (core) Betahistine dihydrochloride 5mg (shell layer) The molar ratio of lactic acid:glycolic acid in PLGA is 60:40, and PLGA is dissolved in ethyl acetate with a concentration of 5mg / mL.
[0032] The concentration of PLGA hydrophobic segment in external water phase is 0.5% w / v.
[0033] Steps: Mixing HA aqueous solution with BDNF and curcumin to get W1 phase; W1 phase was added to PLGA / ethyl acetate solution, and emulsified by ultrasound (100W, 30s) to form W1 / O emulsion; W1 / O emulsion was injected into the external water phase containing PLGA hydrophobic segment and betahistine, and emulsified by ultrasound (200W, 60s) to form (W1 / O) / W2 emulsion; Stirring at 3℃ for 2.5h, then warming to 20℃ for 1.5h, and finally centrifugation and washing.
[0034] Example 7 Formulation (per 100mg PLGA): BDNF 1mg + HA 1mg + 5mg curcumin (core) Betahistine dihydrochloride 30mg (shell) The molar ratio of lactic acid:glycolic acid in PLGA was 80:20, and PLGA was dissolved in ethyl acetate with a concentration of 15mg / mL.
[0035] The concentration of PLGA hydrophobic segment in the external water phase was 3% w / v RGD-TAT peptide 2mg.
[0036] Steps: The HA aqueous solution was mixed with BDNF and curcumin to form W1 phase; W1 phase was added to PLGA / ethyl acetate solution, and emulsified by ultrasound (100W, 30s) to form W1 / O emulsion; W1 / O emulsion was injected into the external water phase containing PLGA hydrophobic segment and betahistine, and emulsified by ultrasound (200W, 60s) to form (W1 / O) / W2 emulsion; Stirring at 5℃ for 1.5h, then warming to 25℃ for 0.5h, and finally adding RGD-TAT peptide for covalent coupling and centrifugation and washing.
[0037] Comparative Example 1 Compared with Example 1, BDNF was directly mixed with betahistine, and the other parts were the same as Example 1.
[0038] Comparative Example 2 Compared with Example 1, HA was not added, and only BDNF was loaded into the core, and the other parts were the same as Example 1.
[0039] Comparative Example 3 Compared with Example 1, BDNF was not added, and only HA was loaded into the core, and the other parts were the same as Example 1.
[0040] Experiment 1: Release behavior The product of each example and comparative example was placed in 5 mL of release medium, and 1 mL of supernatant was taken at intervals for HPLC / BCA detection of drug concentration. The test results are shown in Table 1.
[0041] Table 1 Test results of each example and comparative example As can be seen from the above table, the release behavior is mainly dominated by the core structure design of the delivery system, which is the basic framework for protecting BDNF, controlling initial burst release, and achieving long-term sustained release. The specific data show that the key to controlling release is to encapsulate BDNF in the core composed of hyaluronic acid (HA) and use PLGA as the carrier. This structural design successfully controls the burst release of BDNF at a low level and achieves sustained release for several weeks. In sharp contrast, any design that destroys this protection mechanism (such as no HA core protection or using physical mixing instead of double emulsion method) leads to severe burst release and rapid inactivation of BDNF. In addition, the ratio of lactic acid-glycolic acid of PLGA also has an impact on drug release, especially the sustained release phase. When the ratio of lactic acid-glycolic acid is 80:20, the drug release rate can be significantly slowed down, showing not only a lower burst release level, but also a significantly lower 7-day cumulative release amount than other groups; when the ratio of lactic acid-glycolic acid is 60:40, the release rate is accelerated, showing higher burst release and higher 7-day cumulative release.
[0042] Experiment 2: Meniere's disease treatment test Healthy guinea pigs weighing 250-300 grams were selected, and 20 μL / ear of 3M NaCl solution was injected into the tympanic cavity for modeling. The modeled guinea pigs were divided into a blank control group, a model group, and a drug administration group, with 30 guinea pigs in each group. The drug administration of each group is shown in Table 2.
[0043] Table 2 Drug administration of each group No stimulation was performed from the first modeling to the 1st-21st day, and 20 μL / ear of 3M NaCl solution was injected into the tympanic cavity of the model group and the drug administration group for the second time on the 21st day. The spin platform test was performed every 6 hours within 24 hours after the stimulation. When the falling platform duration was longer than that of the blank control group, it was determined to be recurrence. The test results are shown in Table 3.
[0044] Table 3 Recurrence rate results of each group BDNF can provide sustained neuroprotection, resulting in a significant reduction in disease recurrence rate. The design of the core-shell structure allows BDNF to maintain activity, and the group successfully constructing the core-shell has the lowest recurrence rate of 13.3%, while the recurrence rate of the group with a failed structure increases linearly.
[0045] Furthermore, it should be understood that although the specification is described in terms of embodiments, the specification as a whole, and each embodiment individually, does not contain only one independent technical solution, and the specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.
Claims
1. A betahistine-containing nanosphere, characterized by, Comprising: a carrier consisting of polyglycolic acid-hydroxyacetic acid copolymer; a hydrophilic core encapsulated inside the carrier, the hydrophilic core comprising brain-derived neurotrophic factor and hyaluronic acid; a hydrophobic shell encapsulated on the surface of the carrier, the hydrophobic shell comprising betahistine or a pharmaceutically acceptable salt / derivative thereof.
2. The betahistine-containing nanospheres according to claim 1, characterized in that The amount of brain-derived neurotrophic factor is 1-10 mg, the amount of hyaluronic acid is 1-5 mg, and the amount of betahistine or a salt / derivative thereof is 5-30 mg per 100 mg of polyglycolic acid-hydroxyacetic acid copolymer carrier.
3. The betahistine-containing nanospheres according to claim 2, characterized in that The mass ratio of brain-derived neurotrophic factor to hyaluronic acid is 1-5:
1.
4. The betahistine-containing nanospheres according to claim 1, characterized in that The hydrophilic core further comprises an antioxidant curcumin, and the amount of curcumin is 0.5-5 mg per 100 mg of polyglycolic acid-hydroxyacetic acid copolymer carrier.
5. The betahistine-containing nanospheres according to claim 1, characterized in that The molar ratio of lactic acid to hydroxyacetic acid in the polyglycolic acid-hydroxyacetic acid copolymer is 60-80:20-40.
6. The betahistine-containing nanospheres according to claim 1, characterized in that The surface of the nanosphere is modified with an inner ear blood-labyrinth barrier penetrating peptide.
7. The betahistine-containing nanospheres according to claim 6, characterized in that The amount of modification of the inner ear blood-labyrinth barrier penetrating peptide is 0.1-2 mg of peptide per 100 mg of polyglycolic acid-hydroxyacetic acid copolymer carrier.
8. A process for the preparation of betahistine-containing nanospheres according to any one of claims 1 to 7, characterized in that, A hyaluronic acid aqueous solution is mixed with brain-derived neurotrophic factor to obtain a complex W1, which is added to an ethyl acetate solvent containing polyglycolic acid-hydroxyacetic acid copolymer to form a W1 / O emulsion through primary emulsification; the W1 / O emulsion is dispersed in an external water phase containing betahistine or a pharmaceutically acceptable salt / derivative thereof and a water-soluble segment of polyglycolic acid-hydroxyacetic acid copolymer to form a (W1 / O) / W2 emulsion through secondary emulsification, and the solvent is volatilized and solidified under stirring to obtain multifunctional nanospheres.
9. The method of claim 8, wherein the betahistine-containing nanospheres are prepared by, The specific operation of stirring and volatilizing the solvent to solidify is to stir at 3-5°C for 1.5-2.5 hours, then increase the temperature to 20-25°C and stir for 0.5-1.5 hours.
10. The method of claim 8, wherein the betahistine-containing nanospheres are prepared by, The concentration of polyglycolic acid-hydroxyacetic acid copolymer in the ethyl acetate solvent containing polyglycolic acid-hydroxyacetic acid copolymer is 5-15 mg / mL.