CN113384537A

CN113384537A - Levalbutine sustained-release microspheres and preparation method thereof

Info

Publication number: CN113384537A
Application number: CN202110447997.5A
Authority: CN
Inventors: 镇学初; 曹青日; 何丽雅
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-04-25
Filing date: 2021-04-25
Publication date: 2021-09-14
Also published as: WO2022228585A1

Abstract

The invention relates to a preparation method of levo-stepholidine sustained-release microspheres, which comprises the following steps: dissolving levo-stepholidine and degradable polymer containing hydrophobic chain segments in an organic solvent to obtain a drug-containing polymer solution; adding the drug-containing polymer solution into an aqueous phase under the condition of stirring to form O/W type emulsion, and removing an organic solvent in the emulsion; wherein the water phase contains a stabilizer, and the proportion of the stabilizer to the water phase is 0.1-5% (w/v); and under the condition of stirring, adding the obtained dispersion system into water for solidification to obtain the levo-stepholidine sustained-release microspheres. The levo-stepholidine sustained-release microspheres have proper particle size, round shape, no burst release phenomenon and long-acting antidepressant effect.

Description

Levalbutine sustained-release microspheres and preparation method thereof

Technical Field

The invention relates to the technical field of pharmaceutical preparations, in particular to a levo-stepholidine sustained-release microsphere and a preparation method thereof.

Background

Schizophrenia (schizophrenia) is a group of psychoses characterized by a group of disorders characterized by a lack of coordination between thinking, emotion, behavior, and mental activities. Generally including depression, alzheimer's disease, parkinson's disease, and the like. With the accelerated pace of life and the continuous aggravation of social competition, people are under greater pressure, the mental health problems are increasingly highlighted, particularly, the incidence rate of mental diseases such as depression, Alzheimer's disease, Parkinson and the like is increased year by year, the recurrence rate is very high, and meanwhile, patients cannot work normally, and heavy economic burden is brought to families.

The chemical name of L-stephanine (L-SPD) is (-) -2, 10-dihydroxy-3, 9-dimethoxy-5, 8,13,13 a-tetrahydro-6H-dibenzo [ a, g%]Quinolizine belongs to the class of tetrahydroprotoberberine compounds (THPBs). It has dopamine D₁Agonism, D₂Antagonism, D₃Antagonism and other multiple effects, however, the water solubility and the fat solubility are poor, so that the oral administration is difficult to absorb, the bioavailability is extremely low, and the oral administration is difficult to become a medicament, thereby limiting the further development of the oral administration as the medicament. The compound can stimulate dopamine D₁Receptor antagonism D₂A receptor having anti-schizophrenia activity. The structural formula is shown as follows.

In general, oral administration has many disadvantages, such as high frequency of administration and low bioavailability due to first pass effect. The microspheres are skeleton-type entities formed by dissolving or dispersing drugs in polymer materials, and the particle size of the microspheres is between 1 and 250 mu m. Generally prepared into suspension for injection or oral administration. The medicament has the following characteristics after micro-spheroidization: covering up the unpleasant odor of the medicine, improving the stability of the medicine, reducing the stimulation to the stomach or the inactivation of the medicine in the stomach, solidifying the liquid medicine for convenient storage or further preparing into other dosage forms, controlling the release rate of the medicine, and the like. Moreover, in the market, no microsphere formulation has emerged for levostepholidine.

Therefore, in order to further prepare a pharmaceutical dosage form for more effectively treating mental diseases, it is necessary to provide a sustained-release preparation with a longer acting time.

Disclosure of Invention

In order to solve the problems, the invention aims to provide the levo-stepholidine sustained-release microsphere and the preparation method thereof, the preparation method is simple and quick, and the problems of poor oral absorption, low bioavailability and the like can be effectively solved.

In order to achieve the purpose, the invention provides the following technical scheme:

in one aspect, the invention provides a preparation method of sustained-release microspheres of L-stepholidine, which comprises the following steps:

(a) dissolving levo-stepholidine and degradable polymer containing hydrophobic chain segments in an organic solvent to obtain a drug-containing polymer solution;

(b) dissolving a stabilizer in water to obtain an external water phase;

(c) adding the drug-containing polymer solution into an aqueous phase under the condition of stirring to form O/W type emulsion, and removing an organic solvent in the emulsion;

(d) under the condition of stirring, adding the dispersion system obtained in the step (c) into water for solidification to obtain the levo-stepholidine sustained-release microspheres.

Further, in the step (a), the degradable polymer containing the hydrophobic chain segment is selected from one or more of polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polylactic-polyethylene glycol (PLA-PEG) and Polycaprolactone (PCL).

Further, in the step (a), the degradable polymer containing the hydrophobic chain segment is polylactic acid-glycolic acid copolymer, the relative molecular mass of the copolymer is 5000-150000 g/mol, and the molar ratio of lactide to glycolide is 85: 15-50: 50.

Preferably, the relative molecular mass of PLGA is 12000-90000 g/mol, more preferably 12000-21000 g/mol.

Preferably, the molar ratio of PLGA lactide to glycolide is 75: 25-50: 50, more preferably 75: 25.

Further, in the step (a), the organic solvent is one or more selected from dichloromethane, ethyl acetate, dimethyl sulfoxide, acetone, methyl ethyl ketone and tetrahydrofuran.

Preferably, the organic solvent is dichloromethane, ethyl acetate, dimethyl sulfoxide and acetone.

Further, in the step (a), the weight ratio of the levo-stepholidine to the degradable polymer containing the hydrophobic chain segment is 1: 2-1: 100; preferably 1:25 to 1: 50.

Further, in the step (a), the degradable polymer containing the hydrophobic chain segment accounts for 0.5-20% of the total mass of the drug-containing polymer solution; preferably 1 to 15%, more preferably 1 to 10%. When the concentration of the polymer is too high, the polymer is easy to precipitate in the preparation process, and the preparation of the microspheres is influenced.

Further, in the step (b), the stabilizer is one or more selected from polyvinyl alcohol (PVA), polysorbate 20, polysorbate 80, polyethylene glycol and sodium lauryl sulfate.

Preferably, the stabilizers are PVA and tween 80. The ratio of PVA to the aqueous phase is preferably 0.5% to 5% (w/v); more preferably 2% (w/v). The proportion of the Tween 80 to the water phase is preferably 0.1-0.3% (w/v); more preferably 0.1% (w/v).

Further, in step (c), the stirring rate is 50-1000 rpm; in step (d), the stirring rate is 50 to 1000 rpm.

Further, the volume ratio of the external water phase in the step (b) to the solidified phase in the step (d) is 1: 2-1: 100, preferably 1: 2-1: 50.

On the other hand, the invention also provides the levo-stepholidine sustained-release microsphere prepared by the preparation method, the average grain diameter of the microsphere is 20-100 mu m, and the encapsulation rate is 50-80%.

Furthermore, the medicine in the prepared levo-stepholidine sustained-release microspheres can be slowly released for one month.

Dissolving levo-stepholidine and degradable polymer containing hydrophobic chain segments in an organic solvent, dissolving a stabilizer in water to form an external water phase, adding the formed oil phase into the water phase to form an O/W emulsion, volatilizing the solvent, and further curing with water to obtain the levo-stepholidine sustained-release microspheres. Firstly, levo-stepholidine is a hydrophobic drug, and is co-dissolved in an organic solvent with a degradable polymer containing a hydrophobic chain segment, so that the drug loss can be reduced, and the drug loading rate and the encapsulation rate can be improved; secondly, the reasonable stirring speed is adopted, so that the microspheres can be controlled to have proper particle size; in addition, the microspheres are further cured by water, so that the encapsulation efficiency of the microspheres is effectively improved, the microspheres are harder, the microspheres are prevented from deforming in the collection process, and the microspheres are good in shape.

By the scheme, the invention at least has the following advantages:

the preparation method is simple and quick, and the prepared levo-stepholidine sustained-release microspheres have proper particle size, round shape, no burst release phenomenon, can be slowly released for one month, and effectively improve the compliance of patients.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood and to be implemented according to the content of the specification, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIGS. 1-4 are scanning electron micrographs (400X) of the sustained-release microspheres of L-stepholidine in examples 11, 16, 17 and 18;

FIG. 5 is the in vitro release curve of the sustained release microspheres of L-stepholidine in examples 11, 16 and 17;

FIG. 6 is the in vitro release curve at 37 ℃ and 45 ℃ of the sustained-release microspheres of L-stepholidine in example 16;

FIG. 7 is the in vivo pharmacodynamic graph of the sustained release microspheres of L-stepholidine of example 16.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

100mg of PLGA (molar ratio of lactide to glycolide: 75:25, Mw: 1.2wg/mol) was weighed out, dissolved in 0.5mL of dichloromethane, 50mg of levo-stepholidine was weighed out, and the drug was added to a solution of PLGA in dichloromethane and pulverized by ultrasound (200W, 2s, 45s) to be uniformly dispersed. Dropwise adding the mixture into 2.5mL of 1% PVA solution under stirring, quickly emulsifying by using a vortex instrument, then adding the mixture into 62.5mL of water under stirring to solidify the microspheres and volatilize the organic solvent, collecting the microspheres after 2h, washing the microspheres with water for 3-5 times, suspending, pre-freezing in a refrigerator at-80 ℃ for 8h, and freeze-drying for 48h to obtain microsphere powder.

Example 2

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed, dissolved in 5mL of dichloromethane, 50mg of levo-stepholidine was weighed, dissolved in 0.5mL of DMSO, the drug-containing DMSO solution was added to the PLGA dichloromethane solution, vortexed to disperse the drug uniformly, and dropped into 2.5mL of 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 625mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and then freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 3

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 5mL of dichloromethane, 20mg of levo-stepholidine was weighed out, dissolved in 0.5mL of DMSO, the DMSO solution containing the drug was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 25mL of a 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 625mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and then freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 4

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 5mL of dichloromethane, 12.5mg of levo-stepholidine was weighed out and dissolved in 0.5mL of DMSO, the drug-containing DMSO solution was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 25mL of a 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 625mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and then freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 5

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 10mL of dichloromethane, 50mg of levo-stepholidine was weighed out, dissolved in 0.5mL of DMSO, the DMSO solution containing the drug was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 50mL of a 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 1250mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 6

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 15mL of dichloromethane, 50mg of levo-stepholidine was weighed out, dissolved in 0.5mL of DMSO, the DMSO solution containing the drug was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 75mL of a 1% PVA solution with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 1875mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 7

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 5mL of dichloromethane, 50mg of levo-stepholidine was weighed out, dissolved in 0.5mL of DMSO, the DMSO solution containing the drug was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 15mL of a 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 375mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is put into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and then freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 8

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 5mL of dichloromethane, 50mg of levo-stepholidine was weighed out, dissolved in 0.5mL of DMSO, the DMSO solution containing the drug was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 50mL of a 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 1250mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 9

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 5mL of dichloromethane, 50mg of levo-stepholidine was weighed out, dissolved in 0.5mL of DMSO, the DMSO solution containing the drug was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 25mL of a 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 125mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and then freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 10

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 5mL of dichloromethane, 50mg of levo-stepholidine was weighed out, dissolved in 0.5mL of DMSO, the DMSO solution containing the drug was added to the PLGA in dichloromethane and vortexed to disperse the drug uniformly. The mixture was added dropwise to 25mL of a 1% PVA solution while stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 250mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and then freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 11

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the DMSO solution containing the drug was added to the methylene chloride solution of PLGA and vortexed to disperse the drug uniformly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 2% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 1250mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 12

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the DMSO solution containing the drug was added to the methylene chloride solution of PLGA and vortexed to disperse the drug uniformly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 3% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the system is added into 1250mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 13

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, 125mg of NaCl was weighed out and dissolved in 50mL of aqueous phase containing 2% (w/v) of stabilizer PVA, the drug-containing DMSO solution was added to the PLGA in dichloromethane solution and vortexed to disperse uniformly. The drug-containing PLGA solution was added to 50mL of PVA external aqueous phase with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-. After the organic solvent is completely volatilized, the system is added into 1250mL of water to be solidified for 4h, microspheres are collected and washed by water for 3-5 times, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 14

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out, dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out, dissolved in 0.5mL of dimethylsulfoxide, 31.2mg of levostepholidine was weighed out, dissolved in 50mL of aqueous phase containing 2% (w/v) stabilizer PVA, the drug-containing DMSO solution was added to the PLGA in dichloromethane solution and vortexed to disperse the drug uniformly. The drug-containing PLGA solution was added to 50mL of the outer aqueous PVA phase with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-. After the organic solvent is completely volatilized, the system is added into 1250mL of water to be solidified for 4h, microspheres are collected and washed for 3-5 times by water, the suspension is placed into a refrigerator with the temperature of-80 ℃ for pre-freezing for 8h, and then freeze drying is carried out for 48h, so that microsphere powder is obtained.

Example 15

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the DMSO solution containing the drug was added to the methylene chloride solution of PLGA and vortexed to disperse the drug uniformly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 2% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. And (3) adjusting the pH value of 1250mL of water to 7.8-8.0 by using phosphate, volatilizing the organic solvent, adding the system into 1250mL of water, solidifying for 4h, collecting microspheres, washing for 3-5 times by using water, suspending, pre-freezing for 8h in a refrigerator at-80 ℃, and freeze-drying for 48h to obtain microsphere powder.

Example 16

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the DMSO solution containing the drug was added to the methylene chloride solution of PLGA and vortexed to disperse the drug uniformly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 2% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. And (3) adjusting the pH value of 1250mL of water to 8.0 by using 1mol/LNaOH, volatilizing the organic solvent, adding the system into 1250mL of water, solidifying for 4 hours, collecting microspheres, washing for 3-5 times by using water, suspending, pre-freezing for 8 hours in a refrigerator at-80 ℃, and freeze-drying for 48 hours to obtain microsphere powder.

Example 17

100mg of PLGA (molar ratio of lipid-terminated lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the DMSO solution containing the drug was added to the dichloromethane solution of PLGA and vortexed to disperse evenly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 2% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-. And (3) adjusting the pH value of 1250mL of water to 8.0 by using 1mol/LNaOH, volatilizing the organic solvent, adding the system into 1250mL of water, solidifying for 4 hours, collecting microspheres, washing for 3-5 times by using water, suspending, pre-freezing for 8 hours in a refrigerator at-80 ℃, and freeze-drying for 48 hours to obtain microsphere powder.

Example 18

100mg of PLGA (molar ratio of lactide to glycolide 50:50, Mw 6wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the drug-containing DMSO solution was added to the PLGA in dichloromethane solution and vortexed to disperse uniformly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 2% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. And (3) adjusting the pH value of 1250mL of water to 8.0 by using 1mol/LNaOH, volatilizing the organic solvent, adding the system into 1250mL of water, solidifying for 4 hours, collecting microspheres, washing for 3-5 times by using water, suspending, pre-freezing for 8 hours in a refrigerator at-80 ℃, and freeze-drying for 48 hours to obtain microsphere powder.

Example 19

100mg of PLGA (molar ratio of lactide to glycolide 50:50, Mw 15g/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levo-stepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the drug-containing DMSO solution was added to the PLGA in dichloromethane solution and vortexed to disperse the drug uniformly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 2% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. And (3) adjusting the pH value of 1250mL of water to 8.0 by using 1mol/LNaOH, volatilizing the organic solvent, adding the system into 1250mL of water, solidifying for 4 hours, collecting microspheres, washing for 3-5 times by using water, suspending, pre-freezing for 8 hours in a refrigerator at-80 ℃, and freeze-drying for 48 hours to obtain microsphere powder.

Example 20

100mg of PLGA (molar ratio of lactide to glycolide 75:25, Mw 1.2wg/mol) was weighed out and dissolved in 10mL of dichloromethane, 50mg of levostepholidine was weighed out and dissolved in 0.5mL of dimethylsulfoxide, the DMSO solution containing the drug was added to the methylene chloride solution of PLGA and vortexed to disperse the drug uniformly. The drug-containing PLGA solution was added to 50mL of an aqueous phase containing 2% (W/v) of the stabilizer PVA with stirring to form an O/W emulsion. Stirring was continued at room temperature for 3h to volatilize dichloromethane, wherein the stirring rate was 300-500 rpm. After the organic solvent is completely volatilized, the microspheres are collected and washed by water for 3-5 times, the suspension is put into a refrigerator with the temperature of 80 ℃ below zero for pre-freezing for 8 hours, and then the freeze drying is carried out for 48 hours, so as to obtain the microsphere powder.

Test example 1: particle size of the microspheres

An appropriate amount of the microspheres prepared in examples 1 to 20 was divided into three portions, each of which was suspended in an appropriate amount of 0.1% tween 80 aqueous solution, and the particle size of the microspheres was measured with a laser particle size distribution meter, and the results are shown in table 1, and the obtained microspheres had a particle size (in particular, median size) in the range of 20 to 100 μm, and were suitable for injection.

TABLE 1 particle size of microspheres from different examples

Examples	Mean median diameter (. mu.m). + -. SD
		1	19.98±0.18
2	59.75±0.30
		3	50.29±0.01
4	42.27±0.09
		5	49.86±0.03
6	53.15±0.04
		7	65.89±1.48
8	376.11±8.49
		9	52.53±0.10
10	40.60±0.04
		11	48.89±0.12
12	40.43±0.04
		13	46.32±0.69
14	39.57±0.13
		15	82.63±1.44
16	41.43±0.03
		17	22.41±0.07
18	33.35±0.02
		19	53.73±1.40
20	44.63±0.09

Test example 2: microsphere morphology

A proper amount of the microspheres prepared in example 11, example 16 and examples 17 to 18 was used to determine the morphology of the microspheres by scanning electron microscopy, and the results are shown in FIGS. 1 to 4. As can be seen from FIGS. 1 to 4, the sustained-release microspheres of L-stepholidine have uniform particle size, round microspheres and good shape, and some flakes are not formed due to the excessive precipitation speed when PLGA is separated out.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Test example 3: microsphere drug loading and encapsulation efficiency

5mg of the levo-stepholidine sustained-release microspheres prepared in the embodiments 1-20 are respectively weighed, 0.5mL of DMSO is added, vortex dissolution is carried out, 5mL of methanol is added, centrifugation is carried out (10000rpm, 10min), supernatant is collected, the volume of the methanol is determined to be 50mL, HPLC analysis is carried out, the total drug amount can be obtained, and the total drug amount is further converted into the drug loading amount and the encapsulation rate of the microspheres. The results are shown in Table 2. The calculation formula of the drug loading rate and the encapsulation efficiency is as follows:

TABLE 2 drug loading and encapsulation efficiency for different examples

Comparison of the results of examples 1 and 2 shows that when the method of directly dispersing the solid powder of the drug is adopted in example 1, the encapsulation efficiency is significantly higher than that of the method of dissolving the drug in the organic solvent in example 2, but at the same time, the encapsulation efficiency result of example 1 is more than 100%, because the levo-stepholidine microsphere prepared by the method has more free drug and the encapsulation efficiency result is more than 100%.

A comparison of the results of examples 3-10 shows that the encapsulation efficiency is highest when the drug loading ratio is 1:2, the DMSO: DCM is 1:20, the PLGA concentration is 10 mg/mL, the DCM: PVA is 1:5, the PVA: solidified water is 1:25, and the PVA concentration is 1%.

A comparison of the results of example 5 and examples 11 and 12 shows that the encapsulation efficiency is highest when the PVA concentration is adjusted from 1% (w/v) to 2% (w/v) without changing other factors. This may be related to the stability of the emulsion, and PVA is both a stabilizer and an emulsifier, when the amount is small, the molecules of the emulsifier are not enough to cover the whole oil-water interface, the interfacial tension is not minimized, the emulsion is unstable, the amount is too large, and although the stable emulsion is obtained, the stable emulsion may cause increased foaming, which affects the quality of the emulsion, and may cause certain influence on the process of the polymer separating out and wrapping the drug into balls.

Comparison of the results of example 13 and example 14, example 15 and example 16, example 17 and example 18, example 19 shows that when levostepholidine microspheres are prepared, the external water phase saturated NaCl and the drug have no significant effect on the encapsulation efficiency, and the use of lipid-capped PLGA to adjust the molecular weight of PLGA has no significant effect on the encapsulation efficiency. However, when the pH is adjusted by the solidification phase, the encapsulation efficiency is obviously improved, which may be related to the property of the medicine. Because the levo-stepholidine is an alkaline compound which is difficult to dissolve in water, when the pH of the levo-stepholidine is adjusted to be alkaline by a solidified phase, the medicine is difficult to dissolve into a water phase from the microspheres, and the encapsulation efficiency is improved.

Comparison of the results of example 11 with example 20 shows that the step of solidifying the microspheres after preparation of the microspheres to form colostrum provides a significant increase in the encapsulation efficiency of the microspheres, indicating that solidification provides better formability of the microspheres.

Test example 4: in vitro release profile of microspheres

6.8g of dipotassium hydrogenphosphate and 0.79g of sodium hydroxide were dissolved in 1000mL of water, and 0.5% (w/v) of Tween 20 was added to obtain a release medium PBS-Tween 20 solution.

About 15mg of the sustained-release microspheres of L-stepholidine prepared in examples 11, 16 and 17 were weighed, placed in a 50mL Erlenmeyer flask, added with 50mL of 0.5% Tween 20 in PBS (pH7.4), and placed in a water bath constant temperature shaking box. Oscillating at the speed of 100rpm at the temperature of 45 ℃, taking out 1mL of sample for centrifugation (10000rpm, 10min) for 1h, 4h, 1, 4, 7, 11, 14, 18 and 21 days respectively, and taking 700 mu L of supernatant for sample injection analysis; example 16 samples were taken for 1mL centrifugation (10000rpm, 10min) at 37 deg.C with shaking at 100rpm for 4h, 1, 4, 7, 14, 21, 28 days, respectively, and 700. mu.L of the supernatant was injected for analysis. The cumulative percent release was calculated and the release profile was plotted as cumulative percent release versus time, leaving the microspheres to be resuspended in fresh medium and run out. The results are shown in fig. 5 and fig. 6, from which it can be understood that levo-stepholidine is slowly released from the microspheres without burst release, the release curve is flat, and the levo-stepholidine can be released continuously for one month.

Test example 5: pharmacodynamic experiment of microspheres in animal body

The experiment adopts C57 male mice to preliminarily examine the in vivo anti-schizophrenia effect of the levo-stepholidine sustained-release microspheres of the example 16. Mice were randomly divided into three groups, and (1) a suspension of sustained-release microspheres loaded with l-stepholidine (example 16) was subcutaneously injected in a volume of 10mL/Kg at a concentration of 12.75mg/mL (2) a suspension of sustained-release microspheres loaded with l-stepholidine in a volume of 10mL/Kg at a concentration of 37.5mg/mL (3) as a control group. On the fifth day, injecting PCP inducer (5mg/Kg) into the abdominal cavity of each mouse, placing the mice into a multifunctional spontaneous activity box for PCP-induced spontaneous movement test, and continuously collecting animal activity videos for 70 minutes; the video file is analyzed to obtain the total movement distance of the animal within 70 minutes, and the data of 60 minutes is statistically analyzed.

The result analysis shows that the large-dose experimental group injected with the levo-stepholidine sustained-release microspheres has obvious inhibition effect compared with the control group, and the high-dose group is obviously higher than the control group (p is less than 0.05), which shows that the levo-stepholidine sustained-release microspheres have certain inhibition efficiency in vivo and can achieve certain effect of resisting schizophrenia.

Claims

1. A preparation method of levo-stepholidine sustained-release microspheres is characterized by comprising the following steps:

(b) dissolving a stabilizer in water to obtain an external water phase;

(d) and (c) adding the dispersion system obtained in the step (c) into solidification phase water under the stirring condition for solidification to obtain the levo-stepholidine sustained-release microspheres.

2. The method of claim 1, wherein: in the step (a), the degradable polymer containing the hydrophobic chain segment is selected from one or more of lactide-glycolide copolymer, polylactic acid-polyethylene glycol and polycaprolactone.

3. The method of claim 1, wherein: in the step (a), the degradable polymer containing the hydrophobic chain segment is a lactide-glycolide copolymer, the relative molecular mass of the lactide-glycolide copolymer is 5000-150000 g/mol, and the molar ratio of lactide to glycolide is 85: 15-50: 50.

4. The method of claim 1, wherein: in step (a), the organic solvent is one or more selected from dichloromethane, ethyl acetate, dimethyl sulfoxide and acetone.

5. The method of claim 1, wherein: in the step (a), the weight ratio of the levo-stepholidine to the degradable polymer containing the hydrophobic chain segment is 1: 2-1: 100.

6. The method of claim 1, wherein: in step (b), the stabilizer is one or more selected from polyvinyl alcohol, polysorbate 20, polysorbate 80, polyethylene glycol and sodium lauryl sulfate.

7. The method of claim 1, wherein: in step (b), the ratio of the stabilizer to the aqueous phase is 0.1% to 5% (w/v).

8. The method of claim 1, wherein: in the step (d), the volume ratio of the water phase to the solidified phase is 1: 5-1: 50, and the pH of the solidified phase is 7.8-8.0.

9. The method of claim 1, wherein: in the step (d), the average particle size of the levo-stepholidine microspheres is 20-150 μm.

10. The sustained-release microspheres of levo-stepholidine prepared by the preparation method of any one of claims 1-9.