阅读说明：本技术 一种纳曲酮微球 (Naltrexone microspheres ) 是由 蒋志君 杜欢欢 曹青日 陆小娟 于鹤云 于 2020-04-08 设计创作，主要内容包括：本发明公开了一种纳曲酮微球,其制备方法包括以下步骤：将纳曲酮和含疏水链段的可降解聚合物溶于有机溶剂中,得到含药聚合物溶液；将所述含药聚合物溶液和水相使用微流控装置进行混合,形成O/W型乳剂后,搅拌条件下去除乳液中的有机溶剂；其中,所述水相中含有稳定剂,所述稳定剂与水相的比例为0.1%～5%(w/v)；挥净有机溶剂后,将以上所得的分散体系加至水中进行固化,得到所述纳曲酮微球。本发明所述的微球改善了传统制备方法制备的微球粒径分布不均一、微球形态不规整、释放突释高等缺点。并且,本发明所述的微流控技术的粒子合成过程工艺参数高度可控,有利于微球的生产放大。(The invention discloses a naltrexone microsphere, and a preparation method thereof comprises the following steps: dissolving naltrexone and a degradable polymer containing a hydrophobic chain segment in an organic solvent to obtain a drug-containing polymer solution; mixing the drug-containing polymer solution and the water phase by using a microfluidic device to form an O/W type emulsion, and removing the organic solvent in the emulsion under the stirring condition; wherein the water phase contains a stabilizer, and the proportion of the stabilizer to the water phase is 0.1-5% (w/v); and after the organic solvent is completely volatilized, adding the obtained dispersion system into water for solidification to obtain the naltrexone microspheres. The microsphere overcomes the defects of nonuniform particle size distribution, irregular microsphere shape, high release burst and the like of the microsphere prepared by the traditional preparation method. In addition, the particle synthesis process of the microfluidic technology has highly controllable technological parameters, and is beneficial to the production and amplification of microspheres.)

1. A naltrexone microsphere, characterized by being prepared by the following steps:

a: dissolving naltrexone and a degradable polymer containing a hydrophobic chain segment in an organic solvent to obtain a drug-containing polymer solution; wherein the mass ratio of the naltrexone to the degradable polymer is fixed to be 1:1.97, and the concentration of the degradable polymer in the organic solvent is 2.5-50% (w/v);

b: dissolving a stabilizer in water to obtain an external water phase; wherein the concentration of the stabilizer is 0.5-5% (w/v);

c: respectively taking the drug-containing polymer solution and the water phase as A, B pump solutions of an advection pump, setting A, B pumps of the advection pump at a certain flow rate, generating O/W type emulsion by a microfluidic device, and removing the organic solvent in the emulsion under the stirring state; wherein, the phase A flow rate is set: the flow rate of the phase B is 1: 2-1: 5, the two phases enter a micro mixer through a constant flow rate through a pipeline by an advection pump and are mixed to form an emulsion, and the emulsion is collected and then subjected to volatilization of an organic solvent;

d: c, under the condition of stirring, adding the dispersion system obtained in the step c into water for curing to obtain the naltrexone sustained release microspheres; wherein the volume ratio of the dispersion system to water is 1: 2-1: 100, and stirring and curing are carried out at the speed of 300-500rpm for 4 hours at room temperature.

2. The naltrexone microspheres according to claim 1, wherein in step a, said degradable polymer containing hydrophobic segments is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol and polycaprolactone.

3. The naltrexone microspheres according to claim 1, wherein in step a, the degradable polymer containing the hydrophobic chain segment is polylactic acid-glycolic acid copolymer with relative molecular mass of 5000-100000, wherein the molar ratio of polylactic acid-glycolic acid is 50: 50-85: 15.

4. The naltrexone microspheres according to claim 1, wherein said organic solvent is selected from one or more of dichloromethane, ethyl acetate and acetone.

5. The naltrexone microspheres according to claim 1, wherein in step b, said stabilizer is selected from one or more of polyvinyl alcohol, polysorbate 20, polysorbate 80, polyethylene glycol and sodium lauryl sulphate.

6. The naltrexone microspheres according to claim 1, wherein in step d, the volume ratio of said dispersion to water is 1: 25.

7. The naltrexone microspheres according to claim 1, wherein the ratio of said stabilizer to said aqueous phase is between 0.1% and 5% (w/v).

8. The naltrexone microspheres according to claim 1, wherein in step c, the flow rate of phase a is 1:3 as compared to the flow rate of phase B.

9. The naltrexone microspheres according to claim 1, wherein in step c, the microfluidic device has an internal pore size in the range of 50 μm to 2 mm.

10. Use of naltrexone microspheres as claimed in any one of claims 1 to 9 in the manufacture of an opioid receptor antagonist.

Technical Field

The invention relates to the technical field of pharmaceutical preparations, in particular to naltrexone microspheres.

Technical Field

Naltrexone is an opioid receptor antagonist, has pharmacodynamics similar to that of naloxone, has blocking effect on kappa-, delta-, mu-and other opioid receptors, can obviously weaken or completely block the opioid receptors, and even reverses the effect generated by intravenous injection of opioid drugs. It can block the effect of drug re-taking and prevent or reduce re-taking. The structural formula is shown in the following formula.

Naltrexone preparation is mainly in form of tablet, and the treatment course lasts for half a year and 50mg should be taken daily. Therefore, the treatment course is long, and the medicine has certain toxicity to the liver. In order to improve the compliance of patients, the development of the long-acting microsphere of naltrexone has wider application prospect. The microsphere is a skeleton-type entity formed by dissolving or dispersing a drug in a polymer material, and the particle size of the microsphere is between 1 and 250 micrometers. 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.

Common microsphere preparation methods include spray drying, emulsion-solvent evaporation, hot melt extrusion, and phase separation. The CN200710099359.9 patent uses an O/W type emulsification-solidification method to prepare naltrexone microspheres with higher encapsulation efficiency, and the protected microspheres contain 10 to 50 percent of naltrexone base, 50 to 90 percent of polylactic acid-glycolic acid copolymer (PLGA) and 0.1 to 10 percent of polyvinyl alcohol (PVA); according to the results reported by hongkong et al (CN02145144.3), the encapsulation efficiency of naltrexone microspheres prepared by 35% feeding was 75% when microspheres were prepared by a single emulsion solvent evaporation method, however, the microspheres had poor uniformity and morphology. Therefore, the traditional process still has some defects at present, such as poor reproducibility among batches, non-uniform particle size distribution, difficult control of microsphere morphology, difference of embedding capacities of drugs with different properties, complex preparation process, low production efficiency and the like.

Disclosure of Invention

In view of the above disadvantages, the present invention aims to provide a naltrexone microsphere, which has a simple and rapid preparation method, and can effectively solve the problems of poor uniformity, irregular shape, etc. of the naltrexone microsphere.

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

a naltrexone microsphere is prepared by the following steps:

a: dissolving naltrexone and a degradable polymer containing a hydrophobic chain segment in an organic solvent to obtain a drug-containing polymer solution; wherein the mass ratio of the naltrexone to the degradable polymer is fixed to be 1:1.97, and the concentration of the degradable polymer in the organic solvent is 2.5-50% (w/v);

b: dissolving a stabilizer in water to obtain an external water phase; wherein the concentration of the stabilizer is 0.5-5% (w/v);

c: respectively taking the drug-containing polymer solution and the water phase as A, B pump solutions of an advection pump, setting A, B pumps of the advection pump at a certain flow rate, generating O/W type emulsion by a microfluidic device, and removing the organic solvent in the emulsion under the stirring state; wherein, the phase A flow rate is set: the flow rate of the phase B is 1: 2-1: 5, the two phases enter a micro mixer through a constant flow rate through a pipeline by an advection pump and are mixed to form an emulsion, and the emulsion is collected and then subjected to volatilization of an organic solvent;

d: c, under the condition of stirring, adding the dispersion system obtained in the step c into water for curing to obtain the naltrexone sustained release microspheres; wherein the volume ratio of the dispersion system to water is 1: 2-1: 100, and stirring and curing are carried out at the speed of 300-500rpm for 4 hours at room temperature.

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

Further, in the naltrexone microspheres, in the step a, the degradable polymer containing the hydrophobic chain segment is polylactic acid-glycolic acid copolymer (PLGA), the relative molecular mass of the PLGA is 5000-100000, and the molar ratio of polylactic acid to glycolic acid is 50: 50-85: 15.

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

Preferably, the molar ratio of polylactic acid to glycolic acid in the PLGA is 50: 50-75: 25, and more preferably 75: 25.

Further, in the naltrexone microspheres, in the step a, the organic solvent is one or more selected from dichloromethane, ethyl acetate, acetone, methyl ethyl ketone and tetrahydrofuran.

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

Further, in the naltrexone microspheres, in the step a, the degradable polymer containing the hydrophobic chain segment accounts for 0.5-20% (w/v) of the total mass of the drug-containing polymer solution; the preferable concentration is 2.5-20%, and when the concentration of the polymer is too low, the medicine is easy to leak in the preparation process, so that the preparation of the microsphere is influenced.

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

Preferably, in the naltrexone microspheres, the stabilizing agents are polyvinyl alcohol PVA and Tween 80, and the ratio of the PVA to the water phase is preferably 0.5-5% (w/v); more preferably 1% (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 flow rate of the phase A is 1:3 that of the phase B; the flow rate range of the microfluidic A, B pump is 1-100 mL/min, more preferably the flow rate of the phase A is 6mL/min, the flow rate of the phase B is 18mL/min, the two-phase flow speed ratio directly influences the size of the droplet particle size, and when the AB phase is set as the above speed, the prepared microspheres have the highest encapsulation rate, good uniformity and regular shape.

Further, the volume ratio of the dispersion system in the step b to water is 1: 2-1: 50; further preferably 1:25, and when the volume ratio is set to such a value, the encapsulation ratio of the microspheres is high.

Further, the diameter of the inner hole of the micro-fluidic controller in the step c is between 50 μm and 2mm, and is preferably between 100 μm and 1 mm. The diameter of the inner bore of the microflow controller directly affects the particle size of the final microsphere.

Furthermore, the mean particle size of the naltrexone microspheres prepared by the method is 50-100 microns. When the microcapsule or microsphere with the grain diameter of less than 200 mu m is orally taken, no foreign body feeling is caused in the oral cavity, the smaller grain diameter has larger specific surface area, and the relative drug-loading capacity is large.

Furthermore, the drug in the naltrexone microspheres prepared by the invention can be slowly released for one month.

Furthermore, the naltrexone microspheres prepared by the invention are used for preparing an opioid receptor antagonist.

The scheme shows that the invention at least has the following beneficial effects: dissolving naltrexone and a degradable polymer containing a hydrophobic chain segment in an organic solvent, dissolving a stabilizer in water to form an external water phase, respectively using the formed oil phase and the external water phase as microfluidic A, B phase solutions, setting a A, B pump at a certain flow rate and a certain proportion, generating an O/W type emulsion through a microfluidic device, and removing the organic solvent in the emulsion under a stirring state; further curing with water to obtain naltrexone microspheres; the naltrexone microspheres prepared by the method have the advantages of high encapsulation rate, good slow release effect, uniform particle size, round shape and good reproducibility; meanwhile, the preparation method disclosed by the invention is low in production cost and high in efficiency.

Drawings

FIGS. 1-6 are micrographs of the emulsions of examples 2, 4, 5, 8, 9, and 12, in that order;

FIG. 7 is a scanning electron micrograph (left 200X, right 400X) of microspheres obtained in example 4;

FIG. 8 is a scanning electron micrograph (left 200X, right 400X) of microspheres obtained in example 9; FIG. 9 is an in vitro cumulative release profile of naltrexone microspheres prepared in examples 4, 5, 9 and 12; fig. 10 is a graph showing the release of plasma drug concentration when the naltrexone microspheres prepared in example 9 were subjected to animal experiments.

Detailed Description

The following describes in detail a specific embodiment of the present invention with reference to the drawings, examples, and test examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

1. Comparative preparation example

The following method was used for the preparation of microspheres by the conventional emulsion solvent evaporation method, and the examples 1 to 7 were prepared as comparative examples.

203.2mg of naltrexone and 400mg of polymer are weighed and dissolved in an organic solvent to form a polymer solution containing naltrexone, microspheres are prepared by adopting a traditional method, namely an emulsion solvent volatilization method, the microspheres are dropwise added into a water phase containing a stabilizer under the stirring state to form an O/W emulsion, the emulsion is added into curing water at room temperature, the stirring is continued for 4 hours to volatilize the organic solvent, wherein the stirring speed is 300-500 rpm. Volatilizing organic solvent, standing, removing supernatant after the suspended matter settles, washing with water for three times, centrifuging, collecting microsphere, and freeze-drying. The recipe is given in table 1 below.

TABLE 1 recipe for different comparative examples

Particle size and encapsulation efficiency of microspheres of comparative example:

particle size: an appropriate amount of the microspheres prepared in examples 1 to 7 was divided into three portions, each of which was suspended in an appropriate amount of 0.1% aqueous tween 80 solution, and the particle size of the microspheres was measured with a laser particle size distribution instrument.

Encapsulation efficiency: preparing 0.1mol/L hydrochloric acid solution, weighing 30mg of naltrexone microspheres, putting the naltrexone microspheres into a 10mL centrifuge tube, adding 6mL of 0.1mol/L hydrochloric acid solution, and oscillating the vortex apparatus for 3min at the intensity of 5; centrifuging by a centrifuge at 12000rpm for 15min, collecting the clear supernatant, and performing high performance liquid analysis to obtain the concentration of free drug; adding 2mL of dichloromethane into the precipitate for dissolving, transferring the precipitate into a 50mL volumetric flask, using dichloromethane for constant volume, shaking up, measuring 1mL of sample solution from the precipitate in a penicillin bottle, adding 4mL of 0.1mol/L hydrochloric acid solution, oscillating the vortex instrument for 3min at the strength of 5, volatilizing DCM at the water bath temperature of 40 ℃, volatilizing dichloromethane, centrifuging by using a centrifugal machine at 10000rpm for 10min, taking the supernatant into the 50 or 100mL volumetric flask, using hydrochloric acid for constant volume and uniform mixing, and performing high-efficiency liquid phase analysis to obtain the concentration of the drug-coated drug. The results are shown in Table 2, and the envelope ratio is calculated as follows:

TABLE 2 particle size and encapsulation efficiency of microspheres of comparative examples

Examples	Mean median diameter (μm)	Encapsulation efficiency (%)
			1	48.96	67.65
2	42.64	64.98
			3	41.46	65.23
4	78.60	75.63
			5	79.26	75.24
6	72.23	65.67
			7	69.54	67.65

The results in table 2 show that the microspheres prepared by the conventional method have large particle size difference and low encapsulation efficiency, and from the experimental data, the concentration of PLGA has a large influence on the encapsulation efficiency of the microspheres under the same polymer condition, and when the concentration is high, the polymer solution can better wrap the drug, thereby improving the encapsulation efficiency; after comparing other factors, such as stabilizer type, curing fold, etc., the present study identified the preferred conditions in comparative example 4, which was applied in microfluidic technology as conditions for the examples of the present invention.

2. Preparation examples of the present invention

A naltrexone microsphere is prepared by the following steps:

a: dissolving 2.03g of naltrexone and 4g of degradable polymer containing hydrophobic chain segments in dichloromethane to obtain a drug-containing polymer solution; the concentration of the degradable polymer in the organic solvent is 2.5-50% (w/v);

b: dissolving a stabilizer PVA in water to obtain an external water phase; wherein the concentration of the stabilizer is 1-5% (w/v);

c: respectively taking the drug-containing polymer solution and the water phase as A, B pump solutions of an advection pump, setting A, B pumps of the advection pump at a certain flow rate, generating O/W type emulsion by a microfluidic device, and removing the organic solvent in the emulsion under the stirring state; wherein, the phase A flow rate is set: and the flow rate of the phase B is 1: 2-1: 5, the two phases pass through an advection pump and enter a micro mixer through a pipeline at a constant flow rate to be mixed to form an emulsion, and the emulsion is collected and then subjected to volatilization of an organic solvent.

d: c, under the condition of stirring, adding the dispersion system obtained in the step c into water for curing to obtain the naltrexone sustained release microspheres; wherein the volume ratio of the dispersion system to water is 1: 2-1: 100, and stirring and curing are carried out at the speed of 300-500rpm for 4 hours at room temperature.

The formulations are as described in table 3 below.

TABLE 3 recipe for embodiments of the invention

The particle size and encapsulation efficiency of the microspheres in the preparation examples of the present invention were measured by referring to the methods of comparative examples, and the results are shown in Table 4 below.

TABLE 4 particle size and encapsulation efficiency of microspheres of examples of the invention

Examples	Mean median diameter (μm)	Encapsulation efficiency (%)
			8	78.96	78.65
9	76.34	89.23
			10	77.60	85.93
11	61.60	85.21
			12	92.26	87.51
13	71.23	82.01
			14	67.21	84.00
15	84.96	86.61
			16	62.00	82.90
17	79.71	71.02
			18	68.00	69.53

As shown in Table 4, the microspheres prepared by the method of the invention have uniform particle size and high encapsulation efficiency, and most of the microspheres exceed the encapsulation efficiency of 80% specified by pharmacopoeia.

3. Test example 1

Emulsion morphology and microsphere appearance.

The emulsion in the microsphere preparation process of examples 2, 4, 5, 8, 9 and 12 is taken, and the emulsion droplet shape is observed by a microscope, and the result is shown in attached figures 1-6. As can be seen from the attached drawings 1-6, compared with the comparative proportion, the emulsion prepared by the scheme of the invention in the examples 8, 9 and 12 has uniform and moderate particle size.

Taking a proper amount of the microspheres prepared in the embodiments 4 and 9, observing the surface morphology of the microspheres by using a scanning electron microscope, wherein the results are shown in the attached drawings 7 and 8, and the embodiment 9 means that the naltrexone sustained release microspheres prepared by the scheme provided by the invention have uniform particle size, round microspheres and good morphology.

4. Test example 2

Naltrexone microsphere cumulative release profile

The microsphere in-vitro release medium is PBS (pH7.4) buffer solution containing 0.02% Tween 20, and the release volume is 50 mL; 50mg of the microspheres prepared in examples 4, 5, 9 and 12 were taken and placed in a 50mL conical flask, 50mL of release medium was added, 1mL was sampled at the time points of 4h, 1d, 2d, 3d, 7d, 14d, 21d and 28d … …, centrifugation (12000rpm, 15min) was performed to take the supernatant for high performance liquid phase analysis and 700. mu.L of sample injection, the remaining microspheres were resuspended in 300. mu.L of fresh medium and placed back in the 50mL conical flask for continued shaking release, and all release medium was replaced every 7 days. The cumulative percent release was calculated and a graph of cumulative percent release versus time was plotted, the results of which are shown in figure 9.

It can be seen from the figure that the microspheres obtained by the conventional preparation method, i.e. examples 4 and 5, have high burst release and poor release profile, while in contrast, the microspheres prepared by the microfluidic process parameters of the present invention, i.e. examples 9 and 12, have slow release of the drug from the microspheres, low burst release, gentle release profile, sustained slow release for one month and good in vitro release.

5. Test example 3

And testing the blood concentration of naltrexone microspheres in animals.

3 beagle dogs with the body mass of about 12kg are fasted for 12h, naltrexone microspheres prepared in the best example 9 are taken, injected into beagle dogs in a hip muscle deep injection mode according to the dose of 11.9mg/kg, blank blood is taken before administration, 3mL of blood is taken from forelimb veins after administration for 4h, 24 h, 48 h, 72 h, 168 h, 336 h, 504 h, 672 h, 840 h and 960h, the blood is placed in a test tube added with heparin, centrifuged for 10min at 4000r/min, plasma is divided, high performance liquid phase analysis is carried out, the concentration of the naltrexone in the blood is detected, and a blood concentration release curve graph is drawn, as shown in figure 10, the naltrexone microspheres disclosed by the invention have very good slow release effect in animals. If the naltrexone microsphere microspheres disclosed by the invention are used for preparing an opioid receptor antagonist, the frequency of taking the drug can be obviously reduced.

The results of the above examples and test examples show that the invention uses the microfluidic technology, optimizes the microfluidic parameters, and improves the defects of nonuniform particle size distribution, irregular microsphere shape, high release burst and the like of the microspheres prepared by the traditional preparation method. In addition, the particle synthesis process of the microfluidic technology has highly controllable technological parameters, and is beneficial to the production and amplification of microspheres.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.