CN118615253A - Perampanel microspheres based on multiple emulsion process and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of biomedicine, and specifically to a perampanel microsphere based on a double emulsion process, and a preparation method and application thereof. The preparation method is to use water-in-oil-in-water type double emulsion droplets as a template, and prepare the perampanel long-acting controlled-release microsphere by a microfluidic emulsion template method. The present invention introduces an internal water phase containing an amphiphilic polymer material sustained-release regulator based on microfluidic technology, which can not only prepare perampanel microspheres with a porous structure, but also realize multi-component inclusion (i.e., compound microsphere drugs) at the same time, increase the release channel of the drug, weaken the hysteresis effect, and realize the precise controlled release of perampanel and/or sodium valproate.

Description

Perampanel microspheres based on multiple emulsion process and preparation method and application thereof

Technical Field

The present invention relates to the field of biomedicine, and in particular to perampanel microspheres based on a double emulsion process, and a preparation method and application thereof.

Background Art

Epilepsy, commonly known as epilepsy or epilepsy, is a chronic disease caused by abnormal discharge of brain neurons due to various reasons, leading to temporary brain dysfunction. The cause of the disease is usually incurable, and patients face long-term or even lifelong irregular disease attacks, which seriously affect their normal life, and the disease attacks will cause irreversible damage to the central nervous system.

There are various types of anti-epileptic drugs (AEDs), which are divided into traditional anti-epileptic drugs and new anti-epileptic drugs according to the order of their appearance. Anti-epileptic drugs used in clinical practice before the 1980s are usually called traditional anti-epileptic drugs, including valproic acid, carbamazepine, phenobarbital, etc. Those that appeared after the 1980s are called new anti-epileptic drugs, including lamotrigine, levetiracetam, oxcarbazepine, topiramate, etc. However, the therapeutic effects of many anti-epileptic drugs can no longer meet the increasing demand for refractory epilepsy, and combination therapy is often required. However, with the increase in the number of drug applications, the increase in dosage and the influence of diet, the side effects caused by fluctuations in drug concentrations have become a problem that needs to be solved urgently. Reports have pointed out that irregular drug use and large fluctuations in drug concentrations are one of the causes of refractory epilepsy; at the same time, for patients, taking multiple drugs at the same time every day is prone to missed doses and drug interruptions, poor compliance, and many anti-epileptic drugs have large gastrointestinal reactions, which is also one of the key factors for patients to resist medication.

Perampanel is a new type of anti-epileptic drug developed and marketed by Eisai of Japan, mainly for refractory epilepsy. It inhibits downstream signaling pathways by non-competitive antagonism of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, thereby reducing the increase in intracellular calcium ions and achieving the effect of treating epilepsy. Original research data show that perampanel has good anti-epileptic effects. At the same time, perampanel has strong AMPA selectivity (IC50 = 93nM), and even at a concentration of up to 30μM, it only has a weak effect on NMPA, so the risk of side effects is relatively weak. The structure of perampanel is as follows:

The marketed preparations of Perampanel include tablets (2mg, 4mg, 6mg, 8mg,

10mg, 12mg), granules (1%) and oral suspension (0.5mg/ml). However, the above preparations still have disadvantages such as high frequency and long period of medication, poor medication convenience, and large fluctuations in blood drug concentration.

Drug-loaded microspheres are tiny spheres or quasi-spheres formed by dissolving or dispersing drugs in polymer materials, and the particle size is generally in the range of 1 to 250 μm. After the microspheres are injected into the body (due to their large particle size, they are generally injected subcutaneously and intramuscularly), the polymer slowly dissolves and degrades under physiological conditions, and the encapsulated drugs diffuse in the body according to the degradation of the microspheres, slowly releasing the drugs at a certain rate per unit time, maintaining a stable blood drug concentration at the lesion site, and prolonging the half-life of the drug in the body, thereby achieving long-term sustained release, reducing the frequency of administration, and improving compliance. At present, perampanel also has microspheres and other sustained-release dosage forms under development, such as:

Chinese patent application CN117530933A discloses a method for preparing a long-acting sustained-release microsphere of perampanel and its injection. It is mainly prepared by homogenizing and emulsifying the sustained-release microspheres through a water-in-oil (O/W) single emulsion template, using perampanel and perampanel 3/4 hydrate as raw materials, and PLGA as a carrier material. The encapsulation rate of the prepared sustained-release microspheres varies greatly (45.8%-99.8%), that is, the drug quality control is difficult, it is difficult to achieve stable commercial production, and the particle size uniformity is poor, and it is difficult to achieve accurate control of the release rate of the microspheres. And this method uses water-in-oil single emulsion droplets as a template, and it is difficult to prepare perampanel compound microspheres.

Summary of the invention

In view of this, the present invention provides a perampanel long-acting controlled-release microsphere that can accurately control the sustained-release rate and achieve gradient release, as well as a preparation method and application thereof. The perampanel long-acting controlled-release microsphere includes perampanel single microsphere and perampanel compound microsphere.

The active drug ingredients of the perampanel single microspheres are perampanel and its derivatives, and the active drug ingredients of the perampanel compound microspheres are perampanel and its derivatives and other water-soluble anti-epileptic drugs.

To achieve the above object, the first aspect of the present invention provides a method for preparing perampanel long-acting controlled-release microspheres, which uses water-in-oil-in-water type emulsion droplets as templates and prepares the perampanel long-acting controlled-release microspheres by a microfluidic emulsion template method, comprising the following steps:

Step 1, preparing the inner water phase: the inner water phase comprises an amphiphilic polymer material sustained-release regulator and an inner water phase raw material good solvent, and the amphiphilic polymer material sustained-release regulator is dissolved in the inner water phase raw material good solvent to obtain the inner water phase; the mass fraction of the amphiphilic polymer material sustained-release regulator in the inner water phase is 0.05-2.0wt%. The present invention uses the amphiphilic polymer material sustained-release regulator as the inner water phase viscosity regulator, which can improve the shear efficiency and increase the encapsulation rate.

In some embodiments of the present invention, the internal aqueous phase raw material good solvent is water.

In some embodiments of the present invention, the water includes pure water, water for injection, deionized water or double distilled water.

In some embodiments of the present invention, the amphiphilic polymer material sustained-release regulator is selected from at least one of sodium carboxymethyl cellulose, polyvinyl pyrrolidone, poloxamer, and polyethylene glycol. In some embodiments of the present invention, the amphiphilic polymer material sustained-release regulator is sodium carboxymethyl cellulose (CMC-Na). In some embodiments of the present invention, the mass fraction of CMC-Na in the inner aqueous phase is 0.3wt%.

In order to prepare the compound microspheres, in some embodiments of the present invention, the inner water phase further comprises a water-soluble anti-epileptic drug, and the mass fraction of the water-soluble anti-epileptic drug in the inner water phase is 2.0-15.0wt%. The present invention dissolves the water-soluble anti-epileptic drug in the inner water phase and the oil-soluble anti-epileptic drug in the oil phase, and the drugs between the water and oil phases will not merge with each other, thereby improving the drug encapsulation rate of the microspheres and ensuring the quality of the drugs, thereby realizing the preparation of the compound microspheres.

In some embodiments of the present invention, the water-soluble antiepileptic drug is sodium valproate, and the oil-soluble antiepileptic drug is perampanel. In some embodiments of the present invention, the mass fraction of sodium valproate in the internal aqueous phase is 5.0-10.0wt%, and the mass ratio of sodium valproate to perampanel is (1-2):1.

Step 2, preparing an oil phase: the oil phase comprises perampanel and its derivatives, polylactic acid-glycolic acid copolymer and an oil phase good solvent, and perampanel and its derivatives and polylactic acid-glycolic acid copolymer are dissolved in the oil phase good solvent to obtain the oil phase; the mass fraction of the polylactic acid-glycolic acid copolymer in the oil phase is 5.0-20.0wt%, and the mass ratio of the perampanel and its derivatives to the polylactic acid-glycolic acid copolymer is 1:(1-4).

In some embodiments of the present invention, the mass fraction of the polylactic acid-co-glycolic acid in the oil phase is 15 wt %. In some embodiments of the present invention, the mass ratio of the perampanel and its derivatives to the polylactic acid-co-glycolic acid is 1:3.

In some embodiments of the present invention, the oil phase good solvent is selected from any one of dichloromethane, ethyl acetate, chloroform and acetone. In some embodiments of the present invention, the oil phase good solvent is dichloromethane.

The perampanel and its derivatives are active ingredients of anti-epileptic drugs. Polylactic acid-glycolic acid copolymer (PLGA) is randomly polymerized from two polymer monomers, lactic acid and glycolic acid, and is a degradable functional macromolecular organic compound with good biocompatibility, non-toxicity, good capsule and film-forming properties, and is widely used in pharmaceutical, medical engineering materials and modern industrial fields. The present invention uses PLGA as a sustained-release substrate for encapsulating the active ingredients of anti-epileptic drugs, and realizes the sustained-release administration of perampanel.

In some embodiments of the present invention, the molecular weight of the polylactic acid-co-glycolic acid is 5000Da-120000Da, and the molecular weight distribution coefficient is 1-10. In some embodiments of the present invention, the molecular weight of the polylactic acid-co-glycolic acid is 5000Da-60000Da, and the molecular weight distribution coefficient is 1-10.

Step 3, preparing an external aqueous phase: the external aqueous phase comprises a surfactant and a good solvent for an external aqueous phase raw material, and the surfactant is dissolved in the good solvent for the external aqueous phase raw material to obtain the external aqueous phase; the mass fraction of the surfactant in the external aqueous phase is 0.1-2.0wt%.

In some embodiments of the present invention, the mass fraction of the surfactant in the external aqueous phase is 0.5 wt %. In some embodiments of the present invention, the surfactant is selected from at least one of polyvinyl alcohol, polyvinyl pyrrolidone and poloxamer.

In some embodiments of the present invention, the surfactant is polyvinyl alcohol.

In some embodiments of the present invention, the external aqueous phase raw material good solvent is water.

Step 4: Preparation of perampanel long-acting controlled-release microspheres by microfluidic emulsion template method:

The inner water phase, oil phase and outer water phase prepared in the above steps are introduced into the corresponding inlets of the microfluidic chip at a constant flow rate, the inner water phase, oil phase and outer water phase are sheared in the microfluidic chip, and water-in-oil-in-water type double emulsion droplets are obtained at the outlet of the microfluidic chip;

Step 5: collecting and solidifying the water-in-oil-in-water type emulsion droplets to obtain emulsion microspheres; washing and freeze-drying the emulsion microspheres to obtain the perampanel long-acting controlled-release microspheres.

In some embodiments of the present invention, step 5 specifically includes collecting the water-in-oil-in-water type emulsion droplets in an aqueous solution, stirring at a constant temperature of 40°C and a low speed (150 rpm) for 3 hours, and solidifying the water-in-oil-in-water type emulsion droplets to obtain emulsion microspheres; washing with purified water to remove the surfactant on the surface of the emulsion microspheres, and freeze-drying to obtain the finished product of perampanel long-acting controlled-release microspheres.

Prior art (e.g., Chinese patent CN104825405A) prepares sustained-release microspheres through a water-in-oil (W/O/W) emulsion template, which mainly uses ultrasound + stirring dispersion to prepare colostrum and emulsion. This method is difficult to control the size of colostrum and emulsion, that is, it is difficult to control the drug release rate. On the other hand, this method is difficult to use to prepare compound microspheres, because if the method is used to prepare compound microspheres, two kinds of internal water phases need to be used, and the two kinds of internal water phases are very easy to merge during the preparation of the emulsion. After fusion, on the one hand, the shape of the internal water phase is destroyed and it is difficult to control the release rate. On the other hand, some drugs may interact with each other, making it difficult to ensure the quality of the drug.

Compared with the prior art, the preparation method of perampanel long-acting controlled-release microspheres provided by the present invention uses water-in-oil-in-water type emulsion droplets as templates to prepare perampanel long-acting controlled-release microspheres by a microfluidic emulsion template method, thereby obtaining perampanel long-acting controlled-release microspheres with uniform size (e.g., 120±10 μm), stable quality, and the ability to accurately control the sustained-release rate and achieve gradient release, thereby overcoming the problem that the existing preparation process cannot achieve uniform size and accurate controlled release.

It should be emphasized that the present invention introduces an internal aqueous phase containing an amphiphilic polymer material sustained-release regulator based on microfluidic technology, which can not only prepare perampanel microspheres with a porous structure, but also realize the simultaneous encapsulation of multiple components (i.e., compound microsphere drugs), increase the release channel of the drug, weaken the hysteresis effect, and realize the precise controlled release of perampanel and/or sodium valproate. In addition, the internal pore structure of the perampanel microspheres with a porous structure can also be further adjusted by adjusting the internal aqueous phase (such as components, proportions, and flow rates in microchannels) to further ensure the precise controlled release of perampanel and/or sodium valproate.

Furthermore, the flow rate of the inner water phase into the microfluidic chip is 200-600 μL/min, the flow rate of the oil phase into the microfluidic chip is 20-60 μL/min, and the flow rate of the outer water phase into the microfluidic chip is 400-3000 μL/min. The present invention controls the flow rate of each phase so that the perampanel long-acting controlled-release microspheres with uniform particle size (e.g., 120±10 μm) can be efficiently and stably prepared.

In some embodiments of the present invention, the flow rate ratio of the inner water phase to the oil phase into the microfluidic chip includes 5-20: 1. In some embodiments of the present invention, the flow rate ratio of the inner water phase to the oil phase into the microfluidic chip is 10: 1. By increasing the flow rate ratio of the inner water phase to the oil phase into the microfluidic chip, the specific surface area of the microspheres can be increased to regulate the structure of the pores inside the microspheres, thereby accurately regulating the gradient release of the microspheres.

In some embodiments of the present invention, the flow rate ratio of the external water phase to the oil phase into the microfluidic chip is 10-50: 1. In some embodiments of the present invention, the flow rate ratio of the external water phase to the oil phase into the microfluidic chip is 20-50: 1.

The flow rate ratio of the inner water phase and the oil phase passing into the microfluidic chip can be controlled within the above flow rate ratio to further ensure the generation of microspheres with uniform particle size.

It should still be emphasized that, compared with the preparation method of the single emulsion template, the preparation method provided by the present invention can realize the preparation of compound microspheres, overcoming the problem that the existing preparation process cannot achieve multi-component gradient controlled release. In summary, the present invention provides a long-acting controlled-release microsphere of perampanel with uniform size, stable quality, and the ability to accurately control the sustained release rate and achieve gradient release, and a preparation method thereof, which is suitable for the preparation of perampanel microspheres with single prescription or multiple prescriptions. The perampanel microspheres prepared by the present invention can achieve gradient controlled release of single prescription or multiple prescriptions, and the sustained release period is adjustable for 2 to 6 weeks, and the drug encapsulation rate is high, which meets the clinical needs of gradient release, combination therapy and long-term efficacy.

The second aspect of the present invention provides a long-acting controlled-release microsphere of perampanel, which is prepared according to the above-mentioned preparation method, and its particle size is 120±10μm. The long-acting controlled-release microsphere of perampanel obtained by the preparation method provided by the present invention is a round microsphere with a porous structure inside, with a rounded morphology, uniform size, and controllable release. The perampanel microspheres prepared by the present invention can achieve the effect of 2 weeks to several months of medication with a single injection, and the release of the precisely controlled microspheres is stable. In theory, the blood concentration of perampanel in the body will be much lower than repeated high-frequency oral administration, ensuring the efficacy while reducing the risk of side effects. In addition, perampanel was included in the list of Class II psychotropic drugs by NMPA on July 1, 2023, so it has anti-abuse control requirements (the single prescription amount of ordinary preparations does not exceed 7 days), and the perampanel microspheres prepared by the present invention are effective for weeks to months with only a single injection due to their long-acting sustained-release properties, which is expected to solve this demand.

The third aspect of the present invention provides the application of the above-mentioned perampanel long-acting controlled-release microspheres in the preparation of perampanel injection. The perampanel long-acting controlled-release microspheres prepared by the present invention can be further developed into perampanel long-acting controlled-release injections, which can significantly extend the dosing interval, reduce the number of visits to the doctor to get medicine, increase the convenience of medication, improve patient compliance, and reduce the risk of epileptic seizures caused by missed medications. Especially for some patients who are not suitable for oral administration, such as choking, dysphagia, nasogastric feeding, fistula patients, etc., long-acting controlled-release perampanel microspheres and preparations provide a new choice of more convenient and safe administration. In addition, the perampanel long-acting controlled-release microspheres (especially compound microspheres) prepared by the present invention are particularly suitable for the treatment and/or prevention of epileptic seizures in patients with refractory epilepsy.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art,

The following is a brief introduction to the drawings required for use in the embodiments or prior art descriptions. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

FIG1 is a schematic diagram of a microfluidic chip used in the microfluidic emulsion template method of the present invention;

FIG2 is a microscopic structure diagram of the perampanel single square microspheres prepared in Example 1 of the present invention;

FIG3 is a microstructure diagram of the perampanel single square microspheres prepared in Comparative Example 1 of the present invention;

FIG4 is an in vitro release curve of perampanel monogranules prepared in Comparative Example 1 and Example 1 of the present invention;

FIG5 is a microscopic structure diagram of the perampanel compound microspheres prepared in Example 2 of the present invention;

FIG6 is an in vitro release curve of the perampanel single microspheres prepared in Example 1 of the present invention and the perampanel compound microspheres prepared in Example 2;

FIG. 7 is an optical photograph of an existing perampanel microsphere product.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Herein, "and/or" includes any and all combinations of one or more of the associated listed items.

Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

As used in this specification, the term "about" typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

In this specification, some embodiments may be disclosed in a format of being in a certain range. It should be understood that such description of "being in a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

Below in conjunction with embodiment, the scheme of the present invention will be explained and described.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if no specific technology or condition is indicated, it is carried out according to the technology or condition described in the document in this area or according to the product specification.The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained by commercial purchase.

Explanation of the terms involved in the following embodiments and comparative examples:

Theoretical drug loading: The weight percentage of the drug contained in the theoretical microspheres. Theoretical drug loading = (theoretical dosage/(theoretical dosage + theoretical carrier weight)) × 100%.

Measured drug loading: The weight percentage of the drug contained in the measured microspheres. Measured drug loading = (drug content in microspheres/microsphere weight) × 100%.

Encapsulation rate: the weight percentage of the encapsulated drug in the microspheres. Encapsulation rate = (amount of drug encapsulated in the microspheres/total amount of encapsulated and unencapsulated drugs in the microspheres) × 100% = (1-amount of unencapsulated drugs in the liquid/total amount of encapsulated and unencapsulated drugs in the microspheres) × 100%.

Derivatives: Compounds that have a common core structure and are substituted with different groups.

The specific steps for in vitro release test of microspheres are as follows:

(1) Weigh approximately 20 mg of microsphere sample into a 20 mL vial;

(2) Add 20 mL of pH 7.2 PBS solution containing 1.0% PS80 to resuspend the microspheres and seal the bottle mouth by pressing the stopper;

(3) Place the vial in a constant temperature oscillator at 37 μm and 210 rpm for incubation.

To fully suspend the microspheres;

(4) Frequent sampling within one day of incubation, and then once every 3-4 days.

1 mL of supernatant was sampled each time and supplemented with the corresponding volume of fresh release medium;

(5) HPLC was used to detect the content of active ingredients in the samples, and the cumulative release rate was calculated. The in vitro release curve was drawn with time as the horizontal axis and the cumulative release rate of the active ingredients as the vertical axis.

The amounts of raw materials used in the following examples and comparative examples are shown in Table 1 below.

Table 1 Prescription of perampanel microspheres alone and in combination

Example 1: Preparation of Perampanel Microspheres by Emulsion Method

Follow the steps below:

Step 1: Prepare the inner water phase (W ₁ ), the oil phase and the outer water phase (W ₂ ):

a. Weigh CMC-Na and dissolve it fully in water to obtain an inner aqueous phase W ₁ ;

b. Weigh perampanel and PLGA, and fully dissolve them in dichloromethane to obtain an oil phase;

c. Weigh PVA and fully dissolve it in water to obtain the external water phase W ₂ .

Step 2: The inner water phase _W1 , the oil phase and the outer water phase _W2 are introduced into the corresponding inlet of the microfluidic chip (as shown in FIG. 1 ) through a constant pressure pump at a constant flow rate, sheared in the chip, and emulsion droplets are obtained at the emulsion receiving port; the control flow rate of each phase is: oil phase: 20 μL/min;

W ₁ phase: 200μL/min; W ₂ phase: 400μL/min;

Step 3: Collect the emulsion droplets in an aqueous solution and heat at 40°C at a low speed (150

rpm) for 3 h, and the droplets solidified to obtain double emulsion microspheres;

Step 4: Wash the emulsion microspheres with clean water to remove PVA on the surface; freeze-dry to obtain finished microspheres.

The microstructure of the microspheres was observed by scanning electron microscopy, as shown in Figure 2. The microspheres were round in shape, uniform in size (microsphere size was 120±10 μm), and had a porous structure inside. The in vitro release curve is shown in Figure 4.

Comparative Example 1: Preparation of Perampanel Microspheres by Single Emulsion Method

Follow the steps below:

Step 1: Prepare the oil phase and the external water phase:

a. Weigh perampanel and PLGA, and fully dissolve them in dichloromethane to obtain an oil phase;

b. Weigh PVA and fully dissolve it in water to obtain the external water phase.

Step 2: The oil phase and the external water phase are introduced into the corresponding inlet of the microfluidic chip through a constant pressure pump at a constant flow rate, sheared inside the chip, and a single emulsion droplet is obtained at the chip receiving port; the control flow rate of each phase is: oil phase: 20 μL/min; _W2 phase: 400 μL/min;

Step 3: Collect the single emulsion droplets in an aqueous solution and heat at 40°C at a low speed (150

rpm) for 3 h, and the droplets solidified to obtain single emulsion microspheres;

Step 4: Wash the single emulsion microspheres with clean water to remove PVA on the surface; freeze-dry to obtain finished microspheres.

The microstructure of the microspheres was observed by scanning electron microscopy. As shown in FIG3 , the microspheres were round in shape and uniform in size (the size of the microspheres was 120±10 μm) and had no porous structure inside.

The in vitro release curve is shown in Figure 4. The optical photograph of similar products on the market (microsphere size is 20-140 μm) is shown in Figure 7.

From the above results, it can be seen that the present invention can prepare perampanel microspheres by adopting a double emulsion method and introducing an internal water phase containing an appropriate amount of CMC-Na, not only can perampanel single microspheres with uniform size (e.g., 120±10 μm) be prepared, but also the prepared perampanel single microspheres have a porous structure. In other words, the introduction of an internal water phase containing an appropriate amount of CMC-Na not only adjusts the viscosity of the internal water phase and stabilizes the oil-water interface to a certain extent, but also unexpectedly ensures that the porous structure of the prepared perampanel single microspheres is stable and size-controllable.

Furthermore, it can be seen from Figure 4 that the micropores in the porous structure of the perampanel single microspheres can increase the release channels of the drugs in the microspheres, so that the release medium can enter the microspheres to take out the drugs, which helps to regulate the drug release rate of the microspheres in the 2nd to 3rd week, and weakens the hysteresis effect in the early stage of drug release, thereby enabling the release of perampanel to be precisely controlled throughout the release cycle (especially the early release of perampanel).

On the other hand, as shown in FIG4 , the release cycles of the single-sided microspheres of Comparative Example 1 and Example 1 are not much different, that is, the entire release cycle of the microspheres is mainly controlled by the degradation rate of PLGA, and the introduction of the internal aqueous phase containing a suitable amount of CMC-Na does not affect the degradation of PLGA, so the release cycle changes slightly.

Example 2-5: Preparation of Perampanel Compound Microspheres by Emulsion Method

Follow the steps below:

Step 1: Prepare the inner water phase (W ₁ ), the oil phase and the outer water phase (W ₂ ):

a. Weigh sodium valproate and CMC-Na and fully dissolve them in water to obtain an aqueous phase W ₁ ;

b. Weigh perampanel and PLGA, and fully dissolve them in dichloromethane to obtain an oil phase;

c. Weigh PVA and fully dissolve it in water to obtain the external water phase W ₂ .

Step 2: The inner water phase _W1 , the oil phase and the outer water phase _W2 are introduced into the corresponding inlet of the microfluidic chip (as shown in FIG. 1 ) through a constant pressure pump at a constant flow rate, sheared in the chip, and emulsion droplets are obtained at the emulsion receiving port; the control flow rate of each phase is: oil phase: 60 μL/min;

W ₁ phase: 600 μL/min; W ₂ phase: 3000 μL/min;

Step 3: Collect the emulsion droplets in an aqueous solution and heat at 40°C at a low speed (150

rpm) for 3 h, and the droplets solidified to obtain double emulsion microspheres;

Step 4: Wash the emulsion microspheres with clean water to remove PVA on the surface; freeze-dry to obtain finished microspheres.

The microstructure of the microspheres was observed by scanning electron microscopy. The microstructure of the composite microspheres prepared in Example 2 is shown in FIG5 . As can be seen from FIG5 , the composite microspheres prepared in Example 2 have a rounded morphology, uniform size (microsphere size is 120±10 μm), and a porous structure inside.

From the above results, it can be seen that the present invention adopts the double emulsion method to prepare perampanel microspheres and introduces an internal water phase containing an appropriate amount of CMC-Na, and can also prepare perampanel compound microspheres with uniform size (for example, 120±10μm), and the prepared perampanel compound microspheres have a porous structure, that is, the method of the present invention is suitable for the preparation of perampanel compound microspheres. In other words, the introduction of the internal water phase containing an appropriate amount of CMC-Na and sodium valproate not only adjusts the viscosity of the internal water phase and stabilizes the oil-water interface to a certain extent, but also unexpectedly, it can also ensure that the prepared microspheres can simultaneously encapsulate two anti-epileptic drugs, perampanel and sodium valproate, and the porous structure of the prepared microspheres is still stable and the size is controllable.

Furthermore, it can be seen from Figure 6 that the porous structure of the perampanel compound microspheres can increase the release channels of the drugs in the microspheres, so that the release medium can enter the microspheres and take out the drugs, which helps to regulate the drug release rate of the microspheres in the 2nd to 3rd week, and weakens the hysteresis effect in the early stage of drug release, thereby enabling the perampanel compound and sodium valproate to achieve precise control of release throughout the release cycle (especially the early release).

The drug release rate of compound microspheres is faster than that of single microspheres. The reasons may be:

(1) In the composite microspheres, the release rate of sodium valproate was faster than that of perampanel. This may be because sodium valproate has better water solubility. As the porous structure inside PLGA is gradually opened up during degradation, sodium valproate will be released in advance. This can also be corroborated by the accelerated release of sodium valproate in 10-20 days in Figure 6.

(2) The release rate of perampanel from the composite microspheres was faster than that from the single microspheres, especially in the early stage when sodium valproate had not been completely released. This may be because the aqueous solution of sodium valproate is alkaline, which will promote the degradation of PLGA to a certain extent.

The test results of the drug loading amount and encapsulation efficiency of each active ingredient of the microspheres prepared in Examples 1-5 and Comparative Example 1 are shown in Table 2 below.

Table 2 Test results of drug loading and encapsulation efficiency of perampanel microspheres

As can be seen from Table 2, based on the method provided by the present invention, perampanel has a very high encapsulation rate (>90%) in all control examples and embodiments. Only the encapsulation rate of sodium valproate in Examples 2 and 3 can reach more than 80%, which meets the requirements of the pharmacopoeia. In other words, when the theoretical drug loading (Example 4) and the feed amount (Example 5) increase, the encapsulation rate of sodium valproate will be greatly reduced. This may be because when there is too much sodium valproate in the inner aqueous phase (for example, when the ratio or feed is increased), the inner aqueous phase is easy to form through holes inside the microspheres, resulting in the loss of sodium valproate during the preparation process.

In summary, the present invention uses water-in-oil-in-water type emulsion droplets as templates to prepare single microspheres loaded with perampanel and compound microspheres loaded with perampanel and sodium valproate by microfluidic emulsion template method. The present invention can adjust the gradient release mode of the single microspheres and compound microspheres by adjusting the microstructure of the microspheres.

The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims (10)

1. A preparation method of a pirenzenenaphthalene long-acting controlled release microsphere is characterized by comprising the following steps of: the preparation method of the pirenzenepamil long-acting controlled release microsphere takes water-in-oil-in-water type composite emulsion drops as a template and adopts a microfluidic emulsion template method, and comprises the following steps:

step 1, preparing an inner water phase: the inner water phase comprises an amphiphilic high polymer material slow-release regulator and an inner water Xiang Yuanliao good solvent, and the amphiphilic high polymer material slow-release regulator is dissolved in the inner water Xiang Yuanliao good solvent to obtain the inner water phase; the mass fraction of the amphiphilic polymer material slow release regulator in the inner water phase is 0.05-2.0wt%; the amphiphilic polymer material slow release regulator is at least one selected from sodium carboxymethyl cellulose, polyvinylpyrrolidone, poloxamer and polyethylene glycol;

Step 2, preparing an oil phase: the oil phase comprises pirenzenenaphthalene and derivatives thereof, a polylactic acid-glycolic acid copolymer and an oil phase good solvent, and the pirenzenenaphthalene and derivatives thereof and the polylactic acid-glycolic acid copolymer are dissolved in the oil phase good solvent to obtain the oil phase; the mass fraction of the polylactic acid-glycolic acid copolymer in the oil phase is 5.0-20.0 wt%, and the mass ratio of the pirrennet and the derivatives thereof to the polylactic acid-glycolic acid copolymer is 1: (1-4);

Step 3, preparing an external water phase: the external water phase comprises a surfactant and an external water phase raw material good solvent, and the surfactant is dissolved in the external water phase raw material good solvent to obtain the external water phase; the mass fraction of the surfactant in the external water phase is 0.1-2.0 wt%;

and 4, preparing the pirenzepine long-acting controlled release microsphere by a microfluidic emulsion template method: introducing the inner water phase, the oil phase and the outer water phase prepared by the steps into the inlets corresponding to the microfluidic chip at a constant flow rate, shearing the three phases of the inner water phase, the oil phase and the outer water phase in the microfluidic chip, and obtaining water-in-oil type double emulsion droplets at the outlets of the microfluidic chip;

step 5, collecting and solidifying the water-in-oil-in-water type multiple emulsion liquid drops to obtain multiple emulsion microspheres; and cleaning, freeze-drying the re-emulsion microsphere to obtain the pirenzenenaphthalene long-acting controlled release microsphere.

2. The method of manufacturing according to claim 1, characterized in that: the flow rate of the inner water phase in the step 4 is 200-600 mu L/min, the flow rate of the oil phase in the micro-fluidic chip is 20-60 mu L/min, and the flow rate of the outer water phase in the micro-fluidic chip is 400-3000 mu L/min.

3. The method of manufacturing according to claim 1, characterized in that: the inner water phase also comprises water-soluble antiepileptic drugs, and the mass fraction of the water-soluble antiepileptic drugs in the inner water phase is 2.0-15.0 wt%.

4. A method of preparation according to claim 3, characterized in that: the water-soluble antiepileptic drug is sodium valproate.

5. The method of manufacturing according to claim 1, characterized in that: the amphiphilic polymer material slow release regulator is sodium carboxymethyl cellulose.

6. The method of manufacturing according to claim 1, characterized in that: the molecular weight of the polylactic acid-glycolic acid copolymer is 5000Da to 120000Da, and the molecular weight distribution coefficient is 1 to 10.

7. The method of manufacturing according to claim 1, characterized in that: the surfactant is at least one selected from polyvinyl alcohol, polyvinylpyrrolidone and poloxamer.

8. The method of manufacturing according to claim 1, characterized in that: the inner water Xiang Yuanliao good solvent and the outer water phase raw material good solvent are water, and the oil phase good solvent is selected from any one of dichloromethane, ethyl acetate, chloroform and acetone.

9. A pirenzenenape long-acting controlled release microsphere, which is characterized in that: the preparation method according to any one of claims 1 to 8, wherein the particle size of the pirenzepine long-acting controlled release microsphere is 120+/-10 μm.

10. The use of the pirenzepine long-acting controlled release microsphere prepared by the preparation method of any one of claims 1 to 8 or the pirenzepine long-acting controlled release microsphere of claim 9 in the preparation of a pirenzepine injection.